PALACKY UNIVERSITY OLOMOUC

Faculty of Natural Sciences

Department of Biochemistry

Target identification of potential antitumor drugs

inducing changes in the cell cycle

MASTER THESIS

Author: Alžběta Kameníčková

Study program: B1406 Biochemistry

Study branch: Biochemistry

Study mode: Full – time

Supervisor: MUDr. Petr Džubák, Ph.D.

Submitted: 26.4.2010

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I declare that this master thesis has been written solely and all the sources used in this

thesis are cited and included in the reference part.

In Olomouc 2. 5. 2010

…………………………….

Acknowledgments

First of all I would like to express thanks to my supervisor MUDr. Petr Džubák, Ph.D.

for special leading, valuable reminder, advices and gained experiences during my

work. Other thanks are regarded to all employees of Laboratory of Experimental

Medicine, Department of Pediatrics, Faculty of Medicine, Palacký University and

Faculty Hospital in Olomouc for their help. Last but not least, I would like to thank my

parents and roommates for friendly atmosphere in which this work could originate.

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Bibliografická identifikace:

Jméno a příjmení Alžběta Kameníčková

Název práce

Typ práce:

Identifikace cílů potenciálních protinádorových léčiv indukující změny v buněčném cyklu

Diplomová

Pracoviště Laboratoř experimentální medicíny při Dětské klinice LF UP a FN Olomouc

Vedoucí práce MUDr. Petr Džubák, Ph.D.

Rok obhajoby práce 2010

Abstrakt Počet případů onemocnění rakovinou se neustále zvyšuje, proto je vyvíjeno úsilí identifikovat nová léčiva vykazující protinádorovou aktivitu. Předmětem této práce je identifikovat cíle potenciálních protinádorových derivátů kyseliny betulinové u nichž byly pozorovány zajímavé změny v buněčném cyklu. Kyselina betulinová se řadí mezi přírodní triterpenoidní sloučeniny a je prokázána její vysoká cytotoxická účinnost vůči nádorovým buňkám. Vzhledem k jejím farmakologickým vlastnostem, které nejsou příliš výhodné, se laboratoře snaží o syntézu lépe biologicky dostupných a účinějších derivátů. V naší práci jsme identifikovali geny, které byly ovlivněny účinkem derivátu kyseliny betulinové JS8. V předchozí práci byly identifikovány deriváty kyseliny betulinové, které indukovaly bloky v S a G2/M fázi buněčného cyklu, včetně korespondující fosforylace serinu 10 na histonu H3. Také byl pozorován vliv na syntézu DNA a RNA ve smyslu její aktivace/inhibice. V předkládané práci se zabýváme genovou expresí na základě působení derivátu JS8 na buněčnou nádorovou lini lymfoblastické leukemie CEM pomocí Affymetrix technologie a tím zjistit potenciální cíle účinku tohoto derivátu.

Klíčová slova Kyselina betulinová, rakovina, exprese genů

Počet stran 69

Počet příloh 0

Jazyk

Anglický

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Bibliographical identification:

Author’s first name and surname Alžběta Kameníčková

Title Target identification of antitumor drugs inducing changes in the cell cyle

Type of thesis

Master

Department Laboratory of Experimental Medicine, Department of Pediatrics, Faculty of Medicine, Palacký University and Faculty Hospital in Olomouc

Supervisor MUDr. Petr Džubák, Ph.D.

The year of presentation 2008

Abstract The number of cancer disease is still increasing and that’s why new potentially anti-tumor derivates should be tested. The aim of this work is to identify the targets of potential antitumor derivatives of betulinic acid which were observed to induce interesting changes in the cell cycle. Betulinic acid is natural triterpenoid compound and exhibits high cytotoxic activity against several tumor cells. But it’s pharmacological properties aren’t very good and that’s why there’s an effort to synthesize new derivates, which could be more bioavailable and effective. In our study we have identified number of genes that were affected by the action of betulinic acid derivative JS8. In previous work were identified derivatives inducing block in the S and G2/M phase of cell cycle instead of corresponding phosphorylation of the serine 10 on the histone H3. Affected, activated/inhibited DNA and RNA synthesis was observed. In the present work we deal with gene expression by exposure the lymphoblastic leukemia cell line CEM to JS8 derivative using Affymetrix technology, and thereby identify potential targets of action of JS8.

Keywords Betulinic acid, cancer, gene expression

Number of pages 69

Number of appendices 0

Language English

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Table of content

1. THEORETICAL PART .............................................................................................. 5

1.1 Active triterpenoids as antitumor agents ........................................................... - 9 -

1.1.2 Betulinic acid ................................................................................................ - 9 -

1.1.4 Ursolic acid ................................................................................................. - 14 -

1.1.5 Oleanic acid ............................................................................................... - 16 -

1.1.5.1 CDDO, CDDO-Me, CDDO-Im .............................................................. - 16 -

1.1.6 Glycyrrhetinic acid ...................................................................................... - 17 -

1.1.7 Boswellic acid ............................................................................................. - 18 -

1.1.8 Ginsenosides ............................................................................................. - 19 -

1.1.9 Ganoderic acid ........................................................................................... - 19 -

1.1.10 Cucurbitacins ........................................................................................... - 20 -

1.1.11 Avicins ...................................................................................................... - 20 -

1.1.12 Beta-aescin .............................................................................................. - 21 -

1.1.13 Ardisiacrispin (A+B) .................................................................................. - 22 -

1.2 Methods .......................................................................................................... - 23 -

1.2.2 UV-VIS spectroscopic analysis ................................................................... - 23 -

1.2.3 DNA microarray .......................................................................................... - 23 -

1.2.3.1 Affymetrix technology .......................................................................... - 24 -

2. PRACTICAL PART ............................................................................................. - 26 -

2.1 Chemicals, reagents, instruments .................................................................. - 27 -

2.2 Methods ......................................................................................................... - 28 -

2.2.1 Passage of the suspension cells ................................................................ - 28 -

2.2.2 Isolation of RNA ......................................................................................... - 29 -

2.2.3 Measuring of RNA concentration ................................................................ - 30 -

2.2.4 Whole transcript sense target labeling assay .............................................. - 30 -

2.2.5 UV-VIS spectroscopic analysis .................................................................. - 40 -

2.3 Results ........................................................................................................... - 41 -

2.3.1 Determination of RNA, cRNA concentration ............................................... - 41 -

2.3.2 Determination of cDNA concentration ......................................................... - 42 -

2.3.3 cDNA electrophoresis ................................................................................. - 43 -

2.3.4 Statistical evaluation ................................................................................... - 45 -

2.3.5 Identification and annotation of expressed genes ....................................... - 48 -

2.3.6 UV-VIS spectroscopy analysis ................................................................... - 50 -

2.3 Discussion ...................................................................................................... - 54 -

3. Conclusion .......................................................................................................... - 57 -

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References ............................................................................................................. - 59 -

Abbreviations .......................................................................................................... - 67 -

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Aims of the work

1. Try to find target identification of derivates of betulinic acid inducing changes in

the cell cycle.

2. Reveal genes and their changes in expression involved in cancer cell line CEM

by treatment with derivatives of betulinic acid.

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1. THEORETICAL PART

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1.1 Active triterpenoids as antitumor agents

Natural products are great reservoir of biologically active compounds. Extracts

from natural products have been a main source of folk medicines for thousands of

years, and even today, many cultures still use them for medicinal purposes. Among the

classes of identified natural products, triterpenoids, one of the largest families, have

been studied intensively for their diverse structures and variety of biological activities.

This work is aimed on triterpenoids, which antitumor activity was observed in the last

two years (2008-2009). Practical part is focused mainly on the betulinic acid and its

derivative.

1.1.2 Betulinic acid

Betulinic acid is naturally occurring pentacyclic triterpene belonging to lupane

family. It is widely distributed in many terrestrial plant species. First, it was assumed,

that betulinic acid is a selective cytotoxic compound against melanoma cells (Pisha et

al., 1995) but the antitumor cytotoxicity of BetA has been extensively studied over the

last years in a large variety of cancer cell lines, primary tumor samples and xenograft

mouse models. Recent studies showed antiproliferative activity against breast

adenocarcinoma (MCF-7 cells), as well as neuroblastoma (SKNAS),

rhabdomyosarcoma-medulloblastoma (TE671), lung carcinoma (A549), colon

adenocarcinoma (HT-29), multiple myeloma (RPMI8226), cervical carcinoma (HPCC).

(Fulda S., 2009). BetA also showed broad-spectrum cytotoxicity towards lung,

colorectal, breast, prostate and cervical cancer cell lines as well as drug-resistant colon

adenocarcinoma cell lines (Jung et al., 2007). Recent data showed potential antitumor

effect of BetA against AGS cells (gastric adenocarcinoma), where was detected the cell

cycle arrest and induction of apoptosis (Yang et al., 2010). Derivates of betulinic acid

and betulin were also tested against HepG2 (human hepatocellular carcinoma), Jukart

(human leukemia) and HeLa (human cervical adenocarcinoma), where the derivates

markedly inhibited the proliferation of these cell lines (Santos at al., 2009). Betulin, the

reduced congener of BetA, showed apoptotic effect on human lung cancer cells related

characteristic proteins. Recent study revealed some proteins that were differentially

expressed by using nano-HPLC/MS method. There is a list of proteins identify to be

down or up regulated by treating with betulin. Downregualted proteins were heat shock

protein 90 – alpha 2, poly (rC) – binding protein 1, enoyl CoA hydratase, isoform 1 of 3

– hydroxyacyl – CoA dehydrogenate type 2. Proteins that were analyzed to be

upregulated were aconitase hydratase, malate dehydrogenase and splicing factor

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arginine/serine – rich 1 (Pyo 2009). New observation of betulin studies investigate that

this compound has potent anti-tumor activity especially in combination with cholesterol

that sensitized cells to betulin-induced apoptosis. Comparing to betulin acid, there was

no effect of cholesterol on it (Mullauer et al., 2009). There is the effort to synthesise

new derivates of betulinic acid because of their better pharmacological and physico-

chemical properties. The derivate NVX-207 was described to be well tolerated with

significant anti-cancer activity in vivo and in vitro (Willmann et al., 2009).

I II

Figure 1: Structures of betulinic acid (I) and betulin (II)

It was revealed that BetA induces apoptosis via direct mitochondrial

perturbations (Fulda et al., 1997) This pathway is normally regulated by carefully

balanced interplay between pro and anti-apoptotic members of Bcl-2 family. Over

expression of pro-survival molecules, such as Bcl-2, Bcl-xL or Mcl-1 or deletion of pro-

apoptotic members, such as Bak and Bax, or alternatively deregulation of BH-3 only

molecules like Puma or Bim, is often in tumors and causes resistance of these cells to

intrinsic death stimuli (Adams & Cori, 2007). A typical decrease in Bcl-2 and cyclin D1

gene expression and increase on bax gene expression was observed in several cancer

lines treated with BetA (Rzeski et al., 2006). Subsequent studies on anticancer

mechanism of BetA revealed that it is a potent activator of the chymotrypsin like activity

of the proteasome (Huang et al., 2007). In addition, BetA decreases expression or

vascular endothelial growth factor (VEGF) and the antiapoptotic protein survivin in

prostate cancer cells (LNCaP) by activating selective proteasome-dependent

degradation of the transcription factor´s specifity proteins (Chintharlapalli et al., 2007).

Other studies show the relationship between NF-ĸB expression and BetA (Kasperczyk

et al., 2005). The transcription factor, NF-kB is a key mediator of the cellular stress

response and in the cells exposed to an anticancer therapy NF-kB typically activates

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survival pathways (Aggraval et al., 2006).

It was revealed that BetA cooperated with TRAIL to induce apoptosis in tumor

cells (Fulda et al., 2004). Though functional complementation, simultaneous stimulation

of mitochondrial pathway by TRIAL resulted in complete activation of effectors’

caspases, apoptosis and inhibition of clonogenic survival (figure 1). BetA and TRAIL

cooperation leads loss of mitochondrial membrane potential and release cytochrome c

and Smac from mitochondria. These reports suggest that using BetA as sensitizer in

chemotherapy, radiotherapy or TRAIL-based combination regiments may be a novel

strategy to enhance the efficacy of anticancer therapy (Fulda S. 2009). Betulinic acid

also inhibits in vitro an enzyme aminopeptidase N that is involved in the regulation of

angiogenesis, overexpressed in several cancer (Fulda S. 2009).

Figure 2: Activation of effectors’ caspases by TRAIL (Fulda S., 2009).

Recent findings also suggest an inhibitory role of NF-kB in carcinogenesis and

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tumorigenesis (Chen et al., 2007). It has been demonstrated that inhibition of NF-kB

activity in cancer cell lines could reduce cell proliferation and metastatic capabilities in

vivo (Haung et al., 2001). Treatment of cells with BetA resulted in significant inhibition

of NF-kB activation and that this effect was mediated via the IkBa pathway. BetA shows

potential for use as an agent targeting NF-kB blockage in androgen-refractory human

prostate cancer cells (Rabi et al., 2008). BetA is also reported as an inhibitor of human

topoisomerases I and II, but it does not show synergistic effects with other topo-I

inhibitors. In contrast, BetA inhibited the formation of topo-I DNA cleavable complexes

induced by camptothecin, stautosporine and etoposide in prostate cancer cells. This

study also indicated that the tumor death mediated by BetA can be counteracted by the

mitogen-activated protein kinase 1 (MAPK1) inhibitor U0126 in melanoma cells

(Ganguly et al., 2007).

1.1.3 Lupeol

Lupeol is very common lupane-triterpene widely distributed in the plant

kingdom, occurs across a multitude of taxonomically diverse genera and geographical

areas. In earlier studies was investigated induction of apoptosis and cell arrest in

various cancer cells, further molecular-phenomena signaling pathway were also

revealed.

Figure 3: structure of lupeol

The following in vitro and in vivo studies show similar results that lupeol

inhibited the growth of CaP cells and significantly reduced testosterone-induced

prostate changes in mice (Prasad et al., 2008). Lupeol also significantly reduces the

proliferative and clonogenic potential of androgen-sensitive as well as androgen-

intensive CaP cells by modulating ß-catenin-signaling pathway in which lupeol

decreased the level of ß-catenin in nuclei of CaP cells thus inhibiting the transcription

of proliferation-associated genes in CaP cells (Saleem et al., 2009). One way of

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apoptosis induced by lupeol is characteristic by targeting Fas receptor and

consequently activation the extrinsic apoptotic pathway via caspase 8. Lupeol at 20 μM

concentration increased the expression of the FADD protein and Fas receptor on

androgen sensitive prostate cancer cells (Saleem et al., 2005). Apoptosis resistance is

reported to be overcome by activation of tumor suppressor genes and oncogene

inhibition when lupeol is added. When is lupeol at a concentration of 30 μM there is

also significant reduction of Ras protein that is commonly overexpressed and it leads to

subsequent inhibition of the PI3/Akt pathway that is known for promoting cell growth.

Coincidentally, observation of decreased levels of NF-κB and expression of phospho-

p38 MAPK, which triggers an antiapoptotic response, were observed (Saleem et al,

2005). Lupeol is also reported to inhibit the growth and proliferation of highly

aggressive pancreatic cancer and melanoma cells and it also inhibits the skin

tumorigenesis in mouse model through the modulation of signaling pathways such as

PI3K/AKt, nuclear factor kappa B1 and Ras/PKCα (Saleem et al., 2009). Another study

investigated that lupeol causes apoptosis in chemoresistant pancreatic cells by

recombinant TRAIL via suppression on cFLIP (Murtaza et al., 2009).

Figure 4: Multiple signaling pathways induced by lupeol (Chaturvedi et al., 2008).

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1.1.4 Ursolic acid

Ursolic acid is a prevalent pentacyclic triterpenoid carboxylic acid (figure 5).

Figure 5: Ursolic acid

Recent results of pro-apoptotic activity of ursane acid indicated a modulation of the Bcl-

2 protein family due to a suppression NF-kB, CREB, ATF-2, c-Fos and pro-

inflammatory cytokines by ursolic acid (50 μM) in B16 - F10 mouse melanoma cells.

Induction of apoptosis was accompanied by activation of p53 and caspase-3 gene

expression (figure 2) (Manu et al., 2008). Inhibitory effects were also observed on both

the PI3-Akt and MAPK P44/42 pathways, which are associated with cell apoptosis in

endometrial cancer cell lines (SNG-II and HEC108) (Achiwa et al., 2007). Ursolic acid

also inhibited endogenous reverse transcriptase (RT) in melanoma (A375) and

anaplastic carcinoma (ARO) cell lines, in which was shown down-regulation of c-myc

and cyclin D-1 by ursolic acid (Bonaccorsi et al., 2008). Another study investigated that

ursolic acid directly inhibits the interaction between ZIP/p62 and PKC-z and further

reduced matrix metalloproteinase-9 (MMP-9) activity and expression via blockade of

the NF-kB-dependent pathway induced by IL-1β or TNF-α in C6 glioma cells.

Therefore, it is suggested that inhibition of matrix metalloproteinase-9 expression by

ursolic acid is responsible for dysfunctions in tumor invasion. On the other hand, MMP-

9 does not only influence tumor invasion, but it also degrades the extracellular matrix,

including collagens, laminins, and the blood–brain barrier of angiogenesis in glial

tumors (Huang et al., 2009). Furthermore, it was demonstrated that ursolic acid

effectively induces apoptosis, probably via JNK-mediated Bcl-2 phosphorylation and

degradation in the androgen-independent human prostate cancer cell line DU145.

These findings suggest that UA-induced apoptosis could be a potential therapeutic

approach in advanced prostate cancer (Zhang et al, 2009).

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In addition, astilbortriterpenic acid, new triterpenoid compound structurally very

similar to ursolic acid was identified. This compound was tested against human breast

carcinoma Bcap37 cells, human cervical cancer HeLa cells, human hepatoma HepG2

cells, human ovarian carcinoma HO-8910 cells, human leukemia K562 cells, human

lung adenocarcinoma PAA cells, human gastric carcinoma SGC7901 cells, and murine

leukemic P388 cells where has been shown cytotoxic effect and induction of apoptosis

(Zhang et al., 2007). Subsequently, another study revealed mechanism of action of this

compound and investigate the involvement of mitochondria caspase activation and

reactive oxygen species, loss of mitochondrial transmembrane potential,

downregulation of Bcl-2 and up-regulation of Bax during induction of apoptosis by

astilbortriterpenic acid (Zhang et al., 2009).

Figure 6: Effect of ursolic acid of pro- and anti-apoptotic pathway (Manu et al., 2008).

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1.1.5 Oleanic acid

Oleanic acid (80 μM) showed apoptosis induction in leukemia cells (HL60) via

activation of caspase-9 and caspase-3 accompanied by the cleavage of poly (ADP-

ribose) polymerase (Zhang et al., 2007).

Results of another study revealed that ursolic acid, oleanic acid, and a mixture of

ursolic acid and oleanic acid (figure 7) significantly reduced 1,2 dimethylhydrazine-

induced aberrant crypt foci (ACF), which are considered to be an important early step

in colorectal carcinogenesis and tumors of the rat colon (Furtado et al., 2008).

Figure 7: oleanic acid

1.1.5.1 CDDO, CDDO-Me, CDDO-Im

Synthetic A-ring modified oleanic acid analogues with improved cytotoxicity were

pursued and 2-cyano-3, 12-dioxoolean-1,9-dien-28-oic acid (CDDO) as well as its C-28

methyl ester, CDDO-Me were designed and synthesized. Further derivatization of

CDDO was designed (CDDO-Im). CDDO and CDDO-Me are currently in phase I/II

clinical trials for cancer treatment. CDDO-Me and other derivatives with modifications

at the C-28 position induce apoptosis of human myeloid leukemia (Ito et al., 2000),

multiple myeloma (Ikeda et al., 2004) osteosarcoma (Ito et al., 2001), lung cancer (Kim

et al., 2002), breast cancer (Lapillonne et al., 2003) and pancreatic cancer (Samudio et

al., 2005).

CDDO-Me and related derivatives induce apoptosis in vitro by increasing reactive

oxygen species and decreasing intracellular glutathione. CDDO-Me depletes

intracellular GSH, resulting in ER stress. Subsequently, it activates JNK, leading to

CHOP-dependent DR5 upregulation and apoptosis (Zou et al., 2009). Recent results

also showed that CDDO-Me directly inhibits STAT3. These findings thus indicate that

CDDO-Me blocks the JAK1 STAT3 pathway by directly inhibiting both JAK1 and STAT3

(Rahmad et al., 2008). Furthermore, CDDO-Me at nanomolar or low micromolar

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concentrations can induce tumor cell death by a GSK3-mediated pathway indicates

that CDDO-Me could overcome cell death resistance in tumors unresponsive to

conventional chemotherapy (Vené et al., 2008). Another study showed that CDDO-Im

exerted a marked cytotoxic activity against primary ovarian cancer cells and a panel of

ovarian cancer cell lines, including chemoresistant A2780/ADR cells. It is proposed that

the inhibition of STAT3 activation induced by CDDO-Im determines a decrease of c-

FLIP levels, thus making the tumor cells sensitive to caspase-8 mediated cell death

(Petronelli et al., 2009).

1.1.6 Glycyrrhetinic acid

Glycyrrhetinic acid (GA) is a pentacyclic triterpenoid acid that is found as a conjugate

(glycyrrhizin) in licorice extracts. GA (figure 8) is one of the medicinally active

compounds of licorice and exhibits multiple activities which include the enhancement of

corticosterone levels which contributes to decreased body fat index in human studies

with GA (Armanini et al., 2003).

Figure 8: glycyrrhetinic acid

Although several studies have found that glycyrrhetinic acid give rise to cytotoxic or

apoptotic activities, most of the results have shown only moderate or low potency.

Thus, further investigation has focused more on the preparation of active derivatives

and chemosensitizing activity like 2-cyano-3, 11-dioxo-18b-olean-1, 12-dien-30-oic acid

(CDODA) and its methyl ester (CDODA-Me) from glycyrrhetinic acid and it was

demonstrated that these compounds are highly cytotoxic in colon, prostate (Papineni et

al., 2008) bladder, and pancreatic cancer cells (Chadalapaka et al., 2008). The most

active member of these glycyrrhetinic derivatives is CDODA-Me (18b isomer) which

activates peroxisome proliferator-activator receptor g (PPARg) and induces both

receptor dependent and independent responses in colon and prostate cancer cells

(Papineni et al., 2008). In subsequent study was shown that CDODA-Me inhibits

growth and induces apoptosis in pancreatic cancer cells. Although CDODA-Me

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activates PPARg in Panc28 and Panc1 cells where the induction of growth inhibitory

and proapoptotic proteins and activation of multiple kinase activities is receptor-

independent (Jutooru et al., 2009).

1.1.7 Boswellic acid

The most explored boswellic acid (figure 9) is compound commonly known as AKBA (3-

O-acetyl-11-keto-β-boswellic acid).

Figure 9: 3-O-acetyl-11-keto-β-boswellic acid

A recent discovery revealed that AKBA showed moderate to low toxicity against human

skin-derived normal cell lines (Burlado et al., 2008). However, AKBA induced apoptosis

also in PC-3 and LNCaP cells through a death receptor (DR-5)-mediated pathway,

which is a signal transduction, cascade involving the activation of caspase-8 and

caspase-3 in apoptosis (Lu et al., 2008). Another study investigated that AKBA inhibited

STAT 3 activation in human multiple myeloma cancer cells that in turn leads to the

suppression of gene products involved in proliferation (cyclin D1), survival (Bcl-xL, Bcl-

2 and Mcl-1) and angiogenesis (VEGF). According to these results, AKBA is suggested

to be a novel inhibitor of STAT 3 activation and has potential in cancer treatment

(Kunnumakkara et al., 2009). Finally, it was discovered that AKBA inhibits human

prostate tumor growth through inhibition of angiogenesis induced by VEGFR2 signaling

pathways (Pang et al., 2009).

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1.1.8 Ginsenosides

Ginsenosides are a series of compounds comprised of more than 60 triterpenes and

related glycosides isolated from the leaves, stems, berries and roots of different Panax

species. Recently ginsenosides from natural resources were evaluated for their

antitumor activity. But comparing to another active triterpenoids, most ginsenosides

showed very low cytotoxicity against various cancer cells. The anti-angiogenic

properties of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol were evaluated in vivo

angiogenesis assay using HUVECs, in which the compounds showed strong anti-

proliferative activity (Usami et al., 2008). Another type, ginsenosides R3 (GS-R3), were

also tested for their inhibitory effect on the process of tumor angiogenesis and it was

revealed that GS-R3 down-regulate the expression of VEGF and its kinase insert

domain receptor KDR in human lung cancer SK-MES-1 cell line (Wang et al., 2009).

Furthermore, 20(S)-25-methoxyprotopanaxadiol induces apoptosis and cell cycle arrest

in the G1 phase, inhibited proliferation of T98G, HPAC, A549 and PC-3 cell lines and

was 5- to 15-fold potent than 20(S)-protopanaxatriol (Wang et al., 2008). In addition,

three semi-synthetic derivates with a C-20 sugar moiety showed significant cytotoxicity

against MCF, SK-MEL-2 and B16 cancer cell lines (Lei et al., 2007).

1.1.9 Ganoderic acid

Ganoderic acid (figure 9) was assed for anti-proliferation activity and showed an IC50

value of 17,3 μM against human cervical carcinoma cells (HeLa), which were due to

treatment with this compound arrested at the G2/M phase of the cell cycle with

apoptosis.

Figure 9: ganoderic acid

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A simultaneous proteomic study led to the identification of 21 genes regulated by

ganoderic acid (Yue et al., 2008). Structurally related ganoderic acid A, F and H (GA-A,

GA-F, GA-H) were identified and tested on human breast cancer cells where results

suggested that GA-A and GA-H act through the inhibition of transcription factors AP-1

and nuclear factor kappa B, leading to down-regulation of Cdk4 expression and

suppression of secretion of urokinase-type plasminogen activator (Jiang et al., 2008).

The results of one study demonstrated that another ganoderic acid-Me effectively

inhibited tumor invasion, and it might act as a new matrix metalloproteinase 2/9

inhibitor for anti-metastasis treatment of carcinoma cells (Chen et al., 2008).

1.1.10 Cucurbitacins

Cucurbitane-type triterpenes, isomers of lanostanes, constitute a group of diverse

substances, are known for their cytotoxicity and anticancer activity. Cucurbitacins have

been reported to inhibit several types of cancers including those originating in the

prostate, lung and breast as well as choriocarcinoma, glioblastoma multiform, and

myeloid leukemia cells. Among the various cucurbitacins, the most abundant is

cucurbitacin B (CuB).

Recent studies showed, that the antiproliferative activity of CuB against breast cancer

cells in vitro and in vivo, irrespective of their ER, Her-2/neu or p53 status, is intriguing

and is worthy of further investigation (Wakimoto et al., 2008). It was also showed that

cucurbitacin B possessed inhibitory effects on laryngeal squamous cell carcinoma

(Hep-2), which can be due to the inhibition of STAT-3, a transcription activator in cell

growth (Liu et al., 2008). Another recent study investigated that cucurbitacin B has

profound antipancreatic cancer activity in vitro and in vivo. The drug induces cell cycle

arrest and apoptosis in pancreatic cancer cell lines by inhibiting the JAK/STAT pathway,

increasing the expression of p21/WAF1 independently of p53 activity, inducing the

caspase cascade and the drug also supports the proliferative activity of the nucleoside

analogue gemcitabine (Thoennissen et al., 2009).

1.1.11 Avicins

Avicins are a family of plant triterpene electrophiles with ability to trigger apoptosis-

associated tumor cell death, and suppress chemical induced carcinogenesis by its anti-

inflammatory, anti-mutagenic, and antioxidant properties (Xu et al., 2009). In vitro,

avicins inhibit cell growth and induce apoptosis in leukemia and epithelial cancer cell

lines. In a mouse skin carcinogenesis model, avicins have been shown to suppress

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both initiation and promotion phases of chemical carcinogenesis, as well as UV light B

damage with resultant suppression of oxidative DNA and lipid damage (Haridas et al.,

2004).

Avicins induce apoptosis by affecting mitochondrial function and activating the

intrinsic caspase pathway and close the voltage-dependent anion channel,

subsequently leading to lower cell energy metabolism and triggering cell apoptosis by

permeabilization of the outer mitochondrial membrane and release of cytochrome c

(Haridas et al., 2007). Avicins also suppress multiple pro-inflammatory components of

the innate immune system, including the transcriptional factor NF-kB (Haridas et al.,

2001), the phosphoinositide-3 kinase/AKT signaling pathway (Mujoo et al., 2001), as

well as heat shock proteins (Gaikwad et al., 2005). Recent study evaluated the anti-

tumor effects of avicin D on induction of apoptosis and modulation of signal transducer

and activator of transcription-3 (STAT-3) and apoptosis-related proteins in CTCL cell

lines and patients’ Sézary cells. These data suggest that selective induction of tumor T-

cell apoptosis, inhibition of STAT-3 activation and downregulation of bcl-2 and survivin

underline results demonstrate that beta-aescin is a potent natural inhibitor of cell

proliferation and inducer of apoptosis in HL-60 acute myeloid leukemia cells and the

therapeutic potential of avicin D in patients with Sézary syndrome (Zhang et al., 2008).

Another recent study investigated that avicin D might lead to use of raft-dependent and

intracellular activated Fas-mediated killing in cancer chemotherapy, representing a new

way to target tumor cells (Xu et al., 2009). It was also revealed that avicins can induce

not only apoptotic cell death, but also autophagic programmed cell death by depletion

of cell energy supply. These results showed that even when proapoptotic genes are

deleted or caspases are inhibited, avicins are able to continuously trigger cell death,

implicating the potentional therapeutic application of avicins in apoptosis – resistant

cancers (Xu et al., 2007).

1.1.12 Beta-aescin

Beta-aescin, a natural triterpenoid saponin isolated from the seed of Chinese horse

chestnut (Aesculus chinensis), is known to generate a wide variety of biochemical and

pharmacological effects. In recent study was investigating the antiproliferative and

apoptotic effects of beta-aescin in human chronic myeloid leukaemia K562 cell line in

vitro. The results showed that beta-aescin exhibited potent dose- and time-dependent

anti-proliferative effects in K562 cells. Morphological evidence of apoptosis, a

significant increase of annexin Vţ and PI7 cells (early apoptotic) and apoptotic DNA

fragmentation were observed in cells treated with beta-aescin. It was also observed,

- 22 -

that beta-aescin could lead to an accumulation of sub G1 population in K562 cells, and

suggesting a potential G1 phase accumulation in cell cycle profile of K562 cells. These

findings revealed that beta-aescin is a potent natural inhibitor of proliferation and

inducer of apoptosis in K562 cells, and beta-aescin may be a candidate lead

compound to explore potential antileukemia drugs (Niu et al., 2008). Another study

revealed the effect of mechanism of beta-aescin and 5-fluorouracil on human

hepatocellular carcinoma SMMC-7721 cells. This observation led to conclusion that the

mechanism of action could be through the synergistic arrest of the cell cycle, induction

of apoptosis, caspase 3,8 and 9 activation and down-regulation of Bcl-2 expression

(Ming et al., 2010).

1.1.13 Ardisiacrispin (A+B)

Ardisiacrispin (A + B) is a mixture of ardisiacrispins A and B, derived from Ardisia

crenata with a fixed proportion (2:1). The recent study showed that this mixture has

effect on proliferation of several human cancer cells. The (IC50) of ardisiacrispins (A +

B) were in the range of 0.9–6.5mg/ml by sulphorhodamine B-based colorimetric assay,

in which Bel-7402 was the most sensitive cell line. Ardisiacrispin (A + B) could inhibit

the proliferation of human hepatoma cells (Bel-7402) via inducing apoptosis and

disassembling microtubule. The finding that ardisiacrispins (A + B) has a remarked

anticancer activity on Bel-7402 cells also opens interesting perspectives for further

exploration of the triterpenoid saponins and compounds of as potential anticancer

agents (Li et al., 2008).

- 23 -

1.2 Methods

1.2.2 UV-VIS spectroscopic analysis of interactions between cytochrome c

and betulinic acid derivates

This method was used for monitoring of interactions between cytochrome c and

another derivates of betulinic acid. Triterpenoid compounds, especially betulin and

betulinic acid are well known for their property to decrease mitochondrial potential and

release cytochrome c from mitochondria. Previous study revealed that 3β,28-diacetoxy-

18-oxo-19,20,21,29,30-pentanorlupan-22-oic acid (code JS8) compound form non-

covalent complex with cytochrome c (Dzubak et al., 2007) and other several derivates

were analysed. The changes in cytochrome c absorption spectra caused by the

respective interactions with derivates were visualized as the difference spectra of

betulinic acid derivatives (code JS3x) treated samples of cytochrome c. Differences in

the maximum absorption were used to quantify the effects on the formation/stability of

cytochrome c/JS3 complexes. The range of wave length where the maximal absorption

of cytochrome c occurred was from 350 to 575 nm. The figure 10 shows a typical

absorption spectrum of cytochrome c.

Figure 10: absorption spectrum of cytochrome c

1.2.3 DNA microarray

DNA microarrays is said to be modern platform enabling high sophisticated analysis of

the gene expression, expression of specific exons, microRNA, DNA methylation,

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- 24 -

analysis of nucleotide polymorphism and others in the range of the whole genome or

transcriptome. This method is very important for understanding in the complex access

the complicated mechanism and signal pathways that play role in biological processes.

A lot of data can be obtained during this analysis to provide overview on the

complicated processes such as cancerogenesis, metastasis or drug resistant of the

patient on the treatment. One of the most significant contributions of this method is fact

that there are new staging parameters that are able to identify the patients with high or

low risk on the basis of molecular description of the tumor. As for this work, DNA

microarray was used for the potential target identification and clarification the

mechanism of action of new potential antitumor drugs by comparing the gene

expression of tested tumor cell lines in the presence of the drug.

1.2.3.1 Affymetrix technology

Expression microarrays use probes targeting specific genes based on nucleotide

sequence complementarity to quantitatively measure mRNA levels for tens of

thousands of genes. Each set of the probe contains 22 oligonucleotides representing

one gene where 11 of them are perfect matched and precisely copy of gene sequence

is provided. On the other hand, next eleven oligonucleotides are mismatched and they

differ from each other by one nucleotide in central position (Li & Wong, 2001).

Genetic information from the human is transferred into the gene chip probe arrays by

using process called photolithography. This manufacturing process uses a light to

selectively activate the chip surface by the photochemical reaction with the DNA

building blocks. Repeated cycles of selectively activation of the surface in three

determined patterns allow many different DNA probes to be built up in a small number

of steps. As the size of the feature (the square locations on the array) is reduced, the

amount of information contained on the single gene chip array increases exponentially.

As first, high quality of RNA from sample is extracted and then is reverse transcribe

into more stable complementary DNA (cDNA) with subsequent labeling and

fragmentation. Using the gene chip fluidic station, a test sample is introduced into the

DNA probe array where hybridization occurs. Each probe on the chip is a single

stranded DNA. Hybridization occurs when two complementary strands of DNA come

together to form a complex. When a single stranded sample is washed over the

surface, the sample will bind to its complementary strand. After this hybridization step,

the chips are put into the laser scanner to electronically capture the data. The chip is

scanned with the laser activating fluorescence dyes on the sample´s complementary

DNA. The computer captures this information and calculates the ratio of each spot.

- 25 -

Since the sequence of each location on the chip is known, it is possible to determine

the sample DNA sequence and to see if a target gene is present and whether it

contains mutation (http//media.affymetrix.com).

Figure 10: Steps involved in the Affymetrix technology

(www.umassmed.edu/gcf/indes.aspx)

- 26 -

2. PRACTICAL PART

- 27 -

2.1 Chemicals, reagents, instruments

Reagents:

GeneChip® WT sense target labeling and control reagents contains one of each of the

following kits - GeneChip® eukaryotic Poly-A RNA control kit , GeneChip® WT cDNA

synthesis and amplification kit, GeneChip® WT terminal labeling kit, GeneChip®

sample cleanup module, GeneChip® IVT cRNA Cleanup Kit, GeneChip® hybridization

Control Kit (Affymetrix 900652), GeneChip® eukaryotic Poly-A RNA control kit contains

Poly-A control stock and Poly-A control dilution buffer (Affymetrix 900433), GeneChip®

WT cDNA synthesis and amplification kit Sub-kit 1: GeneChip® WT cDNA synthesis

kit contains T7-(N)6 primers, 2.5 μg/μL, 5X 1st strand buffer, DTT 0.1M, dNTP, 10 mM,

RNase Inhibitor, SuperScript™ II, MgCl2 1M, DNA polymerase I, RNase H, random

primers, 3 μg/μL, dNTP+dUTP, 10 mM, RNase-free water, Sub-kit 2: GeneChip® WT

cDNA amplification kit contains 10X IVT Buffer, IVT NTP Mix, IVT enzyme mix, IVT

control (Affymetrix 900673), GeneChip® WT terminal labeling kit contains 10X cDNA

fragmentation buffer, UDG 10 U/μL, APE1 1,000 U/μL, 5X TdT buffer, TdT, 30 U/μL,

DNA labeling reagent 5 mM, RNase-free water (Affymetrix 900671), GeneChip® IVT

cRNA cleanup kit contains IVT cRNA cleanup spin columns, IVT cRNA binding buffer,

IVT cRNA wash buffer 5 mL concentrate, RNase-free water, 1,5 ml collection tubes (for

elution), 2 ml collection tubes (Affymetrix 900547), GeneChip® sample cleanup module

contains cDNA Cleanup Spin Columns, cDNA Binding Buffer, cDNA Wash Buffer 6ml

concentrate, cDNA elution buffer, IVT cRNA cleanup spin columns, IVT cRNA binding

buffer, IVT cRNA wash buffer 5 ml concentrate, RNase-free water, 1,5 ml collection

tubes (for elution), 2 ml collection tubes, 5X fragmentation buffer (Affymetrix 900371),

GeneChip® hybridization control kit contains 20X hybridization controls 3 nM control

oligo B2 (Affymetrix 900454), GeneChip® hybridization, wash and stain kit containing

pre-hybridization mix, 2x hybridization mix, DMSO, nuclease-free water, stain cocktail

1, stain cocktail 2, array holding buffer (Affymetrix 900720), wash buffer A (Affymetrix

900721), wash buffer B (Affymetrix 900722), absolute ethanol (Serva), RNA 6000 nano

kit (Agilent), TRI reagent (Sigma T9242), pyridine (Sigma P3776), cytochrome c from

horse heart (Sigma 105201), Medium RPMI-1640 (Sigma), fetal bovine serum (Biocom,

CZ), streptomycine 100 µg/ml (Sigma S 9137), peniciline 100 µg/ml (Biotika, SK),

propidium iodide (Sigma), ribonuclease A (Sigma).

Instruments:

NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies), GeneChip®

Hybridization Oven 645 (Affymetrix), Centrifuge (Eppendorf 5810R), Picofuge

- 28 -

(Eppendorf), GeneChip® Fluidics Station 450 (Affymetrix), GeneChip® Scanner 3000

(Affymetrix), GeneChip® AutoLoader with external barcode reader (Affymetrix),

thermocycler (Biotech), Bioanalyzer 2100 (Agilent), heating blocks (Eppendorf), vortex

(Genie), biohazard box (Forma Scientific), PowerWave spectrophotometer (BIO-TEK),

pH meter (Metler Toledo), light microscope, CO2 incubator (Jouan)

Solutions:

10 x concentrated PBS

80 g NaCl, 32,1 g Na2HPO4 . 12 H2O, 2 g KH2PO4 dissolved in 700 ml of distilled water,

pH adjusted at 7,4 by HCl, fill with distilled water to reach 1 litre.

1 x PBS

Prepare by dilution of 10 x concentrated PBS with distilled water.

5 mM Pyridine/HCl, pH 6,0 buffer

Concentration of the stock solution is 0,01M. For preparation of 5 mM buffer take 50 ml

of the stock solution and fill with distilled water into the final volume 100 ml. Adjust pH

on 6,0 with HCl.

4 μM cytochrome c

For 50 ml of buffer 2,46 mg of cytochrome c was needed

2.2 Methods

2.2.1 Passage of the suspension cells

Standardise cell line CEM was used for flow cytometry analysis and for DNA

microarray. This line is cancer line of human lymphoblastic leukemia. The cells were

regularly passaged for their later using in experiments.

Protocol:

1. The cells were observed under microscope before passaging to see if the

contamination occurred.

2. The cultivation flask was shaked.

3. The cover of cultivation flask was disinfected.

4. The cells were put into 50 ml tube.

5. 2 ml of the cell suspension was inoculated on another 175 ml flask and 30 ml of

the culturing medium were added.

6. The flask was disinfected and put back into the incubator for other cultivation.

7. The removed cells were counted and used for experiments.

- 29 -

2.2.2 Isolation of RNA

Protocol:

1) Cells were counted and to reach the final concentration of 1 x 106/1 ml,

according amount of cultural medium RPMI was added.

2) 5,6 ml of the cell suspension was pipette into 25 cm2 culture flask.

3) Cells were incubated for 24 hours.

4) After cultivation time, the derivate of betulinic acid JS8 was added.

5) The incubation with derivate last 90 minutes.

6) After incubation, cells were centrifugated at 4oC for 10 minutes at 2000 rpm,

supernatant was aspirated.

7) Cells were washed with 20 ml of 1x PBS at the same speed, time and

temperature, supernatant was aspirated.

8) Cells were suspended in 1 ml of 1x PBS and transferred into 1,5 ml tube.

9) Samples were centrifugated at 2000 rpm for 4 minutes.

10) 1 ml of TRI reagent was added to each sample, mixed carefully and the mixture

was stored for 5 minutes at room temperature.

11) 200 µl of chloroform were added and then vortex was used until homogenous

mixture results.

12) Samples were incubated for 10 minutes and then centrifugated at 12000 rpm

for 15 minutes at 4oC.

13) The mixture was separated into three phases: 1. lower red (containing

proteins) 2. interphase (containing DNA) 3. upper aqueous phase (containing

RNA).

14) The upper phase was transferred into fresh tubes and 500 µl of isopropanol

was added to 500 µl of aqueous phase. Tubes were mixed by turning up and

down.

15) Samples were incubated at room temperature for 5 minutes and centrifugated

at 12000 rpm for 10 minutes at 4oC.

16) The supernatant was removed and 1,5 ml of 75% ethanol was added and the

pellet was washed by gentle shaking without degradation.

17) Samples were centrifugated at 12000 rpm for 5 minutes at 4oC.

18) The ethanol was removed and the pellet was left to dry.

19) Next step involved dissolving the pellet in water. Water addition was according

to the size of the pellet.

20) Samples were incubated at 60oC in dry bath incubator for 5 minutes.

- 30 -

2.2.3 Measuring of RNA concentration

Concentration of RNA was measured by nanodrop method. 1,5 µl of RNA solution was

used for measuring and water was used as a blank. Final concentration of RNA was

given in ng/µl.

Nanodrop spectrometer operation

1) The adapter power was switch on and the programme was opened by clicking

ND on the desktop.

2) The programme RNA-40 was selected.

3) 1,5 µl of DEPC water was loaded on nanodrop.

4) The blank was measured.

5) Water was removed by pipette and the surface of loading area was cleaned

with tissue paper.

6) 1,5 µl of sample was loaded, the name was given and clicked measure.

7) The value of RNA concentration was counted.

8) The sample of RNA was removed with pipette and cleaned with tissue paper.

9) Water was loaded and measured again.

10) The last step involved pressing exit, escape and save.

2.2.4 Whole transcript sense target labeling assay

This protocol was provided by Affymetrix.

1st day

Preparation of dilutions of poly-A RNA controls

Total RNA labeling protocol

First cycle, first-strand cDNA synthesis

First cycle, second-strand cDNA synthesis

First cycle, cRNA synthesis and cleanup

2nd day

Second cycle, first-strand cDNA synthesis

Hydrolysis of cRNA and cleanup of single-stranded DNA

Fragmentation of single-stranded DNA

Labeling of fragmented single-stranded DNA

Hybridization

3rd day

Washing, staining and scanning

1st day

- 31 -

A) Preparation of dilution of poly-A RNA controls

For this procedure, GeneChip® Eukaryotic Poly-A RNA Control Kit

1) 2 µl of poly-A RNA control stock were added to 38 µl of poly-A control dilution

buffer to make the first dilution (1:20)

2) The solution was mixed and spined to collect it at the bottom of the tube.

3) 2 µl of the first dilution were added to 98 µl of poly-A dilution buffer to make the

second dilution (1:50)

4) The solution was mixed and spined to collect it at the bottom of the tube.

5) 1 µg of total RNA was used as a starting material. 2 µl of the second dilution

were added to 98 µl of poly-A dilution buffer to make third dilution (1:50).

B) Preparation of T7-(N)6 primers/poly-A RNA controls

1) A fresh 250 ng/µl T7-(N)6 primers dilution was prepared (from a 2,5 µg/µl stock)

by adding the concentrated T7-(N)6 primers to the diluted poly-A RNA controls

using non-stick RNase-free microfuge tube as follows:

Table 1: first-cycle, primer/poly-A RNA controls

component

T7-(N)6 primers, 2,5 µg/µl 2 µl

diluted poly-A RNA controls 6 µl

RNase free water 12 µl

total volume 20 µl

2) The solution was flick-mixed, spined down and placed on ice.

C) Preparation of total RNA/T7-(N)6 primers/poly-A RNA controls

1) Total RNA and T7-(N)6 primers/poly-A RNA controls solution was mixed as listed

in table 2.

Table 2: first-cycle, total RNA/primer/poly-A RNA controls

component volume in 1 Rxn

Total RNA, 300 ng 3 µl

T7-(N) primers/poly-A RNA controls

solution 2 µl

Total volume 5 µl

2) The solution was flick-mixed, spined down the tube and incubated for 5 minutes

at 70oC. Then, the sample was cooled for 2 minutes at 4oC and spined down

and placed on ice.

D) First cycle, first strand cDNA synthesis

- 32 -

This procedure requires the use of the GeneChip® WT cDNA Synthesis Kit.

1) First-cycle, first strand master mix was prepared as shown in table3. The

enzyme SuperScript II was added last to the master mix and proceeded

immediately to aliquot into the tubes from procedure C.

Table 3: first-cycle, first-strand master mix

component volume in 6 Rxn

5x 1st strand buffer 12 µl

0,1 M DTT 6 µl

10 mM dNTP mix 3 µl

Rnase inhibitor 3 µl

SuperScript II 6 µl

Total volume 30 µl

2) 5 µl of the first-cycle, first-strand master mix were added to the tube containing

the concentrated rRNA reduced total RNA/poly-A controls/T7-(N)6 primers mix

from procedure C, flick mixed and spined down.

3) The reaction was incubated at:

25 oC for 10 minutes

42 oC for 60 minutes

70 oC for 10 minutes

4) The reaction was cooled to 4 oC at least 2 minutes before immediately

continuing to the first-cycle, second-strand cDNA synthesis.

E) First cycle, second strand cDNA synthesis

This procedure requires the use of the GeneChip® WT cDNA Synthesis Kit.

1) Fresh dilution of 17,5 mM MgCl2 was made by mixing 2 µl of 1 M MgCl2 with 112

µl of RNase-free water.

2) The first-cycle, second-strand master mix was prepared as described in table 4.

The RNase H and DNA polymerase I enzymes were added to the master mix

last and proceeded immediately to aliquot into the tubes from procedure D.

Table 4: first-cycle, second-strand master mix

component volume in 6 Rxn

Rnase-free water 28,8 µl

17,5 mM MgCl2 24 µl

10 mM dNTP Mix 2,4 µl

DNA polymerase I 3,6 µl

Rnase H 1,2 µl

Total volume 10 µl

- 33 -

3) 10 µl of the first-cycle, second-strand master mix were added to the reaction

tube from the first-strand cDNA synthesis reaction in procedure B for a total

reaction volume of 20 µl. The tubes were gently vortexed and spined down.

4) The reaction was incubated in a thermal cycler at:

16 oC for 120 minutes without heated lid

75 oC for 10 minutes with heated lid

5) The sample was cooled at least 2 minutes at 4oC before immediately proceeding

to the next procedure.

F) First-cycle, cRNA synthesis and cleanup

This procedure requires the use of the GeneChip® WT cDNA Amplification Kit and

the GeneChip® Sample Cleanup Module.

1) The IVT master mix was assembled in a separate tube at room temperature as

listed in table 5. The IVT enzyme mix was added to the master mix last and

proceeds immediately to aliquot into the tubes from procedure E.

There can be problem if a white precipitate is still present in the 10x IVT buffer

after thawing. It is recommended to incubate the tube at 37 oC until the

precipitate gets dissolved.

Table 5: First-cycle, IVT master mix

component volume in 6 Rxn

10x IVT Buffer 30 µl

IVT NTP Mix 120 µl

IVT enzyme mix 30 µl

Total volume 180 µl

2) 30 µl of the IVT master mix were added to each first-cycle cDNA synthesis

reaction sample from procedure E to final volume of 50 µl. The solution was

flick-mixed and briefly spined in a microfuge.

3) The reaction was incubated for 16 hours at 37 oC in a thermal cycler.

2nd day

1) The cleanup procedure for cRNA was proceeded using the cRNA cleanup spin

columns from the GeneChio Sample Cleanup Module.

2) 20 ml of ethanol (100%) were added to the cRNA wash buffer supplied in the

GeneChip Sample Cleanup Module.

3) 50 µl of RNase-free water were added to each IVT reaction to a final volume of

100 µl.

4) 350 µl of cRNA binding buffer were added to each sample and vortexed for 3

seconds.

5) 250 µl of 100% ethanol were added to each reaction and flick-mixed.

- 34 -

6) The sample was applied to the IVT cRNA cleanup spin column sitting in a 2 ml

collection tube.

7) The sample was centrifugated for 15 seconds at more than 8000 x g. The flow-

through was discarded.

8) The IVT cRNA cleanup spin column was transferred to a new 2 ml collection

tube. 500 µl of cRNA wash buffer were added to celumn and centrifugated for

15 seconds at more than 8000 x g. The flow-through was discarded.

9) The next washing was performed with 500 µl of 80% (v/v) ethanol. Samples

were centrifugated for 15 seconds at more than 8000 x g and the flow-through

was discarded.

10) The column cap was opened and spined at maximum speed for 5 minutes with

the caps open.

11) The IVT cRNA cleanup spin column was transferred to a new 1,5 ml collection

tube and 15 µl of RNase-free water were added directly to the membrane.

Incubation proceeded at room temperature for 5 minutes and then spined at

maximum speed for 1 minute.

12) The flow-through in the collection tube (something about 13,5 µl) was eluted a

second time by pipetting back onto the spin column membrane. The spin

column was placed back into the collection tube and incubated at room

temperature for 5 minutes and then spined at maximum speed for 1 minute.

13) The volume of eluted cRNA was approximately 13,5 µl and the concentration

was determined by the NanoDrop.

G) Second-cycle, first-strand cDNA synthesis

1) The volume of cRNA to 10 µg was determined.

2) cRNA samples were mixed with the random primers in a strip tubes as listed in

table below.

Table 6: Second-cycle, cRNA/random primers mix

component volume in 1 Rxn

10 µg of cRNA variable

3 µg/µl Random pirmers 1,5 µl

Rnase free water up to 8 µl

Total volume 8 µl

3) The mixture was flick-mixed and spined down the tubes.

4) The second-cycle, cRNA/random primers mix was incubated at:

70 oC for 5 minutes

25 oC for 5 minutes

5) The samples were cooled at 4 oC at least 2 minutes.

- 35 -

6) The second-cycle, reverse transcription master mix was prepared in a separate

tube and the components are described in the table 7.

Table 7: second-cycle, first-strand cDNA synthesis master mix

component volume in 6 Rxn

5x 1st strand buffer 24 µl

0,1 M DTT 12 µl

10 mM dNTP+dUTP 7,5 µl

SuperScript II 28,5 µl

Total volume 72 µl

7) 12 µl of the second-cycle, first-strand cDNA synthesis master mix were

transferred to the second.cycle, cRNA/random primers mix from previous

procedure for a total reaction volume of 20 µl and briefly centrifugated.

8) The reaction was incubated at:

25 oC for 10 minutes

42 oC for 90 minutes

70 oC for 10 minutes

4 oC for at least 2 minutes

H) Hydrolysis of cRNA and cleanup of single.stranded DNA

This procedure requires the use of GeneChip® WT cDNA Synthesis Kit and the

GeneChip® Sample Cleanup Module

1) 1 µl of RNase H was added to each of samples and incubated at:

37 oC for 45 minutes

95 oC for 5 minutes

4 oC for 2 minutes

2) The cleanup step was proceeding by using the cDNA cleanup spin columns from

the GeneChip Sample cleanup module.

3) 80 µl of RNase-free water were added to each sample, followed by 370 µl of

cDNA binding buffer and vortexed for 3 seconds.

4) The entire sample was applied to a cDNA spon column sitting in a 2 ml

collection tube.

5) The samples were spined at more than 8000 x g for 1 minute and the through

flow was discarded.

6) The cap of the cDNA clean up spin column was left to be opened and then the

samples were spined at the maximal speed for 5 minutes. The flow through was

discarded and the column was placed in a 1,5 ml collection tube.

7) 15 µl of the cDNA elution buffer were pipetted directly to the column membrane

and incubated at room temperature for 1 minute and subsequently spined at

maximum speed for 1 minute.

- 36 -

8) The elution step was repeated by pipetting another 15 µl of the cDNA elution

buffer directly to the column membrane and incubated at room temperature for 1

minute, then spined at maximum speed for 1 minute.

9) The total volume of the eluted single stranded DNA was approximately 28 µl. 2 µ

l were taken from each sample to determine the yield by using the NanoDrop to

measure the concentration.

I) Fragmentation of single-stranded DNA

This procedure requires the use of the GeneChip® WT Terminal Labeling Kit.

1) The fragmentation reaction was set up in 0,2 ml tube.

2) The fragmentation master mix was prepared according to table 8.

Table 8: Fragmentation master mix

component volume in 6 Rxn

RNase-free water 60 µl

10x cDNA fragmentation buffer 28,8 µl

10 U/µl UDG 6 µl

1000 U/µl APE 6 µl

Total volume 100,8 µl

3) 16,8 µl of the above fragmentation master mix were added to the samples

prepared on step 1. The tubes were gently vortexed and spined down.

4) The reaction was incubated at:

37 oC for 60 minutes

93 oC for 2 minutes

4 oC for at least 2 minutes

5) The samples were flick-mixed, spined down the tubes and 45 µl of the sample

were transferred to a new 0,2 ml strip tube. The remainder of the sample was

used for size analysis using a RNA 6000 Nano assay kit supplied with Agilent

2100 bioanalyzer. The range of peak size of fragmented samples should be

approximately 50 to 100 nt.

Procedure:

One of the wells of electrode was fill with 350 µl of RNAseZAP and put in

the Agilent 2100 bioanalyzer, the lid was left to be closed for 1 minute.

Another well of electrode cleaner was filled with 350 µl of RNase-free

water, placed in the bioanalyzer, the lid was left to be closed for 10

- 37 -

seconds, then opened, the electrode cleaner was removed and then left

for 10 second to evaporated.

For the gel preparation, 550 µl of RNA Nano gel matrix were pipetted

directly on the filtr and spined at 1500 g for 10 minutes at room

temperature.

65 µl of the gel were transferred into an RNase-free 1,5 microfuge tubes

and 1 µl of RNA dye concentrate was added. To reach the mixture to be

homogenous, it was centrifugated at 13 000 g for 10 minutes at room

temperature.

The samples for denaturation were prepared, 1,2 µl of the sample and

ladder was pipetted into 0,2 ml tubes, then denaturated in cycler for 2

minutes at 70 oC and subsequently cooled on the ice.

A new RNA chip was taken and placed in the Chip Priming Station.

9 µl of the gel-dye mix were drawn up with a pipette and placed at the

bottom of the marked well. The gel-dye mix was dispensed. After

ensurance the plunger is at 1 ml, the priming station was closed.

After 30 seconds, the plunger was released with the clip release

mechanism.

The plunger was pulled back to the 1 ml position, the priming station was

opened and 9 µl of the gel-dye mix were pipetted in each of the wells

marked.

5 µl of marker were dispensed into each of the 12 sample wells.

1 µl of denaturated samples was pipetted into the wells 1-12 and 1 µl of

denaturated ladder was pipetted into the ladder-marked well.

The chip was vortexed for 1 minute at 2000 rpm and then placed in the

analyzer where the analysis was immediately started.

J) Labeling of single-stranded DNA

This procedure requires the use of the GeneChip® WT Terminal Labeling Kit.

1) The labeling reactions were prepared as listed in table 9. A master mix using 5x

TdT buffer, TdT and DNA labeling reagent were prepared just before aliquoting

15 µl into the 0,2 ml strip tubes containing the 45 µl of fragmented single-

stranded DNA.

- 38 -

Table 9: Labeling reaction

component volume in 1 Rxn

Fragmented single-stranded DNA 45 µl

5x TdT Buffer 12 µl

TdT 2 µl

5 mM DNA labeling reagent 1 µl

Total volume 60 µl

2) After the labeling reagents were adding to the fragmented DNA, the samples

flick-mixed and spined down.

3) The reaction was incubated at:

37 oC for 60 minutes

70 oC for 10 minutes

4 oC for at least 2 minutes

K) Hybridization

This procedure requires the GeneChip® Hybridization, Wash and Stain Kit.

1) The hybridization coctail was prepared in a 1,5 ml RNase-free microtube as

shown in table 10.

Table 10: Hybridization coctail

component

volume for one 169 format

array

fragmented and labeled DNA target 27 µl

3 nM control oligonucleotide B2 1,7 µl

20x eucaryotic hybridization

controls 5 µl

2x hybridization mix 50 µl

DMSO 7 µl

Nuclease-free water 9,3 µl

Total volume 100 µl

It is necessary to heat the frozen stock of 20x eukaryotic hybridization control to

65 oC for 5 minutes to completely resuspend the cRNA before aliquoting.

2) The tubes were gently vortexed and spined down.

3) The hybridization coctail was heated at 99 oC for 5 minutes and subsequently

cooled to 45 oC for 5 minutes and centrifugated at maximum speed for 1 minute.

4) The GeneChip ST array was equilibrated at room temperature immediately

before use. The array was labeled with the name of the sample.

5) 80 µl of the sample were injected into the array through one of the septa.

- 39 -

Two pipette tips are necessary to use when filling the probe array cartridge. The

bubble is important to get contact with hybridization coctail and all portions of the

array.

6) The array was placed in 45 oC hybridization oven at 60 rpm and incubated for 17

hours.

3rd day

Before following procedures washing, staining and scanning, the samples must be

registered in Affymetrix GeneChip Command Console (AGCC).

A) Priming the fluidics station

1) The intake buffer reservoirs were filled with the appropriate Wash A and Wash

B solutions.

2) The water reservoir was filled with deionized water.

3) 3 empty 1.5 ml microfuge tubes were placed into the stain holder positions 1, 2

and 3.

4) The wash block lever was placed into the engaged/closed position and the

needle lever was pushed into the down position.

5) The Prime_450 maintenance protocol was set and run.

B) Wash and stain

1) The array was removed after 16 hours of hybridization from the hybridization

oven.

2) The hybridization coctail was extracted and the probe array was completely

refilled with 100 µl of Wash Buffer A.

C) The stain reagents preparation

1) The reagents were aliquoted as described in table 11.

Table 11: volume of stain reagents

compound volume ( µl)

Stain coctail 1 600

Stain coctail 2 600

Array holding buffer 800

2) All the vials were spined down to remove the presence of any air bubble.

D) Using the fluidics station

1) The barcode of the chip was scanned with an external barcode reader.

2) The fluidics protocol was selected.

3) The appropriate probe array was inserted into the designated module of the

fluidics station.

- 40 -

4) The vial containing 600 µl of stain coctail 1 was placed in sample holder 1, the

vial containing 600 µl of stain coctail 2 was placed in sample holder 2 and the

last vial containing 800 µl of array holding buffer was placed in sample holder 3.

5) The needle was pressed down and the run was begun.

6) After complete protocol, the probe arrays were removed from the fluidics

station.

E) Scanning

1) One tough-spots was applied to each of two septa on the back of the probe

array cartridge.

2) The cartridge was inserted into the scanner.

3) The scanning protocol was set and start of analyse begun.

2.2.5 UV-VIS spectroscopic analysis of interactions between cytochrome c

and JS3 triterpenoid derivates

Protocol:

1) 6 µl of certain derivates were taken.

2) 2,46 g of cytochrome c were dissolved in the 5mM pyridine/HCl buffer.

3) Derivatives were mixed with cytochrome c in the buffer, 200 µl were prepared

for each derivative.

4) The mixture was pipetted in the final volume of 50 μl into 384 well plate.

5) Samples were measured by using the spectrophotometer, where the wave

length was set in the range from 200 to 750 nm.

6) The data were analyzed into charts.

- 41 -

2.3 Results

2.3.1 Determination of RNA, cRNA concentration

For further whole transcript sense target labeling assay, 300 ng of RNA were required.

The final concentration of RNA is listed in the table 12. The volume of RNA and H2O to

reach 300 ng is listed in table 13. As the concentration of control 3 was under limit, the

sample was not analyzed. Another measurement of cRNA was done to ensure if

appropriate amount of cRNA was in each sample. Subsequently the volume of cRNA

was determined to reach 10 µg per sample (table 14). cRNA was diluted with water to

the final volume of 6,5 µl (table 15).

Table 12: The final concentration of RNA

the sample RNA concentration (ng/ µl)

control 1 666,7

control 2 588,23

control 3 12,6 (under limit)

JS8 a 1200

JS8 b 652,17

JS8 c 461,54

Table 13: The volume of RNA and H2O to reach 300 ng of RNA

the sample volume of RNA (µl) Volume of H2O (µl)

control 1 0,45 2,55

control 2 0,51 2,5

JS8 a 0,25 2,75

JS8 b 0,46 2,54

JS8 c 0,65 2,35

Table 14: The final concentration of cRNA

the sample concentration of cRNA µg/µl

control 1 2111,57

control 2 4162,92

JS8 a 4350,15

JS8 b 4358,98

JS8 c 4294,7

- 42 -

Table 15: The volume of cRNA and H2O

2.3.2 Determination of cDNA concentration

cDNA concentration had to be measured to see if there was enough cDNA in the

sample and if was able to continue with experiment. The right concentration of cDNA

was needed for the fragmentation procedure (tab). The table shows volumes of cDNA

of each sample and RNase free water addition for the final volume of 30 µl needed for

fragmentation reaction.

Table 16: cDNA concentration

the sample cDNA concentration (ng/µl)

control 1 460,29

control 3 282,54

JS8 a 232,24

JS8 b 419,44

JS8 c 316,43

Table 17: Fragmentation reaction

the sample

volume of c DNA

(µl)

volume of Rnase free

water(µl)

control 1 23,68 7,52

control 3 19,43 11,73

JS8 a 11,95 19,25

JS8 b 13,11 18,09

JS8 c 17,3 13,82

the sample

µl of cRNA needed for 10

µg

µl of H2O needed up to 6,5

µl

control 1 4,74 1,76

control 2 2,4 4,1

JS8 a 2,3 4,2

JS8 b 2,3 4,2

JS8 c 2,33 4,17

- 43 -

2.3.3 cDNA electrophoresis

Fragmented single-stranded DNA samples were used for size analysis using a

Bioanalyzer to see the peak size of fragmented samples. The Agilent Rna chip was

used for successfully control of the fragmentation of single stranded DNA for Affymetrix

GeneChip which is very important for further analysis. After fragmentation of DNA, then

labeling is performed by deoxynucleotidyl transferase that is covalently linked to biotin

anticipating the sufficient target to be generated for hybridization to a single array.

Agilent RNA chip involves appropriate dye and the setting of electrical parameters of

the electrophoresis that is convenient for a single stranded DNA. The area of the peaks

should be in the range of 50-100 nucleotides (nt) thus ensuring the fact of the

successful fragmentation. The figures (11-16) show the right peak size, although there

is a sample JS8a showing another strange peak, but we decided to use all samples

for loading them on the Affymetrix GeneChip.

Figure 11: Electrophoresis run summary

- 44 -

Figure12: Bioanalyzer profile of single-stranded DNA control 1.

Figure 13: Bioanalyzer profile of single-stranded DNA of control 3.

Figure 14: Bioanalyzer profile of fragmented single-stranded DNA of JS8 a.

- 45 -

Figure 15: Bioanalyzer profile of fragmented single-stranded DNA of sJS8 b.

Figure 16: Bioanalyzer profile of fragmented single-stranded DNA of JS8 c.

2.3.4 Statistical evaluation

Array-array correlation method was performed to see the correlation of all arrays from

the experiment with each other. As was predicted, our results show high correlation

among the samples, because all the samples were from the same source, cancer cell

line CEM and no significant changes occurred. Another statistical evaluation, RLE

(relatively long expression) and NUSE (normalized unscaled standard error) was

accomplished. RLE confirmed the fact that only relatively few genes were differentially

expressed that we can see in boxes which are similar in range and are centred close to

value 0. The graphical representation NUSE represents normalized standard error

which estimates are normalized for each probe set and the median standard error

across all arrays is equal to 1. The cluster dendogram provides the information

concerning which observations are grouped together at various level of similarity.

- 46 -

Histogram of p-value was also established. As for p-values characteristic, minimal

value was 2,099 x 10-6, maximum was equal to 1, median reached 0,453 and mean

0,743. According to p-values, 314 genes were detected with p < 0,01 and 38 genes

with p < 0,001.

Figure 17: Array – array intensity correlation

- 47 -

Figure 18: RLE and NUSE statistical evaluation

Figure 19: Cluster dendogram

- 48 -

Figure20: Histogram of p-values

2.3.5 Identification and annotation of expressed genes

The genes of interest were chosen according to the treated/control samples ratio to see

which ones were over/underexpressed. As for underexpressed genes, the ratio <0,7

was established and for overespressed genes was the ratio >1,3. All the genes were

selected on the basis of their p-value = < 0,05. Searching for the genes annotation was

performed by Affymetrix and Ensembl databases. The tables show the genes whose

expression was changed by action of JS8 derivate.

- 49 -

Table 18: Identification of the overexpressed genes

gene ID ratio p-value chromosome location

official

full name

8021584 1,312348 0,021597 18 18q21.3

serpin peptidase

inhibitor, clade B

(ovalbumin), member 5

7905691 1,316503 0,027153 1 1q21 ribosomal

protein S27

8015230 1,319223 0,036293 17 17q12-

q21

keratin

associated

protein 4-11

7893796 1,331488 0,012632 6 6p21.33 ATP-binding cassette

sub-family F member 1

8178841 1,468844 0,032302 6 6p21.3

Transporter 2, ATP-binding

cassette, sub-family B

(MDR/TAP)

8125042 1,575965 0,04101 6 6p21.33

lymphocyte

antigen 6

complex, locus

G6C

8171087 1,619679 0,038681 X,Y Xp22.33;

Yp11.3

protein phosphatase 2

(formerly 2A), regulatory

subunit B'', beta

7895347 1,668704 0,011198 12 12q24.31

transmembrane emp24

domain trafficking

protein 2

8124855 2,490548 0,01321 6 6p21.33 surfactant

associated 2

8095364 3,080356 0,042023 4 4q13.2

transmembrane

protease, serine

11E

- 50 -

Table 19: Identification of the underexpressed genes

gene ID ratio p-value chromosome location

official full

name

8014633 0,570255 0,013925 17 17q12 TBC1 domain family,

member 3C

7894743 0,611395 0,024558 1 1q21.3

interleukin

enhancer binding

factor 2, 45kDa

7893795 0,624477 0,015187 2 2p13-p12

C1D nuclear

receptor co-

repressor

7892639 0,631789 0,029428 X Xq13.1

non-POU domain

containing, octamer-

binding

7892501 0,648596 0,009867 18 18q21 small nucleolar

RNA, C/D box 58B

7894401 0,679641 0,038444 22 22q13.1

eukaryotic translation

initiation factor 3,

subunit D

8020349 0,680136 0,002009 2 2q11.1 ankyrin repeat

domain 20B

2.3.6 UV-VIS spectroscopy analysis of interactions between cytochrome c

and derivates of betulinic acid

This method was performed in connection with the fact that betulinic acid and its

derivates are known to interact with cytochrome c. Absorption spectra of resulting

mixture consists of cytochrome c and derivates were recorded by this analysis. The

measurement was done in triplicate and 63 derivates were analyzed. The changes in

cytochrome c absorption spectra caused by their respective interactions with the

derivates were visualized as the difference spectra (∆A) of derivates treated samples of

cytochrome c. The absorption was measured three times and the average differences

in the maximum absorption (∆Amax) were used to quantify the effects of derivates on

cytochrome c. The final results are listed in the tables 20 and 21. The red labeled

values represent a similar or higher interaction of derivatives with cytochrome c,

comparing to the positive control, JS8. Example graphs (figure 21) show the typical

absorption spectrum of cytochrome c, where the range of wavelength was from 350-

- 51 -

750 nm. The blue line represents cytochrome c and the red one belongs to the certain

derivate.

Table 20: Final result of cytochrome c/derivative interaction

derivate max absorbance (A max)

wave

lenght ∆A max SD IC50

cyt c 0,329 0,339 0,327 405,5

JS8 0,248 0,257 0,231 405,5 0,08633 0,00839

BetA 0,303 0,305 0,297 405,5 0,03 0,004

1382 0,281 0,306 0,271 405,5 0,04567 0,01168 20,796

1384 0,293 0,295 0,284 405,5 0,041 0,00436 198,56

1385 0,274 0,294 0,276 405,5 0,05033 0,00503 10,7

1386 0,276 0,301 0,283 405,5 0,045 0,00755 12,386

1387 0,272 0,285 0,268 405,5 0,05667 0,00252 11,65

1411 0,319 0,332 0,331 405,5 0,00433 0,00737 27,96

1413 0,274 0,301 0,267 406 0,051 0,01153 14,63

1414 0,29 0,311 0,297 405,5 0,03233 0,00586 118,56

1415 0,264 0,282 0,29 405 0,053 0,01442 21,92

1416 0,274 0,289 0,282 405,5 0,05 0,005 24,37

1417 0,273 0,29 0,28 405,5 0,05067 0,00473 15,027

1418 0,277 0,302 0,279 405,5 0,04567 0,00777 37,94

1419 0,269 0,299 0,288 405 0,04633 0,01185 11,67

1420 0,276 0,3 0,284 405,5 0,045 0,00721 3,16

1421 0,284 0,3 0,272 405,5 0,04633 0,00808 249,4

1422 0,278 0,296 0,262 405,5 0,053 0,01114 15,6

1423 0,283 0,304 0,287 405,5 0,04033 0,00551 222,66

1424 0,295 0,304 0,301 405 0,03167 0,00493 39,85

1425 0,298 0,306 0,299 405,5 0,03067 0,00252 79,13

1426 0,288 0,312 0,282 405 0,03767 0,00945 18,92

1427 0,295 0,323 0,286 405,5 0,03033 0,0129 135,11

1428 0,284 0,303 0,289 405 0,03967 0,00473 14,71

1429 0,292 0,293 0,288 405 0,04067 0,00473 69,08

1431 0,314 0,323 0,309 405 0,01633 0,00153 29

1432 0,29 0,309 0,294 405 0,034 0,00458 27,47

1433 0,283 0,308 0,289 405,5 0,03833 0,00751 34,8

1434 0,285 0,302 0,284 405 0,04133 0,00379 9,64

1443 0,29 0,296 0,296 405,5 0,03767 0,00611 16,26

1444 0,217 0,235 0,238 406,5 0,10167 0,01168 24,58

- 52 -

Table 21: Final results of cytochrome c/derivate interaction

derivate max absorbance (A max)

wave

lenght ∆A max SD IC50

1445 0,287 0,299 0,288 405,5 0,04033 0,00153 14,96

1446 0,282 0,302 0,292 405,5 0,03967 0,00643 16,5

1447 0,288 0,303 0,29 405,5 0,038 0,00265 20,661

1448 0,284 0,293 0,294 405,5 0,04133 0,00723 5,73

1449 0,267 0,291 0,276 405,5 0,05367 0,00737 5,99

1450 0,284 0,302 0,287 405,5 0,04067 0,00404 23,07

1451 0,296 0,296 0,295 405,5 0,052 0,02571 2,62

1452 0,294 0,305 0,298 405,5 0,03267 0,00321 5,89

1453 0,308 0,322 0,299 405,5 0,022 0,00557 38,98

1454 0,289 0,295 0,289 405 0,04067 0,00306 37

1455 0,215 0,229 0,174 406,5 0,12567 0,02376 4,95

1456 0,282 0,298 0,27 405,5 0,04833 0,00808 15,11

1457 0,284 0,297 0,279 405,5 0,045 0,003 24,95

1458 0,287 0,3 0,283 405,5 0,04167 0,00252 9,22

1459 0,279 0,29 0,284 405,5 0,04733 0,00379 20,33

1460 0,306 0,326 0,308 405 0,01833 0,00503 14,096

1461 0,286 0,307 0,298 405,5 0,03467 0,00737 3,98

1462 0,278 0,303 0,27 405,5 0,048 0,01082 11,3

1463 0,289 0,296 0,288 405 0,04067 0,00208 15,17

1464 0,943 0,921 0,894 413 -0,5877 0,02401 15,57

1465 0,28 0,288 0,29 405,5 0,04567 0,00757 4,055

1466 0,28 0,302 0,288 405,5 0,04167 0,00643 28,44

1467 0,279 0,3 0,29 405,5 0,042 0,007 17,31

1468 0,29 0,304 0,302 405,5 0,033 0,00721 67,53

1469 0,282 0,298 0,29 405,5 0,04167 0,00503 22,27

1470 0,29 0,304 0,299 405,5 0,034 0,00557 60,147

1471 0,293 0,29 0,292 405,5 0,04 0,00781 53,1

1472 0,278 0,289 0,277 405,5 0,05033 0,00058 3,699

1473 0,283 0,3 0,283 405,5 0,043 0,00361 14,43

1474 0,267 0,303 0,261 405,5 0,05467 0,01629 3,93

1475 0,309 0,332 0,306 405 0,016 0,00781 51,099

1476 0,285 0,299 0,281 405,5 0,04333 0,00306 8,28

1477 0,284 0,276 0,29 405 0,04833 0,01332 3,88

- 53 -

Figure 21: Graphs showing different absorption maximum of cytochrome c and

derivates.

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

35

0

35

9

36

8

37

7

38

6

39

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41

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57

5

cyt c vs JS8

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0,2

0,25

0,3

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cyt c vs BetA

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

35

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35

9

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cyt c vs 1422

- 54 -

2.3 Discussion

The received results given more knowledge about the mechanism of action of

3β,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlupan-22-oic acid (code JS8) on the

lymphoblastic leukemia cell line CEM. In our study we tried to identify target genes

whose expression was changed by JS8 action. Previous study suggested that primary

target of JS8 lays in mitochondria and that direct incubation of JS8 with cytochrome c

form non-covalent complex thus indicating cytochrome c to be a primary target for JS8

(Dzubak et al., 2007).

Our results from spectroscopic analysis confirm the fact that JS8 interacts with

cytochrome c. Other derivatives were also tested and the obtained results lead to

conclusion that they may have a similar effect of action like JS8, thus suggesting their

potential interaction with cytochrome c.

On the other hand, Affymetrix microarray analysis revealed several genes being

under/overexpressed in the base of JS8 action. One of the underexpressed gene was

C1D encodes nuclear-matrix associated protein C1D. C1D is nuclear matrix non-

histone protein which sequence level was characterized (Nehls et al., 1997). C1D was

also found to play role as a component of the complex involved in transcriptional

repression because of its association with the trancsriptional repressor RevErb and the

nuclear corepressors N-cor and SMRT (Zamir et al., 1997). C1D is also appears to

serve as a DNA-dependent protein kinase activator (Yavuzer et al., 1998), which plays

an important role in DNA double strand break (Jackson, 1997). Overexpression of C1D

leads to induction of apoptosis by increasing levels of p21Cip1 (WAF1), correlate with

specific interaction of C1D with DNA-PK that phosphorylates p53 protein on Ser 15

and Ser 37, resulting in p53 activation (Shieh et al., 1997). It was also revealed that

trancriptional and postranscriptional levels of C1D are tightly regulated. The

transcriptional activity of the basal C1D promoter is repressed by cis-acting sequences

comprised in a LINE-1 upstream element (Rothbarth et al., 2001) and proteasome-

mediated degradation appears to avoid random protein levels (Rothbath et al., 2002).

Taking into account all these information and comparing them with our result, where

the level of this gene was downregulated it can be said, that our derivative may play

role as C1D promoter repressor or protein degradation by ubiquitin and subseqeunt

caspase activation can occur. Another detected uderexpressed gene was interleukin

enhancer binding factor 2 ILF2, also known as NF 45. NF 45 together with NF 90 is

associated with the regulation of RNA gene at the levels of transcription, translation,

splicing and export (Zhao et al., 2004). This complex have been shown to stabilize the

association between Ku 70, Ku 80 and the DNA-dependent protein kinase catalytic

- 55 -

subunit DNA-PKcs, suggesting an involvement in DNA repair and chromosome stability

(Ting et al., 1998). It was revealed that increased expression of these proteins occurs

in transformed lymphoma, leukemia and hepatocellular carcinoma cells, suggesting

that aberrant upregulation of NF45 may contribute to the malignant phenotype,

especially in leukemias and lymphomas (Zhao et al., 2004). Taking into account this

information, it can be said that JS8 may play role in association with NF 45 in cancer

cell where DNA repairs fail or it can regulate protein at the transcriptional level with its

subsequent degradation. Another uderexpressed gene involved in DNA repair is

NONO non-POU domain containing, octamer-binding, also known as p54. p54 was

found to form complex with PSF that bind directly to DNA substrates of the end joining

reaction and subsequent cooperate with Ku protein, leading to functional complex

establishment. PSP and p54 are both phosphoproteins. Phosphorylation may be

involved in the relocalization of PSF during apoptosis and in regulating the binding

properties of p54 during mitosis. As this gene was uderexpressed, it can be suggested

that JS8 could again play role in DNA repair.

Next gene found to be underexpressed was TBC1 domain family, member 3C

(TBC1D3C) that includes TBC domain and it has been reported to posses GAP activity

towards RAB5A (Pei et al., 2002). Some in vitro studies and human tumor tissues

analysis revealed that TBC1D3C plays a role as an oncogene. In one study with

prostate cancer cells was this oncogene highly overexpressed and its oncogenic

activity is supposed to influence a tumorigenic phenotype of prostate cancer. The

mechanism of action of these oncogenity is said to be because of its overexpression

that leads to direct interaction of RAB5 due to the stimulation of its GTP hydrolysis (Pei

et al., 2002). In our study, the TBC1D3C gene was underexpressed comparing ratio

treated/control (p=0,014). Hypothetically, our derivative can play role in the mechanism

of TBC1D3C and its interaction with RAB5. Another explanation of the underexpressed

genes is the fact that most of them are regulated by transcription pathways which

should be affected by ubiquitin and caspases.

As for overexpressed genes, metallopanstimulin (MPS-1) was detected. MPS-1

is an ubiquitous multifunctional ribosomal S27/nuclear "zinc finger" protein which high

expression was revealed in a wide variety of cultured proliferating cells and tumor

tissues, including melanoma. MPS-1 is also thought to be involved in DNA repair and

recognition of altered mRNA. It has been investigated that the MPS-1 mRNA was

ubiquitously expressed in normal tissue and the increase message of the MPS-1 gene

was found to be increased in cancer cell lines (Fernandez et al., 1996). It was reported

that MSP-1 protiein contains ine zinc finger domain allowing to bind specifically the

DNA oligomer containing cAMP responsive elements sequence, considering MPS-1

- 56 -

protein might be involve in regulating the transcription of specific genes associated with

growth control and apoptosis (Fernandez, 1994). Another gene found to be

overexpressed is ATP-binding cassette sub family F, member 1 (ATP50) belongs to

ABC family proteins which have been characterised to lack recognizable

transmembrane domains. ATP50 was reported to increased its expression by

treatment of synoviocytes with tumor necrosis factor (Richard et al., 1998) and

subsequent study reveal that ATP50 plays an essential role in iniciation of translation

by binding to eIF2 and to both initiating and elongating ribosomes (Paytubi et al.,

2009). Another gene found to be overexpressed is ATP-binding cassette sub family F,

member 1 (ATP50) belongs to ABC family proteins which have been characterised to

lack recognizable transmembrane domains. ATP50 was reported to increased its

expression by treatment of synoviocytes with tumor necrosis factor (Richard et al.,

1998) and subsequent study reveal that ATP50 plays an essential role in iniciation of

translation by binding to eIF2 and to both initiating and elongating ribosomes (Paytubi

et al., 2009). Serpin B5 also known as Maspin was detected to be overexpressed.

Maspin belongs to serine protease inhibitor family and is known as class II tumor

suppressor, because although it is downregulated in many metastatic carcinoma cells,

no mutation occurs (Sager at al., 1996). Maspin also induce production of angiostatin

and inhibits vascular endothelial cell migration and tube formation to keep

angiogenesis in check (Narayan and Twining, 2009). There were revealed two

mechanisms of altered function of maspin in cancer. As first, the gene encodes maspin

is silenced in tumor by hypermethylation at Cp6 islands. In the second mechanism,

maspin was shown to be regulated by p53, in the case of abnormal function of p53,

maspin is not expressed (Zou et al., 2000). One study also revealed that maspin can

modulate tumor cell apoptosis through regulation of Bcl-2 family protein, where

reduced level of antiapoptotic protein but an increased proapoptotic protein Bax was

observed. This change in Bcl-2 family protein resulted in an increase release of

cytochrome c from mitochondria and subsequently the apoptosis is increased in

maspin-expressing tumor cells (Zhang et al., 2005). In our study was obviously

overexpressed level of maspin gene. This may be due to the cellular compensatory

action in the reaction on the described massive activation of the intracellular proteases.

Another overespressed gene was PPP2R3B protein phosphatase 2 (formerly 2A),

regulatory subunit B'', beta, PR48. It was revealed, that P48 mediated interaction

between Cdc6 and PP2A (serin/threonin phosphatase). This interaction is important for

Cdc6 dephosphorylation, ensuring the right cell cycle progression and DNA replication

control. Overexpression of PR48 causes the cell cycle arrest and has negative impact

on DNA replication (Yen et al., 1999). Comparing with our results, where gene encodes

- 57 -

this protein was overexpressed, it can be suggested that phosphatases compensatory

action may play role in the reaction with JS8. Another gene found to be overexpressed

is TAP-2, belonging to TAP family protein, which is involved in the transport of cytosolic

endogenous peptides to the endoplasmic reticulum mediating a link between antigen

processing and presentation. It was also observed that the transcription of this gene is

responsive to interferon gamma (IFN-gamma), indicating a common regulation and

concerted function of this gene in antigen processing. IFN gamma is produced by

mucosal lymphoid tissue exposed to a number of xenobiotic agents which function as

IFN gamma inducers and IFN-gamma encodes MHC proteins, the two specialized

proteasome subunits, leading to signal cell recognition by cytotoxic T-cell (Bocci et al.,

1981). Our result revealed upregulated TAP 2 via JS8 and it can be suggested that

JS8 enhances IFN-gamma to activate TAP 2.

In our study several potential targets of JS8 were revealed. JS8 is supposed to

be involved in DNA repair mechanism, GTP signaling pathway, cell cycle and in

apoptosis. The changes in the cell cycle were difficult to identify because of another

apoptosis mechanisms, which are much faster. For a specific interaction cytochrome c

is considerated, but other experiments are needed to be performed.

- 58 -

3. Conclusion

In this work, the effect of the JS8 on the expressional profile of CEM was analyzed.

JS8 is derivate of betulinic acid that is well known for its antitumor properties and one

of the modes of action is cytochrome c release and subsequent caspase activation

leading to programmed cell death. JS8 in comparison with betulinic acid showed

stronger activity against the tumor cell lines and the new observation were needed to

be revealed. That is why it was chosen for our experiments and by the method of

Affymetrix microarray assay the new potential targets and explanations of previous

experiments were identified.

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Links:

http://media.affymetrix.com/about_affymetrix/outreach/lesson_plan/downloads/student-

manual_activities/activity2_structure_function.pdf

www.umassmed.edu/gcf/indes.aspx

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Abbreviations

A549 lung carcinoma cell line

A2780/ADR ovarian cancer cell line

A375 melanoma cell line

ACF aberrant crypt foci

AGS gastric adenocarcinoma cells

AKBA 3-O-acetyl-11-keto-β-boswellic acid

ARO anaplastic carcinoma cell line

ATF-2 Activating transcription factor 2

B16 - F10 mouse melanoma cell line

Bak Bcl-2 homologous antagonist/killer

Bax Bcl-2–associated X protein

Bcap37 human breast carcinoma cell line

Bcl-2 B-cell CLL/lymphoma 2

Bcl-xL Bcl-2 family member

Bel-7402 hepatoma cell line

Bim Bcl-2 family member

CaP androgen-intensive cells

CDDO 2-cyano-3, 12-dioxoolean-1,9-dien-28-oic acid

Cdk cyclin dependent kinase

CDODA 2-cyano-3, 11-dioxo-18b-olean-1, 12-dien-30-oic acid

cFos proto-oncogene

CHOP ccaat-enhancer-binding protein

CTCL Cutaneous T cell lymphoma cell line

CREB cAMP response element-binding

CuB cucurbitacin B

DNA deoxyribonucleid acid

DU145 androgen-independent human prostate cancer cell line

ER endoplasmatic reticulum

FADD Fas-associated protein with death domain

Fas death receptor

GSH gglutathione synthetase

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GSK3 glycogen synthase kinase 3 alpha

GS-R3 ginsenosides R3

HEC108 endometrial cancer cell line

HeLa human cervical adenocarcinoma

HepG2 human hepatoma cells

HL-60 myeolid leukemia cell line

HO-8910 human ovarian carcinoma cells

HPAC human pancreatic cancer cell line

HPCC cervical carcinoma

HPLC/MS high pressure liquid chromatography/mass spectrometry

HT-29 colon adenocarcinoma cells

HUVECs human umbilical vein endothelial cells

IL-1β interleukin 1, beta

IkBa nuclear factor of kappa light polypeptide gene enhancer in B-cells

inhibitor, alpha

JAK 1 Janus kinase 1

JNK mitogen-activated protein kinase 8

K562 human leukemia cells

KDR kinase insert domain receptor

LNCaP prostate cancer cell line

MAPK1 mitogen activated protein kinase 1

MCF-7 breast adenocarcinoma cells

Mcl-1 induced myeloid leukemia cell differentiation protein

MMP-9 matrix metalloproteinase-9

NF-ĸB nuclear factor kappa B

p21/WAF1 cyclin-dependent kinase inhibitor 1

p53 tumor suppressor protein

p62 ubiquitin binding protein

P388 murine leukemic cell line

PAA human lung adenocarcinoma cells

PC-3 prostate cancer cell lines

PI3 phosphoinositide 3

PKCα protein kinase C alpha

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PPARg proliferator-activator receptor g

Ras family of genes encoding small GTPases

RPMI8226 multiple myeloma cell line

SGC7901 human gastric carcinoma cells

SK-MES-1 lung cancer cell line

SK-MEL-2 human melanoma cell line

SKNAS neuroblastoma cells

SMMC-7721 hepatocellular carcinoma cell line

SNG-II endometrial cancer cell line

STAT3 signal transducer and activator of

transcription 3

T98G glioblastoma cell line

TE671 rhabdomyosarcoma-medulloblastoma

cells

TNF-α tumor necrosis factor alpha

TRAIL TNF-related apoptosis-inducing ligand

VEGF vascular endothelial growth factor A

VEGFR 2 vascular endothelial growth factor

receptor 2

ZIP death-associated protein kinase 3