The Influence Of Testosterone Propionate On Triple-negative MDA-MB-231 Breast Cancer Cell Line

Objective:In recent years,breast cancer has become a threat to health and life of woman as “a public enemy”.While the survival rate of breast cancer has been greatly improved through systematacially and normally comprehensive treatments,there is still certain carcinoma such as triple-negative breast cancer whose survival benefit is not significant.The definition of the triple-negative breast cancer is the expression of ER,PRand HER-2 are all negative.Because of lack of effective targeted drugs,there is few treatment plan for the triple-negative breast cancer.Besides,recurrence rates are high.Estrogen and progesterone and their receptors are vital in terms of the development and progression even the treatment to breast cancer. Anti-estrogen therapy has always been main endocrine therapy.While the research that androgen and androgen receptor treat breast cancer is rarely reported.Androgen is the major reason for the occurrence and development of prostate cancer.The study found that dihydrotestosterone and testosterone could combine with androgen receptor to induce gene transcription and cell proliferation,resulting in prostate cancer.Therefore resistance to androgen receptor has become main treatment of prostate cancer.Moreover,researches about the effect of androgen on breast cancer were launched long ago.In 1958,report declared that testosterone propionate,administered intramuscularly in doses of 150 to 300 mg.weekly,was one of methods in the clinical treatment of breast cancer.The research on effect of DHT on the breast cancer cell line ZR-75-1 also found that androgen inhibited proliferation of luminal breast cancer cells. However,study on effect of androgen on breast cancers is still at exploration stage. The purpose of this study is to observe the proliferation and apoptosis and related protein level of triple-negative MDA-MB-231 breast cancer cell lines which different concentrations of testosterone propionate act on after different time periods.Futher more,we also explore the effect of testosterone propionate on the proliferation and inhibition of the triple-negative MDA-MB-231 breast cancer cell lines and possible mechanism.Methods: We cultivated breast cancer cells MDA-MB-231 and analyzed the expression of the ER,PR,AR,HER-2 in cell lines by using immunocytochemical stain.Cultivated breast cancer cells were inoculated on 96-well plates and divided into several groups and a control group.The concentrations in 10-4mmol/L to 10-12mmol/L of testosterone propionate were added respectively to different groups which had been trained for 24-72 h.Then we detected the inhibition rates by MTT.At the same time,we selected four concentrations whose effects were obvious and detected the expressions of Cyclin D1,m TOR and PTEN protein by flow cytometry.As a result,we could further explore the mechanism of effect of testosterone propionate on the breast cancer cell lines in protein expression level. SPSS16.0 statistical software was used.Results: 1 Slides made through immunocytochemical stain were observed by using inverted microscope.It showed the expressions of the ER,PR and HER-2 in breast cancer cell line MDA-MB-231 were all negative and the expression of AR was positive. 2 The effect of different concentrations of testosterone propionate on breast cancer cell MDA-MB-231 after different time.2.1 The proliferation of breast cancer cell MDA-MB-231 with the effect of different concentrations of testosterone propionate detected by MTT after same time:the effect of testosterone propionate with concentrations in 10-5mmol/L,10-7mmol/L,10-9mmol/L,10-11mmol/L on breast cancer cell line MDA-MB-231 after 24 hours was proliferation.And the rates of proliferation were respectively 1.86%±1.13%,4.70%±0.59%,7.14%±0.07%,9.26%±1.10%. The higher concentration was,the weaker proliferation was.The effect after 48 hours was inhibition.And the rates of inhibition were respectively 9.73%±2.04%,4.47%±1.17%,1.92%±0.23%,0.35%±0.16%.The higher conce-ntration was,the more obvious inhibition was. The effect after 72 hours was also inhibition.And the rates of inhibition were respectively 17.69% ±0.74%,15.41%±0.10%,6.38%±0.23%,1.57%±0.78%.The higher concentratio-n was,the more obvious inhibition was. The statistical analysis showed that there were differences among the proliferation rates and inhibition rates of breast cancer cell MDA-MB-231 with the effect of different concentrations of testosterone propionate after same time.The comparison among groups were of statistical significance.(P<0.05) 2.2 The proliferation of breast cancer cell MDA-MB-231 with the effect of same concentrations of testosterone propionate detected by MTT after different time:the inhibition rates of testosterone propionate with concentration in 10-5mmol/L on breast cancer cell line MDA-MB-231 after 24 h,48h,72 h were respectively-1.86%±1.13%,9.73%±2.04%,17.69%±0.74%. The inhibition rates of 10-7mmol/L were respectively-4.70%±0.59%,4.47% ±1.17%,15.41%±0.10%.The inhibition rates of 10-9mmol/L were respective- ly-7.14%±0.07%,1.92%±0.23%,6.38%±0.23%.The inhibition rates of 10-11mmol/L were respectively-9.26%±1.10%,0.35%±0.16%,1.57%±0.78%. The longer time of the effect continued the more obvious inhibitory effect was.The statistical analysis showed that there were differences among the proliferation rates and inhibition rates of breast cancer cell MDA-MB-231 with the effect of same concentrations of testosterone propionate after different time.The comparison among concentrations in groups were of statistical significance.(P < 0.05). 3 The expression of Cyclin D1,m TOR and PTEN protein in breast cancer cell line MDA-MB-231 with the effect of different concentrations(10-5mmol/L,10-7mmol/L,10-9mmol/L,10-11mmol/L) of testosterone propionate after different time.Detection by flow cytometry:The expression percentages of Cyclin D1 protein in breast cancer cell line MDA-MB-231 control groups cultivated after 24,48,72 h were respectively 99.82% ± 0.02%,99.90% ± 0.04%,99.91% ±0.04%.The expression percentages of m TOR protein were respectively 99.63% ± 0.03%,99.97% ± 0.02%,99.77% ± 0.06%.The expression percentages of PTEN protein were respectively98.90%±0.06%,98.30%±0.31%,97.35%±0.35%.The expression percentages of Cyclin D1 protein and m TOR protein with the effect of four concentrations( 10-5mmol/L,10-7mmol/L, 10-9mmol/L,10-11mmol/L) of testosterone propionate after 24 h were respectively 99.86% ± 0.04%,99.90% ± 0.06%,99.92% ± 0.05%,99.96% ±0.03%;99.78% ± 0.10%,99.82% ± 0.11%,99.88% ± 0.08%,99.92% ±0.09%.The expression percentages of Cyclin D1 protein and m TOR protein after 48 h were respectively99.67% ± 0.06%,99.74% ± 0.10%,99.81% ±0.11%,99.85% ± 0.10%; 99.68% ± 0.16%,99.72% ± 0.15%,99.82% ±0.13%,99.93%±0.03%. The expression percentages of Cyclin D1 protein and m TOR protein after 72 h were respectively 99.42% ± 0.18%,99.66% ±0.16%,99.76% ± 0.12%,99.82% ± 0.15%; 99.41% ± 0.11%,99.49% ±0.15%,99.60% ± 0.10%,99.65% ± 0.13%. The expression percentages of PTEN protein with the effect of four concentrations(10-5mmol/L,10-7mmol/L, 10-9mmol/L,10-11mmol/L) of testosterone propionate after 24 h were respectively 98.80% ± 0.17%,98.76% ± 0.18%,98.61% ± 0.33%,96.18% ±0.20%.The expression percentages of PTEN protein after 48 h were respectively 99.59% ± 0.20%,99.46% ± 0.19%,99.42% ± 0.18%,98.86% ±0.13%.The expression percentages of PTEN protein after 72 h were respectively99.76% ± 0.11%,99.51% ± 0.14%,98.93% ± 0.17%,98.46% ±0.18%.The statistical analysis showed that there were differences among the expression of Cyclin D1、m TOR and PTEN protein in breast cancer cell line MDA-MB-231 with the effect of different concentrations of testosterone propionate after three gradients of time.The comparison of concentration among groups of protein were of statistical significance(P < 0.05).Conclusions:1 The expressions of ER,PR and HER-2 in breast cancer cell line MDA-MB-231 are all negative and the expression of AR is positive.2 The effect of different concentrations(10-5mmol/L,10-7mmol/L,10-9m- mol/L,10-11mmol/L) of testosterone propionate on breast cancer cell MDA-MB-231 after 24 h is profiferation. And the higher concentration is, the weaker proliferation is.While,the effect of those concentrations of testosterone propionate on breast cancer cell MDA-MB-231 after 48 h and 72 h is inhibition.And the higher concentration is,the more inhibition is. More than that,the longer time of the effect of same concentrations of testosterone propionate continues the more obvious inhibitory effect on breast cancer cell line MDA-MB-231 is.3 The expressions of Cyclin D1 protein and m TOR protein in breast cancer cell line MDA-MB-231 with the effect of testosterone propionate after 24 h are up regulated;The expression of PTEN protein is down regulated.The expression of PTEN protein in breast cancer cell line MDA-MB-231 with the effect of testosterone propionate afer 48 h and 72 h are up regulated.The expressions of Cyclin D1 protein and m TOR protein are down regulated.Therefore testosterone propionate probably act on breast cancer cell line MDA-MB-231 through regulating proliferin Cyclin D1,m TOR and apoptin PTEN.