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目的探讨生理浓度(10-9mol/L)褪黑素和药理浓度(10-5mol/L)褪黑素对阿霉素抑制雌激素受体阴性乳腺癌细胞MDA-MB-231作用影响及其机制。方法 (1)应用四甲基偶氮唑蓝(MTT)法检测经不同浓度褪黑素孵育后阿霉素对MDA-MB-231的抑制率和IC50变化。(2)应用流式细胞学方法观察不同浓度褪黑素、阿霉素以及两药联用时对MDA-MB-231细胞周期分布和凋亡的影响。(3)应用western blot法检测不同浓度褪黑素、阿霉素单药和两药联用对MDA-MB-231细胞p53和bcl-2蛋白表达的影响。结果 (1)阿霉素对乳腺癌MDA-MB-231细胞具有明显的抑制作用,且呈剂量时间依赖性。IC50值为(1.00±0.09)μg/ml,经生理浓度和药理浓度褪黑素孵育后IC50分别降为(0.85±0.06)μg/ml和(0.47±0.03)μg/ml,前者与孵育前相比未,差异无统计学意义(P>0.05),后者相比差异无统计学意义(P<0.01)。(2)流式细胞学检测结果显示,阿霉素对细胞MDA-MB-231有凋亡促进作用,随浓度增高凋亡率未见增加(P>0.05)。生理浓度褪黑素联合阿霉素与相应浓度的阿霉素单药相比,凋亡率未见明显增加(P>0.05)。药理浓度褪黑素联合阿霉素与相应浓度阿霉素单药比较,凋亡率明显提高(P<0.05),但联合组随着阿霉素浓度增加凋亡率并未见明显变化(P>0.05)。(3)MDA-MB-231乳腺癌细胞株中呈p53蛋白低表达、bcl-2蛋白高表达。药理浓度褪黑素可显著增高细胞p53蛋白并降低bcl-2蛋白表达(P<0.01)。药理浓度褪黑素联合不同浓度阿霉素对两蛋白表达未见显著性差异(P>0.05);阿霉素对两种蛋白表达未见明显影响(P>0.05)。结论 (1)生理浓度褪黑素(10-9mol/L)对阿霉素的抗雌激素受体阴性乳腺癌细胞作用未见明显影响,药理浓度(10-5mol/L)以上褪黑素表现出对阿霉素明显的增敏作用。(2)阿霉素较低浓度时,凋亡促进作用可能是褪黑素对其增敏机制的一部分,随着阿霉素浓度的提高,褪黑素的细胞毒增敏机制可能占主要地位。(3)药理浓度褪黑素能提高雌激素受体阴性乳腺癌细胞p53蛋白表达并降低bcl-2蛋白表达,并具有剂量依赖性。涉及p53和bcl-2的凋亡通路可能是褪黑素对阿霉素增敏机制的一部分。
Objective To investigate the effect of melatonin (10-9mol / L) and melatonin (10-5mol / L) on the inhibitory effect of adriamycin on estrogen receptor-negative breast cancer cell line MDA-MB-231 . Methods (1) The inhibitory rate and IC50 of adriamycin on MDA-MB-231 after incubation with different concentrations of melatonin were detected by MTT assay. (2) Flow cytometry was used to observe the effects of different concentrations of melatonin, adriamycin and their combination on the cell cycle distribution and apoptosis of MDA-MB-231 cells. (3) The effects of different concentrations of melatonin, doxorubicin and two drugs on p53 and bcl-2 protein expression in MDA-MB-231 cells were detected by western blot. Results (1) Adriamycin significantly inhibited breast cancer MDA-MB-231 cells in a time-and dose-dependent manner. The IC50 values were (1.00 ± 0.09) μg / ml, IC50 decreased to (0.85 ± 0.06) μg / ml and (0.47 ± 0.03) μg / ml respectively after incubation with physiological and pharmacological concentrations of melatonin. The difference was not statistically significant (P> 0.05), but there was no significant difference between the latter (P <0.01). (2) The results of flow cytometry showed that doxorubicin could promote the apoptosis of MDA-MB-231 cells, and the apoptosis rate of MDA-MB-231 cells did not increase with increasing concentration (P> 0.05). Physiological concentrations of melatonin combined with doxorubicin and the corresponding concentration of doxorubicin monotherapy, apoptosis rate was not significantly increased (P> 0.05). Pharmacological concentration of melatonin combined with doxorubicin and the corresponding concentration of doxorubicin monotherapy, the apoptosis rate was significantly increased (P <0.05), but the combination group with the doxorubicin concentration increased apoptosis rate did not change significantly (P > 0.05). (3) The expression of p53 protein and bcl-2 protein were high in MDA-MB-231 breast cancer cell lines. Pharmacological concentration of melatonin can significantly increase cell p53 protein and reduce bcl-2 protein expression (P <0.01). Pharmacological concentration of melatonin combined with different concentrations of doxorubicin on the expression of the two proteins showed no significant difference (P> 0.05); doxorubicin on the two protein expression was not significantly affected (P> 0.05). Conclusions (1) The physiological concentration of melatonin (10-9mol / L) has no obvious effect on the effect of doxorubicin on the anti-estrogen receptor negative breast cancer cells. The pharmacological concentration (10-5mol / L) The obvious sensitization of doxorubicin. (2) At the lower concentration of doxorubicin, the promotion of apoptosis may be part of the sensitization mechanism of melatonin. With the increase of doxorubicin concentration, the mechanism of cytotoxic sensitization of melatonin may be dominant . (3) Pharmacological concentration of melatonin can increase p53 protein expression and reduce bcl-2 protein expression in estrogen receptor-negative breast cancer cells in a dose-dependent manner. Apoptotic pathways involving p53 and bcl-2 may be part of the mechanism of sensitization of doxorubicin to doxorubicin.