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Journal of Ethnopharmacology

journal homepage: www.elsevier.com/locate/jethpharm

Cytotoxic effect of aqueous ethanolic extract of Luffa cylindrica leaves oncancer stem cells CD44+/24- in breast cancer patients with variousmolecular sub-types using tissue samples in vitro

Ibrahim Mohamed Abdel-Salama, A.A. Abou-Bakrb, Mohamed Ashourc,∗

a Cancer Biology Department, National Cancer Institute, Cairo University, Egyptb Pathology Department, National Cancer Institute, Cairo University, EgyptcMedical Research Department, National Institute of Occupational Safety and Health, Egypt

A R T I C L E I N F O

Keywords:Luffa cylindricaBreast cancer stem cellsCD44+/24-

A B S T R A C T

Ethnopharmacological relevance: Luffa cylindrica (L.) M.Roem is a climbing plant its parts have been used astraditional medicine for the treatment of different types of diseases including diarrhea, inflammation, cancer andviral infections. The parts used include fruit, seeds and leaves.

Aim of the study: Our study aims to investigate the effect of the aqueous-ethanol extract of Luffa cylindricaleaves on breast cancer stem cells CD44+/24- and other cell sub-populations using clinical samples with differentmolecular sub-types of breast cancer in vitro.

Materials and Methods: Breast tissues were obtained from patients undergoing surgery for the removal ofbreast tumors after complete clinical and pathological investigations. Tissue samples were processed to cellsuspensions and treated with the extract in the tissue culture laboratory. Percentages of cell sub-populationswithin tumors and viability were measured by flowcytometry using clusters of differentiation as cell markers.Results: Our results revealed that there were decreases in the total cell viability, CD44+/24- and total CD24+

cell sub-populations percentages after treatment with the extract, this may be an important indication of usingLuffa leaves extract in the treatment of breast cancer or in combination with the traditional treatments.

Conclusion: Luffa cylindrica has proven to have anticancer activity on three different subtypes of breast cancerincluding luminal A, luminal B and Her2/neu enriched more over it has cytotoxic effect on both bulk tumor cellsas well as cancer stem cells sub population CD44+/24- which possess high tumorigenic potency, these resultswere confirmed by measuring their viable number after treatment and sphere formation assay results.

1. Introduction

Breast cancer represents one of the most abundant types of malig-nant diseases in women all over the world, the role of early detectionand new methods of diagnosis open more opportunities for therapy, thecriteria of this therapy must consider first the efficacy of the drug used,safety, prevention of recurrence, complete eradication of tumor cells,ability to be used in combination with other known chemotherapeuticagents and low cost (Sorlie et al., 2001). The main problems in breastcancer treatment are the recurrence and metastasis which may be re-ferred by many studies to a group of cells called cancer stem cells(Sotiriou and Pusztai, 2009; Weigelt et al., 2010).

Cancer stem cells (CSCs) are defined as subtypes of cells within atumor, which may represent from 1 to 5 percent from the total bulk oftumor cells, these cells can produce new tumors as they divide to give

CSCs and neoplastic cells generating tumor tissue (Wang et al.,2017).CSCs in solid tumors are identified using a list of markers (Maket al., 2011). None of the known markers solely mark CSCs (O'Brienet al., 2007), as they routinely exhibit much broader expression thancould be expected from the low CSC numbers identified in tumors byxenotransplantation assays (Ricci-Vitiani et al., 2007). The definition ofCSCs as the only self-renewing tumor cells capable of seeding a newtumor implies that CSCs are also responsible for initiation of metastases(A-P et al., 2008), a notion strengthened by the connection betweenCSCs and Epithelial–mesenchymal transition (Mani et al., 2008). CSCsare identified in many cancer types like breast cancer, Hepatocellularcarcinoma, colorectal cancer and lung cancer. Breast cancer stem cells(BCSCs) are considered as pivotal base for formation of breast carci-noma and they are related to metastasis and chemotherapeutic re-sistance, so CSCs eradication may reduce tumor drug resistance to

https://doi.org/10.1016/j.jep.2019.111877Received 17 February 2019; Received in revised form 6 April 2019; Accepted 10 April 2019

∗ Corresponding author. 156-El Hegas Street, Cairo, Egypt.E-mail address: mohamed_ashour_mohamed@yahoo.com (M. Ashour).

Journal of Ethnopharmacology 238 (2019) 111877

Available online 14 April 20190378-8741/ © 2019 Elsevier B.V. All rights reserved.

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anticancer drugs (Nilendu et al., 2018). The main pathways of CSCsrenewal and proliferation include Notch, Hedgehog, and Wnt signalingpathways, these are the major pathways involved in the self-renewaland differentiation of BCSCs (Song et al., 2017), identification markersinclude CD44+/CD24-/low, ALDH1, EpCAM/ESA, and nestin; however,standard method to detect BCSCs is not found, BCSCs may be a base forstruggling resistance to chemotherapy in breast cancer (Shima et al.,2017). The subpopulations CD44+/CD24-/low, ALDH1+ andALDH1+CD44+/CD24-/low represent the most sub-populations withhigh tumorigenic activity, ALDH1+CD44+/CD24-/low cells have potentability of self-renewal, invasion, proliferation and tumorigenicity incomparison with the other cell subpopulations (Shao et al.,2016).Targeting cancer stem cells (CSCs) is a key strategy to preventcancers from developing drug resistance and metastasis. A study carriedout by Lin et al. showed that when doxycycline used with che-motherapy they have eradicated BCSCs as well as other tumor cells (Linet al., 2018).The main target pathways for eradication of CSCs includeWnt, Hedgehog, Notch and miRNAs regulating CSCs genes (Kaur et al.,2018).

Luffa cylindrica is a type of climbing plant that grows in high tem-perature countries in Asia and Africa and used in many Chinese medi-cine formulations. In Asia, it is known as ‘tori’ (Sangh et al., 2012).Seeds called Si Gua Zi are used by Chinese folk medicine practitioners(Jun-Ping, 2017). Also known as Suakwa Seed (Chongyun et al., 2005).The fruit is called Si Gua Luo and has hepatoprotective properties(Hempen and Fischer, 2009), also it is used in treatment of cough,dyspnea, rheumatic diseases and skin inflammations (Xinrong et al.,2003), the fruit decreases swellings in the breast and has mild antic-ancer activity. The formula used for anticancer activity Consist of chaihu (Bupleuri Radix; Bupleurum) 10 gm., qing pi (Citri reticulatae viridePericarpium; Citrus reticulata green pericarp) 10 gm., zhi zi (GardeniaeFructus; Gardenia fruit) 10 gm., mu dan pi (Moutan Cortex; Paeoniasuffruticosa root cortex) 10 gm., gua lou shi (Trichosanthis Fructus;Trichosanthes fruit) 15 gm, si gua luo (Luffa cylindrica fruit) 15 gm.,maiya (Hordei Fructus germinatus; Hordeum vulgare; barley sprouts;malt) 15 gm., pu gong ying (Taraxaci Herba; Taraxacum mongolicum)15 gm., chen pi (Citri reticulatae Pericarpium; Citrus reticulata pericarp)6 gm. where the luffa fruit is used in combination with other herbs asanticancer remedy (Lahans, 2007).

Several studies were carried out using Luffa cylindrica leaves whichhave shown anticancer activity against various types of breast cancer,these studies have identified the main ingredients as well as the an-ticancer activity of the leaves extract against breast cancer cell lines(Garai et al., 2018; Habibah et al., 2017; Sharma et al., 2015). Otherparts of the plants have shown cytotoxic effect like seeds which containthe protein called Luffin; this protein has inhibitory effect on ribosomalfunctions (Liu et al., 2010; TB et al., 1992). The fruit contains anti-oxidants include 1-O-feruloyl-β-d-glucose, diosmetin-7-O-β-d-glucur-onide methyl ester, apigenin-7-O-β-d-glucuronide methyl ester and lu-teolin-7-O-β-d-glucuronide methyl ester(Du et al., 2006a), Apigenintriggers apoptosis and autophagy (Yan et al., 2017), luteolin acts on theintrinsic and extrinsic apoptotic pathways in many cancers (Amin et al.,2010; Tuorkey, 2016) and diosmetin has shown cytotoxic effects onprostate cancer cells via cell cycle arrest (Oak et al., 2018), also it in-hibits cell proliferation in HepG2 cell line and induces apoptosis (Liuet al., 2016).

The main constituents of the aqueous-ethanol extract of the leavesinclude apigenin 7 glucuronide, eriodictyol-7 glucoside, kaemferide,luteolin-O-diglucoside, neodiosmin, diosmin, kaempferol 3-[2‴,3‴,4‴-triacetyl-α-L-arabinopyranosyl-(1–6)-glucoside] and Lucyoside K, theextract has shown cytotoxic effect on MCF-7, BT-474, MDA-MB 231breast cancer cell lines, it exerts its effects via induction of caspase 8,alteration of proliferation and cell cycle arrest (Abdel-Salam et al.,2018).

Another study has shown cytotoxic effect of the water extract of thewhole plant on the circulating cancer stem cells derived from blood

samples of hepatocellular carcinoma patients (Abdel-Salam et al.,2019).

According to the data obtained from different studies using tissuesamples there is a correlation between breast cancer recurrence andmetastasis and the presence of certain types of cancer cells sub-popu-lations (Chen et al., 2015; Lin et al., 2012) that retain high renewablepotency like CD44+/CD24- (Li et al., 2017; Shima et al., 2017), tar-geting these cells may decrease recurrence and metastasis, as they areresistant to most therapeutic strategies, introducing new agents thatdecrease their percent in the bulk of the tumor may be beneficial eitheras an additional treatment beside the traditional treatments or as aprophylactic regimen for high risk subjects. Breast cancer showssomewhat a type of heterogeneity in its cells phenotypes, this hetero-geneity is attributed to the representation of hormone receptors likeestrogen, progesterone and Her2/neu receptors, this heterogeneity inrepresentation of receptors leads to different outcome for therapy andindeed the prognosis so we carry our study using different sub-types ofbreast cancer subjects.

Based on the previous studies carried out using Luffa cylindrica, ourstudy aims to investigate the effect of aqueous-ethanol extract of Luffacylindrica leaves on breast cancer stem cells derived from tumors ofbreast cancer patients in vitro.

2. Materials and methods

2.1. Research design

Plant material collection and authentication, aqueous-ethanol ex-tract preparation, HPLC analysis for active ingredients and screening ofthe potency of the extract against breast cancer cell lines were carriedout according to Abdel-Salam et al. (2018). Patients were selected anddiagnosed by clinical and histopathological methods, after surgical re-moval of the tumors from patients the excised tissues were processedinto single cell suspension to obtain primary cell culture from the tu-mors cells, these primary cell cultures were used for testing the effect ofthe extract on the total tumor cells and tumor cells sub-populationsincluding cancer stem cells sub-population CD44+/24- using flow cy-tometry. Mammospheres test was carried out using the primary cellculture cells to confirm the results of the flowcytometry.

2.2. Plant collection, authentication and extract preparation

Plant material was collected from Orman garden, Giza, Egypt andidentified by Prof. Dr Abdel Salam El Noyehy, Professor of Taxonomy,Botany Department Faculty of Science, Ain Shams University (Cairo,Egypt). The plant was deposited at the herbarium of Pharmacognosydepartment, Faculty of Pharmacy, Ain Shams University, Cairo – Egyptunder code (PHG-P-LC-251). Extraction procedure was as described byAbdel-Salam et al. (2018).

2.3. Patients and tissue samples collection

Twenty five patients were recruited and fully diagnosed for breastcancer by the Department of Medical Oncology, Surgery and SurgicalPathology at National Cancer Institute, Cairo University, Egypt. Thestaging of patients followed the criteria of the tumor-node-metastases(TNM) classification of the International Union Against Cancer (2002).Breast tissues were obtained from the department of pathology aftercomplete histopathological examinations. After removal of breast tu-mors from patients, breast tumor tissues were stored at 2–8 °C for up to24 h in 15ml sterile tubes in culture media DMEM supplemented with10% heat-inactivated fetal bovine serum (FBS) and 50 μg/ml genta-micin. Tumor tissues were collected following the protocol approved bythe institutional review board of National Cancer Institute, Cairo Uni-versity. Approved consent was signed by all patients. Patients handlingand samples collection followed the rules of the World Medical

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Association Declaration of Helsinki.

2.4. Immunohistochemical diagnosis of patients using tissues

2.4.1. ChemicalsPrimary antibodies against ER (Lab Vision, Fremont, CA, USA), PR

(Lab Vision, Fremont, CA, USA), Herceptin (Her2) kit (Dako, Glostrup,Denmark), Primary antibodies against CD44 (Lab Vision, Fremont, CA,USA) and CD24 (Lab Vision, Fremont, CA, USA) were imported viaEgyptian international scientific center (Cairo, Egypt). Bound sec-ondary antibiotics were visualized by standard avidin-biotin-peroxidasetechniques using diaminobenzidine as a chromogen.

2.4.2. Staining estrogen, progesterone and Her2/neu receptorsParts of tumor tissue samples were initially fixed in 50% ethanol for

6 h, followed by fixation in 10% buffered formol saline for 6 h. Finallytissues were formalin-fixed within 48 h. Immunohistochemistry wasperformed using mouse monoclonal antibodies for estrogen receptors(ER), progesterone receptors (PR) (Lab Vision, Fremont, CA, USA), ER(1D5; 1:50), PR (PgR636; 1:400), and Herceptin (Her2) kit (HercepTest,Dako, Carpinteria, CA) according to the manufacturer's instructions. ForER and PR, antigen retrieval was performed as follows: sections weredeparaffinized and rehydrated with deionized water. Then they wereheated in citrate buffer (pH 6.0), using an electric pressure cooker for3min at 12–15 pounds per square inch (PSI) approximately at 120 °C,and cooled for 10min prior to immunostaining. Slides were exposed to3% H2O2 for 5min, incubated with labeled primary antibody for 30minand stained with hematoxylin as counterstain for 5min. These in-cubations were performed at room temperature. Sections were washedwith Tris-buffered saline (TBS) in between incubations (Williams et al.,2009).

HercepTest kit was used for Antigen retrieval for HER2/neu re-ceptors by immersing and incubating the slides in 10mmol/L citratebuffer in a water bath (95-99 °C) for 40min. After decanting the solu-tion, the sections were rinsed in the wash buffer, and later soaked in thebuffer for 5–20min prior to staining (Fonseca-Alves et al., 2017).

2.4.3. Staining Ki67 proteinParaffin wax was removed from slide sections using xylene and

rehydrated in ethanol. For antigen retrieval, slides were incubated withcitrate buffer (pH 6.0) in a pressure cooker then treated with freshlyprepared 3% hydrogen peroxide in methanol for 20min and furtherwashed in Tris-buffered saline. Slides were incubated with the Ki67primary antibodies overnight at 4 °C (Fonseca-Alves et al., 2017).

2.4.4. Diagnostic criteriaER, PR and Her2/neu diagnostic criteria were determined by im-

munohistochemistry using ASCO/CAP guidelines (Fitzgibbons et al.,2010). Tumors with> 1% positively stained tumor cells are classifiedas positive for ER and PR. HER2-positive tumors were defined as +3and HER2-negative tumors were defined as 0 or 1 (Rakha et al., 2015).

2.5. In vitro expansion of progenitor cells from breast tumor specimens

Breast tumor tissues were transferred into sterile glass Petri dish andminced into small pieces of about 2mm×2mm, the minced pieces ofthe breast tumors were digested enzymatically using solution of col-lagenase (Roche Diagnostics GmbH, Mannheim, Germany) and in-cubated at 37 °C for 30min. The digested tissues were forced through a40 μm pore cell strainer; single cells suspensions were obtained. Cellsuspensions were centrifuged at 1000 rpm for 3min and the cell pelletswere collected and washed with PBS three times. Cell pellets were re-suspended in complete DMEM media and checked for viability withtrypan blue dye exclusion method, re-suspended at a concentration of0.5–1×106 cells/mL of DMEM (Biowest, MA, USA), supplementedwith 20% FBS, 2mM L-glutamine, 50μg/mL gentamicin (complete

medium) (all from Life Technologies Inc.) and divided into three ali-quots, the first was cultured in 25 cm2 tissue culture flasks (Corning,Stone Staffordshire, UK) at 37 °C and 5% CO2. Tumor cells having goodviability attached to the bottom of the tissue culture flask within12–24 h. Cultures at 75%–100% confluence were selected for sub-culture by trypsinization with 0.25% trypsin. The other two aliquotswere preserved and used for carrying the other experiments (Turinet al., 2014). To increase the purity of epithelial tumor cells we carriedout differential trypsinization to destroy fibroblasts that may crosscontaminate the primary culture (Jones, 2008).

2.6. Treatment of primary cultures by the Luffa cylindrica extract

Cells were obtained from the flasks containing adherent primaryculture cells and subcultured in 25mL tissue culture flasks, the cellswere divided for each sample on two flasks; one flask served as controland the other was treated by the herbal extract at a dose of 100 μg/mLfor 48 h then the cells were collected and transferred to flowcytometricanalysis. The treatment concentration was calculated as the mean of thevalues of the IC50s of three different subtypes of breast cancer cell linesas described by Abdel Salam et al. (Abdel-Salam et al., 2018).

2.7. Flowcytometry

Cell suspensions obtained from the primary culture were made intopellets then the pellets were resuspended in 1 mL HBSS +2% FBS.Conjugated antibody solutions were prepared in the dark and on ice asthe following: CD24-Fitc 1:20 dilution in HBSS +2% FBS and CD44-APC 1:20 dilution in HBSS +2% FBS. 200 μL of the antibody solutionswere added to each test and control tubes and mixed well and in-cubated for 30min in dark place then transferred to the flowcytometrystation.

2.8. Mammospheres assay

The samples were transferred into 100mm tissue Petri dishes withsmall volumes of media (2mL). The adipose tissues were removed usingsterile scissors, scapel and tweezers. The samples were minced into1mm3 pieces with a sterile scapel or razor blade in petri dishes con-taining 2ml of DMEM/F12 until no large pieces remain. The tissuepieces were resuspended in pre-warmed 10ml DMEM containing pro-teolytic enzyme (3000 U/ml collagenase) and incubated at 37 °C in arotary shaker until all the tissue fragments are digested. Usually di-gestion takes 30min. The fragments were allowed to sediment for5min, then the supernatant transferred in a 15ml conical poly-propylene tube and centrifuged at 1000 rpm for 5min at room tem-perature. The supernatant was carefully decanted off and cells wereresuspended in 1–5ml of mammosphere media.

Mammosphere media DMEM/F12 supplemented with 2mM L-glu-tamine, 100 U/ml penicillin and 100 U/ml streptomycin. Completemedia were prepared immediately before use by adding 20 ng/ml re-combinant human epidermal growth factor (EGF; Sigma), 10 ng/mlrecombinant human basic fibroblast growth factor (bFGF; R&DSystems) and 1x B27 supplement.

Primary culture cells were obtained from flasks containing adherentprimary culture cells (70–80% confluence) by aspirating media,washing twice in 1x PBS, and trypsinization of cells. Primary culturecells suspensions were centrifuged at 1000 rpm at room temperature for5min. Supernatants were decanted and cells were resuspended in1–5ml of mammosphere media. The cells were cultured in ultra-lowattachment sex well plates at 37 c° and 5% CO2.The number of cells isabout 1000 cells per well. Formation of mammospheres was monitoredevery three days (Shaw et al., 2012).

After the culture period, mammospheres greater than 40 μm indiameter were counted under a microscope at 40X magnification.Images of 5 random fields were taken using a digital camera on a light

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microscope and mammospheres size was determined using the ImageAnalysis Software (ImageJ for Windows, National Institute of MentalHealth, Bethesda, Maryland, USA). Mammosphere Forming Efficiency(MFE %) was calculated using the following equation:

= ×MFENo of mamospheres per well

No of cells seeded per well(%)

..

100

2.9. Statistical analysis

Data was represented as the mean ± Standard error, and the dif-ference between two means was tested by student-t test for pairwisecomparison using GraphPad Prism 7 software (GraphPad Software, Inc.,San Diego, California, USA). The level of significance p < 0.05.

3. Results

3.1. Clinicopathological data

Clinicopathological data of the patients includes age, grade of thetumors, hormone receptors and the proliferation protein Ki67.According to this data patients can be differentially diagnosed as lu-minal A, luminal B and Her2/neu enriched breast cancer subtypes asshown in Table 1.

3.2. Flocytometric data of the untreated samples

Flocytometric analysis of the single cell suspensions obtained frompatient tissues has shown that the percentages of total CD44−, totalCD44+, total CD24+, total CD24− and CD44+/24- are85.55 ± 2.94%, 7.36 ± 1.27%, 27.62 ± 2.9%, 66.76 ± 2.62% and1.2 ± 0.2% respectively in Grade II samples while in Grade III, thepercentages of total CD44−, total CD44+, total CD24+, total CD24−

and CD44+/24- are 84.21 ± 4.36%, 6.52 ± 1.3%, 20.19 ± 3.63%,55.73 ± 4.28% and 1.307 ± 0.24% respectively. Flocytometric dataof the samples before treatment with the extract has shown that thereare no significant differences in the percentages of cell subpopulationsbetween Grade II and Grade III except for the subpopulation CD24−

which is lower about 11.03 ± 3.57% (N=13) in Grade III (N= 9)samples (p=0.0129) Fig. 1.

Fig. 1 Cell sub-populations percentages in grade I and II. The dis-tribution of cell sub-populations based on the representation of the

cluster of differentiation CD 44 and CD 24. Most tumor cells in theuntreated samples represent CD24 while breast cancer stem cells re-present CD 44 and low or no CD 24. The percentage of total CD24−

cells is significantly lower in Grade III than Grade II in the untreatedsamples (p= 0.0129).

3.3. Effect of the extract on the viability of total tumor cells after treatment

The percentage of total viability of the total cancer cells derivedfrom the tumors of untreated samples is 90.49 ± 1.32% and in thetreated samples is 55.31 ± 4.74%. Treatment of tumor cells derivedfrom breast tumor with the extract for 48 h has shown a significantdecrease in the total viability of cells of about 35.19 ± 4.1%(p= 0.009, N= 25) Fig. 2.

Fig. 2 Effect of the extract on the total cell viability. The figure il-lustrates the effect of the extract on the viability of total cells in thetumor. (A) The viability in the untreated cells. (B) The viability in thetreated cells. The region (Q1) at the left side of the dotplot representsthe live cells that don't take the dye 7AAD (7-Amino Actinomycin D).The region (Q2) shows the dead cells which take the dye. X-axis isfluorescence of the dye and the y-axis is the number of cells. The per-centages of viable cells in the control and after treatment are90.4 ± 1.32% (N=16) and 55.3 ± 4.74% (N=16) respectively,p= 0.009.

3.4. Differential cytotoxic effect of the extract on the tumor cells sub-populations

The percentages of cells sub-populations total CD44+, total CD24+,total CD44−, total CD24− and CD44+/24- are 7.4 ± 0.95%,24.37 ± 2.07%, 85.18 ± 2.21%, 61.39 ± 2.34% and 1.29 ± 0.13%respectively in the untreated samples. In the treated samples theCD44+, CD24+, CD44−, CD24− and CD44+/24- cells sub-populationspercentages are 6.62 ± 1.28%, 16.08 ± 1.65%, 86.97 ± 2.21%,57.88 ± 4.89%, 0.76 ± 0.11% respectively. Treatment with the ex-tract affects mainly the sub-populations CD24+ and CD44+/24-. Thereare significant decreases in the viability of the sub populations CD24+

and CD44+/24- equal to 8.2 ± 0.14% and 0.5 ± 0.1% respectively(p= 0.01, N=25) on the other hand there are no significant differ-ences in the other cell sub-populations at this treatment dose comparedto the untreated cells Fig. 3.

Fig. 3 Cytotoxicity of the extract in different cells sub-populations.(A) Distribution of cells sub-populations before and after treatment oftumor cells derived from patients with aqueous-ethanol extract of Luffacylindrica. (B) Dotplot of the untreated cells (control) and (C) Dotplot ofthe treated cells (test). The total viability, CD24+ and CD44+/24- sub-populations viabilities were decreased after treatment. The four quad-rants, upper left, upper right, lower left and lower right represent thecell populations CD44-/24+, CD 44+/24+, CD 44-/24- and CD 44+/24-

respectively. The X-axis represents the fluorescence of the CD44-APCconjugate and the Y-axis represents the fluorescence of the CD24-Fitc

Table 1Patients clinicopathological data.

Parameter Mean or Percentage

Age 53 ± 1.6Grade Grade I 8%

Grade II 52%Grade III 40%

Estrogen receptor + ve 76%- ve 24%

Progesterone receptor + ve 56%- ve 44%

Her2/neu receptor + ve 56%- ve 44%

Ki67 <20 68%>20 32%

Luminal A a [ER+|PR+] HER2-KI67- 40%Luminal B a [ER+|PR+] HER2-KI67+ 44%Her2 enriched a [ER-PR-] HER2+ 16%

a Luminal A subtype represents estrogen[ER], progesterone [PR] hormonereceptors and don't represent Her2 receptor nor Ki67 protein ([ER+|PR+]HER2- KI67-). Luminal B represents estrogen[ER], progesterone [PR] hormonereceptors, Ki67 protein and don't represent Her2 receptor. Her2 enriched don'trepresent estrogen[ER], progesterone [PR] hormone receptors and representsKi67 protein.

Fig. 1. Distribution of tumor cells sub-populations in grade II and grade III inthe untreated samples.

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conjugate. ∗ The difference from the control is significant at p < 0.05.

3.5. Cytotoxic effect of the extract on the tumor cells sub-population CD44+/24- in grade II and III tumor cells

Treatment of tumor cells derived from Grade II (N=13) and III(N= 9) has shown a significant decrease in the number of viable cellssub-population CD 44+/24- (P=0.002, P=0.003) Fig. 4.

Fig. 4 Effect of the extract on the viability of CD 44+/24- cells sub-population (Breast cancer stem cells) after treatment among patientswith Grade II and III. ∗ The difference from the untreated (Control) issignificant at p < 0.05.

3.6. Cytotoxic effect of the extract on the tumor cells sub-population CD44+/24- in luminal A, luminal B and Her2 enriched breast cancer sub-types

Treatment of tumor cells derived from luminal A(N=3), luminal B(N=10) and Her2 enriched (N=3) subtypes leads to decreases in thepercentages of the viable CD 44+/24- subpopulation from1.56 ± 0.27% to 0.78 ± 0.16% (p=0.018),1.33 ± 0.19% to0.65 ± 0.09% (p= 0.003) and 1.83 ± 0.17% to 0.65 ± 0.12%(p= 0.03) respectively.

Fig. 5. The effect of the extract on the viability of breast cancer stemcells CD 44+/24- sub-population derived from different molecular sub-types of breast cancer. ∗ The difference from the untreated is significant

Fig. 2. Cytotoxic effect of Luffa cylindrica extract on total cell population.

Fig. 3. Effect of Luffa extract on the distribution and viability of cells sub-populations.

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at p < 0.05. s.

3.7. Cytotoxic effect of the extract on cancer stem cells confirmed by themammosphere formation assay

The Mammosphere Forming Efficiency (MFE %) was decreased aftertreatment from 8.11 ± 0.07% to 3.48 ± 0.56% (p < 0.05). Spherediameter was significantly decreased from 66.3 ± 1.8 μm to45 ± 2.8 μm (p=0.04) Fig. 6.

Fig. 6. The mammospheres formed from the total bulk tumor cellsderived from the clinical samples of luminal A, luminal B and Her2/neuenriched photographed after 21 days from the start of culture usingOlympus IX53 inverted phase contrast microscope. The untreated cells

(control) and treated cells at magnification 40×.The diameters of thespheres after treatment are smaller than that in the control p= 0.04.

4. Discussion

Luffa cylindrica plant has shown different pharmacological actions;the plant has hepatoprotective, anti-inflammatory, anthelminthic, an-timicrobial, anticancer and enzyme inhibition properties (NK et al.,1972; Peters et al., 1997; SK and V, 1968). The seeds have anti-in-flammatory, antimicrobial, cytotoxic and bronchodilator effects(Muthumani et al., 2010). Vegetable of Luffa cylindrica has antioxidantactivity(Du et al., 2006b). Luffa cylindrica leaves extract (aqueous-ethanol) has been proved to be effective as anticancer agent in differentsub-types of breast cancer cell lines via induction of apoptotic pathwaysand alteration of cell cycle (Abdel-Salam et al., 2018). Water extract ofthe whole plant has shown anticancer activity towards circulatingcancer stem cell isolated from the blood of hepatocellular carcinomapatients(Abdel-Salam et al., 2019).

As the common treatments for cancers target the bulk of the tumorcells and cannot attack CSCs due to the high resistance properties ofthese cells, leading to metastasis and tumor recurrence, so introducingnew agents to decrease their numbers may create a new way for com-plete eradication of cancer. CD24 is represented in 84% of breast car-cinoma patients and is related to the tumor aggressiveness (Kristiansenet al., 2003), as a result of this many studies try to focus on CD24 cellsas a therapeutic target (Ayob and Ramasamy, 2018; Cong et al., 2017).CD44 and CD24 are expressed in 61.4%, 44% of tumor cells, respec-tively (Riaz et al., 2018). Breast cancer cells subpopulation CD44+/

CD24- has stem cell like properties, patients with tumors containinghigh percentages of CD44+/CD24- cells develop bone metastases athigher rate than those with less percentages (Aulmann et al., 2010).

Few studies that use natural extracts to study their effects on thecancer stem cells subpopulation, so investigation of Luffa Cylindricaaqueous ethanol extract may introduce new way to develop a newtherapeutic management for breast carcinoma.

From the previous studies on cancer stem cells in breast cancer weconduct our study and concentrate our research target on CD44 andCD24 containing cells and the cells have a combination of these CDs.Tumor cells were derived from patient samples were cultured andtreated by the extract, treatment of live cells leads to a significant de-crease in the total viability of the whole cell population but on the otherhand there is different rate of killing between the cell sub populationswithin the tumor, CD44−, CD 44+, CD 24+, CD 24- and CD 44+/24- arethe four determined cell subpopulations detected by two monoclonalantibodies and flowcytometry.

The most prominent cytotoxic effect of the extract was on theCD24+/44- sub-population and the CD 44+/24- subpopulation, theformer represented mainly the bulk of tumor cells while the later re-presented the cancer stem cells of breast tumor. To confirm the effect ofthe extract on cancer stem cells, mammosphere assay was carried out.Formation of mammosphere depend on cancer stem cells as they re-present the main component of the spheres, after treatment of thespheres with the extract the number and diameter of the spheres formedhas been significantly decreased. Testing of the extract on grade II andIII derived cells leaded to a significant decrease in the viability of thecancer stem cells CD 44+/24-, also the same results were obtained inthe three sub-types of breast cancer (luminal A, luminal B and Her2enriched) derived cells.

The effect of the extract on cancer stem cells may be attributedmainly to the ingredients apigenin and luteolin which have been provedto affect stem like cells in different types of cancers, apigenin blocks thephosphorylation of c-Met and mitogen-activated protein kinase inGlioblastoma cancer stem cells (Kim et al., 2016). Another proposedmechanism includes disruption of the YAP/TAZ-TEADs protein–proteininteraction and their target CTGF and CYR61 genes (Li et al., 2018).Apigenin decreases the expression of the proteins CK2α and Gli1

Fig. 4. Effect of Luffa extract on CD 44+/24- cells sub-population in Grade IIand III.

Fig. 5. Effect of Luffa extract on CD 44+/24- cells in the molecular sub-types ofbreast cancer.

Fig. 6. Mammosphere formation from total bulk tumor cells derived from tu-mors tissues.

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proteins in the ovarian cancer stem cells of ovarian cancer SKOV3 cellline (Tang et al., 2015). Luteolin interferes with Wnt signaling pathwayby up regulation of FZD6 (frizzled class receptor 6) (Han et al., 2018).

According to this study Luffa cylindrica has shown cytotoxic effecton breast cancer stem cells sub-population CD44+/24- which wereconfirmed by the results of flowcytometry and mammosphere assay.This makes the extract of high value either as a therapeutic agent todecrease recurrence and metastasis or as a prophylactic nutrition forhigh risk subjects. A future research about the use of this extract in theclinical situation must be put in consideration.

Conclusion: Luffa cylindrica has proven to have anticancer activityon three different subtypes of breast cancer including luminal A, lu-minal B and Her2/neu enriched more over it has cytotoxic effect onboth bulk tumor cells as well as cancer stem cells sub populationCD44+/24- which possess high tumorigenic potency, these results wereconfirmed by measuring their viable number after treatment and sphereformation assay results.

Author contribution statement

All authors certify that they have participated sufficiently in thework to take public responsibility for the content, including participa-tion in the concept, design, analysis, writing, or revision of the manu-script. I.M. Abdel-Salam established the research design, methodology,data collection and statistical analysis. Amany Abou Bakr collectedsamples, carried out histopathological examination for tissues. M.Ashour carried out cell culture procedures, flowcytometry and writingmanuscript.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jep.2019.111877.

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