in vitro and in vivo studies of a new class of anticancer … · 2016. 3. 2. · 1 in vitro and in...

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In Vitro and In Vivo Studies of a New Class of Anticancer Molecules for Targeted Radiotherapy of Cancer

Chun-Rong Wang1

, Javed Mahmood3, 4, Qin-Rong Zhang1, Ali Vedadi3, Jenny Warrington1, Ning Ou1, Robert G. Bristow3,5, David A. Jaffray3,5 and Qing-Bin Lu1,2

1Department of Physics and Astronomy and 2Departments of Biology and Chemistry, University

of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada; 3Princess Margaret Cancer Centre and Ontario Cancer and Techna Institutes, University Health Network, Toronto, Ontario, Canada; and 4Department of Radiation Oncology, School of Medicine, University of Maryland, Baltimore, USA and 5Departments of Radiation Oncology and Medical Biophysics,

University of Toronto, Toronto, Ontario, Canada

Running title: Discovery of targeted radiotherapies through femtomedicine Keywords: Small molecule therapeutics; Radiation therapy; Targeted therapy; Chemical biology; Femtomedicine Financial support: This study was supported by an operating grant (#102483) to Q.B. Lu, D.A. Jaffray and R.G. Bristow and a New Investigator Award (#112642) to Q.B. Lu from the Canadian Institutes of Health Research, a Discovery grant (#299089) to Q.B. Lu from Natural Science and Engineering Research Council of Canada (NSERC), and an Early Researcher Award (# 41477) to Q.B. Lu from the Ontario Ministry of Research and Innovation. Requests for reprints: Qing-Bin Lu, Department of Physics and Astronomy, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1, E-mail: [email protected]; David Jaffray and Robert Bristow, Princess Margaret Cancer Centre, University Health Network, 610 University Avenue, Toronto, Ontario, Canada M5G2M9, E-mails: [email protected] and [email protected]

Potential conflicts of interest: Q.B. Lu, R.G. Bristow, D.A. Jaffray, C.R. Wang have ownership interest in Gashu Femtomedicine Inc.. No potential conflicts of interest were disclosed by the other authors.

The word count (excluding references): 6898; the total number of figures and tables: six.

on December 10, 2020. © 2016 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on February 26, 2016; DOI: 10.1158/1535-7163.MCT-15-0862

http://mct.aacrjournals.org/

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Abstract: There is a compelling need to develop anti-cancer therapies that target cancer cells and tissues. Arising from innovative femtomedicine studies, a new class of non-platinum-based halogenated molecules (called FMD molecules) that selectively kill cancer cells and protect normal cells in treatments of multiple cancers have been discovered. This paper reports the first observation of the radiosensitizing effects of such compounds in combination with ionizing radiation for targeted radiotherapy of a variety of cancers. We present in vitro and in vivo studies focused on combination with radiation therapy of cervical, ovarian, head and neck, and lung cancers. Our results demonstrate that treatments of various cancer cells in vitro and in vivo mouse xenograft models with such compounds led to enhanced efficiencies in radiotherapy, while the compounds themselves induced no or little radiotoxicity toward normal cells or tissues. These compounds are therefore effective radiosensitizers that can be translated into clinical trials for targeted radiotherapy of multiple types of cancer. This study also shows the potential of femtomedicine to bring breakthroughs in understanding fundamental biological processes and to accelerate discovery of novel drugs for effective treatment or prevention of a variety of cancers.

Introduction Radiotherapy remains a major curative therapy for cancer (1-4). A number of chemical

compounds, termed radiosensitizers, have been tested in clinical trials to enhance the

radiosensitivity (2, 4). The presence of hypoxia in tumors has important impacts on tumor

progression and treatment outcome (2, 5); hypoxic cells are refractory to radiation treatment.

Thus, discovery of hypoxic radiosensitizers that target tumor hypoxia is in large demand.

In liquid water, a free electron resulting from ionizing radiation (IR) is rapidly solvated by

surrounding H2O molecules to form the well-known hydrated electron (ehyd−), which was first

discovered in the 1960s and its reactivity was well determined (6, 7). With the advent in the

1980s of femtosecond (fs) (1fs=10−15 s) time-resolved laser spectroscopy (fs-TRLS), which is a

direct technique to visualize molecular reactions in real time (8), the ultrashort-lived precursor to




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ehyd− (the so-called pre-hydrated/solvated electron, epre

−) was directly observed (9-11). Our

careful fs-TRLS measurements (12) led to resolving the long-standing controversies about the

lifetime and physical properties of epre−. After identifying and removal of the coherent ‘spike’

effect in fs-TRLS measurements, we demonstrated that epre− has a lifetime of about 500 fs and is

a weakly-bound excited state of the well-bound hydrated electron (12). Moreover, our fs-TRLS

studies demonstrated the high reactivity of epre− via direct real-time observations of the formation

and dissociation kinetics of the intermediate state AB*− of dissociative electron transfer (DET)

reactions: epre−+AB→AB*−→A−+B• (12-18). Previous reviews on ultrafast DET reactions of epre

−

with various biological and environmental molecules can be found in refs. (19-21).

Through fs-TRLS studies, we found that weakly-bound epre− plays a key role in IR-induced

DNA damage in an aqueous environment. Particularly we discovered that the ultrafast DET

reaction of epre− leads to chemical bond breaks at the guanine base (17) and DNA strand breaks

(22). Our results challenged the conventional notion that damage to the genome by IR is mainly

induced by an oxidizing OH• radical, and might lead to improved strategies for radiotherapy of

cancer and radioprotection (19, 23). Moreover, we have recently demonstrated a reductive

damaging mechanism in living cells, which may be related to the pathology of diseases,

especially cancer (24).

We also discovered the DET reaction mechanisms of cisplatin as both a widely-used

chemotherapeutic drug and a radiosensitizer (14, 15) and of halopyrimidines (XdUs, X=Cl, Br or

I) as potential radiosensitizers (13, 16, 18). Our findings have provided a molecular basis for the

success and failure in clinical use/trials of cisplatin and halopyrimidines, respectively.

The integration of fs-TRLS technique with molecular biology and cell biology methods to

advance fundamental understanding and therapies of human diseases, notably cancer, might lead

to a new transdisciplinary frontier called femtomedicine (FMD) and advances in cancer therapy

(19). Our mechanistic studies in FMD (12-19, 22) provide remarkable opportunities to improve

the therapies of existing drugs and to develop new effective drugs. Indeed, our studies have led

to the development of novel cisplatin-based combination therapies that could significantly

improve the therapeutic efficacy of cisplatin against human ovarian, cervical and lung cancers

(25). Based on our new understanding of differential intracellular environments between normal

and cancerous cells, we have also discovered a new family of non-platinum-based molecules




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(called FMD molecules) that selectively kill cancer cells and protect normal cells, effective for

targeted chemotherapy of multiple cancers (26).

The newly discovered FMD compounds, such as 4,5-dichloro/dibromo/diiodo-1,2-

diaminobenzene (benzenediamine/ phenylenediamine) and 4(3)-chloro/bromo/iodo-1,2-

diaminobenzene (benzenediamine/phenylenediamine) (shortened as FMD-nX-DABs or

B(NH2)2Xn with X=Cl, Br or I, and n=1, 2), act essentially as cisplatin analogues, whose

molecular structures were reported previously (26). They are expected to be highly effective in

DET reactions with either weakly-bound electrons intrinsic in the more reductive intracellular

environment of abnormal (cancer) cells or ultrashort-lived epre− produced by ionizing radiation of

biological systems. The resultant radical [B(NH2)2Xn-1]• is highly reactive and can effectively

lead to DNA damage and cell death. An advantage of these FMD compounds over cisplatin and

halopyrimidines (XdUs) is that the DET reaction of FMDs is sufficiently effective but they are

far less toxic due to the absence of the heavy metal (Pt). Such FMD compounds alone have

shown highly desirable targeting properties as anti-tumor agents for targeted chemotherapy of

multiple types of cancer (26). Moreover, the DET reaction with a FMD molecule will be more

effective in the microenvironment of tumor due to its hypoxia, which reduces the competitive

attachment of epre− to oxygen. Thus, the FMD compounds are expected to be effective hypoxic

radiosensitizers. This paper presents results of in vitro and in vivo studies of the FMD

compounds in combination with ionizing radiation to achieve targeted radiotherapy of human

cervical, ovarian, head and neck, and lung cancers.

Materials and Methods Chemicals and reagents. Details on all the chemicals and reagents used in this study have been

given previously (26). FMD-nX-DABs have excellent solubility of >40 mM in ethanol (EtOH),

and limited solubility ranging from 0.1 to 10 mM in MEM (cell culture medium). Thus, their

stock solutions in 20 or 40 mM were prepared by dissolving the compounds in pure EtOH. For in

vitro cell line experiments, small volumes of the compound stock solutions were added into the

culture wells while the vehicle (EtOH) concentration was kept below 1.5%. For in vivo mouse

experiments, our studies were focused on FMD-2Br-DAB and its solubility in MEM (cell culture




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medium) was well determined to be 0.85±0.07 mM by spectrophotometric measurements. The

stock solution of FMD-2Br-DAB was diluted in MEM to have 10% ETOH, and 100 μl of the

diluted solution was injected into each mouse (~20 g) to make a concentration at 7 mg/kg of

FMD-2Br-DAB in mice, corresponding to an estimated in vivo concentration of 0.35±0.03 mM.

Cell lines and culture conditions. In the Waterloo laboratory (24-26), a human skin diploid

fibroblast (GM05757 cell line) was directly obtained from the Coriell Cell Repository (CCR);

human cervical cancer cell line (HeLa, ATCC® CCL-2TM; or ME-180, ATCC® HTB-33TM),

human ovarian cancer cell line (NIH:OVCAR-3, ATCC® HTB-161TM) and human lung cancer

cell line (A549, ATCC® CCL-185™), together with RPMI 1640, F-12K, McCoy’s 5A and L-15

culture media, were directly obtained from the American Type Culture Collection (ATCC).

Fetal bovine serum (FBS) was obtained from Hyclone Laboratories (UT, USA). The GM05757

normal cells and HeLa cells were cultivated with MEM (Hyclone) supplemented with 10% FBS,

100 units/mL penicillin G and 100 μg/mL streptomycin (Hyclone). ME-180, NIH:OVCAR-3,

and A549 cells were cultured with the ATCC-formulated McCoy’s 5A Medium supplemented

with 10% FBS, RPMI 1640 medium with 20%, F-12K medium with 10% FBS, and L-15

Medium (Leibovitz) with 10% FBS, respectively. The cells were maintained at 37 °C in a

humidified atmosphere containing 5% CO2. In the STTARR facility in Toronto, 74B cells were

acquired through a generous donation by Dr. Brad Wouters (PMH, UHN) and were cultured in

Iscove’s MDM media with 10% FBS, while ME180 and A549 cells were obtained from the

ATCC directly.

The above-listed cells obtained directly from the CCR or ATCC were passaged in our

laboratories for fewer than 6 months after receipt or resuscitation. The CCR performed cell line

characterizations by karyotypic and chromosome analyses, while the ATCC performed cell line

characterizations by karyotypic analysis, morphology check, isoenzymology and short tandem

repeat (STR) analysis (DNA fingerprinting). The 74B cells obtained from Dr. Brad Wouters

were tested by mycoplasma testing using PCR and genotyping within 6 months after receipt.

In vitro cell viability and clonogenic assays. The cell growth and survival rates with various

treatments were respectively determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl

tetrazoliumbromide (MTT) assay (Invitrogen), one of the most commonly used cell viability




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assays, and the clonogenic assay. The details have been given previously (24-26). Briefly, cells

were plated at a density of 7×103 cells/well in 96-well plates. Following overnight incubation,

the culture medium was replaced by fresh culture medium. For in vitro radio-toxicity tests, the

cells were incubated for 12 h with varying drug concentrations, then irradiated by 225 kV X-ray

(Precision, X-RAD IR 225) at a dose rate of 2 Gy/min with various X-ray doses, and after

irradiation, the cells were incubated for 12 days. The MTT assays of cell viability were then

conducted. For in vitro tests of the radiosensitizing effects of the FMD compounds, similar MTT

assays were conducted at 6 days post-irradiation. For clonogenic assays (24, 27), various

numbers of the cells ranging from 200 up to 2×105 were seeded to 100 mm tissue culture dishes

(Thermo Scientific Biolite), depending on types of cancer cells and treatments. After X-ray

irradiation, the cells were incubated for 15-17 days to form colonies. The cell colonies were then

were fixed with glutaraldehyde (6.0%), stained with crystal violet (0.5% w/v) and counted.

In vitro DNA double-strand break (DSBs) assay. The phosphorylated H2AX foci are a

biomarker of DNA DSB. The HCS DNA Damage Kit (Invitrogen) was developed to quantify the

population of apoptotic cells by specific antibody-based detection of phosphorylated H2AX

(γH2AX) in the nucleus. In addition, the kit included Hoechst 33342, which is a DNA-binding

dye (blue fluorescence) and allows the observation of nuclear morphology of all normal and

damaged cells. Briefly, the cells were treated with different concentrations of the compound for

8 h, followed by IR (225 keV X-rays). At 12 hr after x-ray irradiation, the cells were fixed and

stained. Following the detailed Protocol provided by the manufacturer, we performed the HCS

DNA Damage Assay of the treated cells, as described previously (24-26). The images of cells

were acquired with a Nikon Eclipse TS100 fluorescence microscope; quantitative analyses of

activated γ-H2AX (DNA DBS yield) in the cells were performed using an Image J software.

Xenograft mouse cancer models. ME180 cervix, 74B head and neck, and A549 lung xenografts

were derived at STTARR (UHN, Toronto) for in vivo growth delay studies involving a FMD

compound in combination with a large single dose IR (X-rays). 7-8 week old female SCID mice

were injected s.c. in the flank with 1.5×106 cells; the treatments were started when xenografts

reached a volume of ~100 mm3. Mice were randomly divided into four groups with 5 mice per

group: (a) injection vehicle only; (b) FMD-2Br-DAB (7 mg/kg); (c) IR 15 Gy; (d) the FMD




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compound plus IR. The FMD compound at 7 mg/kg was administrated by IP injection into the

mice, and at 1 hr after injection, the mice received given doses of X-rays from a X-RAD 225 kV

irradiator (Precision X-Ray, Connecticut, USA), either targeted to the tumor or the gut using a

collimator. Animals were weighed and flank tumors measured every 2-5 days with calipers;

measurements were converted into tumor volume. Growth delay was calculated as the time

difference (in days) between treatment and control groups to grow to 1000 mm3.

Similar mouse xenograft models (26) of ME-180 cervical cancer and A549 lung cancer were

established in the Central Animal Facility of the University of Waterloo to investigate tumor

growth delays caused by a FMD compound in combination with multiple small dose fractionated

IR (X-rays). Namely, 6-8 week old female SCID mice were injected s.c. in the flank with

1.5×106 ME-180 cells or 5×106 A549 cells. The treatments were started when xenografts reached

a volume of ~100 mm3. For each treatment, mice were randomly divided into four groups with 5

mice per group: (a) control (vehicle only); (b) FMD-2Br-DAB alone at 7 mg/kg was

administrated for totally 3 IP injections at days 1, 3, and 5 (7 mg/kg ×3); (c) ionizing radiation

alone (2 Gy×3); and (d) for the combination of a FMD with radiation. The FMD at 7 mg/kg was

administrated by IP injection into the mice, and at 1 hr after injection, the targeted tumor in mice

received radiation (2 Gy, 225 keV x-rays), with totally 3 treatments at Day 1, 3 and 5 (7 mg/kg

FMD×3 + 2 Gy×3). Mice were weighed, and flank tumors were measured every 2-5 days with

calipers, and tumor volumes were calculated.

All animal experiments were conducted in accordance with the guidelines of the University

Health Network Animal Care Committee or the University of Waterloo Animal Care Committee.

Immunohistochemistry. All serial sections were cut from paraffin-embedded tumor tissue, and

stained with hematoxylin and eosin (H&E). Sections were also stained for EF5 (provided by Dr.

Cameron Koch, University of Pennsylvania), CD31 (Dr. C. Koch), γH2AX (Bethyl Laboratories,

Rabbit polyclonal), 53BP1 (Bethyl Laboratories, Rabbit polyclonal), CC3 (Cell Signaling

Technology, Rabbit polyclonal), and Ki67 (Thermo Scientific, Rabbit monoclonal). Secondary

antibodies were used alone to control for nonspecific background. Sections were counterstained

with 1ug/mL DAPI to outline the nuclear area. Images were scanned on the TS4000 (Huron

Technologies) at 0.5 um/pixel. Regions of tumor, necrosis, stroma, folds were specified, creating




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a training rule-set for tissue recognition. Cellular analyses included nucleus identification and

separation, objects <10 µm2 being excluded.

In vivo radio-toxicity assays in gut, kidney and liver, overall and general toxicity assays. For

gut/kidney/liver radio-toxicity assay, the experimental details were given previously (27). Briefly,

SCID and nude mice were irradiated with targeted small intestine radiation at doses of 5, 10, 12,

or 15 Gy with and without compound FMD-2Br-DAB at 7 mg/kg. Mice were sacrificed after 72

hours and the small intestine was removed for Ki67 staining. After image staining and scanning,

surviving crypts in the intestine were measured compared to those that did not show a positive

Ki67 stain. The overall radio-toxicity was assessed through analysis of proteins, enzymes,

metabolites, and plasma electrolytes in blood. The mice were treated with 0, 5, 7 mg/kg FMD-

2Br-DAB daily for 10 days. In addition, to evaluate radiation-induced toxicity therapy, mice

were also injected with 0, 5, or 7 mg/kg of FMD and underwent 15 Gy of X-ray radiation at 1

hour after injection, in accordance with the pharmacokinetics data obtained. At the end of

treatments, blood samples were collected from the tail vein; if this was not technically possible

the saphenous vein was used as an appropriate substitute. These samples were then centrifuged at

13,000 rpm and serum was removed from the mixture. The hepatotoxicity (ALT, ALP, AST),

nephrotoxicity (blood BUN, creatinine), and electrolytes (Na, K, etc.) were measured from the

samples using an Autoanalyzer (Pulse Instrumentation). Moreover, mice were observed for any

general toxicity over the whole period of treatment. These physical indicators included: body

weight changes, dull sunken eyes, rapid/shallow breathing, hunched back, and lethargy.

Dynamic Contrast Enhanced (DCE) MRI. All MR imaging was performed on the Bruker

ICON high-performance 1T MRI Icon system (Aspect Imaging/Bruker, Germany). Tumors were

visualized 19 days after treatment, using a T2 weighted sequence: RARE (TE/TR=15/2319,

FOV=30mm, Matrix=130, Rare Factor 8, Averages=6, Resolution 233um, Acq. Time 3:28

min:sec). DCE-MRI was performed, on the same day, by injecting 30μl of Gd-HP-DO3A

(ProHance®, Bracco Diagnositics, USA) via tail vein cannulation. T1-FLASH

(TE/TR=3.17/41.7, FOV=30mm, Matrix=120, Resolution 250um, 3 slices acquired every 5

seconds, continued over 300 seconds). Tumor volume was quantified using the semi-automated

tools within the VivoQuant software (inviCRO, USA). DCE-MRI analysis was completed by




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inviCRO using the Kety-Tofts equation, assuming a fixed T1 value and a model input function.

Ktrans and AUC were tabulated along with parametric color maps for both.

Pharmacokinetic studies. SCID mice were injected with 7 mg/kg of FMD-2Br-DAB through

intraperitoneal injection (100 µL injection volume). At previously determined endpoints, mice

were sacrificed through cervical dislocation and tumor tissue was removed for analysis. Blood

was also collected in heparinised tubes using the cardiac puncture technique. The blood was then

centrifuge at 5,000 rpm for 5 minutes at 4oC. The tissue (tumor) or plasma concentration of

FMD-2Br-DAB was determined by HPLC-MS (Applied Biosystems, Foster City, CA). Data

analysis including peak integration and calculations were performed using Applied Biosystem

MDS Analyst 1.4.2 software. The elimination rate constant ke of the compound removed from

the body was determined by the measured elimination half-life (t1/2): ke=ln2/t1/2, and the

clearance (CL) was calculated by: CL=Vd×ke, where Vd is the plasma volume of a mouse.

Radiation treatment. Irradiations of cells and mice were performed at 225 kV. The X-ray unit

at Waterloo (XRAD 225, Precision X-ray, Connecticut, USA) was used for cell and animal

irradiations at a dose rate of 2 Gy/min. For in-vivo studies at STTARR (UHN, Toronto), image-

guided irradiations were targeted using cone-beam CT guidance (XRAD225Cx, Precision X-Ray,

Connecticut, USA) and a 1.0 cm circular collimator (Dose rate: 2.88 Gy/min; parallel-opposed

beam geometry). All mice at UHN received a large single dose of 15 Gy, either targeted to the

tumor or the gut/kidney/liver using the 1 cm collimator, while the cells and mice in the

University of Waterloo were treated with various X-ray doses and multiple fractionated radiation

doses (2 Gy ×3), respectively.

Statistical analysis. When two groups were compared, the appropriate student’s t-test was used.

Multiple groups were compared by ANOVA, extended with an HSD post-hoc test. Statistical

analyses were performed using STATISTICA 8 (StatSoft, Tulsa, Ok, USA). A P value <0.05

was considered statistically significant (P*<0.05; **P<0.01; ***P<0.001).

Results




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In Vitro Radio-Toxicity Tests of FMD-nX-DABs as Novel Radiosensitizers. The in vitro

radio-toxicity of FMD-nX-DABs in combination with IR (225 kV x-rays) was investigated in

human skin fibroblasts (GM05757). The GM05757 cell line has been widely used as human

normal cells in cancer research, particularly in testing new radiosensitizing agents (27). The cells

were treated by FMD-nX-DABs at various concentrations of 0-300/400 μM for 12 hr, followed

by various doses of X-ray irradiation. At 12 days post-irradiation, the viability of the cells in 96-

well plates was measured by MTT assay. First, the data plotted in Figure 1 show that at zero

radiation dose (0 Gy), FMD compounds appeared to have significant ‘toxicity’, but this was to

be expected when the normal cells were incubated with the compounds for 12.5 days (12 hr pre-

irradiation plus 12 days post-irradiation). It was not surprizing that the viability of normal cells

decreased with such a long compound incubation time. This is different from the results for

normal cells incubated with FMD compounds alone for up to 72 hr, exhibiting little or no effect

on the cell viability (26). Second, Figure 1 interestingly shows that the viability of normal cells

pre-treated with FMD compounds up to 400 μM was independent of radiation dose, i.e. no radio-

toxicity was observed on normal cells pre-treated with FMD compounds. This is in marked

contrast to the results for radiosensitizing effects of FMD compounds on cancer cells (see Figure

2 below).

In Vitro Radiosensitizing Effect Tests of FMD-nX-DABs. The in vitro radiosensitizing effects

of FMD-nX-DABs in combination with IR (225 kV x-rays) were investigated in cisplatin-

sensitive human cervical cancer cell line (HeLa or ME-180), cisplatin-resistant human ovarian

cancer (NIH:OVCAR-3, HTB-161) and lung cancer (A549) cell lines. The cells were treated by

FMD-nX-DABs at various concentrations for 12 hr, followed by various doses of X-ray

irradiation. The cell viability at 6 days post-irradiation was measured by MTT, while the cell

survival rates at 15-17 days post-irradiation was measured by the clonogenic assay. As plotted in

Figures 2A-F, together with Figure S1, the results clearly show that FMD-nX-DABs enhanced

the anti-proliferative effects of IR in cancer cells in an IR dose dependent manner (**P<0.01).

The enhancement ratio (ER) was calculated as the ratio of the radiation dose without FMD to

that with FMD for the same level of biological effect for the clonogenic survival curves in

Figures 2D-F. The ERs for these cancer cell lines under air conditions (with an oxygen

concentration typically of a few mM) were determined to be 1.2-1.5 only. However, the ERs of




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FMD compounds are expected to be significantly larger for cells under hypoxic conditions or in

the hypoxic tumor tissue due to the lack of the competitive attachment of epre− to oxygen. Further

confirmation of this property by in vitro cell experiments using a hypoxic chamber will be

interesting, but this will be examined by in vivo tests of the radiosensitizing effects of FMDs in

normal and cancer tissues in mice by combination with both a large single dose radiation and

multiple small dose fractionated radiation (see the results below).

In Vitro DNA DSBs Measurements in Cancer Cells. Given the observed results shown in

Figures 2 and S1, we further measured DNA DSBs in cancer cells treated by FMD-2Br-DAB in

combination with IR (225 kV X-rays), using the HCS DNA Damage (γH2AX) Kit (24-26).

Figure 3 show the fluorescence images of cervical cancer (ME-180) cells with/without the

treatment of 25/200 µM FMD and the yield of DNA DSBs detected by γH2AX foci with various

X-ray doses, respectively. A significant increase in DNA DSB yield was observed with the

presence of the FMD compound. These data clearly show that the radiosensitizing effect of

FMD-2Br-DAB resulted in significant enhancements in DNA DSBs in cancer cells (**P<0.01),

consistent with the cell viability and clonogenic survival results shown above. We therefore

conclude that FMD-2Br-DAB can effectively induce DNA DSBs in cancer cells under IR. The in vitro results presented in Figures 1-3 and S1 demonstrate that the presence of a FMD

compound enhanced the effectiveness of IR leading to tumor cell death while the compound

itself induced no radio-toxicity for normal cells. Thus, the FMD compounds are expected to

enhance the effect of IR while inducing minimal or no radio-toxicity in vivo animals.

In Vivo Pharmacokinetic and Radio-Toxicity Studies of FMD-2Br-DAB. The

pharmacokinetic (PK) characteristics and in vivo radio-toxicity of a representative FMD agent,

FMD-2Br-DAB, in 6-8 week SCID or nude mice are presented in Figure 4. Figure 4A

demonstrates PK profiles of the compound at both the tumor and blood plasma. Significant

levels of FMD-2Br-DAB were detected in the plasma, and the highest concentration was

observed at about 30 min after i.p. injection and then dropped exponentially with time. For the

tumor, the maximum uptake of the compound was observed at ~60 min. We measured the

elimination half-life (t1/2) of the FMD compound in plasma to be about 60 min, and given that

the plasma volume of a mouse is typically ~50 ml/kg, our estimated clearance (CL) is




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approximately 0.7 ml/hr. These results indicate that the compound has promising

pharmacokinetic properties.

FMD-2Br-DAB acute radio-toxicity was first studied in both SCID and nude mouse models

through a gut toxicity assay, as described previously (27). The nude mouse model is more

resistant to radiation therapy, and therefore has a larger radio-resistance towards gut toxicity.

Figure 4B shows the results of these tests in both SCID and nude mice. The crypt survival was

determined through a histological marker Ki67, which marks proliferating cells. Firstly, the

results elucidate a decrease in crypt survival in the small intestine (Jejunum and ileum) as the

radiation doses increased at 0-15 Gy, consistent with those reported previously (27). Second, the

results show that there was no significant difference in crypt survival rate between irradiated

mice in the control and irradiated mice pre-treated with 7 mg/kg FMD-2Br-DAB, indicating that

the FMD compound did not enhance radiation-induced gut toxicity (P>0.05). The histological

images representing the gut tissues from the irradiated SCID mice are also shown in Figure S2.

In addition, we studied the acute radio-toxicity of FMD-2Br-DAB in kidney and liver normal

tissues. The results are shown in Figures 4C and S3, which demonstrates no significant

difference in Ki67 levels between mice injected with FMD and those with vehicle only for both 0

Gy and 15 Gy irradiation. Although the level of Ki67 increased with radiation, this was expected

as after radiation cells begin to repair and proliferate in response to injury. H&E images in

Figure S3 show that there was very little or no radiobiological toxicity associated with the FMD

compound with and without 15 Gy radiation. These data indicate that the FMD as a novel

radiosensitizer is specific to the tumor tissue.

Direct drug (radio-)toxicity was also studied in mice through the overall drug toxicity and

general toxicity. FMD-2Br-DAB was administered by IP injection into mice at 0, 5 and 7 mg/kg

daily for 10 days, due to the non-toxicity observed in vitro cell line experiments (26). The

results displayed in Table S1 show that the FMD compound (even up to 7 mg/kg/day × 10day)

induced no observable overall toxicity, i.e., no hepatotoxicity, no nephrotoxicity, and no changes

in electrolytes. Moreover, Table S1 also shows that there were no considerable changes in the

overall drug radio-toxicity induced by 15 Gy X-rays between the groups pre-treated with 0, 5, or

7 mg/kg of FMD. Nearly all the measured values were within the normal ranges, except the

lower level of globulin. The latter was however also observed in the control group which

received no FMD compound/IR. This is attributed to the phenotype of SCID mice employed in




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the study. Moreover, the survival rate of mice was 100% over a 60 day period with no change in

body weight observed (Figure S4A), demonstrating that the FMD agent exhibited no general

toxicity.

Overall, the substantial in vivo data for toxicity assays in normal tissues and blood shown in

Figures 4B, 4C, S2, S3 and S4, together with Table S1, demonstrate no significant overall, acute

and general radiotoxicities (P>0.05), consistent with the in-vitro results in Figure 1, at the FMD

dose levels tested.

In Vivo DNA DSBs and Apoptosis Measurements in IR of a Cervical Cancer Xenograft Model. The in vivo DNA damage and cell death induced by FMD-2Br-DAB were investigated in

a xenograft mouse tumor model of human cervical cancer (ME-180). As shown in Figure 5, we

measured DSBs and apoptosis in tumor tissue, including hypoxic and non-hypoxic regions, at 1

and 24 hrs post-IR of 15 Gy. Figure 5A shows that higher levels of DNA DSBs (γH2AX foci)

were observed at 1 hour after the combination treatment of the FMD compound at 7 mg/kg

followed by IR (applied 1 hr after drug injection corresponding to the time of highest FMD

concentration in the tumor) in the whole tissue including both hypoxic and non-hypoxic regions.

It is seen from Figure 5B that there was a significant (***p<0.001) enhancement in DSBs

detected by γH2AX of DNA in hypoxic regions identified by the EF5 hypoxia marker (4) at both

1 and 24 hr post-treatment for the FMD+IR combination. Similarly, Figure 5C shows that there

was also an enhancement in the level of the DNA DSB-repair marker at both time-points in the

hypoxic areas (53BP1/EF5 double positivity) for the combination treatment ((*p<0.05).

Moreover, a similar enhancement in apoptosis measured by the CC3 marker was observed, as

shown in Figure 5D. Histological images corresponding to the results shown in Figures 5A-D are

shown in Figures S5 and S6. These results indicate that the presence of FMD-2Br-DAB

enhanced radiosensitivity by increasing DNA DSBs and apoptosis in the tumor, especially in

hypoxic regions. As a result, FMD-2Br-DAB can be used as an effective hypoxic radiosensitizer.

Histological Changes in Xenograft Models Treated by FMD-2Br-DAB plus 15 Gy IR. Histological changes in the tumor tissue at 21 days post-treatment were also measured, as shown

in Figures 5E-H. Figure 5E shows that in the ME180 xenograft model, there was a significant

increase in the necrotic fraction for the treatment of IR in combination with FMD-2Br-DAB




14

(***P<0.001). Figure 5F shows that a decrease in vessel density was observed for the

combination treatment, compared with the IR alone. Figure 5G shows the relationship between

levels of hypoxia following treatment in all 3 tumor models, while Figure 5H shows that Hoechst

vessel perfusion was decreased after treatment with the FMD compound plus IR when compared

to IR alone. This was also confirmed by DCE MRI Area Under the Curve (AUC) analysis.

Detailed histological images related to Figures 5E-H are also shown in Figures S7-9. These

results indicate that the FMD+IR treatment led to a decrease in vessel density and an increase in

tumor cell death (necrosis).

Tumor Growth Inhibition / Regrowth Delay Caused by 15 Gy IR Combined with FMD-2Br-DAB. Tumor growth inhibition and regrowth delay by FMD-2Br-DAB combined with a

larger single IR dose (15 Gy) were investigated in the three xenograft mouse tumor models of

human cervical cancer (ME-180), head/neck (74b) cancer, and lung (A549) cancer through

tumor size measurements using DCE MRI and calipers. As shown in Figures 6A-C for the three

xenograft models, significant growth delays of 41, 35 and 20 days between the IR alone and the

combination of 7 mg/kg FMD-2Br-DAB followed by IR were observed for the ME180, 74B and

A549 models, respectively (***P<0.001). This significant growth delay, which was observed for

all the three tumor models, demonstrates that this novel FMD compound can act as an effective

radiosensitizer with a significant effect and impact on tumor growth. For the ME180 xenograft

model, more detailed analyses of relative tumor volume changes and the drug treatment efficacy

are also given in Figures S10A and B. It is shown that a ≥40% increase in radiation treatment

efficacy was observed when combined with the FMD. Moreover, Figure 6D illustrates the results

of dynamic contrast enhanced (DCE) MRI measurements of the tumor size and perfusion

characteristics. These show that the combination of FMD with IR resulted in more tumor

perfusion and a much smaller tumor region.

Tumor Growth Inhibition / Regrowth Delay Caused by fractionation IR Combined with FMD-2Br-DAB. Conventionally fractionated radiotherapy uses multiple small doses of 1.8-3 Gy

to achieve local tumor control for acceptable normal tissue toxicity. However, the re-oxygenation

effect that occurs during multiple fractionation schemes is not expected to benefit the

radiosensitizing effect of FMD compounds being hypoxic radiosensitizers suggested by the




15

observed results described above. Here, tumor growth inhibition by fractionated radiotherapy

using FMD-2Br-DAB was investigated in the xenograft mouse models of human cervical cancer

(ME-180) and lung cancer (A549). As shown in Figures 6E and F, certain tumor regrowth delays

between the control group, the (7 mg/kg ×3) FMD-2Br-DAB alone, the fractionated radiation (2

Gy ×3) alone and the combination of FMD (7 mg/kg ×3) with radiation (2 Gy ×3) were observed,

respectively. The chemotherapeutic effect of the FMD at 7 mg/kg×3 alone was observed, in

accordance with the previous study (26). The combination of FMD with fractionated irradiation

enhanced the tumor regrowth delays compared with the radiation alone (**P<0.01), but it is not

as significant as the large single dose 15 Gy irradiation (Figure 6A-C). No changes in body

weight over a 90 day period for all four groups of mice were observed (Figure S4B). These

results are consistent with the radiosensitizing properties of FMD compounds acting as effective

hypoxic radiosensitizers, which may particularly benefit the SABR using large single doses.

Discussion

A practical way to improve the efficacy of radiotherapy is to combine it with a chemical

compound (radiosensitizer) (2, 4, 27, 28). Our femtomedicine approach and the discovery of the

key role in causing damage to the DNA of epre−, a novel electron species produced in the

radiolysis of water under IR, have offered unique opportunities to improve existing drugs and to

develop new effective drugs for high-performance therapy of cancer. Enabled by this innovative

strategy and based on the DET mechanism established in our previous studies (12-18, 19, 22),

we have successfully found a series of non-platinum-based halogenated compounds (FMD

compounds) as potent anticancer agents (26) and now as hypoxic radiosensitizers.

The present study offers several important observations. First, our in vitro results show that the

FMD compounds caused essentially no (radio-)toxicity toward normal cells. These compounds

are in contrast to the clinically used Pt-based anticancer drugs, which are highly toxic. Second,

our in vitro results show that a FMD compound significantly enhanced the anti-proliferative

effects of IR in both cisplatin-sensitive and cisplatin-resistant cancer cells. Enhancements in

DNA DSBs in the treated cancer cells were also observed. Thus, these FMD compounds have

significant potential as anticancer agents that achieve radiosensitization while inducing no or

little systemic toxicity and radio-toxicity.




16

Correspondingly, our in vivo results have clearly demonstrated that FMD-2Br-DAB as an

exemplary FMD compound combined with IR exhibited minimal (radio-)toxicity in mice, i.e., no

overall, acute and general toxicities in normal tissues (gut, kidney and liver) and blood. These

results are in good agreement with the in vitro results observed in human normal cells. The

observed results also show that the FMD compound is no longer detectable in the blood after 3-4

hours, exhibiting interesting pharmacokinetic properties that could be exploited for improved

clinical efficacy.

Furthermore, the in vivo results from the xenograft mouse tumor models of human cervical,

head/neck, and lung cancers exhibited significant tumor growth inhibition and regrowth delay

due to the radiosensitizing effect of FMD-2Br-DAB combined with a large single dose radiation

(15 Gy). Our in vivo results also show that the compound induced significant enhancements in

DNA DSBs and apoptosis especially in hypoxic regions, indicating that it is an effective hypoxic

radiosensitizer. Moreover, our histological and MRI data also show that FMD in combination

with IR led to a decrease in vessel density and an increase in tumor cell death (necrosis).

The radiosensitizing effect of FMD-2Br-DAB combined with multiple small dose fractionated

radiation (2 Gy ×3) was also observed, but it was less significant compared with the large single

dose of 15 Gy. This is consistent with the properties of FMD compounds as hypoxic

radiosensitizers, where reoxygenation between multiple fractionated irradiations reduces the

DET reaction efficiency of FMD and the radiosensitizing effect. These results demonstrate that

the FMD compound is indeed a novel and effective (hypoxic) radiosensitizer with significant

radiosensitizing effect and impact on tumor growth, especially in hypoxic tumor areas. This is of

special significance, as there is growing interest in the use of stereotactic ablative radiotherapy

(SABR) using large single IR doses in the clinic (28). The presence of tumor hypoxia is a major

negative factor in limiting the curability of tumors by SABR at radiation doses that are tolerable

to surrounding normal tissues. It has been suggested that this negative effect of hypoxia could be

overcome by the addition of a hypoxic cell radiosensitizer at clinically tolerable doses (28).

Compared with clinically available hypoxic cell radiosensitizers, the FMD compounds have a

significant advantage that they are essential no toxic even at very high doses, inducing no

systemic toxicity in chemotherapy (26) and no radio-toxicity in combination with radiation, as

observed in the present study.




17

The results observed in this study can well be explained by the dissociative-electron-transfer

(DET) mechanism established in our previous studies (12-19, 22): the DET reaction of FMD-nX-

DABs as highly reactive halogenated molecules with the prehydrated electron (epre−) is expected

to be much more effective under the hypoxic conditions due to the absence of the competing

attachment of epre− to O2. In other words, there are a larger number of epre

− available for the DET

reaction with FMD-nX-DABs in the tumor tissue characteristic of hypoxia. Thus, these FMD

compounds can be used as effective hypoxic radiosensitizers.

Finally, it is believed that the in vitro and in vivo results reported in this study have significant

potential to be extended to other cancer cells and models beyond those exemplified here. More

broadly, our efforts in advancing the femtomedicine approach has identified a new and

promising family of anticancer compounds and represents an efficient and powerful alternative

to random screening approaches for drug development. As one of the main outcomes of our

efforts in femtomedicine, the present results have demonstrated its potential as an exciting new

frontier to bring breakthroughs in fundamental understandings of major human diseases such as

cancer and to design more effective drugs.

Acknowledgments

The authors thank Drs. Cameron Koch (U Penn), Brad Wouters (PMH, UHN), and Ming Tsao (PMH, UHN) for their generous providing of biomarkers EF5 and CD31, 74B cells and A549 cells, respectively, to our experiments carried out at PMH/OCI/UHN in Toronto. We also particularly thank Dr. Richard Hill for his very helpful comments on an earlier version of this manuscript.

Footnotes

Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

References 1. Bristow R, Hill RP. Molecular and Cellular Radiobiology. In: Tannock IF, Hill RP, Bristow R,

Harrington L, editors. The Basic Science of Oncology. 4th Ed. New York: McGraw-Hill;

2005. p.261-288.




18

2. Lehnert S. Biomolecular Action of Ionizing Radiation. London: Taylor & Francis Ltd; 2008.

p.399-434.

3. Dawson LA, Jaffray DA. Advances in image-guided radiation therapy. J Clin Oncol 2007; 25:

938-946.

4. Koch CJ, Parliament MB, Brown JM, Urtasun RC. Chemical Modifiers of Radiation

Response. In: Phillips TL, Hoppe RT, Roach III M, editors. Leibel and Phillips Textbook of

Radiation Oncology. 3rd Ed. New York: Elsevier; 2010. p.55-68.

5. Bagley RG, editor. The Tumor Microenvironment, Cancer Drug Discovery and

Development. New York: Springer; 2010.

6. Hart E, Anbar M. The Hydrated Electron. New York: John Wiley & Sons, Inc.; 1970.

7. Buxton GV, Greenstock CL, Helman WP, Ross AB. Critical Review of Rate Constants for

Reactions of Hydrated Electrons, Hydrogen Atoms and Hydroxyl Radicals (OH•/O−•) in

Aqueous Solution. J Phys Chem Ref Data 1988; 17: 513-886.

8. Zewail AH. Femtochemistry: Atomic-Scale Dynamics of the Chemical bond using ultrafast

lasers (Nobel lecture). Angew Chem Int Ed 2000; 39: 2587-2631.

9. Migus A, Gauduel Y, Martin JL, Antonetti A. Excess electrons in liquid water: First evidence

of a prehydrated state with femtosecond lifetime. Phys Rev Lett 1987; 58: 1559-1562.

10. Long FH, Lu H, Eisenthal KB. Femtosecond studies of the presolvated electron--an excited-

state of the solvated electron. Phys Rev Lett 1990; 64: 1469-1472.

11. Laenen R, Roth T, Laubereau A. Novel precursors of solvated electrons in water: Evidence

for a charge transfer process. Phys Rev Lett 2000; 85: 50-53.

12. Wang CR, Luo T, Lu QB. On the lifetimes and physical nature of prehydrated electrons in

liquid water. Phys Chem Chem Phys 2008; 10: 4463-4470.

13. Wang CR, Hu A, Lu QB. Direct observation of the transition state of ultrafast electron

transfer reaction of a radiosensitizing drug bromodeoxyuridine. J Chem Phys 2006; 124,

241102.

14. Lu QB. Molecular reaction mechanisms of combination treatments of low-dose cisplatin

with radiotherapy and photodynamic therapy. J Med Chem 2007; 50: 2601-2604.

15. Lu QB, Kalantari S, Wang CR. Electron transfer reaction mechanism of cisplatin with DNA

at the molecular level. Mol Pharmaceutics 2007; 4: 624-628.




19

16. Wang CR, Lu QB. Real-time observation of a molecular reaction mechanism of aqueous 5-

halo-2’-deoxyuridines under UV/ionizing radiation. Angew Chem Intl Ed 2007; 46: 6316-

6321.

17. Wang CR, Nguyen J, Lu QB. Bond Breaks of Nucleotides by Dissociative Electron Transfer

of Nonequilibrium Prehydrated Electrons: A New Molecular Mechanism for Reductive DNA

Damage. J Am Chem Soc 2009; 131: 11320-11322.

18. Wang CR, Lu QB. Molecular mechanism of the DNA sequence selectivity of 5-halo-2’-

deoxyuridines as potential radiosensitizers. J Am Chem Soc 2010; 132: 14710–14713.

19. Lu QB. Effects of Ultrashort-Lived Prehydrated Electrons in Radiation Biology and Their

Applications for Radiotherapy of Cancer. Mutat Res: Rev Mutat Res 2010; 704: 190-199.

20. Lu QB. Dissociative electron transfer reactions of halogenated molecules adsorbed on ice

surfaces: implications for atmospheric ozone depletion and global climate change. Phys Rep

2010; 487: 141-167.

21. Lu QB. New Theories and Predictions on the Ozone Hole and Climate Change. New Jersey,

London, Singapore: World Scientific Publishing Co.; 2015. p.83-140.

22. Nguyen J, Ma Y, Luo T, Bristow RG, Jaffray DA, Lu QB. Direct Observation of Ultrafast

Electron Transfer Reactions Unravels High Effectiveness of Reductive DNA Damage. Proc

Natl Acad Sci USA 2011; 108: 11778-11783

23. Sanche L. Beyond radical thinking. Nature 2009; 461: 358–359.

24. Lu LY, Ou N, Lu QB. Antioxidant induces DNA damage, cell death and mutagenicity in

human lung and skin normal cells. Sci Rep 2013; 3: 3169(1-11).

25. Luo T, Yu JQ, Nguyen J, Wang CR, Bristow RG, Jaffray DA, et al. Electron transfer-based

combination therapy of cisplatin with tetramethyl-p-phenylenediamine for ovarian, cervical,

and lung cancers. Proc Natl Acad Sci USA 2012; 109: 10175-10180.

26. Lu QB, Zhang QR, Ou N, Wang CR, Warrington J. In Vitro and In Vivo Studies of Non-

Platinum-Based Halogenated Compounds as Potent Antitumor Agents for Natural Targeted

Chemotherapy of Cancers. EBioMedicine 2015; 2: 544-553.

27. Choudhury A, Zhao H, Jalali F, Rashid S, Ran J, Supiot S, et al. Targeting homologous

recombination using imatinib results in enhanced tumor cell chemosensitivity and

radiosensitivity. Mol Cancer Ther 2009; 8: 203-2013.




20

28. Brown JM, Diehn M, Loo Jr. BW. Stereotactic ablative radiotherapy should be combined

with a hypoxic cell radiosensitizer. Int J Radiat Oncol Biol Phys 2010; 78: 323–327.

Figure Legends Figure 1. (A-C) In vitro radio-toxicity assays of FMD compounds (FMD-nX-DABs). Cell viabilities of human normal cells (GM05757) after the 12-hr pre-treatment of FMD Compounds with various concentrations at 0-300/400 μM, followed by 225 keV X-ray irradiation. At 12 days post-irradiation, the viability of the cells in 96-well plates was measured by MTT assay. Figure 2. (A-F) In vitro radiosensitizing assays of FMD compounds (FMD-nX-DABs). Cell viabilities or clonogenic survival rates of human cancer cells after the treatment of the FMD compounds at various concentrations for 12 hr, followed by 0-10 Gy of 225 kV X-ray irradiation. At 6 days post-irradiation, MTT assays of the cells were performed. For clonogenic assays, the cells were incubated for 15-17 days post-irradiation to form colonies. The enhancement ratio (ER), defined as the ratio of the radiation dose without FMD to that with FMD for the same level of biological effect, is shown for the clonogenic assay results in D, E and F (see the text).

Figure 3. In vitro DSB assay of DNA in cancer cells treated by a FMD compound combined with X-ray radiation. ME-180 cancer cells were treated with/without 25/200 µM FMD-2Br-DAB for 8 h, followed by IR (225 keV X-rays) with various IR doses. At 12 hr post-irradiation, the cells were fixed and stained, and γ-H2AX assays were performed. A [(a)-(l)]. Images show γ-H2AX foci in the cells treated with the FMD compound at 25 and 200 µM in combination with IR at 0, 1, 2, 4 Gy; Hoechst 33342 was used to map nuclei. B. The graph shows the γ-H2AX intensity in ME-180 cells versus X-ray dose. Figure 4. In vivo pharmacokinetics and (radio-)toxicity of FMD-2Br-DAB in mice. A. (Note that the y-axis is plotted with a log-scale) The levels of the FMD in blood plasma and tumor over 150 min post-injection. B. The gut radio-toxicity of both SCID and nude mice treated by the vehicle only (control) and FMD at 7 mg/kg was measured as the number of surviving and proliferating crypts, determined through Ki67 staining. C. Kidney and liver (radio-)toxicity. H&E and Ki67 staining were conducted for 4 treatment groups: vehicle only, FMD, radiation therapy (15 Gy), and radiation in combination with FMD. Cell proliferation was presented through the pixel count positivity of Ki67, a ratio of the number of positive cells to the total cell number. Figure 5. A-D: In vivo DNA DSBs and apoptosis caused by FMD-2Br-DAB plus 15 Gy IR in mouse xenograft cervical (ME180) cancer model. The FMD at 7 mg/kg was administrated by IP injection into the mice, and at 1 hr after injection, the mice received IR (15 Gy, 225 kV x-rays) targeted to the tumor. The measurements were performed at both 1 and 24 hr post treatment. A. DNA DSBs (γH2AX foci) in the whole tissue including both hypoxic and non-hypoxic regions. B. DNA DSBs in hypoxic regions identified by the hypoxia marker EF5. C. DNA




21

damage repair marker 53BP1 for hypoxic regions. D. Apoptosis in hypoxic regions. Histological images corresponding to these results (A-D) are given in Figures S5 and S6. E-H: Histological changes in three xenograft models after the combination treatment of IR with FMD-2Br-DAB. All tissues were collected at 21 days post-treatment. E. Necrosis fraction in the xenograft cervical cancer (ME180) model. F. Average vessel density in the ME180 model. G. Hypoxia in all three tumor models with treatments of the FMD compound, IR, and the FMD plus IR. H. Hoechst vessel perfusion in the ME180 model. Figure 6. A-D: Tumor growth delays in three mouse xenograft cancer (ME180, 74B and A549) models treated by FMD-2Br-DAB combined with large single dose IR (15 Gy, 225 kV x-rays). For each treatment of FMD in combination with IR, the FMD at 7 mg/kg was administrated by IP injection into the mice, and at 1 hr after injection, the mice received 15 Gy IR (225 kV x-rays) targeted to the tumor. Tumor growth delays were measured through tumor volume change over time. In D, DCE MRI images illustrate the differences between the combination and the 3 control groups at 19 days post-treatment in the ME180 cancer; the color-bar represents vessel perfusion in the tumor area. E and F: Tumor growth delays in two mouse xenograft cancer models of human cervical (ME-180) cancer and lung (A549) cancer treated by FMD-2Br-DAB combined with fractional IR (2Gy×3, 225 kV x-rays). For each treatment of FMD in combination with IR, the FMD at 7 mg/kg was administrated by IP injection into the mice, and at 1 hr after injection, the targeted tumor in mice received radiation (2 Gy, 225 keV x-rays), with totally 3 treatments done at Day 1, 3 and 5.




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Published OnlineFirst February 26, 2016.Mol Cancer Ther Chun-Rong Wang, Javed Mahmood, Qin-Rong Zhang, et al. Molecules for Targeted Radiotherapy of CancerIn Vitro and In Vivo Studies of a New Class of Anticancer

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