Effect Of Sodium Selenite On Biological Behavior Of Renal Cell Carcinoma And Its Mechanism

BackgroundRenal cell carcinoma(RCC)is one of the most common malignant tumors and the most fatal urinary tumors.Its causes include obesity,hypertension,smoking,heredity and so on.The most common pathological type is clear cell renal cell carcinoma(ccRCC).Although cancer detection is improving due to advances in imaging,more patients with early-stage renal cancer are being detected and treated effectively.However,between 20%and 30%of patients still have metastasis,known as metastatic renal cell carcinoma(mRCC),at the time of diagnosis.Furthermore,around 20%and 40%of patients have recurrence and metastasis after surgery.RCC is not sensitive to radiotherapy and chemotherapy,it undoubtedly increases the difficulty of treatment and leads to poor prognosis of patients with advanced renal cancer.Although targeted therapy drugs such as Sorafenib,Sunitinib and Tyrosine kinase inhibitor(TKI),which inhibit angiogenesis,have been developed,their improvement in patients’ survival is still very limited.This requires us to continue to explore and study the clinical treatment of advanced renal cancer,which is an important and urgent problem.Trace elements are vital to human health and play an important role in the constitution of the body and the maintenance of physiological functions.Normally,the body’s trace elements are in balance.But when this balance is disrupted for some reasons,multiple organs aren’t at their best,resulting in a variety of diseases.For example,lack of selenium can lead to Keshan disease,hypothyroidism and other diseases,too much manganese can lead to polycythemia,cirrhosis and other diseases.In addition,the epidemiology shows that disturbance of trace elements is associated with a variety of tumors,such as breast cancer,prostate cancer,etc.In the preliminary work,our research group has tested and analyzed the content of trace elements in serum of healthy people and patients with renal cancer,and found that the content of selenium in serum of patients with advanced renal cancer is lower than that of normal people and patients at early stage.Therefore,we consider that selenium may affect the biological behavior of renal cell carcinoma,and selenium supplementation may be helpful in the treatment of patients with advanced renal cancer.In this study,selenite,the most common form of selenium,was used to verify the influence of sodium selenite on the biological behavior of RCC cells,and the mechanism of sodium selenite on RCC cells was initially studied.Finally,we verified whether sodium selenite has inhibitory effect on the growth of RCC transplanted tumor models in nude mice and whether there are obvious toxic and side effects on nude mice,hoping to provide some help for the treatment of patients with advanced renal cancer.Part Ⅰ Effects of sodium selenite on biological behavior of RCC cellsObjective:To verify the effect of sodium selenite on the biological behavior of RCC cells by researching the effect of sodium selenite on RCC cells proliferation,apoptosis and metastasis.Methods:RCC cells(786-O cells and ACHN cells)were treated with different concentrations of sodium selenite(0,2.5um,5uM,10uM,20uM,40uM),and observed the changes of cell morphology and number at different time by optical microscope.MTT assay and clony formation assay were used to verify the effect of sodium selenite on the proliferation of RCC cells.The apoptosis of RCC cells was determined by flow cytometry.The effect of sodium selenite on the metastasis of RCC cells was verified by scratch wound healing assay and Transwell assay.Results:Through optical microscope observation,we found that with the increase of sodium selenite concentration and the extension of treatment time,the number of cells decreased,the cell morphology atrophied,and the cytoplasm became darker than before.By MTT assay,We found that sodium selenite could effectively inhibit the proliferative activity of RCC cells in a time-dependent and concentration-dependent manner.Clonal formation assay showed that sodium selenite could inhibit the clonal formation ability of RCC cells.Flow cytometry showed that sodium selenite could induce apoptosis of RCC cells,and the apoptosis rate increased with the increase of concentration.Scratch wound healing assay and Transwell assay showed that sodium selenite inhibited the migration and invasion of RCC cells.The above results were statistically significant(P<0.05).Conclusion:Sodium selenite can inhibit the proliferation and induce the apoptosis of RCC cells.In addition,sodium selenite can inhibit the metastasis of RCC cells.Part Ⅱ Molecular mechanism of sodium selenite promoting apoptosis and inhibiting metastasis of RCC cellsObjective:To explore the molecular mechanism of sodium selenite promoting apoptosis and inhibiting metastasis of RCC cells.Methods:In order to detect whether sodium selenite causes RCC cells apoptosis through endogenous pathway,we used fluorescence spectrophotometer to measure the changes of mitochondrial membrane potential and reactive oxygen species.Furthermore,Real-time PCR was used to detect the changes of apoptosis related mRNA levels,Western Blot to detect the changes of apoptosis related proteins.Real-time PCR and Western Blot were used to detect changes in mRNA and protein levels of MMP-9.E-cadherin and VEGF which was related to tumor cell metastasis.The regulation of ROS content by NAC was used to verify whether sodium selenite induced apoptosis and inhibited metastasis of RCC cells through ROS.Searching the String database for ROS interacting proteins.Western Blot was used to detect the changes of NF-κB pathway related proteins in RCC cells at different concentrations of sodium selenite.Finally,the ROS levels was regulated by NAC,and the effect of sodium selenite on NF-κB pathway through ROS was observed by Western Blot and immunofluorescence assay.Results:We found that the mitochondrial membrane potential(MMP)decreased and reactive oxygen species(ROS)increased with the increase of sodium selenite concentration.This suggests that sodium selenite causes endogenous apoptosis of RCC cells.The changes of endogenous apoptosis-related mRNA(Bcl-2,Bax,cIAP,xIAP)levels and associated proteins(Bcl-2,Bax,Cleaved Caspase3)levels were fully suggested that sodium selenite can induce endogenous apoptosis of RCC cells.We found that with the increase of sodium selenite concentration.The mRNA level and protein content of E-cadherin increased gradually,while MMP-9 decreased gradually,they are associated with epithelial mesenchymal transition(EMT).Besides,MMPs was associated with tumor angiogenesis,which plays an important role in tumor metastasis.VEGF is also related to angiogenesis.So we detected the changes of mRNA levels and protein content of VEGF at different concentration of sodium selenite,the result was that they both decreased gradually with the increase of sodium selenite concentration.When reactive oxygen scavenger NAC was added to RCC cells treated with sodium selenite,we found that the ability of sodium selenite inducing apoptosis and inhibiting metastasis on RCC cells was reduced.These results indicated that sodium selenite affected apoptosis and inhibited metastasis of RCC cells through ROS.ROS is associated with the regulation of various signaling pathways.The String database suggested that ROS may interact with proteins related to the NF-κB pathway.By further experiment,we found that sodium selenite inhibited the NF-κB pathway related proteins(P65 and IκB-α)phosphorylation,and inhibited the entry of p-p65 from cytoplasmic to cell nucleus,thereby promoting apoptosis and inhibiting metastasis of RCC cells,The addition of NAC could reduce the inhibition of sodium selenite on NF-κB pathway.Conclusion:Sodium selenite induces apoptosis of RCC cells through endogenous apoptotic pathway and influences RCC cells metastasis through EMT and microangiopathy related protein and mRNA levels.Sodium selenite can promote apoptosis and inhibit metastasis of RCC cells through ROS mediated inhibition of NF-κB pathway.Part Ⅲ Effect of sodium selenite on renal cell carcinoma transplanted tumor modelObjective:Investigate the effect of sodium selenite on renal clear cell carcinoma transplanted tumor model.Methods:We constructed a subcutaneous xenograft model of BALB/c nude mice and divided into sodium selenite group and control group.After the tumor grew to a certain volume,the sodium selenite group was injected intraperitoneally with 2mg/kg sodium selenite every two days,and the control group was injected with the same dose of normal saline.The long and short diameters of the transplanted tumor and the weight of the nude mice were measured every 3 days after injection.After 2 weeks,the mice were sacrificed and the tumor weight was measured.TUNEL staining was used to detect tumor apoptosis,and lung tissue was examined by pathological section.Western Blot assay was used to detect the changes of apoptosis related protein Cleaved caspase-3,metastasis related protein E-cadherin,VEGF and NF-κB pathway related protein p-p65.Results:The tumor volume and weight in sodium selenite group were lower than those in control group,but there was no significant effect on the body weight of mice.TUNEL experiment showed that sodium selenite induced apoptosis of transplanted tumor.There were no obvious abnormalities in the pathological sections of lung tissue of mice in the two groups,no obvious metastasis was found,no obvious drug toxicity changes such as interstitial pneumonia and pulmonary fibrosis.Western Blot assay showed that sodium selenite induced increased expression of Cleaved Caspase-3 and E-Cadherin,and decreased expression of VEGF in transplanted tumor cells.Sodium selenite decreased NF-κB pathway related protein p-p65 in transplanted tumor cells.Conclusion:Sodium selenite inhibited the growth of transplanted tumor in nude mice.Sodium selenite induced apoptosis of transplanted tumor,induced changes of apoptosis,metastasis and NF-κB pathway related proteins.There was no significant difference in body weight and lung tissue slices between the two groups,suggesting that sodium selenite had no obvious toxic and side effects on mice.