Figure 3.
ZA reduces CCL3 expression required to support the CSC phenotype in OSCC. (A) The effect of ZA on CCL3 expression in OSCC was determined by quantitative PCR. Decreased expression of CCL3 was confirmed in 3 OSCC cell lines (SCC4, UM17B and BapT). The cells were treated with the indicated ZA concentrations for 2 days. *P<0.05 and **P<0.01 vs. control (CTL) in one-way ANOVA. (B) The level of CCL3 expression was determined in CSC-enriched population and non-CSC population derived from SCC4 cells. Monolayer adherent cells (Mono., non-CSC population) vs. spheres (Sph., CSC-enriched population); ALDHlow (non-CSC population) vs. ALDHhigh (CSC-enriched population); parental cells (Pa., non-CSC population) vs. cisplatin-resistant cells (CispR., CSC-enriched population). *P<0.01 vs. corresponding non-CSC populations by t-test. (C) The effect of CCL3 on self-renewal capacity was assessed in multiple OSCC cell lines by tumorsphere formation assay. Tumorsphere formation assay was performed without CCL3 (CTL) or with 10 ng/ml CCL3 (CCL3). *P<0.01 and **P<0.05 compared to CTL by t-test. (D) The effect of CCL3 on the migration ability of OSCC cells was determined by Transwell assay. *P<0.01 and **P<0.05 compared to CTL by t-test. (E) The effect of maraviroc (MVC) on CCL3-induced self-renewal capacity of SCC4 cells was assessed by tumorsphere formation assay. The assay was performed in medium containing no treatment (−), 10 ng/ml CCL3 (+), or 100 µM MVC (+) for 7 days. **P<0.05 in one-way ANOVA. (F) The effect of MVC on CCL3-induced migration in SCC4 cells was assessed by Transwell assay. The assay was performed with no treatment (−), 10 ng/ml CCL3 (+), or 100 µM MVC (+) for 48 h. **P<0.05 in one-way ANOVA. ZA, zoledronic acid; CSC, cancer stem-like cells; OSCC, oral/oropharyngeal squamous cell carcinoma; CCL3, chemokine (C-C motif) ligand 3; ALDH, aldehyde dehydrogenase; IL, interleukin; VEGF, vascular endothelial growth factor.