Figure 6.

Combinatorial interaction of curcumin and UV-radiation on cell proliferation and survival. (A) Effect of curcumin and UV-irradiation on cell proliferation. Representative immunofluorescence image of day10 cervicospheres derived from CaCxSLCs treated with curcumin(24 hr) or UV-irradiation(4 hr) or in-combination by acridine orange/ethidium bromide (AO/EtBr) staining as described in Methods. Scale bar = 50 μm. (B) Effect of curcumin and UV-irradiation on cell cycle. Representative histogram showing cell cycle distribution as assessed by propidium iodide (PI) staining. (C) Flowcytometric analysis of day10 cervicospheres derived from CaCxSLCs treated as mentioned above and analyzed using AnnexinV-FITC apoptosis detection kit as per manufacturer’s instructions described in Methods. (D) Assessment of cervicosphere forming ability. Representative photomicrograph of day10 cervicospheres derived from treated CaCxSLCs under low adherence environment in DCM (original magnification-100x) (i). Representative confocal immunofluorescence image of day10 CaCxSLC cultures treated with curcumin(24 hr) and/or UV-radiation(4 hr). Each cultures were fixed and stained with primary (c-Fos and c-Jun) and (Alexa-488 or Alexa-594 conjugated goat anti-mouse or anti-rabbit) secondary antibodies and counterstained with DAPI. Merged photograph indicate co-localization of AP-1 in nuclei. (E) Tumor weight derived from post-treated CaCxSLCs (20 × 10³) measured after 4-weeks post-injection in the athymic nude mice (i). Data presented as mean ± SE, *p value < 0.05 w.r.t. vehicle treated tumor. Tumor growth curves of post-treated CaCxSLCs (20 × 10³ each) at 4-weeks post-injection in the athymic nude mice (ii). Data presented as the mean ± SE, *p value < 0.05 w.r.t. vehicle treated tumor. (F) H&E staining and IHC of tumors derived from curcumin or UV treated CaCxSLCs (original magnification-400x). (G) Representative cropped blots showing expression level of ABCG2 in extracts prepared from CaCxSLCs treated with different doses of curcumin(24 hr). β-actin was used as input control (i). The gels were run under the same experimental conditions. The abundance ratio of ABCG2 to β-actin was analyzed by densitometry. Data are expressed as the mean ± SD of three independent experiments (ii). *p-value < 0.05 vs. vehicle treated cells from CaCxSLC-derived cervicospheres. (iii). Representative 3D-ribbon structure of ABCG2(grey) and curcumin(yellow) complex. Red square showing binding area of curcumin in ABCG2 receptor. Close-up view showing ABCG2 amino-acid residues involved with curcumin binding.