Nitidine Chloride Induces Apoptosis, Cell Cycle Arrest, And Synergistic Cytotoxicity With Doxorubicin In Breast Cancer Cells

ObjectiveBreast cancer has become one of the most common malignant tumors, in recent years, breast cancer has been the major cause of cancer-related deaths. Medicinal plant extracts have been widely used for cancer treatment. Nitidine chloride (NC) is a natural bioactive alkaloid that has recently been reported to have diverse anticancer properties. It has been found that NC exhibits several types of biological activities, including anti-inflammatory, antimalarial, antifungal, antiangiogenesis, and anticancer activity. It has been reported that NC can inhibit the proliferation of hepatocellular carcinoma and renal cancer and the metastasis of breast cancer through suppressing the c-Src/focal adhesion kinase (FAK)-associated signaling pathway.MethodsUsing two different breast cancer cell lines, MCF-7 andMDA-MB-231, the cytotoxicity of nitidine chloride was determined by MTT assay and Colony formation assay in vitro. Pl-annexinV assays and TUNEL assays were used to examined induced apoptosis effect of nitidine chloride in breast cancer cell lines. Flow cytometry assay was used to determine whether Nitidine chloride inhibits cell cycle arrest in both cells. The apoptosis induced by nitidine chloride was detected by MMP assays. In addition, the protein level of P53, AKT and apoptosis-associated proteins were determined by Western blot analysis. This study demonstrates that the combination of NC and doxorubicin has a synergistic inhibitory effect on the proliferation in breast cancer.ResultsWe aimed to investigate the cytotoxic effects of NC and the effectiveness of combinatorial treatment including NC and doxorubicin in breast cancer cells. Using MTT and flowcytometry assays, we found that NC induced cell growth inhibition and G2/M cell cycle arrest in time and dose-dependent manner both in MCF-7 and MDA-MB-231 breast cancer cell lines. Cancer cell growth inhibition was associated with increased levels of the p53 and p21 proteins. Apoptosis induction by NC treatment was confirmed by JC-lmitochondrial membrane potential, annexin V-positive cell, and TUNEL staining. Using western blot analysis, we found that NC upregulated the pro-apoptotic proteins Bax, cleavedcaspase-9 and -3 and cleaved PARP and that it down regulated the anti-apoptotic proteins Bcl-2 and PARP. By using thePI3K/Akt inhibitor LY294002, we further demonstrated that NC-induced apoptosis might be Akt-specific or dependent. In addition, NC exhibited a synergistic effect with doxorubicin on the growth inhibition of the human breast cancer cell linesMCF-7 and MDA-MB-231.ConclusionsOur study demonstrated the anticancer effect of NC on breast cancer and highlighted the potential clinical application of NC. This effect is mediated through the induction of G2/M phase cycle arrest and triggering apoptosis. NC induced apoptosis through the upregulation of proapoptotic proteins and downregulation of anti-apoptic proteins. In addition, NC-induced apoptosis is likely to act through the intrinsic, mitochondrial-dependent caspase pathway. Furthermore, NC exhibited a synergistic effect with doxorubicin on the growth inhibition of the human breast cancer cell lines MCF-7 and MDA-MB-231.