| Objective: Gastric cancer is a cause of significant morbidity and cancer-related mortality worldwide and one of the major leading causes of cancer death in China.Surgery remains the most effective curative treatment for early-stage gastric cancer,but it is usually diagnosed at a late stage and most of them lost the opportunity of surgery.Only chemotherapy,molecular targeted therapy and immunotherapy can be given to them.Chemotherapy is the major treatment for advanced gastric cancer,but the outcome is very poor,with median overall survival(OS)of less than 12 months and a 5-year OS rate only 4%.Meanwhile combined chemotherapy with cytotoxic drugs usually leads to severe toxicity which lowers the quality of life of patients.In recent years,some achievements have been made in molecular targeted therapy and immunotherapy of gastric cancer.Despite these treatment options,the prognosis of advanced and metastatic gastric cancer is still poor.Therefore,new agents with high anti-tumor activity but low side effects are urgently needed.Traditional Chinese medicine treatment has a history of thousands of years.In recent years,Chinese medicine preparation in the treatment of cancer has made a lot of progress.In this study,we investigated the antitumor mechanism of two kinds of drugs,one is bufalin,extracted from the venom of toad,and the other is a Chinese herbal medicine Kang'ai.Toad venom is a traditional Chinese medicine.It is the secretions of the posterior ear glands and epidermal glands of toads.Bufalin is one of the active ingredients of an anticancer Chinese medicine called “Chan su”,an extract of dried toad venom from the skin glands of Bufo gargarizans or Bufo melanostictus.Experimental research has suggested that bufalin exhibited significant antitumor activity against various tumors such as leukemia,prostate cancer cells,gastric cancer and osteosarcoma through inhibition of cell proliferation,induction of cell differentiation,induction of apoptosis,disruption of the cell cycle,inhibition of cancer angiogenesis,reversal of multi-drug resistance and regulation of the immune response.Huachansu injection,an injectable form of Chan su in physiological saline solution,has been approved officially,and widely used in clinical therapy for various cancers in China.Moreover,data suggest huachansu injection could enhance response rate(RR)of chemotherapy on advanced gastric cancer,meanwhile it may also enhance quality of life for patients with cancer.So the Chan su-related drugs have clinical application prospect.Bufalin,as a major active ingredient of Chan su,the molecular mechanisms of which are complex,still need to be further elucidated.Autophagy is a hot topic in the field of biology,and it is a cellular process that engulfs organelles and cytoplasmic contents to digest and recycle these materials to sustain cellular metabolism.Autophagy is an active process of gene regulation.Autophagy induced under pathological conditions functions as an adaptive cell response,allowing the cell to survive bioenergetic stress.However,extensive or persistent autophagy also results in cell death,thereby it is also known as II programmed cell deaths.Recent years the role of autophagy and its regulation in cancer cells continues to be a concern.The role of autophagy in cancer is determined by many factors,and autophagy can be tumor-suppressive or tumor-promoting in different contexts in cancers.The process and molecular mechanisms of autophagy are very comlex,which may involve a number of signal pathways,and the detailed molecular mechanisms need to be further studied.Autophagy and apoptosis usually coexist in the same cell,and they interact with each other.Autophagy has been shown to have a complex relationship with apoptosis,especially in tumor cell lines.So autophagy plays an important role in the balance between cell death and survival.The relationship between autophagy and apoptosis is reported inconsistently,which may depend on the cell type and stress stimuli.The molecular mechanisms by which autophagy regulates survival and death need to be further studied.Bufalin is reported to be able to induce both apoptosis and autophagy in gastric cancer cells.However,very limited is known about the regulation of autophagy in gastric cancer cells,and the detailed molecular mechanisms need to be further studied.Therefore,in this study,we focused on clarifying the mechanisms of bufalin-induced autophagy and the effect of bufalin-induced autophagy on apoptosis,so as to provide theoretical basis for the further development of bufalin as a treatment of cancer.The results showed that bufalin induced protective autophagy in gastric cancer MGC803 cells via Cbl-b regulating m TOR inhibition and ERK1/2 activation.The second part of this study is to explore a Chinese herbal medicine preparation –Kang'ai,the main ingredients of which are Astragalus membranaceus,Ginseng and Sophora flavescens.The main function of Kang'ai is to benefit the right Qi and enhance cellular immune functions,and meanwhile it can inhibit tumor growth,reduce the side effects of chemotherapy and improve the general state of the body.It has been widely used in the treatment of a variety of tumors,such as primary liver cancer,lung cancer,lymphoma,gynecological malignancies,colorectal cancer,gastric cancer,esophageal cancer and so on.Basic and clinical studies have shown that Kang'ai can improve the quality of life,enhance cellular immune functions,and it can improve disease control rate,reduce side effects of chemotherapy and prolong PFS and MST when combined with chemotherapy,in addition,Kang'ai can also inhibit the proliferation of tumor cells.Therefore,Kang'ai has a high clinical value in the treatment of tumors,but its efficacy and mechanism is still not very clear,which is worth of further study and discussion.In this part,we take MGC803 cells and BGC823 cells as models to study whether Kang'ai has anti-tumor effect in gastric cancer cells,and the related mechanisms involved.The results showed that Kang'ai could inhibit the proliferation of gastric cancer cells by inhibiting IL-6/STAT pathway and induced cell cycle arrest.Methods: 1.MTT was used to measure the viability of cells.2.Apoptosis was analyzed by flow cytometry following Annexin V–PI staining,and cell cycles was analyzed by flow cytometry following PI staining.3.Autophagy was determined by fluorescence microscopy and transmission electron microscopy.4.Western blot was used to detect the expression of protein.5.RT-PCR was used to detect the expression of the target gene.6.Statistical analysis: The experiments were repeated at least three times.Data are expressed as the means ± SD.Differences in the results for two groups were evaluated by the Student's t-test.P < 0.05 was considered to be statistically significant.Results:The first part: 1.Bufalin inhibited the viability of gastric cancer cells in a doseand time-dependent manner.2.Bufalin can induce apoptosis and autophagy in gastric cancer cells.3.Bufalin induced autophagy by altering the expression levels of Atg proteins in human gastric cancer cells.4.The induction of autophagy in MGC803 cells after bufalin treatment is mediated through inhibition of the PI3K/Akt/m TOR/p70S6 K signaling pathway and the activation of the ERK1/2 signaling.5.Cbl-b promoted autophagy induced by bufalin through the suppression of m TOR activation and enhancement of ERK1/2 activation.Cbl-b was time-dependently increased after treatment of MGC803 cells with bufalin.We found that knockdown of Cbl-b significantly reduced the expression of LC3 II in bufalin-treated cells compared with that of bufalin-treated control cells.Further investigation showed that sh RNA knockdown of Cbl-b significantly enhanced the expression of phospho-m TOR and reduced the expression of phospho-ERK.6.Bufalin induced autophagy protected MGC803 cells from undergoing apoptosis.The second part: 1.Kang'ai inhibited the viability of gastric cancer cells in a dosedependent manner.2.Kang'ai induced cell cycle arrest at G1 phase,and decreased the number of cell in S phase.3.Kang'ai inhibited the expression of STAT3,but had on effect on the expressions of Akt and ERK.Conclusion: The first part: 1.Bufalin could inhibited the viability of gastric cancer cells,and induce apoptosis and autophagy in gastric cancer MGC803,BGC823 and SGC7901 cells.2.Beclin 1,Atg12-Atg5 conjugated protein,Atg9 as well as Atg16L1 might contribute to the induction of autophagy by bufalin.3.Autophagy induced by bufalin in gastric cancer cells is mediated via PI3K/Akt/m TOR/p70S6 K inhibition and ERK1/2activation.4.Cbl-b promoted autophagy induced by bufalin through the suppression of m TOR activation and enhancement of ERK1/2 activation.5.Autophagy induced by bufalin prevents human gastric cancer cells from undergoing apoptosis and is cytoprotective.The second part: 1.Kang'ai inhibited the viability of gastric cancer cells in a dosedependent manner.2.Kang'ai inhibited the viability of gastric cancer cells by inducing cell cycle arrest at G1 phase.3.Through inhibiting IL-6 gene expression,Kang'ai inhibited STAT3,and therefore inhibited the proliferation of gastric cancer cells. |
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