Background: Qingjie Fuzheng granules (QFGs) are part of a traditional Chinese medicine formula, which has been widely used and found to be clinically effective with few side effects in various cancer treatments, including colorectal cancer (CRC). However, the precise mechanisms and molecular signaling pathways involved in the activity of QFGs' anticancer effect have not been reported in the literature. In this study, we hypothesized that QFGs can inhibit the growth of colorectal cancer cells, and that its mechanism is closely related to one or more intracellular signal transduction pathways.
Aim: To better evaluate the mechanism underlying the anti-cancer effect of QFGs on the CRC cell lines HCT-116 and HCT-8.
Method: First, we measured cell viability and cytotoxicity by performing MTT and lactate dehydrogenase (LDH) assays. We evaluated the role of QFGs in cell proliferation and apoptosis by assessing colony formation and analyzing Hoechst 33258 staining. Second, cell cycle and apoptosis rates were measured by fluorescence activated cell sorting, and the expression levels of survivin, cyclin D1, CDK4, p21, Bax, Bcl-2, Fas, FasL, and cleaved-caspase-3/-8/-9 were measured by performing western blots and caspase activity assays. Furthermore, inhibitors of caspase-3/-8/-9 were used to elucidate the specific apoptosis pathway induced by QFGs in cancer cells. Finally, activation of the PI3K/AKT and ERK signaling pathways was examined using the western blot assay to investigate the possible mechanism.
Results: MTT and LDH assays revealed that after 0.5-2.0 mg/mL of QFGs treatment, cell viability was reduced by (6.90% ± 1.03%)-(59.70% ± 1.51%) (HCT-116; P < 0.05) and (5.56% ± 4.52%)-(49.44% ± 2.47%) (HCT-8; P < 0.05), and cytotoxicity was increased from 0.52 ± 0.023 to 0.77 ± 0.002 (HCT-116; P < 0.01) and from 0.56 ± 0.054 to 0.81 ± 0.044 (HCT-8; P < 0.01) compared with the non-QFGs treatment groups. Additionally, colony formation and Hoechst 33258 staining assays showed that QFGs inhibited proliferation and induced apoptosis in CRC cells. QFGs also increased the expression levels of Bax, Fas and FasL, decreased the level of Bcl-2, and stimulated the activation of caspase-3/-8/-9, which were revealed by western blot and caspase activity assays. In contrast, when adding the three caspase inhibitors, the suppression effect of QFGs on cell viability and apoptosis were markedly inhibited. Moreover, QFGs suppressed the phosphorylation levels of PI3K, AKT and ERK.
Conclusion: These results demonstrated that QFGs can inhibit CRC cell proliferation and induce apoptosis by suppressing the PI3K/AKT and ERK signaling pathways.
Keywords: Apoptosis; Colorectal cancer; ERK; PI3K/AKT; Proliferation; Qingjie Fuzheng granules.