Role Of Vitamin C As A Sensitizer For Paclitaxel In Breast Cancer Cells And Its Mechanism

Background and PurposeFrom the latest epidemiological survey,the incidence of breast cancer is still rising year by year.With the continuous development of economy in our country,the diet structure has also begun to approach to western developed countries.The estrogen level in our country is rising.The advancement and prolongation of menstrual period make the incidence of breast cancer and gynecologic cancer increase in our country.More and more young women are diagnosed with breast cancer.According to foreign statistics,the median incidence of breast cancer of women in China in the past three years is 10 years earlier than that of developed countries in Europe and the United States.The incidence of breast cancer in China has leapt to the first place in malignant tumors.With the continuous improvement of modern medical technology,the more accurate the early diagnosis of breast cancer,the use of different therapies,such as:radiotherapy,chemotherapy,surgery,endocrine and molecular targeted therapy make the overall survival rate of breast cancer has been significantly improved.Although the overall survival rate has improved,30%-40%of patients will have recurrence and metastasis.At present,the treatment of breast cancer is still based on the comprehensive treatment of surgery,but only surgery is unable to improve long-term survival.Radiotherapy and chemotherapy is an important aid in the treatment of breast cancer.Patients can achieve a better long-term survival rate through radiotherapy and chemotherapy.However,the side effects after radiotherapy and chemotherapy are often unacceptable to patients.Many patients are poisoned by chemotherapy Response and forced to stop treatment caused by secondary recurrence of breast cancer,long-term radiotherapy and chemotherapy will increase the resistance of breast cancer cells.Therefore,we hope to find more effective adjuvant therapy to improve the long-term survival of breast cancer,while reducing the side effects of the adjuvant therapy on the human body.Vitamin C（VitC）is a clinically common auxiliary drug,but the dose used in chemotherapy is generally relatively small,according to some foreign literature reported that high-dose intravenous injection（10g/d）is effective for cancer treatment.Paclitaxel（PTX）is one of the commonly used chemotherapy drugs in clinical breast cancer,and is an anti-cancer active ingredient extracted from the bark of Taxus chinensis.The use of PTX chemotherapy will produce common chemotherapy side effects,such as:nausea,vomiting,hair loss,dizziness,headache and so on.This is also the main reason that patients with chemotherapy are difficult to adhere to.Looking for a can not only improve the curative effect of chemotherapy but also reduce the toxic side effects of chemotherapy is imminent.Materials1.Research materialsBreast cancer MCF-7 cells,Vitamin C injection,PTX2.Research methodsPart 1:Cultured breast cancer MCF-7 cells in vitro,The inhibitory rates of VitC,PTX,VitC combined with PTX on the proliferation of breast cancer cells were detected by MTS method and the IC₅₀ values were calculated.Calculate the tumor cell proliferation inhibition rate,the use of King’s correction formula to calculate whether the synergy between the two drugs.Part 2:Logarithmic growth phase of breast cancer MCF-7 cells,after cell counting,plating,each hole about 1×10⁵ cells.Still placed in 37℃,5%CO2incubator 24 hours to be synchronized cell cycle.According to the instruction manual of Annexin V-FITC Apoptosis Detection Kit reagent kit,the reagents were added successively,and finally detected by flow cytometry.The experiment was repeated 3 times.Part 3:According to the experimental design,different drugs were added after breast cancer MCF-7 cells was synchronized cell cycle,respectively.VitC single drug,PTX single drug,PTX0.625mmol/L,respectively,plus VitC high and low Three groups,each set 3 complex hole.Total protein was extracted according to the kit instructions,and the expression of Caspase-3 and Bcl-2 was detected.Β-actin was used as an internal control.The protein band gray value was analyzed by Image J software and the experiment was repeated 3 times.ResultsPart 1:VitC,PTX and VitC combined with PTX inhibited breast cancer MCF-7cells cultured in vitro to a certain extent,and from the experimental results,they were concentration-dependent.Low doses of VitC monotherapy（<1.25 mmol/L）had almost no effect on MCF-7 cells.High-dose VitC monotherapy（>1.25 mmol/L）had a concentration-dependent inhibitory effect on proliferation of MCF-7 cells.Proliferation of MCF-7 was inhibited by PTX monotherapy in a concentration-dependent manner.Proliferation inhibition rates of 1mmol/LVitC in combination with 0.00475μmol/L,0.00237μmol/L and 0.00118μmol/L were 82%,67%and50%.Part 2:Normal logarithmic phase of breast cancer MCF-7 cells and epithelial cells,epithelial-like growth under the microscope,cell pleomorphism,clear outline,the number of apoptotic and apoptotic cells are less.After the single dose of VitC,PTX monotherapy,different concentrations of VitC combined with PTX,the apoptotic rate of each group was detected by flow cytometry.One-way ANOVA was used among the groups.SNK-q test was used for any pairwise comparison.There was no significant difference in apoptotic rate between 0.5mmol/L VitC group and control group（P>0.05）.Compared with the control group,PTX group and VitC combined with PTX group showed significant difference in apoptosis rate（P<0.05）.Compared with PTX group,there was a significant difference in apoptosis rate between VitC group and PTX group（P<0.05）.Part 3:The expressions of Caspase-3 and Bcl-2 were detected.Breast cancer MCF-7 cells were treated with PTX alone and with different concentration of VitC.With the increase of VitC concentration,the expression of Bcl-2 protein decreased gradually and the expression of Caspase-3 protein increased gradually.Compared with PTX group and VitC 0.5mmol/L group,PTX group and VitC 1mmol/L group,PTX group and VitC 2mmol/L group,there was significant difference in the relative expression of protein between the PTX group and the blank control group（P<0.05）.Conclusions1.VitC,PTX and VitC combined with PTX all inhibited the proliferation of breast cancer cells,whereas the concentration of VitC was more than 1.25 mmol/L.2.VitC,PTX and VitC combined with PTX all can promote the apoptosis of breast cancer MCF-7cells.3.VitC can sensitize PTX,so that the expression of Bcl-2 is gradually decreased and the expression of apoptosis-promoting protein Caspase-3 is gradually increased.