Zoledronic acid overcomes chemoresistance by

Biotechnic & Histochemistry

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Zoledronic acid overcomes chemoresistance by sensitizing cancer stem cells to apoptosis H Rouhrazi, N Turgan & G Oktem To cite this article: H Rouhrazi, N Turgan & G Oktem (2018): Zoledronic acid overcomes chemoresistance by sensitizing cancer stem cells to apoptosis, Biotechnic & Histochemistry, DOI: 10.1080/10520295.2017.1387286 To link to this article: https://doi.org/10.1080/10520295.2017.1387286

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Date: 09 January 2018, At: 05:27

Zoledronic acid overcomes chemoresistance by sensitizing cancer stem cells to apoptosis H Rouhrazi1, N Turgan2, G Oktem3,4 1

Department of Medical Biochemistry, Ege University Institute of Health Sciences, Faculty of Medicine, Bornova, Izmir, Department of Medical Biochemistry, Near East University Faculty of Medicine, Nicosia, 3Department of Histology and Embryology, Ege University Faculty of Medicine, Bornova, Izmir, and 4Department of Stem Cell, Ege University Institute of Health Sciences, Bornova, Izmir, Turkey 2

Downloaded by [Dokuz Eylul University ] at 05:27 09 January 2018

Abstract Unlike low tumorigenic bulk tumor cells (non-CSCs), cancer stem cells (CSCs) are a subset of tumor cells that can self-renew and differentiate into different cancer subtypes. CSCs are considered responsible for tumor recurrence, distant metastasis, angiogenesis, and drug or radiation resistance. CSCs also are resistant to apoptosis. Zoledronic acid (ZA) is a third generation bisphosphonate that reduces cell proliferation and exhibits anti-tumor effects by inducing cell death in some malignancies; however, the effects of ZA on CSCs are unclear. We investigated the anti-cancer effects of ZA on two epithelial cancer cell lines, prostate DU-145 and breast MCF7, focusing primarily on induction and activation of apoptosis. Cluster of differentiation (CD) 133+/CD44+ prostate CSCs and CD 44+/ CD24 breast CSCs were isolated from the DU-145 human prostate cancer and MCF-7 human breast cancer cell lines, respectively, using FACSAria flow cytometry cell sorting. CSCs and non-CSCs were exposed to increasing concentrations of ZA for 24, 48 and 72 h to determine the IC50 dose. Annexin-V assay for detecting cell death and cell cycle was performed using the Muse™ Cell Analyzer. Prostate CSCs and non-CSCs were assayed by quantitative reverse transcription PCR (qRT-PCR) array for detecting 84 key apoptosis related genes. Gene regulation at the protein level was investigated by immunofluorescence. ZA caused a dose- and time-dependent decrease in cell viability. Treatment with ZA resulted in a concomitant increase in apoptosis and cell cycle arrest at S-phase in CSCs. Significant over/under-expressions were detected in seven of the genes of ZA-treated DU-145 CSCs cells. Expressions of CASP9, CASP4, BAX and BAD genes increased, while the expressions of BIRC3, BIRC2 and BCL2 genes decreased. In the DU-145 non-CSCs, five genes exhibited changes in gene expression after ZA treatment, two exhibited increased expression (CASP7 and BAD) and three exhibited decreased expression (BIRC3, BIRC2 and BCL2). ZA caused cell death of drug resistant breast MCF-7 and prostate DU-145 cancer stem cells by activating apoptosis. ZA can facilitate the intrinsic pathway of apoptosis in human prostate CSCs by down-regulating anti-apoptotic genes and up-regulating pro-apoptotic genes. ZA may be an effective therapeutic agent for targeting chemoresistance in CSCs. Key words: apoptosis, bisphosphonates, cancer, drug resistance, stem cells, zoledronic acid

There is evidence for a small population of cancer cells that can self-renew and initiate new tumors. These cells are known as cancer stem cells (CSCs)

Correspondence: Gulperi Oktem, Department of Histology and Embryology, Ege University Faculty of Medicine, Bornova, 35100, Izmir, Turkey. e-mail: [email protected] © 2017 The Biological Stain Commission Biotechnic & Histochemistry 2017, Early Online: 1–12

DOI: https://doi.org/10.1080/10520295.2017.1387286

and they are responsible for resistance to cytotoxic drugs and radiation. CSCs can differentiate; they are slow cycling cells that can reconstitute tumors. CSCs were identified first in human acute myeloid leukemia and later have been found in many solid tumors (Clevers 2011, Valent et al. 2012, Magee et al. 2012, Visvader and Lindeman 2012, Singh 2013). Studies using lineage tracing techniques for epidermal tumors, intestinal adenomas and

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glioblastoma have shown that these tumors originate from CSCs (Driessens et al. 2012, Chen et al. 2012, Schepers et al. 2012). The CSC theory offers potential strategies to target CSCs instead of traditional approaches for cancer treatment. So far, no drugs have been identified that target CSCs efficiently (Singh 2013). The ability of CSCs to resist cell death is an important factor for treatment of cancer (Hanahan and Weinberg 2011). Significant advances have been made in discovery and validation of novel cancer therapeutics designed to stimulate apoptosis. Such novel therapeutics may prove significant for treating cancers that are resistant to conventional therapies (Schuler and Meiler 2006, Hassan et al. 2014). Apoptosis is programmed, physiological cell death that occurs naturally in cells; it is characterized by morphological changes including chromatin condensation and nuclear fragmentation with the formation of apoptotic bodies. The signal that triggers apoptosis arises from a subtle balance between apoptotic and anti-apoptotic proteins. Bisphosphonates (BPs) are the synthetic analogues of pyrophosphates that are used clinically for treating abnormal bone absorption diseases such as osteoporosis and hypercalcemia associated malignancies (Yuasa et al. 2007). There are two classes of BPs that differ in their mechanism of action and structure (Hosfield et al. 2004). One class is pyrophosphate-resembling BPs that act as inhibitors of ATP-dependent enzymes; etidronate and clodronate are two examples. A second class is more potent and includes nitrogen-containing BPs (NBPs) including ibandronate, risedronate, pamidronate, alendronate and zoledronic acid (ZA). The N-BPs, or so-called second and third generation BPs, inhibit a key enzyme in the mevalonate pathway, farnesyl pyrophosphate synthetase, which subsequently causes depletion of isoprenoid pools and decreased prenylation of small guanine nucleotide-binding regulatory proteins (small G proteins) (Luckman et al. 1998). These lipid modifications are essential for most small G proteins to bind to cytoplasmic and organelle membranes where prenylated small G proteins become functional (Wennerberg et al. 2005). The third generation BP, ZA is considered the most potent nitrogencontaining BP available clinically and it is widely used against skeletal metastases of various tumor types including prostate and breast cancer (Corey et al. 2003, Almubarak et al. 2011). Evidence is accumulating that ZA also has anti-tumor activities such as antiproliferative and cytostatic effects on some types of human malignancies by mechanisms including cell adhesion, invasion, anti-angiogenic

effects and apoptosis (Dumon et al. 2004, Lee et al. 2001, Oades et al. 2003). We investigated the inhibitory effect of ZA on CSCs using two epithelial cancer cell lines, prostate DU-145 and breast MCF7, as models to explore in vitro the anti-cancer effect of ZA. Our focus was primarily on induction and activation of the apoptotic pathway.

Material and methods Cell lines and cultures We performed all experiments using the established human prostate cancer cell line, DU-145, and human breast cancer cell line, MCF-7. The cell lines were purchased from American Type Culture Collection (Manassas, VA). These cell lines were selected for their high tumorigenic potency and drug resistance. Cells were cultured in RPMI 1640 (Lonza, Basel, Switzerland) containing 10% fetal bovine serum (FBS) (Gibco, Invitrogen Life Technologies, Paisley, UK), 1% penicillin and streptomycin (Sigma-Aldrich, St. Louis, MO) and grown at 37º C in a humidified atmosphere with 5% CO2. Growth and morphology were checked microscopically each day to ensure cell health. Cells were split passaged upon reaching approximately 80% confluency, harvested using 0.05% trypsin (Gibco, Invitrogen) and centrifuged at 180 x g for 5 min at room temperature after adding RPMI 1640 to inactivate trypsin. After centrifugation, they were re-suspended in culture medium.

Fluorescence-activated cell sorting (FACS) For cell sorting, the cells were detached using nonenzymatic cell dissociation solution (SigmaAldrich) and re-suspended in Dulbecco’s phosphate-buffered saline (DPBS) (Gibco, Invitrogen). Approximately 3 × 105 cells were incubated with an antibody diluted 1:100 with FACS containing 0.5% bovine serum albumin (BSA), 5 mM EDTA and 2 mM NaN3 for 15 min at 4º C. For DU-145 cells, phycoerythrin (PE) labeled monoclonal antibody (Miltenyi Biotec Ltd., Bisley, UK) was used and the samples were labeled with PE labeled CD133 (clone AC133/1; Miltenyi Biotec Ltd.) and fluorescein isothiocyanate (FITC) labeled CD44 antibody (clone G44-26; BD Biosciences, San Jose, CA). For MCF7 cells, PE labeled CD24 and FITC labeled CD44 monoclonal antibodies were used. Cells were washed and subsequently re-suspended

2 Biotechnic & Histochemistry 2017, Early Online: 1–12

after incubation for 5 min. The DU-145 cells were sorted as CD 133high/CD44high population (sorting cells) and non-sorting counterparts. MCF7 cells were sorted as CD 44high/CD24low population as sorting cells and the rest as non-sorting counterparts using the FACS machine (FACSAria; BD Biosciences, San Jose, CA). Purities were confirmed by FACSAria flow cytometry with post-sort analysis and samples with > 90% purity were used for further experiments. Sorted cell populations were cultured in monolayer culture for treatments.


Cell cytotoxicity analysis We counted cells before and after exposure to increasing doses of ZA using the Muse Count and Viability kit (Muse™ Cell Analyzer; Millipore, Billerica, MA) according to the manufacturer’s instructions. This is based on an in house nuclear staining method for assessment of cell viability. Briefly, 50 μl treated cell suspension was added to 450 μl MUSE Count and Viability Reagent diluted 1:10, incubated for 5 min at room temperature, then analyzed. Viability of the cells following treatment also was determined using this method. The cells were cultured at a density of 105 cells/well in 6-well flat-bottomed tissue culture plates (Becton Dickinson, San Jose, CA) with complete culture medium and allowed to adhere to the plate overnight. Then the cells were incubated with increasing concentrations [0 (control)−140 µM] ZA for an additional 24, 48 and 72 h at 37º C in an incubator with humidified atmosphere and 5% CO2. Complete culture medium was used for control cells. After incubation, all cells were collected and diluted with phosphate-buffered saline (PBS). Cell viability was evaluated using the Muse™ Count and Viability kit. Data were presented as proportional viability (%) by comparing the treated group with the untreated cells, the viability of which was assumed to be 100%. The drug concentration that reduced the viable fraction of cells in each well by 50% compared to the control constituted the IC50 dose. All experiments were performed in triplicate. Apoptosis analysis The Muse Annexin V & Dead Cell Assay Kit (Muse™ Cell Analyzer; Millipore) and the Muse Cell Analyzer (Muse™ Cell Analyzer; Millipore) were used for quantitative analysis of live, early/ late apoptotic and dead cells according to the manufacturer’s instructions. The assay uses Annexin V to detect phosphatidylserine (PS) on the external membrane of apoptotic cells and a dead cell

marker (7-AAD). Briefly, following treatment with ZA, all cells were harvested and diluted in PBS containing 1% BSA and 1% FBS to a concentration of 5 × 105 cells/ml. Then, 100 μl of Annexin V and Dead Reagent and 100 μl of single cell suspension were mixed in a microtube and incubated in the dark for 30 min at room temperature prior to analysis using the Muse Cell Analyzer. The apoptotic proportion was determined by discrimination of four distinct populations: 1) Annexin V negative and 7-AAD negative = live cells with no detectable apoptosis; 2) Annexin V positive and 7-AAD negative = early apoptotic cells; 3) Annexin V positive and 7-AAD positive = apoptotic cells; 4) Annexin V negative and 7-AAD positive = dead cells that were dying by a non-apoptotic route. All experiments were performed in triplicate. Cell cycle analysis Cell cycle analysis was performed using the Muse™ cell cycle kit Muse™Cell Analyzer (Millipore) according to the manufacturer’s instructions. Briefly, cells were seeded into 6-well plates at a density of 2 × 105 cells/well for 24 h. Then, the cells were treated with an IC50 dose of ZA for 72 h. Cells then were harvested by trypsinization and washed twice with ice-cold PBS. For rapid cell fixation, the cell suspensions were added drop-wise to 1 ml 70% ethanol at −20º C for 5 h. After washing with PBS and centrifugation, the cell pellet was resuspended in 200 μl Muse cell cycle reagent and incubated for 30 min at room temperature, protected from light. The percentage of cells in G0/ G1, S and G2/M phases then was calculated by the Muse cell analyzer. Rt2profilertm human apoptosis polymerase chain reaction (PCR) array Total RNA was isolated and purified using RNeasy Mini Kit (SABiosciences-Qiagen, Frederick, MD) according to the manufacturer’s instructions. Total RNA then was eluted in nuclease-free water and quantified using MaestroNano Micro-Volume Spectrophotometer (Maestrogen MN-913, Hsinchu, Taiwan). RNAs with an OD 260 nm/OD 280 nm absorbance ratio of at least 2.0 were used. Samples were stored at −80º C until further analysis. Total RNA was reverse-transcribed into cDNA using the RT2 First Strand Kit (SABiosciences-Qiagen) using a GeneAmp PCR 9700 (Applied Biosystems, Foster city, CA). Prepared cDNA then was combined with RT2 qPCR master mix with Sybr Green (SABiosciences) and aliquoted in equal volumes to each well of the

Zoledronic acid sensitizes cancer stem cells to apoptosis 3

real-time PCR arrays. Arrays were run on an Applied Biosystems ABI 7900HT Fast Real-Time PCR System (Life Technologies, Carlsbad, CA) using universal cycling conditions (10 min at 95º C, 40 cycles of 15 sec at 95º C, 1 min at 60º C). The Human Apoptosis RT2ProfilerTM PCR Array (SA Biosciences) examines 84 genes related to the apoptotic pathway (Table 1). All cycling thresholds (CT) for individual wells ≥ 35 were adjusted to 35. Data analysis was conducted using SABioscience’s proprietary online program to calculate relative gene expression using ΔΔCt.

(BIRC2), Baculoviral IAP repeat containing-3-protein (BIRC3), caspases 9, 7 and 4, B-cell lymphoma-2 (Bcl2), BCL2 associated agonist of cell death (BAD) and BCL2 associated X (BAX) proteins (All from Abcam, Cambridge, UK) overnight, then with the corresponding secondary anti-rabbit and anti-mouse fluorescein (FITC)-conjugated antibodies (both from Invitrogen) for 1 h at room temperature. The immunostained cells were mounted in mounting medium containing 4,6diamidino-2-phenylindole (DAPI), and visualized using a fluorescence microscope equipped with a camera (Olympus BX-51 and the Olympus C-5050 digital camera, Tokyo, Japan).


Immunofluorescence staining Immunofluorescence staining was performed to investigate the levels of the proteins whose associated gene expressions indicated were significantly altered by treatment with ZA. Cells were cultured on special coverslips at 104 cells/coverslip and placed in 6-well flat-bottomed tissue culture plates (Becton Dickinson) with complete culture medium and allowed to adhere to the plate overnight. Subsequently, cells were treated with an IC50 dose of ZA and fixed in 4% paraformaldehyde for 15 min. The cells then were permeabilized with 0.1% Triton X-100 for 10 min and rinsed three times with PBS. Nonspecific binding sites were blocked using 5% BSA for 1 h. The preparations were incubated at 4º C with primary antibodies against Baculoviral IAP repeat containing-2-protein

Statistical analysis All experiments were carried out in triplicate. Comparisons between groups were made using independent sample t-test and one-way ANOVA, followed by Tukey post hoc test. Differences were considered significant if the p value was ≤ 0.05.

Results Purity and sorting rates of cscs and non-cscs subpopulations DU-145 human prostate cancer cells were sorted for CD133 and CD44 surface expression and

Table 1. Apoptosis related genes investigated Gene symbols 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

AKT1 APAF1 AVEN BAD BAG1 BAK1 BAX BBC3 BCL2 BCL2L1 BCL2L10 BCL2L11 BCL2L13 BCL2L2 BID BIK BIRC2 BIRC3 BIRC5 BOK CAD

22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.

CASP1 CASP10 CASP12 CASP14 CASP2 CASP3 CASP4 CASP5 CASP6 CASP7 CASP8 CASP8AP2 CASP9 CFLAR CRADD DFFA DIABLO ENDOG FADD FAM96A FAM96B

43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63.


FAS FASLG HMGB1 HRK HSP90B1 HTRA2 LRDD MCL1 NFKB1 NFKB2 NGFR PMAIP1 PTEN REL RELA RELB SOCS2 SOCS3 STAT1 STAT5A STAT5B

64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84.

TNF TNFRSF10A TNFRSF10B TNFRSF10C TNFRSF11B TNFRSF1A TNFRSF1B TNFRSF21 TNFRSF25 TNFSF10 TNFSF11 TNFSF8 TP53 TP53I3 TRAF1 TRAF2 TRAF3 TRAF4 TRAF5 TRAF6 TRAF7

MCF7 human breast cancer cells were sorted for CD44 and CD24 by FACS. The rates of DU-145 CSCs and non-CSCs were 5.7 and 94.3%, respectively. In MCF7 cells, rates were 4.4% for sorting cells and 95.6% for non-sorting cells. The purities of the sorted cell populations were > 85%.


Cytotoxicity effects of ZA on prostate and breast cscs Prostate DU145 CSCs and bulk population (DU145 non-CSCs), breast MCF7 CSCs and bulk population (MCF7 non-CSCs) were treated with increasing concentrations of ZA (0 − 140 µM) for 24, 48 and 72 h, and cell viability was measured using the MuseTM cell counter. ZA induced a dose- and time-dependent decrease in cell viability as shown in Fig. 1. The inhibitory IC50 concentration of ZA for DU-145 CSCs and nonCSCs was 108 µM and 96 µM in 72 h, respectively. The IC50 of ZA for MCF7 CSCs and nonCSCs was 94 µM and 92 µM in 72 h, respectively (Fig. 1). There was a significant difference in IC50 dose between DU-145 CSCs and non-CSCs (p = 0.004). DU-145 non-CSCs exhibited great sensitivity to ZA, which resulting in less viability compared to the DU-145 CSCs. The IC50

dose in MCF7 cells was less than for DU-145 cells. IC50 doses for MCF7 CSCs and non-CSCs were not significantly different.

ZA induced apoptosis in cscs both prostate and breast cancers To elucidate the mechanisms underlying ZA induced growth inhibition, we used the Muse™ Annexin V and Dead Cell assay for prostate DU145 CSCs and bulk population (DU-145 nonCSCs), breast MCF7 CSCs and bulk population (MCF7-non-CSCs) cells exposed to ZA at their individually determined IC50 doses. ZA significantly induced apoptosis in all four groups of cells. In DU-145 CSCs, we observed a significant difference in total apoptosis between ZA treated cells compared to controls (42.78 ± 1.24 vs. 3.13 ± 0.47, respectively) (p < 0.001). We found a significant difference in total apoptosis between ZA treated cells compared to controls; 50.43 ± 0.74 vs. 3.47 ± 0.44, respectively) (p < 0.001) in DU-145 non-CSCs. Therefore, ZA accelerated apoptosis significantly in DU-145 non-CSCs compared to DU-145 CSCs (p = 0.006). In MCF7 CSCs, we found a significant increase in total apoptosis between ZA

Fig. 1. Assessment of cell cytotoxicity and inhibitory concentration of IC50. Viability was determined using the Muse cell analyzer after 24, 48 and 72 h exposure of human prostate DU145 CSCs, non-CSCs and human breast MCF7 CSCs and non-CSCs to increasing concentrations of ZA. Values are means ± SD of three experiments.


Altered cell cycle distribution in cscs and noncscs caused by ZA We tested the effect of ZA on the cell cycle to determine whether ZA induced apoptosis of cells was caused by alterations of the cell cycle. Cells were exposed to IC50 doses of ZA and cell cycles were investigated using the Muse™ cell cycle kit. The percentages of cells in the G0/G1, S and G2/M


treated cells compared to controls (52.72 ± 1.16 vs. 2.74 ± 0.24, respectively) (p < 0.001). We also found a significant difference in total apoptosis between ZA treated cells and controls (52.70 ± 1.69 vs. 3.26 ± 0.21, respectively) (p < 0.001) in MCF7 non-CSCs (Fig. 2A, B). The number of total apoptotic cells in MCF7 CSCs and non-CSCs were similar.

Fig. 2. Apoptotic effect of ZA measured by Annexin V and Dead assay. A) ZA induced apoptosis in cells treated with IC50 doses of ZA. Data are means ± SD of three experiments. B, C) Representative images of result of Muse Annexin V and Dead Cell assay, respectively.


and 62.92 ± 1.01, 31.75 ± 1.11 and 5.43 ± 0.70%, respectively, for untreated CSCs. For the MCF-7 non-CSCs group, the percentages were 51.60 ± 0.64, 40.85 ± 0.58 and 7.24 ± 0.33%, respectively, in ZA-treated cells and 58.68 ± 1.23, 36.32 ± 0.72 and 5.36 ± 0.32%, respectively, for untreated cells (Fig. 3A, B). Generally, ZA treatment increased the percentage of cells in S-phase in all four groups. In DU-145 CSCs, we observed a noteworthy increase in the number of cells in S-phase and a reduction of the number of cells in


phases were 40.89 ± 1.56, 53.35 ± 0.91 and 3.66 ± 0.38%, respectively, in DU-145 CSCs compared to 65.23 ± 1.05, 31.32 ± 1.40 and 2.96 ± 0.09% in untreated DU-145 CSCs. For DU-145 non-CSCs, the percentages were 51.51 ± 0.83, 40.64 ± 0.55 and 6.60 ± 0.32%, respectively, for ZA treated and 64.96 ± 1.46, 28.47 ± 0.52 and 4.44 ± 0.29%, respectively, for untreated cells. For MCF-7 CSCs, the percentage of cells in the G0/G1, S and G2/M phases were 55.34 ± 0.87, 37.49 ± 0.73 and 7.53 ± 0.78%, respectively, for ZA treated CSCs,

Fig. 3. Cell cycle analysis following ZA treatment using the Muse cell cycle assay. A) Cell cycle phase alterations caused by IC50 doses of ZA in prostate DU-145, breast MCF-7 CSCs and non-CSCs. The percentage of cells in G0/G1, S and G2/M phases were calculated by the Muse cell analyzer. B, C) Representative histograms showing the effect of ZA on cell cycle.


G0/G1 phase. Our findings suggest that ZA downregulates CSCs viability and survival by causing cell cycle arrest at S-phase.


ZA induced alterations in apoptosis related gene expression in DU-145 cscs and non-cscs DU-145 CSCs and DU-145 non-CSCs were treated with the IC50 dose of ZA for 72 h. After treatment, total RNA was extracted and cDNA was reversetranscribed from mRNA for real-time PCR of 84 apoptosis related genes. A three-fold change in the mRNA expression level was used as the cut-off requirement to determine significant regulatory effects on genes. Among the genes studied, seven genes in ZA treated DU-145 CSCs exhibited over/ under-expression consistently compared to controls. Overexpression was observed in CASP9, CASP4, BAX and BAD genes (FC = 4.507 ± 0.73, 4.339 ± 0.07, 3.589 ± 0.23 and 3.458 ± 0.38, respectively) and under-expression was observed in BIRC3, BIRC2 and BCL2 genes (FC = −4.755 ± 0.30, 4.449 ± 0.32, -4.545 ± 0.47, respectively) (Fig. 4A). In the DU-145 non-CSCs, five genes exhibited changes in gene expression after ZA treatment, CASP7 and BAD exhibited increased expression (FC = 4.981 ± 0.74 and 3.442 ± 0.24, respectively) and BIRC3, BIRC2 and BCL2 exhibited decreased expression (FC = −3.160 ± 0.20, −3.151 ± 0.40 and −4.488 ± 0.15, respectively) compared to controls (Fig. 4B). The functional grouping and comparison of changes of the most responding genes in ZA

treated DU-145 CSCs and non-CSCs is presented in Table 2. Confirmation of apoptotic proteins related to significant expression of genes affected by ZA with immunofluorescence staining We used Immunofluorescence staining to investigate alterations in the regulation of genes at the protein level. In DU-145 CSCs (Fig. 5A), ZA treatment caused a significant increase in CASP4 and BAD, and a significant decrease in BIRC2, BIRC3 and BCL-2 immunofluorescence compared to controls. In DU-145 non-CSCs, CASP3, BAD and BAX were increased and BIRC2, BIRC3 and BCL-2 were decreased (Fig. 5B).

Discussion We believe ours is the first report of the inhibitory effect of ZA directly on CSCs. We used two epithelial cancer cell lines, prostate DU-145 and breast MCF7, as models to explore in vitro the anti-cancer effect of ZA by focusing primarily on induction and activation of apoptosis pathway. The evidence is clear that BPs decrease the proliferation and viability of tumor cell lines in vitro, reduce skeletal tumor burden and slow the progression of bone lesions in animal models (Corey et al. 2003, Croucher et al., 2003, Alvarez et al. 2003). BPs exhibit anti-cancer activities in a variety of cancer cells including multiple myeloma, pancreas, prostate and breast cancer (Green 2004). We

Fig. 4. Gene expression profile associated with the apoptosis signaling pathway. Fold changes (FC) in gene expression in prostate CSCs (A) and non-CSCs (B) treated with IC50 doses of ZA compared to untreated groups.


0.20 0.40 0.56 0.01 0.74 0.15 0.24 0.47 ± ± ± ± ± ± ± ± -3.160 -3.151 2.392 1.087 4.981 -4.488 3.442 2.488 0.30 0.32 0.73 0.07 0.07 0.47 0.38 0.23 Only genes responding the most to treatment are listed in the table. A fold change >3 was defined as a significant difference in expression.

± ± ± ± ± ± ± ± -4.755 -4.449 4.507 4.339 1.241 -4.545 3.458 3.589 Baculoviral IAP Repeat Containing 3 Baculoviral IAP Repeat Containing 2 Caspase 9 Caspase 4 Caspase 7 B-Cell CLL/Lymphoma 2 BCL2-Associated Agonist of Cell Death BCL2-Associated X Protein BIRC3 BIRC2 CASP9 CASP4 CASP7 BCL2 BAD BAX

IAP family IAP family Caspase family Caspase family Caspase family BCL2 protein family BCL2 protein family BCL2 protein family

non-CSCs FC: ZA/Control CSCs FC: ZA/Control Functional gene grouping Description Gene Symbol

Table 2. The functional grouping and changes of apoptotic genes in CSCs and non-CSCs following exposure to ZA


found that ZA inhibited the growth of both breast and prostate cancer cells directly in vitro. Our finding is consistent with earlier reports of the inhibitory effect of ZA (Brown et al. 2004, Zekri et al. 2014). The IC50 of ZA differed among the groups studied. We found that the breast MCF7 cells were more sensitive to ZA than prostate DU-145 cells; the breast cancer cells were inhibited by a lower concentration of ZA. ZA exhibited a greater cytotoxic effect on non-CSCs than on CSCs in DU-145 cells, which suggests that prostate CSCs are more resistant to ZA than non-CSCs. Although ZA showed a greater cytotoxic effect on breast MCF7 cells, we found no significant differences in cytotoxicity between breast CSCs and non-CSCs. Our findings indicate that the survival capability and drug resistance differs among CSCs. It has been reported that ZA exerts a direct apoptotic effect (Zekri et al. 2014). Our findings are consistent with earlier reports concerning the antiapoptotic effects of ZA on breast and prostate cancer cells. We used the Annexin V test to determine the mechanism of the reduced viability of cancer cells caused by ZA treatment. We treated both CSCs and non-CSCs in both breast and prostate cancer cell lines with their specific IC50 doses. The increased amount of total apoptosis in prostate DU-145 non-CSCs compared to CSCs suggests that the mechanism of resistance to cytotoxicity in prostate CSCs may be related to their ability to resist apoptosis. By contrast, we found no differences in total apoptosis between CSCs and non-CSCs in breast MCF7 cells. Therefore, ZA exhibits different effects on apoptosis in different cancer stem cell types. In mammalian cells, caspases trigger apoptosis by regulating the extrinsic and intrinsic pathways. Caspase-9 is a critical upstream component of the apoptotic protease cascade. We found that ZA treatment significantly up-regulated the expression of caspase-4 and caspase-9 genes in prostate DU-145 CSCs, but not in non-CSCs, which means that induction of apoptosis by ZA is promoted by these two caspases. Activation of caspase-7, a component of the downstream apoptosis pathway, also is essential for induction of apoptosis (Lamkanfi and Kanneganti 2010). In support of this, we found that caspase-7 promoted apoptosis in prostate DU-145 non-CSCs. BCL-2 protein and its family members play critical roles in maintaining the balance between cell survival and apoptosis. Their contribution to chemoresistance in CSCs and to primary or secondary oncogenic events during tumorigenesis is well established (Abdullah and Chow 2013). Bcl-2 family proteins are composed of anti-apoptotic proteins (Bcl-2, Bcl-XL and Mcl-1) (He et al. Zoledronic acid sensitizes cancer stem cells to apoptosis 9


Fig. 5. Immunofluorescence staining of apoptotic proteins related to significant expression of genes affected by ZA. Prostate CSCs (A) and non-CSCs (B) following treatment with IC50 doses of ZA. Caspase-9, caspase-7, caspase-4, BAX, Bad, Bcl-2, BIRC2 and BIRC3 were visualized using FITC-conjugated secondary antibody (green). Nuclear staining was visualized using DAPI (blue) staining. Images are representative of three experiments. Scale bar = 40 μm. .

2014) and pro-apoptotic molecules (Bax, Bak, Bid, Bim, Bik, Noxa and Puma (He et al. 2014). We found that treatment of CSCs with ZA caused down-regulation of the BCL-2 gene and reduced its protein product in both prostate CSCs and non-CSCs. Our findings are consistent with earlier reports (Liu et al. 2006, Madjd et al. 2009, He et al. 2014). BCL-2 exerts its pro-survival effects by binding to the pro-apoptotic proteins, BAX and BCL-2, homologous antagonist killer (BAK) and decreasing the release of apoptogenic proteins such as cytochrome c from the mitochondria (Kim et al. 2006). We found that the basal levels of pro-apoptotic BAX and BAD proteins in both prostate CSCs and non-CSCs were low and that treatment with ZA caused overexpression of these proteins in both CSCs and non-CSCs. It is noteworthy that the up-regulation of these proapoptotic proteins was significantly greater in CSCs than in non-CSCs, which may indicate that ZA has a more potent apoptotic effect on CSCs than on non-CSCs. Taken together, overexpression of pro-apoptotic proteins and downregulation of BCL-2 protein with subsequent loss of its survival effect could explain the induction of apoptosis by ZA in these cells. Our observations are consistent with the increased apoptosis detected by the Annexin V assay. Similarly, Ma et al. (2013) reported activation of the pro-apoptotic protein, Bax, and inhibition of the anti-apoptotic protein, Bcl-2, release of cytochrome c and activation of caspase-9 and caspase-3 enzymes in breast CSCs treated with the anticancer agent,

berberine (Ma et al. 2013). Wang et al. (2013), using morusin, another anticancer agent, reported significantly decreased Bcl-2 and increased Bax and caspase-3 in a dose-dependent manner in cervical cancer stem cells. The inhibitor of apoptosis protein (IAP) family participates in apoptosis by regulating caspase activity, cell division and cell survival. It has been suggested that APs may contribute to oncogenesis and resistance to antitumor treatment (Smolewski and Robak 2011). Increased IAP expression has been reported for a variety of human cancers (Fulda and Vucic 2012). Among the IAPs that we evaluated, BIRC2 (IAP1) and BIRC3 (IAP2) were significantly down-regulated by treatment with ZA in both CSCs and non-CSCs. The mitochondrial apoptosis pathway begins with alteration of mitochondrial permeability. Bcl-2 family proteins regulate this change by interacting with mitochondrial channels (Tsujimoto and Shimizu 2002). This is followed by the release of pro-apoptotic factors, such as cytochrome c, which can activate the initiator, caspase-9, to form apoptosome complexes, which lead to activation of downstream effector caspases (Hengartner 2000, Susin et al. 1999, Li et al. 1997). Pro-apoptotic proteins, such as Bad and Bax, facilitate the release of apoptotic factors to the cytosol where anti-apoptotic proteins, such as Bcl-2, prevent their release (Rostovtseva et al. 2004, Susin et al. 1999). We found that treatment of prostate CSCs with ZA decreased Bcl-2 expression and increased caspase-9, caspase-4, BAX and BAD expressions, which suggests that ZA induced



apoptosis of prostate cancer stem cells occurs by the mitochondrial (intrinsic) pathway by alteration of caspase-9 and Bcl-2 family member expression. Isoprenoid synthesis appears to be inhibited by nitrogen-containing BPs, which inhibit farnesyl pyrophosphate synthase (Luckman et al. 1998). This results in inhibition of post-translational farnesylation of small G proteins including Rac, Rho and Ras family proteins (Dunford et al. 2006). It has been proposed that inhibition of the mevalonate pathway is the mechanism of apoptosis induced by BPs (Senaratne et al. 2002, Shipman et al. 1998, Rogers 2003, Segawa et al. 2005). Based on these studies, we postulate that the induction of apoptosis detected in CSCs in our study resulted from the inhibition of the mevalonate pathway by ZA. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

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