The Roles Of Wogonin In The Malignant Behavior And Resistance To Targeted Drug Of Renal Cell Carcinoma

With the changes in modern human lifestyle and living environment,the incidence of kidney cancer has gradually increased in recent years.The incidence of kidney cancer is higher in economically developed countries.Among urinary system tumors,the incidence of kidney cancer is second only to bladder cancer and prostate cancer,ranking third.The incidence of kidney cancer in men is higher than that in women,and the ratio is about 3:2.The incidence of kidney cancer ranks sixth among men and ninth among women of all cancer.In 2021,there are estimated to be more than 76,000 new cases in the United States alone,and more than 13,000 patients will die of kidney cancer.Renal cell carcinoma(RCC)originates from renal tubular epithelial cells in the renal parenchyma.It is the most common type of renal tumor.The incidence of RCC accounts for about 90%of all renal malignancies.For patients with advanced metastatic RCC,systemic drug therapy is the main clinical treatment,and palliative surgery or radiotherapy for the primary or metastatic tumor is supplemented.Clinical trials showed that molecular targeted drugs can significantly prolong the survival period of patients with RCC and improve the prognosis.Multiple targeted drugs including sunitinib and sorafenib,are the first-line treatments for advanced metastatic RCC.However,with the widespread application of molecular targeted drugs,the development of drug resistance has seriously affected the therapeutic efficacy in RCC patients and limited its clinical application.Therefore,development of novel drugs for the treatment of RCC,increasing the sensitivity of RCC to targeted drugs and delay ing or reversing the drug resistance remain critical clinical problems that need to be resolved urgently.In recent decades,various forms of complementary and alternative drug therapy have been widely used in the clinical treatment of cancer all over the world.Traditional Chinese Medicine(TCM)has now been widely accepted as a complementary and supportive treatment method that is beneficial to the survival of various cancer patients.In Asia,TCM has been widely used for thousands of years.Among them,the most commonly used are Chinese herbal medicines,including sliced herbs and Chinese patent medicines.Flavonoids are an important component of polyphenol compounds,and polyphenol compounds naturally exist in various fruits,vegetables and some medicinal plants.Wogonin is a natural flavonoid extracted from Scutellaria baicalensis Georgi,and its molecular formula is C16H12O5.Researches have showed that wogonin has potent anti-tumor activities such as inhibiting tumor cell viability and suppressing tumor cell proliferation in a variety of malignancies.Wogonin can exert its anti-tumor effects through many different ways,such as increasing the intracellular reactive oxygen species(ROS),inducing cell apoptosis,blocking the cell cycle and reversing tumor drug resistance.The multiple anti-tumor effects of wogonin may be closely related to its ability to regulate multiple cell signaling pathways,including serine-threonine kinase Akt signaling pathway,AMP-activated protein kinase(AMPK)signaling pathway,p53-dependent/independent apoptosis and inhibition of telomerase activity.However,the roles and mechanisms of wogonin in RCC,as well as the effects of wogonin on the sensitivity and drug resistance of RCC to targeted drugs,have not yet been reported.Based on the above research background,I carried out the following research work:Firstly,I explored the effects of wogonin on the proliferation,invasion and migration of RCC cells in vitro,and explored the influences of wogonin on the tumorigenesis of RCC cells in vivo;later,the effects of wogonin on the cell cycle progression,DNA damage and apoptosis of RCC cells were tested;then the specific molecular mechanism of wogonin underlying its anti-tumor effects in RCC cells was explored;finally,the effects of wogonin on the sensitivity and drug resistance of RCC cells to targeted drugs were explored.Part One:The roles and mechanisms of wogonin in RCC1.OS-RC-2 and 786-O cells were first treated with different concentrations of wogonin or DMSO.Then MTT assays,colony formation assays and EdU incorporation assays were performed to detect the effects of wogonin on the cell proliferation,colony formation and DNA replication in RCC cells.The results showed that wogonin can significantly inhibit the proliferation of RCC cells.The number of clones in the wogonin treatment group are significantly suppressed compared with the control group,and the proportion of EdU-positive cells is significantly lower than that of the control group.These results suggest that wogonin could significantly inhibit the proliferation,colony formation and DNA replication of RCC cells2.Tumor-bearing model with 786-O cells in nude mouse were first established and the mice were randomly divided into two groups(control group and wogonin group).Then the mice were given saline or wogonin intragastrically every day,and the tumor volume was measured every two days.The mice were sacrificed two weeks later and the tumors were excised,pictured and weighed.The results showed that wogonin suppressed tumor growth of RCC cells in vivo,and the tumor volume and weight in the wogonin-treated group were significantly lower than those of the control group at the end point.3.Wound healing assays and the Transwell assays were performed to explore the effects of wogonin on the migration and invasion of RCC cells.The results showed that wogonin can significantly inhibit the wound healing rate and reduce the cell number that invade through the Matrigel,indicating that wogonin could inhibit the migration and invasion of RCC cells.Western Blot was then performed to detect the protein expression of multiple EMT markers,and the results showed that wogonin can upregulate the expression level of E-Cadherin and suppress the expression levels of N-Cadherin and Vimentin,indicating that wogonin can inhibit the EMT process in RCC cells.4.Flow cytometry assays were performed to examine the effects of wogonin on cell cycle,and the results demonstrated that wogonin can induce G2/M phase arrest of RCC cells.The morphological changes of RCC cells induced by wogonin suggested the possibility of cell apoptosis.Western Blot experiments were performed to detect the expression of apoptosis-related proteins,and the results showed that the expression levels of Cleaved Caspase-3 and Cleaved PARP significantly increased in the wogonin-treated RCC cells.The results of TUNEL assays also showed that the TUNEL positive percentage of RCC cells in the wogonin-treated group was significantly higher than that in the control group,indicating that wogonin can induce apoptosis of RCC cells.5.The alkaline comet assays were used to detect effects of wogonin on the DNA damage of RCC cells and the results demonstrated that the cells treated with wogonin had obvious higher tailing moment compared with the control group.Subsequently,Western Blot experiments were performed to further detect the protein expression level of DNA damage marker yH2A.X.The results showed that wogonin can significantly increase the expression of yH2A.X in RCC cells,indicating that wogonin can induce DNA damage in RCC cells6.The molecular mechanisms underlying the anti-tumor activities of wogonin in RCC cells were then explored.Firstly,Western Blot experiments were used to detect the effects of wogonin on the expression of various cell cycle-related proteins in RCC cells.The results showed that wogonin can significantly inhibit the protein expression levels of CDC6,MCM2 and MCM6 in a concentration-dependent manner in RCC cells.In order to further verify whether CDC6 is an important target of wogonin for its anti-tumor activities in RCC,plasmid transfection was performed with CDC6 expression vector to overexpress CDC6 in RCC cells and then treated with wogonin.Western Blot results showed that plasmid transfection effectively restored the expression level of CDC6 in RCC cells.Subsequently,EdU incorporation assays and alkaline comet assays were used to detect the DNA replication and DNA damage of RCC cells.The results showed that exogenous CDC6 can partially reverse the effects of wogonin on DNA replication and DNA damage of RCC cell7.We further explored the specific mechanism of wogonin regulating the expression of CDC6 in RCC cells.Real-time PCR(RT-PCR)was first used to detect the effects of wogonin on the mRNA level of CDC6,and the results showed that wogonin could inhibit the transcription of CDC6.Subsequently,the effects of wogonin on the expression of upstream pathway of CDC6,including CDK4,CDK6,RB and CyclinDl were tested.The result showed that wogonin can inhibit the expression of CDK4,p-RB and CyclinDl in RCC cells,indicating that wogonin can inhibit the CDK4-RB pathway to down-regulate the transcription of CDC6,thereby exerting anti-tumor activities in RCC.Further study results show that wogonin can inhibit the transcription of CDK4 and reduce the mRNA level of CDK4,and its down-regulation of CyclinD1 protein expression is achieved by accelerating protease-mediated protein degradation and shortening its half-life.Part 2 The effects of wogonin on the sensitivity and drug resistance of RCC cells to targeted drug1.We first explored the effects of wogonin on the sensitivity of RCC cells to targeted drug.OS-RC-2 and 786-O were treated with wogonin or sunitinib alone or a combination of two drugs.The results of the MTT assays showed that combination treatment demonstrated significantly higher inhibitory effects on the cell viability of both RCC cells than sunitinib alone,indicating that wogonin can increase the sensitivity of RCC cells to targeted drug.2.By culturing 786-O cells with medium containing low concentration of sunitinib and gradually increasing the concentration of sunitinib to a final concentration of 10?M,a sunitinib-resistant renal cancer cell line was established.MTT assays were then performed to evaluate the sensitivity of wildtype and sunitinib-resistant 786-O cells to sunitinib treatment.The results of MTT assays showed that the sensitivity of sunitinib-resistant 786-O cells to sunitinib is significantly reduced,with an IC50 more about 5 times of the wild-type cells.3.Western Blot were performed detected the CDK4-RB signaling pathway including protein expression of CDK4,p-RB and CyclinDl in wild-type and sunitinib-resistant 786-O cells.The results showed that the expression levels of CDK4 and p-RB in drug-resistant cells were significantly higher than those in wild-type cells after sunitinib treatment.RT-PCR was used to detect the mRNA expression of other downstream targets of CDK4-RB signaling pathway,and the results showed that the mRNA levels of CDC25A,CCNE2 and CCNA2 in sunitinib-resistant 786-O cells were significantly higher than those in wild-type cells after sunitinib treatment,indicating that the CDK4-RB signaling pathway in sunitinib-resistant 786-O cells is over-activated.4.The sunitinib-resistant 786-O cells were treated with wogonin and Western Blot results showed that wogonin can effectively inhibit the expression of CDK4,p-RB and CyclinDl in drug-resistant cells,and reverse the over-activation of CDK4-RB signal pathway.The results of the MTT experiments proved that the combination of wogonin and sunitinib can effectively inhibit the cell viability of sunitinib-resistant 786-O cells and reverse the resistance to sunitinib.5.Tumor-bearing nude mouse models were established with sunitinib-resistant 786-O cells.The mice were randomly divided into sunitinib group and combination therapy group,and were given sunitinib or sunitinib and wogonin intragastrically every day.Two weeks later,the mice were sacrificed and the tumor volumes and tumor weights were measured.The results showed that the combined treatment could significantly suppress the tumor growth compared with the sunitinib alone,with significantly smaller tumor volume and lighter tumor weight.Conclusions1.Wogonin can inhibit the proliferation,colony formation and DNA replication of RCC cells.2.Wogonin inhibites the migration and invasion of RCC cells by suppressing the EMT pathway.3.Wogonin can block the cycle progression and induce DNA damage and apoptosis of RCC cells.4.Wogonin exerts its anti-tumor activities by down-regulating the expression of CDC6 in RCC cells.5.Wogonin regulates the transcription of CDC6 by inhibiting the CDK4-RB signaling pathway.6.Wogonin can increase the sensitivity of RCC cells to sunitinib and effectively reverse the resistance of RCC cells to sunitinib.