| Gastric cancer is one of the most common cancers of the digestive system in clinical practice.Currently,the main treatment methods for gastric cancer are adjuvant chemoradiotherapy and traditional drugs after resection of the lesion.However,the severe and unavoidable gastric injury caused by chemoradiotherapy and traditional drugs makes it difficult to further improve the quality of life of patients.Therefore,finding new drugs to treat stomach cancer is urgent.The occurrence,development,diffusion and metastasis of gastric cancer are closely related to its microenvironment.Hypoxia is a hallmark of tumor microenvironment（TME）that cannot be ignored,which enhances the stem cell-like properties of tumor cells to adapt to hypoxia,resulting in a more aggressive and drug-resistant tumor phenotype,leading to epithelial-to-mesenchymal transition（EMT）of tumor cells,which promotes invasion and metastasis,and more malignant behavior（such as resistance to apoptosis,treatment resistance）.Notably,ion channels and transporters are also closely related to hypoxic tumor microenvironment,such as acidic p H,metabolism changes,etc.Therefore,regulating ion channels may be a target for cancer treatment.Kv1.5 channel is a transmembrane protein encoded by the KCNA5 gene that acts as an oxygen sensor to alter intracellular potassium levels to adapt to changes in redox status and helps regulate cell proliferation and survival under hypoxic conditions.In recent years,a number of studies have shown that Kv1.5channel protein is aberrantly expressed in a wide range of tumors and significantly increased in gastric cancer compared to normal cells.These results suggest that Kv1.5channel is closely associated with gastric cancer initiation and progression,and further investigation of the molecular mechanisms between Kv1.5 and gastric cancer progression is warranted.Cinobufagin is currently widely used in clinical anticancer studies.A number of studies have shown that Cinobufagin can not only inhibit tumor angiogenesis and improve tumor microenvironment,but also participate in the regulation of ion channels.By establishing hypoxia gastric cancer model,this study explored the influence of Cinobufagin on hypoxia microenvironment of gastric cancer and the preliminary mechanism of Kv1.5 channel in inhibiting migration and invasion of gastric cancer by Cinobufagin.ObjectiveTo study the effects of Cinobufagin on the hypoxia microenvironment of gastric cancer and the mechanism of inhibiting the proliferation,migration and invasion of gastric cancer cells,and to clarify the role of Kv1.5 channel in Cinobufagin’s influence on the hypoxia microenvironment of gastric cancer and inhibiting migration and invasion of gastric cancer.MethodsThe tumor model of gastric cancer was established in nude mice,and the tumor size was observed and recorded.Immunohistochemistry and ELISA assays were used to determine the effects of Cinobufagin on HIF-1αlevels in transplanted tumor and serum of nude mice with gastric cancer.The effect of Cinobufagin on gastric cancer transplantation tumor and heart was detected by HE staining,and the protein changes associated with gastric cancer proliferation and metastasis were detected by immunohistochemistry.The model of hypoxic gastric cancer MGC-803 cells was constructed in vitro,and the concentration of Cinobufagin on hypoxic MGC-803 cells was screened by CCK-8assay.The effect of Cinobufagin on the expression of hypoxia-inducible factor in hypoxic human gastric cancer cells MGC-803 was detected by Western blot.Cell scratch assay and transwell assay were used to detect the effects of Cinobufagin on the migration and invasion ability of hypoxic MGC-803 cells.The expression levels of EMT-related proteins in hypoxic human gastric cancer cells MGC-803 were determined by Western blot.The effects of Cinobufagin on Kv1.5 channel protein in hypoxic gastric cancer cells in vitro and in vivo were detected by Western blot and immunohistochemistry.The effect of blocking Kv1.5 channel on the survival rate of hypoxic gastric cancer cells was detected by CCK-8 assay.The effect of blocking Kv1.5 channel on HIF-1αlevel in hypoxic gastric cancer cells was detected by Western blot.The effects of blocking Kv1.5channel on migration and invasion ability of hypoxic MGC-803 cells were detected by cell scratch assay and transwell assay.Western blot assay was used to detect the effect of blocking Kv1.5 channel on EMT of hypoxic gastric cancer cells.Results1.Cinobufagin affects the hypoxic microenvironment of gastric cancer and inhibits EMT of Gastric cancer transplant tumour.（1）The effect of Cinobufagin on gastric cancer transplantation was observed by establishing a nude mouse model of gastric cancer transplantation with 5-FU as a positive control drug.The results showed that Cinobufagin inhibited the growth of gastric cancer transplantation tumor（P<0.05）;（2）The level of HIF-1αin tumor and serum of nude mice was determined by immunohistochemistry and ELISA kit,and the results showed that Cinobufagin inhibited the expression of HIF-1αin gastric cancer（P<0.05）;（3）HE staining and immunohistochemical results indicated that Cinobufagin inhibited proliferation and EMT of gastric cancer cells in nude mice.2.Cinobufagin can affect the hypoxic microenvironment of gastric cancer and inhibit migration and invasion of MGC-803 cells.（1）The effect of hypoxia duration on the morphology of MGC-803 was observed through cell morphology.The results showed that,with the extension of hypoxia duration,the cell morphology gradually became thinner and longer.The number and state of cell proliferation were the best at 24h,and then the cell morphology began to round and gradually died;（2）The effect of hypoxia duration on the survival rate of MGC-803 was detected by CCK-8 assay,and the results were consistent with cell morphology.The results showed that the survival rate of MGC-803 cells was the highest and the condition was the best when cultured at 1%oxygen concentration for 24h;（3）The cell scratch assay and transwell assay detected the effects of 1%oxygen concentration culture for 24h on the migration and invasion of MGC-803.The results showed that 1%oxygen concentration culture for 24h significantly promoted the migration and invasion of gastric cancer cells（P<0.05）,the experiment confirmed that 1%oxygen concentration culture MGC-803 for 24h is suitable to simulate the state of gastric cancer cells in the hypoxia microenvironment;（4）The effect of Cinobufagin on the survival rate of hypoxia MGC-803 was detected by CCK-8,and the results showed that Cinobufagin inhibited the proliferation of hypoxia gastric cancer cells（P<0.05）,IC50 was 8.4 mg/m L;（5）Western blot analysis of the effect of Cinobufagin on HIF-1αin hypoxia MGC-803cells showed that Cinobufagin inhibited HIF-1αprotein expression（P<0.05）;（6）The effects of Cinobufagin on the migration and invasion ability of hypoxia gastric cancer cells were detected by cell scratch assay and transwell assay.The results showed that Cinobufagin inhibited the migration and invasion ability of hypoxia gastric cancer cells（P<0.05）;（7）Western blot analysis of the effect of Cinobufagin on the expression of EMT-related proteins in hypoxic MGC-803.The results showed that Cinobufagin inhibited the EMT of hypoxic MGC-803（P<0.05）.3.The regulation of Kv1.5 channel by Cinobufagin affects the hypoxic microenvironment of gastric cancer and inhibits its migration and invasion.（1）Western blot and immunohistochemistry were used to detect the influence of Cinobufagin on Kv1.5 channel protein level in hypoxia MGC-803 cells in vitro and in vivo,and the results showed that Cinobufagin could significantly inhibit the expression of Kv1.5 channel protein（P<0.05）in a dose-dependent manner;（2）The effect of specific Kv1.5 channel inhibitor on the survival rate of hypoxic MGC-803 cells was detected by CCK-8 assay,and the results showed that the inhibitor had no significant effect on the survival rate of hypoxia MGC-803 cells（P>0.05）;（3）Western blot assay was used to detect the effect of blocking Kv1.5 channel on HIF-1αprotein in hypoxic gastric cancer cells.The results showed that Kv1.5 channel is involved in regulating the expression of HIF-1αand blocking Kv1.5 channel improved the hypoxia state of gastric cancer cells（P<0.05）;（4）The effects of blocking Kv1.5 channel on the migration and invasion ability of hypoxia gastric cancer cells were detected by cell scratch assay and transwell assay.The results showed that blocking Kv1.5 channel inhibited the migration and invasion of hypoxia gastric cancer cells（P<0.05）;（5）Western blot assay detected the effect of blocking Kv1.5 channel on EMT of hypoxic gastric cancer cells,and the results showed that blocking Kv1.5 channel could inhibit EMT of hypoxic gastric cancer cells（P<0.05）.ConclusionThe results showed that Cinobufagin blocked the Kv1.5 channel,affected the hypoxia microenvironment of gastric cancer and inhibited its proliferation,migration and invasion. |
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