Figure 2.

(i–iv) Curcumin inhibits cell proliferation rate, spheroid formation and miRNA-21 expression in oral cancer stem cells. (i) Cell proliferation rate: Parental, SP and NSP cells of (A) UD-SCC2 (HPV16+ve), (B) UPCI:SCC131 (HPV–ve), and (C) UPCI:SCC84 (HPV–ve) were incubated with increasing concentrations of curcumin (0–50 μM) for up to 24 h. and analyzed for cell proliferation rate. Curcumin treatment resulted in a significant dose dependent decrease in cell proliferation in all three cells when compared with untreated controls. Results are representative of three independent experiments. (ii) Spheroid formation ability: (A) CSCs from UD-SCC2 (HPV16+ve), (B) UPCI:SCC131(HPV–ve) and (C) UPCI:SCC84 cells were grown in low adherent plates and treated with increasing concentrations of curcumin (0, 10, 20, 30 and 50 μM) and performed spheroid assay to assess the effect of curcumin on orosphere forming ability. (iii) Primary sphere formation: Effect of curcumin on OSCC-SP spheroid formation ability was analyzed by counting spheroid and sphere forming efficiency was calculated and bar diagram was drawn. Curcumin treatment significantly inhibited orosphere formation in HPV+ve and HPV–ve OSCC-SP orospheres. The experiments were performed at least three times and data are presented here as mean ± standard errors. P-value is determined with respect to their vehicle controls (*p < 0.05). (iv) Downregulation of miRNA-21 expression: The expression level of miR-21 in orosphere forming CSCs of UD-SCC2 (HPV+ve) and UPCI:SCC131 and UPCI: SCC 84(HPV–ve) cell lines after 24 h of 30 μM curcumin treatment. RT-qPCR was used to detect miRNA expression and RNU6B was used to normalize data. Each set is normalized with their respective vehicle control. The experiments were performed at least three times and data are presented here as mean ± standard errors. ***P < 0.0001, *P < 0.01.