Study On The Inhibitory Effect And Radiosensitivity Of Berberine On Cervical Cancer Cells

The incidence of cervical cancer ranks first among women with malignant tumors of reproductive organs,it seriously endanger women’s life and health.At present,the common treatment methods for cervical cancer are surgery,radiotherapy and chemotherapy.According to statistics,more than 80%of cervical cancer patients need radiotherapy,and its curative effect is not ideal,in patients with stage Ⅰ and Ⅱ,the survival rate was only 65%-85%after radiation therapy,the survival rate of patients with stage Ⅲ and Ⅳ after cervical cancer treatment was 20%-50%in 5 years.Radiation therapy for cervical cancer patients can cause systemic adverse reactions,such as bone marrow hematopoietic dysfunction,gastrointestinal discomfort,radiation therapy can also cause local damage,such as radiation proctitis,radioactive cystitis,ovarian atrophy,pelvic fibrosis Etc,this caused varying degrees of pain and multiple injuries to the patient.In clinical medicine,radiotherapy and chemotherapy are often combined,If radiation therapy sensitivity testing is performed before radiation therapy and radiation dose application is guided based on the test results,it can minimize physical trauma and reduce treatment costs.therefore,the study of cervical cancer cell radiation sensitivity and to find ways to improve the radiosensitivity of cervical carcinoma cells is a hotspot and the focus of the study.Berberine,also known as berberine,is a natural alkaloid extracted from rhizomes such as Coptis chinensis and Phellodendron chinense in the Ranunculaceae family.It belongs to isoquinolines and has a wide range of biological functions.Berberine was originally mainly used for the treatment of gastrointestinal related diseases,with detoxification and anti-bacterial anti-inflammatory effects,with the continuous deepening of research,berberine also has anti-inflammatory,lowering blood pressure,lower blood pressure,anti-arrhythmia,lowering blood sugar,improve Insulin resistance,lowering of androgen,etc.,have a certain value in the treatment of various diseases such as digestive tract diseases,cardiovascular diseases,endocrinology,and gynecology.Recent studies have shown that berberine also has an anti-tumor effect.In vitro cell experiments found that berberine can inhibit the growth of many kinds of cancer cells within a safe and non-toxic dose range.On the one hand,berberine can inhibit various carcinogens such as nitrosoguanidine,cyanobacteria,carbon tetrachloride and other damage to the body,and inhibit the normal physiological function of cancer cells by affecting the activity of N-acetyltransferase(NAT);On the other hand,berberine can block the cell cycle of cancer cells,inhibit the synthesis of deoxyribonucleotides and proteins in tumor cells,activates the cysteine-containing aspartate proteolytic enzyme cascade to inhibit cancer cell growth and induce cancer cell apoptosis,which has different mechanisms of action for different types of tumor cells.In addition to the direct killing of tumor cells,berberine can further inhibit tumor growth by increasing the regulation of other related pathways.For example,berberine can enhance the inhibitory effect of paclitaxel on gastrointestinal related malignancies.For example,berberine can enhance the inhibitory effect of paclitaxel on gastrointestinal related malignancies.Berberine has a synergistic effect on arsenic trioxide-induced apoptosis of neuroblastoma,and berberine combined with daunorubicin can treat leukemia.Berberine can also increase the radiotherapy sensitivity of breast cancer cells and nasopharyngeal cancer cells.Therefore,it is very important for the clinical treatment of cervical cancer to clarify the effect and mechanism of berberine on the growth,apoptosis and radiosensitivity of cervical cancer cells.In this study,cervical cancer cells as the main research object,after treatment with different concentrations of berberine,CCK-8 method was used to detect the effect of berberine on the proliferation of three cervical cancer cells,a cell with obvious effect was selected as the research object.Flow cytometry was used to detect the effect of berberine on apoptosis and cell cycle of cervical cancer cells.The level of STAT3 and p-STAT3 in cervical cancer cells was detected by Western blot.Finally,the cell survival curve was drawn by cell cloning experiments,and the radiosensitivity ratio was calculated based on the multi-target model parameters,in order to clarify the effect of berberine on the radiosensitivity of cervical cancer and to explore its possible mechanism of action.It is of great significance to study the anti-tumor effect and radiosensitization of berberine.Part I Effects of berberine on proliferation of cervical cancer cells MethodsThree cervical cancer cells,Siha,HeLa,and Caski,were cultured in vitro.Three cells were cultured in cell culture medium containing berberine at final concentrations of 5,10,15,and 20 μmol/L,At the same time,three cells were cultured in cell culture medium without berberine.After 48 hours,cell proliferation activity was measured by CCK-8 method,and half inhibition concentration was calculated.ResultsThe proliferation activities of Siha,HeLa,and Caski in cervical cancer cells treated with 5 μ mol/L,10 μ mol/L,15 μ mol/L,and 20 μ mol/L berberine were significantly lower than those in the control group.The difference was statistically significant(P<0.05).Berberine inhibited the proliferation of Siha,HeLa,and Caski cells,and the half inhibitory concentrations were(16.84±3.52)μmol/L,(23.54±8.67)μ mol/L,and(21.86 + 6.35)μ mol/L,respectively.Berberine inhibited the growth of Siha cells more strongly than the other two cells.ConclusionsBerberine inhibits the proliferation of cervical cancer cells Siha,HeLa,and Caski,and its effect on inhibiting the growth of Siha cells is stronger than that of the other two cells.Subsequent use of 17 μmol/L berberine can be applied to Siha cells for further study.Part Ⅱ Effects of berberine on cell cycle and apoptosis in cervical cancer cellsMethods1.Using half the inhibitory concentration of berberine on cervical cancer Siha cells for 48h,apoptosis was detected by flow cytometry.2.Using half the inhibitory concentration of berberine on cervical cancer Siha cells for 48h,Western blot was used to detect the level of apoptosis-related protein Cleaved Caspase-3.3.Using half the inhibitory concentration of berberine on cervical cancer Siha cells for 48h,the cell cycle was detected by flow cytometry.4.Using half the inhibitory concentration of berberine on cervical cancer Siha cells for 48h,Western blot was used to detect the expression of Cyclin B1 and CDK1.Results1.The apoptosis rate of Siha cells after half-inhibitory concentration of berberine was(32.25 ± 1.25)%,but The apoptosis rate of Siha cells after treatment without berberine was(9.32 ± 0.95)%,the difference was statistically significant(P<0.05).The apoptosis of cervical cancer Siha cells increased after berberine treatment.2.The level of Cleaved Caspase-3 in Siha cells after half-inhibitory concentration of berberine was increased,using GAPDH was used as an internal reference,the expression of Cleaved Caspase-3 was 0.97 ± 0.09,but the expression of Cleaved Caspase-3 in Siha cells without berberine treatment was 0.23±0.06,compared with the two group,the difference was statistically significant(P<0.05).Berberine may promote the apoptosis of cervical cancer cells by increasing the expression of Cleaved Caspase-3.3.The G2/M phase ratio of Siha cells after half-inhibitory concentration of berberine was(42.91±4.57)%,while the ratio of G2/M phase of Siha cells without berberine treatment was(7.68 ± 1.23)%.The difference was statistically significant(P<0.05).That berberine can cell arrest in the G2/M phase.4.Cyclin B1 and CDK1 protein levels in Siha cells with half-inhibitory concentration of berberine decreased,GAPDH was used as an internal reference,CyclinB1 protein expression was 0.85 ± 0.07,and CDK1 protein expression was 0.36 ± 0.04 without berberine.The expression of CyclinB1 protein in treated Siha cells was 1.14 ±0.08,and the CDK1 protein expression was 1.09 ±0.12.The difference was statistically significant(P<0.05).It is suggested that berberine may affect the cervical cancer cell cycle by decreasing the levels of CyclinBl and CDK1 proteins.Conclusions1.Berberine induced apoptosis of Siha cells in cervical cancer by increasing Cleaved Caspase-3 expression.2.Berberine blocks cervical cancer Siha cells in the G2/M phase by reducing CyclinB1 and CDK1 protein levels.Part III Effects of berberine on the levels of STAT3 and p-STAT3 in cervical cancer cellsMethodsUsing half the inhibitory concentration of berberine on cervical cancer Siha cells for 48h,Western blot was used to detect the levels of STAT3 and p-STAT3 in STAT3 signaling pathway.ResultsThe expression of STAT3 protein in Siha cells after half-inhibitory concentration of berberine did not change significantly,but the level of p-STAT3 decreased.Using GAPDH as an internal reference,the expression level of p-STAT3 protein was 0.15±0.04 in Siha cells treated with berberine at half inhibitory concentration,and the expression of p-STAT3 protein was 0.82±0.06 in Siha cells not treated with berberine.The difference was statistically significant(P<0.05).Berberine may affect cervical cancer cell growth and apoptosis by decreasing STAT3 phosphorylation.ConclusionsThe expression of STAT3 protein in Siha cells after half-inhibitory concentration of berberine did not change significantly,but the level of p-STAT3 decreased.Berberine may affect cervical cancer cell growth and apoptosis by decreasing STAT3 phosphorylation levels.Part IV Effects of berberine on radiosensitivity of cervical cancer cells MethodsSiha cells were cultured in berberine medium containing half inhibitory concentration and irradiated with 0,2,4,6,and 8 Gy X-rays.The cell clone formation assay was used to detect radiosensitivity.ResultsThe survival fraction of Siha cells after half-inhibitory concentration of berberine was reduced,and berberine can increase the radiotherapy sensitivity of cervical cancer with a sensitization ratio of 1.550.ConclusionsBerberine can inhibit the proliferation of cervical cancer cells,promote apoptosis,block cell cycle,increase the radiosensitivity of cervical cancer cells.