The Effect And Mechanism Of Ginsenoside CK On Glutamine Metabolism In Triple-negative Breast Cancer

Breast cancer has replaced lung cancer as the number one malignant tumor worldwide.Triple-negative breast cancer(TNBC)is the most dangerous of all breast cancer subtypes,and its prevention and treatment are of great concern to researchers and medical practitioners at home and abroad.Ginsenoside CK,a deglycosylated product of the original ginsenoside,has multiple effects.For metabolically active cancer cells,there are few reports on the role and mechanism of ginsenoside CK in metabolism against triple-negative breast cancer.In order to clarify the anti-breast cancer effects of CK,this study systematically investigated the potential mechanism of its inhibition in TNBC using the prepared CK.The findings were as follows:(1)Ginsenoside CK was prepared by converting ginsenoside Rb1 using β-glucosidase,and the converted mixture was separated and purified by preparative chromatography column,and its structure was identified by nuclear magnetic resonance.The results showed that almost all Rb1 was converted to CK when transformed for 48 h,and the structure was identified as the same as that of CK standard after purification and separation.The purity of the obtained by high performance liquid chromatography ginsenoside CK was 98%.(2)The MTT assay was performed to verify the glutamine(Gln)addiction of different breast cancer cell lines and to investigate whether the anti-proliferative effect of CK on breast cancer in vitro was correlated with glutamine addiction.The results showed that triple-negative breast cancer cells with high glutamine-dependent were more sensitive to ginsenoside CK compared with low glutamine-dependent breast cancer cells.(3)A SUM159 breast cancer xenograft model was established to investigate the anti-tumor effect of ginsenoside CK in vivo.After successful modeling and intraperitoneal administration for 4 weeks,the inhibitory effects of CK on tumors were observed by tumor volume,weight,H&E and TUNEL staining.Meanwhile,the side effects of CK on nude mice were analyzed by detecting liver,kidney,immune function indexes and organ H&E staining.The results showed that ginsenoside CK significantly inhibits SUM159 tumor growth without obvious side effects on the organism.(4)The inhibitory effects and the way of cell death induced by CK on TNBC cells were further demonstrated by lactate dehydrogenase(LDH)release,inhibitors co-culture,Hoechst 33342 staining,and western blot assays.The results showed that ginsenoside CK inhibited the proliferative in vitro and in vivo by inducing apoptosis in TNBC cells.(5)The effects of CK on the glutamine metabolic pathway in TNBC cells and its mechanism of inducing cell apoptosis were investigated by metabolomics,western blot,q RT-PCR,ATP production,glutathione(GSH)synthesis,reactive oxygen species(ROS)levels,immunohistochemistry,immunofluorescence,glutaminase 1(GLS1)silencing,and glutaminase(GLS)activity.These results showed that ginsenoside CK inhibited glutamine metabolism in TNBC cells by suppressing the expression of enzymes involved in glutamine metabolism and decreasing amino acid utilization related to glutamine metabolism,resulting in decreased ATP production and GSH synthesis,and stimulating ROS accumulation;supplementation with glutamine attenuated the inhibitory effect of CK on TNBC cells and restored GLS1 expression,and GLS1-SiRNA transfection increased CKinduced cell death,decreased GLS activity,and enhanced cell apoptosis induced by ginsenoside CK,indicating that ginsenoside CK inhibited the proliferation of TNBC cells mainly by decreasing the expression of GLS1.In summary,this study clarified that CK inhibited the proliferation of TNBC cells by targeting glutamine metabolism and inducing cell apoptosis in TNBC via reducing the expression of GLS1,which provides a strong theoretical basis for the application of CK in TNBC as a glutamine metabolism inhibitor.