| Background:Renal cell carcinoma is one of the top ten the most common malignancies worldwide.According to the statistics,about 290,000 patients are newly diagnosed with renal cancer every year,and its mortality rate is about 2% of the global cancer mortality rate.At present,the main treatment of renal cancer is the surgery.Patients with metastases,larger volume or complexity of the anatomy,are often treated with targeted drugs.Sorafenib,sunitinib are commonly used in clinic practice.Targeted drugs significantly promote the survival and decrease the mortality of renal cancer patients.However,patients should be faced with an inevitable problem,namely,drug resistance.Hence,how to solve the drug resistance or enhance the sensitivity of targeted drugs become an a central clinical problem.A different approach prefers the intake of foods with anticancer effects in the daily diet and prevents or treats the cancer.For example,apigenin and allicin have been shown an certain antitumor effect.Similarly,methoxyflavones compounds which are mainly present in vegetables and citrus fruits,were also demonstrated to have anti-inflammatory,antiangiogenetic and proapoptotic effects.Nobiletin is a methoxyflavonoid compound which is isolated from oranges and lemons.Its anticancer mechanisms include inhibition of the angiogenic Src/FAK/STAT3 pathway,inhibition of the proliferative pathways such as ERK,AKT,inhibition of MMP2,MMP9.At present,its anti-tumor effect is reported in liver cancer,breast cancer,ovarian cancer,etc.Howerver,it is rarely reported in renal cell carcinoma.Therefore,it has clinical significance to explore the anti-tumor effect of nobiletin on the renal cell carcinoma and whether it can enhance the sensitivity of the sorafenib.Objects:(1)To explore the anti-tumor effect of nobiletin on the renal cell carcinoma in vitro.The xenograft tumor nude were used to explore the antitumor effect of nobiletin in vivo.Finally,an attempt is made to decipher its anti-tumor mechanism.(2)Combined nobiletin with sorafenib to explore whether it can enhances the sensitivity of sorafenib.The synergy was verified in xenograft tumor nude in vivo and in the renal carcinoma cell in vitro.Methods:(1)The CCK8 assay and cloning experiments were used to assess cell viability.Cell cycle and apoptosis were analyzed by flow cytometry.(2)Transwell assays and scratch test were used to assess migration and invasion ability(3)We used western blotting to detect the expression of proteins which were influenced by drugs.The AKT protein agonist IGF-1was used to explore whether it can reverse the biological effects of nobiletin,and attempted to explain its molecular mechanism.The protein was stained by immunofluorescence and observed by confocal laser scanning microscopy.A renal cell carcinoma tissue chip was stained by immunohistochemistry.(4)The renal carcinoma cell line was implanted under the dorsal skin of the nude mice and experimental group was administered the drug via gastric lavage.The volume of tumors were dynamiclly monitored and the tumors were excised and weighed.The tumor tissues were embedded in paraffin and cut into paraffin sections.Tissue sections were stained by immunofluorescence to detect the protein expression.The C57 mice was administered the nobiletion via gastric lavage,and the body weight was dynamically monitored.The main organs were embedded in paraffin and cut into paraffin sections.Results:(1)The results of CCK8 showed that nobiletin significantly inhibited the proliferation of renal cancer cells in a concentration-and time-dependent.The results of plate cloning showed that the colonies of the tumor treated with nobiletin were less than the control group.The cell cycle showed that nobiletin induces G0/G1 cell cycle arrest in renal carcinoma cells.Apoptosis was detected by flow cytometry,and the results showed that nobiletin promoted the apoptosis.(2)The scratch test showed that the renal cancer cells treated with nobiletin had a shorter migration distance than the control group.Transwell assays showed that nobiletin could significantly inhibit the invasion.(3)Western blot showed that the activation of expression of Src,AKT,STAT3 and YAP was inhibited by nobiletin.Immunofluorescence indicated that nobiletin could decrease the nuclear localization of STAT3.Immunofluorescence indicated that the expression of YAP was reduced in the tumor cell After the renal carcinoma cells were treated with nobiletin,we then treated them with IGF-1.IGF-1 was able to reverse the anti-tumor effect of nobiletin.The expression of p-STAT3,p-AKT,YAP which were inhibited by nobiletin were all reversed by IGF-1.At the same time,the decreased proliferation ability of tumor cells was also improved.We stained renal cell tissue chip by immunohistochemistry and the Kaplan-Meier survival curve was used to analyzed the relationship between YAP expression and renal cancer prognosis.The results showed that patients with high YAP expression had a poor prognosis.(4)The xenograft tumor nude was administered the nobiletin via gastric lavage.The tumor volume and weight of the nobiletin group were smaller than control group.TUNEL showed that the tumor apoptosis increased in the nobiletin group.The tumor tissue sections were detected by immunofluorescence,and the expression of Ki-67,p-STAT3 were decreased in the nobiletin group,and the expression of p-YAP was increased.The body weight of C57 which was administered the nobiletin via gastric lavage was not significantly reduced.Compared with the control group,HE staining of the heart,liver,kidney,spleen and small intestine in nobiletin-treated group showed no significant pathological changes in the tissue structure.(5)Nobiletin and sorafenib were combined to treat the renal cancer cells.We found that the combination therapy inhibited the proliferation of renal cancer cells more significantly than either of the drugs respectively.These results indicated that the synergistic effect of nobiletin and sorafenib.Similarly,in migration,invasion,and apoptosis,the effects of combination therapy were also significantly than either of the drugs respectively.(6)The xenograft tumor nude was administered the saline,nobiletin,sorafenib,nobiletin combined with sorafenib.We found that the tumor volume and weight in the combination group were smaller than another three groups.Tumor tissue sections were analyzed by immunofluorescence staining.Compared with the other three groups,more cell apoptosis and decreased cell proliferation were in the combination group.Conclusion:Our study firstly confirmed the biological effects of nobiletin against renal cell carcinoma.The main mechanism involves the inhibition of the Src/AKT,STAT3,YAP pathways.In addition,nobiletin and sorafenib were also combined to treat the renal cancer cells,and we found that nobiletin could significantly enhance the sensitivity of sorafenib.Therefore,the anti-tumor effect of nobiletin and its synergistic effect with sorafenib could provide a new concept for the treatment of renal cancer.Next,we will explore the optimal ratio of combination therapy and its clinical transformation. |
PDF Full Text Request