| Research background and purpose:Bladder cancer currently ranks as the 10th most common malignancy in the world and it is the 6th in cancer incidence rate and the 9th in mortality rate in men,which are approximately four times those among women respectively.The most common causes of bladder cancer include gene mutations,continuous exposure to urinary bladder carcinogens,long-term exposure to tobacco,cigarette smoke residues and alcohol,occupation exposure to petroleum products,metals,dyes,paints and inorganic arsenic compounds,etc.Bladder cancer can be divided into non-muscle invasive bladder cancer(NMIBC)and muscle invasive bladder cancer(MIBC).NMIBC is often confined to mucosa or submucosa,but accounts for about 75%of all bladder cancer cases.The most important treatment for NMIBC is transurethral resection.However,the 5-year probability of postoperative recurrence for NMIBC is at 31～78%,whereas the probability of postoperative disease progression after 5 years is 0.8～45%.Therefore,postoperative intravesical Bacillus Calmette-Guérin or intravesical chemotherapy,including mitomycin,epirubicin and gemcitabine,are essential for preventing recurrence and progression.In total,20～25%of all cases of BC are MIBC,where～50%of these patients progress within 5 years after cystectomy.Combined treatment with gemcitabine and cisplatin has been applied as the standard chemotherapy regimen for patients with metastatic bladder cancer and MIBC.Gemcitabine is a broad-spectrum antimetabolic substance and a cytosine analogue.Gemcitabine could induce cell cycle arrest and "masked chain termination" to inhibit further DNA synthesis and lead to cell death.At present,gemcitabine is considered to be an important part of first-line chemotherapy for bladder cancer,non-small cell lung cancer and pancreatic cancer,etc,but the rate of disease relief is reported to be only 49%for metastatic bladder cancer and MIBC patients.In addition,these treatment strategies may lead to serious adverse reactions,including nausea,vomiting,neutropenia,thrombocytopenia,mucositis and febrile neutropenia.Patients who apply the above treatment are still prone to recurrence and drug resistance.Therefore,a series of drugs have been studied to sensitize the effects of gemcitabine and reduce side effects.It is reported that everolimus,sunitinib,vitamin C and vitamin K3 can enhance gemcitabine induced bladder cancer cytotoxicity.Traditional Chinese medicine has a long history in China.Now,more and more traditional Chinese medicine derivatives and herbal medicines have also been proved to have bioactive anti-cancer properties.Berberine is an isoquinoline alkaloid,which can be isolated from berberine(Oregon grape)and berberine(baberi).Recently,it has been found that it has a variety of pharmacological properties,including anti-inflammatory,cardiovascular protection,neuroprotection,anti-hyperglycemia,anti-hyperlipidemia,anti-hypertension and anti-tumor effects.In addition,berberine can be cytotoxic to many types of tumor cells,including urogenital tumors,digestive system tumors,gynecological tumors,lung cancer and melanoma,etc.Previous studies have shown that berberine can not only make bladder cancer cells sensitive to epirubicin,but also inhibit the proliferation,promote apoptosis and cell cycle arrest to produce bladder cancer cells.DNA damage usually plays a key role in accelerating cell death,and DNA double strand breaks(DSB)is one of the most cytotoxic types of DNA damage.After DSB,the DNA repair system started quickly.Rad51 recombinase(Rad51)is a key element of DNA homologous recombination(HR)repair and is considered to be an error free repair mechanism that mediates the maintenance of genomic integrity.The high expression level of Rad51 has previously been associated with aggressive and therapeutic resistance in many tumors,including pancreatic cancer,lung cancer,breast cancer and ovarian cancer,and the downregulation of Rad51 has been shown to improve the efficacy of chemotherapy or radiotherapy sensitivity.Other studies reported that both berberine and gemcitabine could induce DSBs,gemcitabine can induce the up regulation of Rad51 and berberine could down regulate Rad51 expression.Therefore,this study hypothesized that berberine can sensitive bladder cancer to gemcitabine by regulating the expression of Rad51.The purpose of this study is to evaluate the potential effect of berberine on gemcitabine induced bladder cancer cytotoxicity and explore possible mechanisms,which may be a new therapeutic agent or target for bladder cancer treatment.Part Ⅰ Berberine enhances gemcitabine-induced cytotoxicity in bladder cancer in vitro Background and purpose:Although gemcitabine has been used clinically for the treatment of bladder cancer for many years,serious adverse drug events or drug resistance often occur.At present,in order to enhance the sensitivity of patients to gemcitabine and reduce its side effects,related drug research has been carried out.Traditional Chinese medicine is a characteristic of the Chinese nation,and many Chinese herbal medicines have been proved to have anti-tumor effects,such as Huaier,baicalein,sinomenine,berberine,etc.In order to evaluate the effect of berberine on the toxicity on the bladder cancer cells induced by gemcitabine,this part uses in vitro cell experiments to treat bladder cancer cells with gemcitabine and/or berberine to carry out related research.Methods:Human bladder cancer cell lines 5637 and T24 were selected,then the cells were treated with different concentrations of gemcitabine and/or berberine,and the activity of bladder cancer cells was evaluated by CCK-8 method.After treatment of appropriate concentrations of gemcitabine or berberine,cell viability assay,cell apoptosis assay,intracellular reactive oxygen species detection,and wound-healing assay were performed to verify the effect of berberine on gemcitabine-induced cytotoxicity.Results:Berberine and gemcitabine could inhibit the viability of 5637 and T24 BC cells in a time-and dose-dependent manner.Cell viability was significantly lower in the combination group than that in the gemcitabine group.Both berberine and gemcitabine monotherapy increased apoptosis rates and intracellular ROS levels,but combination treatment significantly increased apoptosis and ROS compared to cells treated with gemcitabine alone.The woundhealing assay results showed that monotherapy and combination treatment inhibited the migratory ability of 5637 and T24 cells,with the combination group having a lower migration rate than the gemcitabine groupConclusion:Berberine enhanced the inhibitory effect of gemcitabine on the viability of bladder cancer cells in vitro,and enhanced gemcitabine-induced apoptosis,reactive oxygen species generation and inhibition of migration.Part Ⅱ Molecular mechanism of berberine enhancing gemcitabine-induced cytotoxicity in bladder cancerBackground and purpose:The continuous development of network databases is playing an increasingly important role in medical research.Finding the relevant molecules and pathways through the network database,and then being verified by further experiments,could help us clarify the relevant conclusions.This part is verified by network database and cell experiments related to relevant molecules and pathways to clarify the molecular mechanism of berberine enhancing gemcitabine-induced cytotoxicity in bladder cancer.Methods:The Oncomine database and Gene Expression Profile Interaction Analysis(GEPIA)were used to search for Rad51 recombinase(Rad51)mRNA expression.The expression of Rad51,Akt and the phosphorylation of Akt(p-Akt)were assessed using western blot or RTPCR.PI3K inhibitor LY294002 was used to detection effect of PI3K/Akt pathway on gemcitabine induced cytotoxicity in bladder cancer cell.Overexpression plasmids or specific Rad51 small interfering RNAs were used to examine the effect of Rad51 in drug-treated bladder cancer cells.Transfection with the myr-Akt plasmid,which upregulates Akt phosphorylation,in the presence or absence of berberine and/or gemcitabine treatment,was used to examine whether the enhancement of berberine on gemcitabine-induced cytotoxicity and berberineinduced Rad51 downregulation in bladder cancer cells were mediated by the PI3K/Akt pathway.Results:According to Oncomine and GEPIA analysis,Rad51 expression was significantly upregulated in bladder cancer tissues compared with normal tissues,in which there was a weak but positive correlation between Rad51 and Aktl expression(R=0.21;P<0.05).gemcitabine could induce the activation of PI3K/Akt pathway and upregulation of Rad51,which could be reversed by BER.Knockdown of Rad51 enhanced gemcitabine-induced cytotoxicity,while over-expression of Rad51 reversed cell viability downregulation induced by berberine and gemcitabine.Inactivation of the PI3K/Akt pathway by LY294002 or berberine enhanced gemcitabine-induced cytotoxicity and downregulated Rad51 expression,whereas upregulation of Akt phosphorylation by constitutively active Akt,which indicating the activation of PI3K/Akt pathway,restored Rad51 expression and cell viability previously reduced by berberine and gemcitabine.Conclusion:Berberine could downregulate the expression of Rad51 by inactivating the PI3K/Akt pathway,thereby enhancing gemcitabine-induced toxicity in bladder cancer cells,which might be a new target for bladder cancer therapy.Part Ⅲ Berberine enhances the anti-proliferative effects of gemcitabine in bladder cancer in vivoBackground and purpose:There are many differences between in vitro experiments and in vivo experiments.The usage of all drugs must be verified by in vivo experiments.In order to prove that berberine can enhance the toxic effect of gemcitabine on bladder cancer,the study of this part investigated the effect of berberine on gemcitabine-induced cytotoxicity in bladder cancer in vivo.Methods:5637 cells were injected subcutaneously into the flanks of mice,and then the mice were divided into control group,berberine group,gemcitabine group and combination group to observe tumor growth.The volume and weight of the xenograft were measured.The expression of Ki67 in xenograft tissues was detected by immunohistochemical staining to assess cell proliferative activity in vivo.Results:In vivo,compared with the control group,the tumor volume and weight of the mice in the experimental group were significantly reduced,and combination treatment significantly reduced the tumor weight and volume compared with those in mice treated with gemcitabine.Immunohistochemical analysis revealed that the expression levels of Ki67 in the xenografted tumors were significantly decreased in the combination group compared with those in tissues from mice treated with gemcitabine alone.Conclusion:Berberine could enhance the antiproliferative effect of gemcitabine in bladder cancer in vivo. |
PDF Full Text Request