The Inhibitory Effect Of Huaier On Renal Cancer Cells And Its Mechanisms

Renal cancer is one of the most common malignant tumors in human,accounting for about 4%of all malignancies.In urinary tumors,the incidence of renal cancer is second only to bladder cancer,ranking second.In the United States,renal cancer is the 9th most common malignant tumor in the human body.In China,the incidence of renal cancer is also increasing year by year,with a high incidence in 45-65 years old,more men than women.With the continuous improvement of medical and health conditions in China,more and more patients with renal cancer are discovered at an early stage.The typical "triple syndrome" of renal cancer is difficult to find in clinical practice.Currently,the main treatment for renal cancer is surgical treatment,the lesion is removed as much as possible,and supplemented by other treatments.However,due to the deep anatomical location of the kidney,early symptoms are still concealed.About 30%of the newly diagnosed patient are already in the stage of tumor progression with a distant metastasis,missing the best surgical opportunity.Even if the surgical treatment was done,about 20%-30%of patients will experience recurrence or even distant metastasis during the follow-up.At this time,the median survival time of the patient is less than 10 months,and the prognosis is extremely poor.In addition,unlike most other malignant tumors,renal cancer is not sensitive to chemotherapy or radiotherapy,endocrine therapy is not effective,and targeted drugs are expensive.These lead to a high mortality rate of renal cancer worldwide,and renal cancer has become a major hazard to human health.Therefore,finding new natural drugs that are efficient,cheap,easy to obtain,and well tolerated is of great significance for the treatment of renal cancer.Huaier is a kind of raw material in Chinese herbal medicine.The theory of traditional Chinese medicine believes that it has the effect of strengthening the body,strengthening the blood and eliminating blood stasis.It has been applied in China for more than 1,000 years.It is a fungus that grows on the trunk of the Huaier,so it got its name.Modern pharmacy analysis shows that the fungus is complex,containing polysaccharides,proteins,ketone bodies,alkaloids,other biological active ingredients and more than 20 kinds of mineral elements,of which the active ingredients are mainly proteoglycans.At present,the anti-tumor effect of Huaier has gradually attracted widespread attention.More and more basic research and clinical practice have shown that Huaier may be a promising anticancer drug.At present,it has been confirmed that Huaier has inhibitory effects on various malignant tumor cells such as triple-negative breast cancer,lung adenocarcinoma,gastric adenocarcinoma,hepatocellular carcinoma,colorectal cancer,cervical cancer,etc.Huaier is also gradually applied to the adjuvant treatment of some tumors.Its anti-tumor mechanism mainly includes:inhibiting the proliferation of tumor cells,inducing apoptosis of tumor cells,inhibiting migration and invasion of tumor cells,inhibiting the formation of blood vessels in tumor tissues,improving the function of immune cells,and enhancing the sensitivity of tumor cells to radiotherapy and chemotherapy.However,for different types of tumor cells,the effects of Huaier are not the same,and the potential anti-tumor mechanisms may also be different.At present,there is no research on the effect of Huaier on renal cancer cells at home and abroad.In order to study the inhibitory effect of Huaier on renal cell carcinoma and explore its possible mechanism,and provide pharmacological basis for future clinical drug use,this study is mainly explored from the following three parts:PART ? The effect of Huaier on inhibiting the proliferation of renal cancer cells and its mechanismBackgroundsThe most obvious biological feature of malignant tumor cells is their near-infinite growth and proliferation ability.Antitumor drugs should have the ability to inhibit the growth and proliferation of tumor cells.Many in vitro studies have shown that Huaier can inhibit the proliferation of various cancer cells such as breast cancer,lung adenocarcinoma,gastric adenocarcinoma,hepatocellular carcinoma,colorectal cancer and cervical cancer.The mechanism is summarized as follows:regulating cell growth by multiple micro-RNAs,inducing apoptosis by caspase-3 pathway,mediating autophagy via mTOR/S6K pathway,and arresting cell cycle into G2/M phase.For different tumor cells,the underlying mechanism are not the same,and there may be one or more mechanisms involved in the regulation.Combining with domestic and foreign literatures,there is no research report on the mechanism of action of Huaier on renal cancer cells.This part is an in vitro cytology experiment.The renal cancer cells will be treated with different concentrations of Huaier solution(solvent is serum-free cell culture medium)to understand the effect of Huaier on the cell growth,and the possible mechanism is also discussed.MethodsTwo kinds of human renal cell carcinoma lines:786-0 and A498 were selected,and the morphological changes of renal cancer cells were observed after treatment with different concentrations of Huaier for a different period of time.The growth rate of renal cancer cells was tested via CCK-8,plate clone fonnation assay and flow cytometry.The expression level of related factors in the cell growth signal pathway was determined by Western Blot.Results1.Effect of Huaier on the morphology of renal cell carcinomaHuman renal cancer cell lines 786-0 and A498 were treated with different concentrations of Huaier solution(0,4,8,12 mg/ml).After 24 hours,the proliferation and morphological changes of cells were observed.The results showed that compared with the blank control group,Huaier solution can significantly change the density and morphology of the above two kinds of cells,mainly showing a decrease in cell density,cell atrophy,irregular cell shape,"brushed",nuclear lysed and stained weakly,cytoplasmic vacuolation accompanied with abnormal black particles,some cell membrane folds and even "dissolved".The above changes were concentration-time dependent,that is,the higher the concentration of Huaier,the longer the treatment time,and the more obvious the abnormality of the cell morphology.2.Inhibition of proliferation of renal cancer cells by HuaierThe inhibition of proliferation in the two renal cancer cell lines was detected by CCK-8 assay and plate cloning assay.The CCK-8 test showed that Huaier inhibited the proliferation of 786-0 and A498 renal cancer cells.After treated for 48 h,the IC50 in 786-0 cells was 5.88 mg/ml,while the IC50 in A498 cells was 6.80 mg/ml,the inhibitory efftect on the 786-0 cell line was relatively obvious.Treatment of the two cells with different concentrations of Huaier at different times,we found that the inhibition of the two cells in a concentration-and time-dependent manner.The higher the concentration of Huaier,the longer the treatment time,the more obvious the inhibitory effect on the proliferation of renal cancer cells.In the plate cloning experiment,the low-density plated cells were treated with different concentrations of Huaier,the size and number of the clonal communities of the two cell lines were inhibited by the Huaier in a concentration-dependent manner.3.Effect of Huaier on apoptosis of renal cell carcinoma cellsThe effect of Huaier on apoptosis of 786-0 and A498 was detected by flow cytometry.After treating the two cells with different concentrations of Huaier solution(0,4,8,12 mg/ml),the proportions of cells in each state(survival,early/late apoptosis,necrosis)were statistically analyzed.The proportion of apoptotic cells in the cells increased significantly and was concentration-dependent,that is,the higher the concentration of Huaier,the higher the proportion of apoptosic cells.4.Effect of Huaier ear on renal cancer cell cycleThe effect of Huaier on the cell cycle of 786-0 and A498 was examined by flow cytometty.After treating the two cells with different concentrations of Huaier solution(0,4,8,12 mg/ml),the proportion of cells in each division cycle was statistically analyzed.It was found that the cycle proportions of the two cells changed similarly,at G0/G1.The proportion of cells in the period increased significantly,indicating that Huaier can block the renal cancer cell division cycle in the G0/G1 phase.5.Huaier inhibits the proliferation of renal cancer cells by regulating PI3K/AKT/mTOR and Wnt/?-catenin signaling pathwayWestern Blot was used to study the changes of related protein factors in the PI3K/AKT/mTOR signaling pathway in renal cancer cells treated with Huaier.The total amount of PI3K,AKT and mTOR did not change significantly in the 786-0 and A498 cells treated with Huaier solution for 24 h,but their phosphorylation levels(p-PI3K,p-AKT,p-mTOR)were all reduced.Two important target proteins p70S6K and 4E-BP1 in the downstream of this pathway also showed the same trend.In the Wnt/?-catenin signaling pathway,related proteins such as DVL2,p-GSK-3 p/GSK-3?,?-catenin and Cyclin D1 all showed decreased expression levels,indicating that the signaling pathway was also inhibited.The above changes are all concentration dependent.Conclusions1.Huaier can inhibit the proliferation of human renal cancer 786-0 and A498 cells;2.Huaier can induce cell cycle of human renal cancer cell lines arrested in the S phase.3.Inhibition of proliferation in human renal cancer cell lines by Huaier is achieved by modulating the PI3K/AKT/mTOR and Wnt/?-catenin signaling pathwayPART ? Effect of Huaier on inhibiting migration and invasion of renal cancer cells and its mechanismBackgroundsMigration and invasion are one of the most obvious characteristic biological behaviors of malignant tumor cells,renal cancer is no exception.The main treatment for early renal cancer is surgical treatment.However,due to the concealment of early symptoms of renal cancer,about 30%of patients were in the stage of tumor progression on the first visit,and there was a distant metastasis,missing the best surgical opportunity.Even after surgery,about a quarter of patients will have local recurrence and metastasis during postoperative follow-up.This group of patients have a very poor prognosis,with a median survival of less than 10 months.The reason for the high mortality rate of renal cancer worldwide is early metastasis.More and more experiments have shown that Huaier can inhibit the migration and invasion of a variety of malignant tumors(liver cancer,cholangiocarcinoma),and can be used as an effective adjuvant for tumor resection to reduce the probability of tumor recurrence and metastasis.The mechanism also involves various aspects,including inhibiting the growth of blood vessels inside the tumor,enhancing the adhesion ability between tumor cells,and reversing the epithelial-mesenchymal transition of malignant cells.Whether Huaier can inhibit the migration and invasion of renal cancer cells is essential for the treatment of renal cancer in the future.However,we can not find research reports in this area.The purpose of this part of the experiment is to study whether Huaier has a certain impact on the migration and invasion ability of renal cancer cells,and further explore the molecular mechanism of its role.In this part,we studied whether Huaier can inhibit the migration and invasion of renal cell carcinoma cells under in vitro laboratory conditions,and to explore its mechanism.MethodsThis section continues to use two human renal cancer cell lines:786-0 and A498,using scratch test and Transwell migration and invasive experiments.Treat the above renal cancer cells wieh different concentrations of Huaier solution(0,2,4mg/ml),observed the "healing" condition of the scratch over a period of time,as well as the number of cells crossing the cell's Transwell chamber basement membrane.The expression levels of protein factors in tumor metastasis-associated signaling pathways were determined by Western Blot and cellular immunofluorescence techniques.Results1.Huaier can inhibit the migration ability of renal cancer cells.The 786-0 and A498 cells were treated with different concentrations of Huaier solution(0,2,4 mg/ml)for scratch test.The results showed that the "healing" ability of both cells was significantly decreased in a concentration-dependent manner.The higher the concentration of Huaier,the more suppressed the ability of the cell to"heal".The results of Transwell chamber migration experiments showed that the ability of renal cancer cells treated by Huaier pass through the basement membrane of the chamber was decreased.The above in vitro experiments show that Huaier can inhibit the migration ability of renal cancer cells.2.Huaier inhibits the invasive ability of cervical cancer cells in vitro.786-0 and A498 cells were treated with different concentrations of Huaier solution(0,2,4 mg/ml)for Transwell chamber invasion experiments.The results showed that the number of renal cell carcinoma cells treated by Huaier was significantly reduced in passing through the Matrigel membrane at the bottom of the chamber,which showed that the invasive ability of the cells was decreased.3.Huaier inhibits migration and invasion of renal cancer cells by regulating epithelial-mesenchymal transition(EMT)Western Blot and cellular immunofluorescence were used to study the changes of related protein factors in EMT during renal cell carcinoma treatment.The expression levels of N-cadherin,Vimentin,Twist and other proteins were decreased in 786-0 and A498 cells treated with Huaier solution for 24 hours,while the expression level of E-cadherin was the above,indicating that Huaier can inhibit the expression of proteins involved in metastasis-promoting and suppress the EMT process,the above changes are concentration dependent.Conclusion1.Huaier can inhibit the migration and invasion ability of human renal cancer cells in vitro;2.The effect of Huaier on the migration and invasion ability of renal cancer cells is achieved by regulating EMT process.PART ? Effect of Huaier on tumorigenic ability of renal cancer cells in nude miceBackgroundsThe cytology experiments under laboratory conditions are significantly different from the experiments in vivo.The experimental environment of the two is quite different,which leads to different and even the opposite experimental results.The above two parts are in vitro cytology experiments under laboratory conditions,and the cell growth conditions are controllable and the influencing factors are single.Therefore,the experimental results are stable and obvious,which proves the inhibitory effect of Huaier on the renal cancer cells in vitro.However,the drug should be applied to the clinic in the end,and whether the drug can exert anti-tumor effect in a complex human environment needs further verification.Therefore,it is very important to perform animal experiments which can simulate the human body environment.It will be very helpful for us to further clarify the inhibitory effect of Huaier on human tumors.In this part,the tumor formation experiment was used to observe the effect of Huaier on the tumorigenic ability of renal cancer cells in nude mice.Methods786-O cells were selected to be seeded subcutaneously in nude mice.After the tumor formation was stable,the experimental group was gavaged with 100 ?1 Huaier aqueous solution(containing 2 mg of Sophora japonica),the experimental group was gavaged with water,once every other day.The body weight and tumor size of the nude mice were recorded,and after 7 weeks of recording,the nude mice were sacrificed and the tumors were removed.The differences between the experimental group and the blank control group were compared.Immunohistochemical technique was used to detect the difference in the expression levels of related protein factors in the nude mice.Results1.After inoculation of tumor cells,there was no abnormaity in the living state of the two groups of nude mice,and the body weight of the nude mice did not change significantly,but the volume of the nude mice in the experimental group was lower than that of the control group at each stage,and the difference in tumor volume and weight between the two groups was statistically significant;2.Immunohistochemical staining showed that the expression of N-cadherin,Vimentin were decreased in nude mice,while E-cadherin staining deepened,which further confirmed the second part of the in vitro experiment that Huaier suppressed EMT process.ConclusionsHuaier can inhibit the growth of renal cancer cells in nude mice.Maj or Innovations1.This study was the first to verify the inhibitory effect of Huaier on two human renal cancer cell lines;2.It was the first study to discovered that Huaier can inhibit PI3K/AKT/mTOR signaling pathway,Wnt/?-catenin signaling pathway and reverse epithelial-mesenchymal transition(EMT)in human renal cancer cells;3.For the first time,it was confirmed that Huaier can inhibit the growth of renal cancer cells in nude mice.