Anti-Breast Cancer Effect And Mechanism Of Ginsenoside Rk1

Breast cancer is one of the most frequently diagnosed cancer and the leading cause of cancer death among women.Breast cancer accounts for about 30%of all cancer cases and 14%of all cancer deaths among females.As in various nations,breast carcinoma has become the most widespread cancer among Chinese women;the cases of new breast cancers and deaths in China account for 12.2%and 9.6%of the world’s total.Presently,surgery,chemotherapy and radiotherapy are the main treatments for breast cancer.While chemotherapeutic treatments have induced a variety of severe side effects,including emesis,nausea,alopecia,myelosuppression and thromboembolism.Therefore,it is necessary to develop new anticarcinogen with lower side effects for breast cancer patients.Panax genus,a traditional medicinal herb,has received extensive attention of researchers.Ginsnosides,the major effective ingredients of Panax genus,have been reputed to possess various biological functions,such as immune regulation,antibacterial,anti-fatigue,anticancer activity and so on.Ginsenoside Rk1 is a component presented in processed ginseng and it possesses anti-insulin resistance,anti-inflammation and anti-cancer activities.However,little is reported about the anti-breast cancer effects of Rk1.Therefore,a series of experiments were performed to explore the anti-proliferatory effect and the potential mechanisms of ginsenoside Rk1 in breast cancer in vivo and in vitro.1.The content of ginsenosides 20（S）-Rg3,Rk1 and Rg5 were improved by single factor and response surface experiments.The optimum conditions were 91.16%ethanol concentration,dissolution 42.31 min,precipitation three times,the ginsenoside content was 86.84±2.20%.Ginsenoside Rk1 was prepared by preparative high performance liquid chromatography.CCK-8 cytotoxicity assay was performed to detect cell viabilities of human hepatoma cell line（SMMC-7221）,lung adenocarcinoma cell line（A549）,colorectal cancer cell line（Caco-2）,gastric cancer cell line（SGC-7901）,breast cancer cell line（MCF-7）,triple negative breast cancer cell line（MDA-MB-231）and normal human mammary epithelial cell line（MCF-10A）.Ginsenoside Rk1 had the most significant inhibitory effect on breast cancer cells.After treatment with 120μM ginsenoside Rk1 for 48h,the cell viability of MCF-7 breast cancer cells decreased to 16.78±3.26%,the cell activity of MDA-MB-231 triple negative breast cancer cells decreased to 17.41±2.94%,and the cell viability of MCF-10A normal mammary epithelial cells decreased to 86.15±2.39%.Ginsenoside Rk1 induced significant cytotoxicity in breast cancer cells with no obvious toxicity to MCF-10A normal mammary epithelial cells.In addition,ginsenoside Rk1 also promoted the release of LDH in breast cancer cells in a dose-dependent manner.Treatment with 120μM ginsenoside Rk1 induced LDH leakage 6.95fold（MCF-7 cells）and 6.18 fold（MDA-MB-231 cells）compared with control group,respectively.Moreover,treatment with 120μM ginsenoside Rk1 decreased colony formation to 2.02±0.67%（MCF-7 cells）and 2.27±0.70%（MDA-MB-231 cells）,respectively.In vivo experiments,ginsenoside Rk1 did not cause a decrease in body weight of tumor-bearing nude mice.Ginsenoside Rk1 had a significant anti-tumor effect on tumor-bearing nude mice.The tumor inhibition rate of 20 mg/kg Rk1 on MCF-7 breast cancer bearing nude mice was68.31%,and MDA-MB-231 triple-negative breast cancer bearing nude mice was 66.40%.H&E staining results showed that ginsenoside Rk1 significantly inhibited the growth of breast cancer cells and there was no obvious damage to the heart,liver,spleen,lung and kidney of breast cancer bearing nude mice compared with the solvent control group.Furthermore,there were no significant differences for ALT,AST or creatinine levels between Rk1 and control group,indicating unimpaired liver and kidney functions.2.Cell cycle distribution detection,Hoechst 33342 fluorescence staining,Annexin V/PI double staining,mitochondrial membrane potential detection,qRT-PCR,western blot and immunohistochemistry were performed to investigate the cell cycle distribution and cell apoptosis of ginsenoside Rk1 on breast cancer cells.The results showed that ginsenoside Rk1induced S phase arrest in MCF-7 breast cancer cells.Treatment with 120μM ginsenoside Rk1induced the proportions of cells in S phase increase to 60.44±1.88%.Ginsenoside Rk1triggered G₀/G₁ phase arrest in MDA-MB-231 triple-negative breast cancer cells.Treatment with 120μM ginsenoside Rk1 induced the proportions of cells in G₀/G₁ phase increase to70.92±1.12%.In MCF-7 breast cancer cells,ginsenoside Rk1 regulated S phase arrest by increasing p21 and p53 levels and decreasing cyclin A and CDK2 expressions.In MDA-MB-231 triple-negative breast cancer cells,ginsenoside Rk1 induced G₀/G₁ phase arrest by upregulating the expression levels of p21 and p53,down-regulating the expression levels of cyclin D1 and CDK4.In the cell apoptosis experiments,exposure to 120μM ginsenoside Rk1caused MCF-7 cell apoptosis increase to 50.27±4.33%,which was 11.88 fold of the control group,and induced MDA-MB-231 triple negative breast cancer cell apoptosis increase to54.87±3.96%,which was 8.95 fold of the control group.In addition,ginsenoside Rk1decreased mitochondrial membrane potential,up-regulated Bax level,down-regulated Bcl-2expression,promoted cytochrome c release,and activated caspases to induce cell apoptosis in MCF-7 and MDA-MB-231 breast cancer cells.In the tumor tissues of breast cancer bearing nude mice,ginsenoside Rk1 also had similar effects to induce tumor cell apoptosis and inhibit tumor growth.The use of the apoptosis inhibitor Z-VAD-FMK further demonstrated that ginsenoside Rk1 induced cell apoptosis through activation of caspases.3.The effects of ginsenoside Rk1 on ROS/PI3K/Akt signaling pathway in breast cancer cells were investigated by DCFH-DA staining,western blotting and immunohistochemistry.The results showed that ginsenoside Rk1 could induce ROS overgeneration in MCF-7 and MDA-MB-231 breast cancer cells.Western blotting showed that ginsenoside Rk1 had no effect on the expression levels of total PI3K,Akt and mTOR of MCF-7 and MA-MB-231 breast cancer cells,but inhibited the phosphorylation of PI3K,Akt and mTOR.In animal experiments of breast cancer-bearing nude mice,ginsenoside Rk1 also suppressed the expression of p-PI3K,p-Akt and p-mTOR,and has the same results as in vitro experiments.The use of the PI3K/Akt signaling pathway inhibitor LY294002 indicated that ginsenoside Rk1 had a similar function to LY294002,and the use of the PI3K/Akt signaling pathway activator insulin further demonstrated that ginsenoside Rk1 blocked PI3K/Akt signaling pathway in MCF-7 and MDA-MB-231 breast cancer cells.The use of ROS scavenger NAC suggested that ginsenoside Rk1 could induce breast cancer cell death via the ROS/PI3K/Akt signaling pathway.In addition,ginsenoside Rk1 repressed glucose uptake,lactate production and ATP formation in MCF-7 and MDA-MB-231 breast cancer cells.Moreover,ginsenoside Rk1inhibited HK2,PKM2 and LDHA in breast cancer cells and inhibited the aerobic glycolysis of breast cancer cells,thereby inhibiting the growth and proliferation of breast cancer cells.Taken together,the present study first demonstrated the anti-breast cancer efficacy and mechanism of Rk1 against breast cancer,providing basic evidence for Rk1 as a potential anti-breast cancer agent.