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  • br Materials and methods br Results br Discussion CUs

    2022-12-02


    Materials and methods
    Results
    Discussion CUs, which have been used as traditional medicine for thousands of years in East Asian countries, have the potential to be used for cancer chemoprevention and chemotherapy [17], [18], [19], [20]. CuB is one of the most promising agents as it is reported to have anticancer effects on a variety of tumors [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34]. The efficacy of CuB against human prostate cancer, however, has not previously been examined. In our present study, we, for the first time, showed that CuB treatment significantly inhibited human prostate cancer cell growth and caused apoptotic cell death both in vitro and in vivo. Our results revealed a novel anti-cancer mechanism of CuB, in which the ACLY signaling pathway is involved in CuB-induced apoptosis in human prostate cancer. Our data indicates that CuB has anti-cancer potential in human prostate cancer GI 254023X as evidenced by the inhibition of cell growth and the induction of apoptotic cell death. The inhibition of cell survival by 24-h treatment with CuB was statistically significant at IC50 ∼0.3μmol/L concentrations, as determined by CellTiter-Glo® luminescent cell viability assays (Fig. 1A). The results of both the clonogenic survival assay and trypan blue dye exclusion assays show that the IC50 concentrations for CuB on cell viability were ∼0.1μmol/L (Fig. 1B and C). These results indicate that the clonogenic survival assay and trypan blue dye exclusion assays are more sensitive than the CellTiter-Glo® luminescent cell viability assays. Previous studies [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34] have reported that CuB inhibited cell growth in other types of cancer cells with IC50 at 0.1–1.0μmol/L concentrations. Our present results indicate that prostate cancer cells are sensitive to cell death induced by CuB because the IC50 0.1–0.3μmol/L concentrations of CuB to prostate cancer cells are within the IC50 0.1–1.0μmol/L range of CuB for other types of cancer cells. Next, we investigated whether CuB treatment has a selective activity on human prostate cancer cells. To address this question, we conducted an experiment to show the effects of CuB on the proliferation of PrEC, a normal human prostate epithelial cell line. Our data clearly showed that CuB-treated PrEC cells displayed significantly less cell growth inhibition than the CuB-treated human prostate cancer cells (Fig. 1). Consistent with our data, Dakeng et al. [20] reported that CuB exerts a strong anticancer activity in human breast cancer GI 254023X cells but only has a slight effect on the proliferation of non-malignant HBL-100 cells. Collectively, these results indicate that the human prostate cancer cells are more sensitive to growth inhibition by CuB than is the normal human prostate epithelial cell line PrEC. Although the prostate cancer cell lines PC3 is androgen-independent whereas LNCaP is androgen-dependent, the present data showed that the LNCaP and PC-3 cells exhibited comparable sensitivity to CuB. We, therefore, concluded that androgen-responsiveness is not a critical factor in CuB-mediated growth inhibition in prostate cancer cells. The present results indicate that the anticancer activity of CuB against human prostate cancer cells is associated with apoptosis induction. This conclusion is based on the following: (a) CuB treatment resulted in a significant increase of Caspase 3/7 activity in human prostate cancer cells, but not in the normal human prostate epithelial cell line (Fig. 2C and E), (b) an increase in the Sub-G0/G1 phase was observed in CuB-treated prostate cancer cells (Fig. 2D), (c) CuB treatment increased an immunoblotting band of cleaved PARP and cleaved Caspase 3 in the prostate cancer cells (Fig. 2A and B) and (d) CuB-induced apoptotic cell death was also determined by flow cytometric analysis of apoptosis using the Alexa Fluor®488-annexin V binding assay (Fig. S1).