The apoptotic effects of zoledronic acid on cancer stem cells

Objective: In contrast to low-tumorigenic bulk tumor cells (non-CSCs), cancer stem cells (CSCs) are a subset of tumor cells with the potential to self-renew and differentiate into different cancer subtypes. CSCs are thought to be responsible for tumor recurrence, distant metastasis, angiogenesis, and drug/radiation resistance. Resistance to apoptosis is one of the most important features of CSCs. Bisphosphonates are a class of compounds designed to treat osteoporosis and skeletal related malignancies. Zoledronic acid (ZA), a third generation and one of the most potent bisphosphonates, has been shown to reduce cell proliferation and has anti-tumor effects through cell death induction in some malignancies. However, the effects of ZA on CSCs remain unclear. The present study aimed to determine the apoptotic effect of ZA on CSCs using prostate and breast CSCs as a model system. Material and Method: Cluster of differentiation (CD) 133+high/CD44+high prostate CSCs were isolated from the DU-145 human prostate cancer cell line and CD 44+high/CD24-low breast CSCs were isolated from the MCF-7 human breast cancer cell line through FACSAria flow cytometry cell sorting. CSCs and non-CSCs were exposed to increasing concentrations of ZA (0-140 μM) for 24, 48 and 72 hours and the viability was examined to determine the IC50 dose. Cell cycle and Annexin-V analyses were performed for the detection of cell death using Muse™ Cell Analyzer. Cell groups was assayed by qRT-PCR array for the detection of 84 key apoptosis related genes and immunofluorescence microscopy performed to validate protein expression of related significantly altered gene experience. Results: Zoledronic acid caused a dose- and time-dependent decrease in cell viability. Interestingly, the half maximal inhibitory concentration (IC50) was significantly different between CSCs and non-CSCs in DU-145 cells but there was not find significant differences between CSCs and non-CSCs in MCF-7 cells. Treatment with ZA caused a concomitant increase in apoptosis and S phase cell cycle arrest. Significant over/under-expressions were detected in seven of the studied 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, 5 genes showed changes in gene expression after ZA treatment, with 2 showing increased expression (CASP7 and BAD) and 3 showing decreased expression (BIRC3, BIRC2 andBCL2). Conclusion: Our data revealed that ZA has the potential to overcome apoptosis resistance in prostate CSCs by induction of apoptosis through up-regulation of some pro-apoptotic and down-regulation of some anti-apoptotic genes. Inhibition of the mevalonate pathway by ZA seems to be the probable underlying mechanism of the apoptotic induction in prostate CSCs. These data suggest that, ZA may be an effective therapeutic approach for targeting apoptosis resistance in CSCs. Keywords: Cancer stem cell; Apoptosis; Drug resistance; Biphosphonate; Zoledronic acid