| Research background and objectiveWith the continuous development of social and economic condition,breast cancer incidence increased year by year,it has become one of the main diseases endangering women's health.According to the survey of relevant studies,the total number of female diagnosed with breast cancer accounts for nearly 30%of all cancer cases every year.Triple negative breast cancer lacks expression of the estrogen receptor(ER),progesterone receptor(PR),and epidermal growth factor receptor-2.Because of these missing receptors,it has characteristics of the most aggressive,early recurrence time,high distant metastasis rate,rapid disease progression,limited treatment options,poor prognoses,relatively low disease free survival rate and overall survival rate,and short total survival time.Therefore,more and more attention was paid by domestic and foreign scholars on studying how to increase the therapeutic response rate,and to explore the treatment options of using low toxic and high effective traditional Chinese medicine extract or vitamin supplements on this subtype of breast cancer.This study first observed the effect of the above methods on the survival time and tumor size of the population of the triple negative breast cancers who had previously used curcumin supplements and vitamin C injections by retrospective analysis.Then,from the cell level,to observe the use of curcumin alone,the use of vitamin C alone,and the combination group on the human triple negative breast cancer MDA-MB-468 cells,whether it can inhibit the cell growth.Finally,we study the mechanism.Research methodStudy 1: Retrospective study diagnosis with triple negative breast cancer patients in the previous 8 years in our hospital,to observe whether chemotherapy in combination with curcumin supplements and vitamin C injection can have any effects on tumor size and survival time.Study 2: Human MDA-MB-468 cell line was selected as the research object.The cells were treated with curcumin,VitC and the combination of curcumin+VitC by using CCK8 method,flow technique and transwell chamber invasion assay.To observe the effect of each group on cell proliferation,cell cycle,apoptosis and cell invasiveness.Study 3: To study the mechanism of different drug groups on the growth inhibition of these cells,the effects of different groups on EGFR / Akt signaling pathway were observed at two time points(24 hours and 48 hours).The expressions of EGFR,Akt,p-Akt,P53,Bax,Bcl-2,caspase-9,caspase-3,P27,MMP-2,MMP-9,and VEGF were analyzed by PCR and Western blot.Research resultsStudy 1: All patients were evaluated for efficacy.There were 2 cases of complete remission(CR),15 cases of partial remission(PR)and 13 cases of stable disease(SD).The effective rate was 49% and the clinical benefit rate was 86%.In the control group,there were no CR cases,PR14 cases,SD14 cases,the response rate(RR)of treatment was 40% and the clinical benefit rate(CBR)was 80%.Compared with the treatment group,the RR and CBR were not significantly different(P> 0.05).The median progression free survival time was 7 months(1.5 to 28.5 months)and the median overall survival time was27 months(4 to 40 months)in treatment group.The median disease free survival time was 4.5 months(1.5 to 8 months)and the median overall survival time was 18 months(3 to 26 months).The disease free progression survival time and the overall survival time of the treatment group were significantly longer than those of the control group(P <0.05).Two groups of patients receiving gemcitabine combined with cisplatin chemotherapy regimens,experienced in adverse reactions in the digestive tract response and myelosuppression.The incidence of adverse reactions was significantly lower in the treatment group compared with the control group(P <0.05).Study 2: 1.This study demonstrated that curcumin,VitC,and the combination of curcumin and vitamin C can inhibit the proliferation and invasion of human triple negative breast cancer cell line MDA-MB-468,and can inhibit the expression of EGFR,MMP-2,MMP-9 and VEGF.The results of this study showed that curcumin,vitamin C and the combination of curcumin and vitamin C can inhibit the proliferation of human triple negative breast cancer cell line MDA-MB-468.Curcumin group,vitamin C group,and the combination group showed an increasing inhibitory effect on MDA-MB-468 cells with an increaseof the concentration.The inhibitory effect of combination 50+20 was significantly stronger than other groups(P<0.05).2.In the combination group50+20,MDA-MB-468 cells had a large distribution in the G1 phase,and the proportion of S phase was decreased,which was the same as curcumin group.All three groups presented the effect of inhibiting the cell invasion ability.To compare the effect of three groups on inhibiting the cell invasion ability in MDA-MB-468: we found the following orders: the combination group >curcumin group >vitamin C group.With the increase of drug concentration,the number of cells through the membrane were less.Study 3: Each group through the regulation of EGFR / Akt signaling pathway,to inhibit the proliferation of MDA-MB-468 cells.1.The expression of EGFR in each group were significantly inhibited at24 hours and 48 hours(P<0.05).The expression of EGFR was statistical significantly lower in the combination group 50+20 at 48 hours' time point in comparison with other groups.(P<0.05).2.Compared with the control group,the expression of P-Akt and Bcl-2 was significantly inhibited at 24 hours and 48 hours in all the three groups(P<0.05),the difference was statistically significant,which was consistent with the effect of Ly294002 drug.Compared with the control group,the expression of P53,Bax,Caspase-3,Caspas-9 and P27 was significantly increased in all three groups in 24 hours and 48 hours(P <0.05).The difference was statistically significant and consistent with the effect of Ly294002.The effect of 50 + 20 on MDA-MB-468 cells was significantly higher than other drugs(P <0.05).3.The effect of three groups on expression of MMP-2,MMP-9 in MDA-MB-468cell: each group can significantly inhibit the expression of MMP-2 at 24 h and48h.The combination group had the strongest inhibitory effect on the expression of MMP-2 at 48 hours.The order of inhibition effect of three intervention groups on MMP-2 expression at 48 h was as the followings: the combination group >Vitamin C group and curcumin group.The combination group can inhibit the expression of MMP-9,while other groups did not have similar effect.4.The expression of VEGF in each group was significantly inhibited at24 h and 48 hours,the difference was statistically significant(Figure 3-5-3and figure 3-5-4,P<0.05).To compare the inhibition of three groups on the expression of VEGF,the inhibition effect was in the following order: the combination group>vitamin C group and curcumin group.ConclusionRetrospective studies showed that there was no significant difference in the efficacy of treatment group compared with the control group;however,the functional status score of the treatment group was significantly higher after the treatment than before the treatment,and significantly higher than that of the control group(P <0.05).Compared with the control group,the treatment group significantly prolonged progression-free survival time and total survival time(P <0.05).Moreover,the incidence of adverse reactions in the treatment group was significantly lower than that in the control group(P<0.05).The results of this study suggest that curcumin supplements combined with vitamin C intravenous infusion therapy,used during chemotherapy,can help reduce the adverse effects of chemotherapy drugs,but also help to extend the late stage patients prolong disease free progression survival time and overall survival time.This study confirmed that curcumin,VitC and combination therapy groups can inhibit the proliferation of human triple negative breast cancer MDA-MB-468 cells.Compared with the control group,the three groups of drugs had inhibitory effect on MDA-MB-468 cells.With the increase of drug concentration in each group,the inhibitory effect was stronger in combination group than that in other groups.(P <0.05).MDA-MB-468 cells were more frequently distributed in the G1 phase and decreased in the S phase.Compared with the control group,the three groups have the ability to inhibit cell invasion in vitro.(P <0.05).With the increase of drug concentration,the number of cells passing through the basement membrane was lower(P <0.05).From the point of time,the role of each group in the 48 h was stronger than in 24 h.The inhibitory effect of curcumin,VitC and combination therapy on the growth of MDA-MB-468 cells was confirmed by regulating the signal transduction pathway of EGFR / PI3 K / Akt cells.Each group can regulate cell apoptosis,cell cycle,invasion and metastasis,and neovascularization by regulating the level of individual genes and downstream target proteins in the signaling pathway.Specific mechanisms of action are as the followings: by inhibitingthe expression of EGFR,decreasing the level of p-Akt,and then upregulatingp53 gene expression,inhibiting Bcl-2 levels.p-Akt was inhibited,the expression of Bax was directly activated,the ratio of Bcl-2 to Bax was decreased,the expression of caspase-9 and caspase-3 was up-regulated,cell death was initiated.The expression of MMP-2,MMP-9,VEGF were inhibited,thus new blood vessel cannot provide blood supply to the tumor.(Fig 4-7)... |
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