光动力疗法联合拉帕替尼抑制人表皮生长因子受体-2阳性乳腺癌细胞的基础研究

Effect of 5-ALA Mediated Photodynamic Therapy Combined with Lapatinib on Breast Cancer Cell:a Potential Strategy to Improve HER2-positive Breast Cancer Therapeutic Efficacy

背景光动力疗法(PDT)对增生性疾病有很好的治疗效果,其在抗肿瘤治疗中表现出高效、毒副作用小等优点。拉帕替尼(LAP)是一种口服片剂,主要作用于表皮生长因子受体-1(HER1)和表皮生长因子受体-2(HER2),属于双靶点受体酪氨酸激酶抑制剂。然而目前PDT与LAP联用的协同效应还不是很明确。目的探讨新型光敏剂5-氨基乙酰丙酸(5-ALA)用于PDT对人HER2阳性乳腺癌细胞系SKBR3的抑制情况,并分析其具体机制,同时讨论PDT联合LAP对HER2阳性乳腺癌的治疗效果,以期为HER2阳性乳腺癌的治疗提供实验依据,为相关研究提供参考。方法本研究时间为2016年5月-2017年12月。将对数生长期的HER2阳性乳腺癌细胞系SKBR3随机分为空白对照组(不做干预)、LAP组[分别加入不同浓度梯度(0.50、1.00、2.00、5.00、10.00μmol/L)的LAP]、PDT组[分别加入不同浓度梯度(0.25、0.50、1.00、2.00、4.00 mmol/L)的5-ALA,后给予激光照射]、LAP+PDT组[分别加入不同浓度梯度(0.50、1.00、2.00、5.00、10.00μmol/L)的LAP,再加入不同浓度梯度(0.25、0.50、1.00、2.00、4.00 mmol/L)的5-ALA,后给予激光照射],分别计算LAP和5-ALA的半数抑制浓度(IC50),确定最佳给药浓度及后续研究LAP组、PDT组、LAP+PDT组给药浓度,分别比较各组在最佳给药浓度下24、48 h时的细胞生长抑制率。将对数生长期的HER2阳性乳腺癌细胞系SKBR3随机分为空白对照组(不做干预)、LAP组(加入1.00μmol/L的LAP)、PDT组(加入1.00 mmol/L的5-ALA,后给予激光照射)、LAP+PDT组(加入1.00μmol/L的LAP,再加1.00 mmol/L的5-ALA,后给予激光照射),采用流式细胞术检测各组细胞凋亡情况,Rhodamine123法检测各组绿色荧光强度及线粒体膜电位,Western blotting法检测各组HER2表达水平。结果 LAP的IC50为1.65μmol/L,选取1.00μmol/L(LAP组细胞的生长抑制率为40.12%)为后续研究LAP组、LAP+PDT组给药浓度。5-ALA的IC50为1.57 mmol/L,选取1.00 mmol/L(PDT组细胞的生长抑制率为41.23%)为后续研究PDT组、LAP+PDT组给药浓度。当LAP为1.00 mmol/L,5-ALA为1.00μmol/L时,LAP+PDT组细胞生长抑瘤率为79.71%。联合治疗Q≈1.23。LAP组、PDT组、LAP+PDT组24、48 h细胞生长抑制率均大于空白对照组(P<0.05);PDT组48 h细胞生长抑制率大于LAP组(P<0.05);LAP+PDT组24、48 h细胞生长抑制率均大于LAP组、PDT组(P<0.05)。与空白对照组相比,LAP组、PDT组的细胞主要表现为凋亡,LAP+PDT组则出现了明显的凋亡和坏死,早期凋亡占比很低。LAP组、PDT组荧光强度有所降低;LAP+PDT组的荧光强度降低最明显。LAP组、PDT组、LAP+PDT组线粒体膜电位低于空白对照组(P<0.05);PDT组、LAP+PDT组线粒体膜电位低于LAP组(P<0.05);LAP+PDT组线粒体膜电位低于PDT组(P<0.05)。LAP组、PDT组、LAP+PDT组HER2表达水平均低于空白对照组(P<0.05);PDT组、LAP+PDT组HER2表达水平均低于LAP组(P<0.05);LAP+PDT组HER2表达水平均低于PDT组(P<0.05)。结论新型光敏剂5-ALA用于PDT对HER2阳性乳腺癌细胞系SKBR3具有明显的抑制作用,且PDT联合LAP的抑制作用更强,其机制与降低肿瘤细胞线粒体膜电位、HER2表达水平有一定关系。因此,PDT联合LAP有望改善HER2阳性乳腺癌患者生活质量,提高其生存率,具有良好的应用前景。

Background Photodynamic therapy(PDT)has a good therapeutic effect on proliferative diseases,and it shows high efficiency and small toxic and side effects in anti-tumor treatment.Lapatinib(LAP)is an oral tablet,which mainly acts on human epidermal growth factor receptor 1 and 2(HER1 and HER2),and belongs to double-targeted receptor tyrosine kinase inhibitors.However,the synergistic effect of PDT and LAP is still not clear.Objective To investigate the inhibitory effect and mechanism of PDT with a novel photosensitizer,5-aminolaevulinic acid(5-ALA)on HER2 positive breast cancer cells SKBR3,and to discuss the therapeutic effect of PDT with LAP on HER2-positive breast cancer,providing evidence for the treatment and research of HER2 positive breast cancer.Methods This study was conducted from May 2016 to December 2017.SKBR3 cells were randomly divided into blank control group(without intervention),LAP group[intervened with LAP of different concentration gradients(0.50,1.00,2.00,5.00,10.00 μmol/L)]and PDT group[first intervened with 5-ALA of different concentration gradients(0.25,0.50,1.00,2.00,4.00 mmol/L),then were exposed to laser],LAP+PDT group[first intervened with different concentration gradients of LAP(0.50,1.00,2.00,5.00,10.00 μmol/L),then with different concentration gradients of 5-ALA(0.25,0.50,1.00,2.00,4.00 mmol/L),and later were exposed to laser],respectively.Half maximal inhibitory concentration(IC50)of LAP and 5-ALA was calculated,respectively,and their optimal concentrations for LAP,PDT and LAP+PDT subgroups were determined.Cell growth inhibition rates in each group at 24,48 hours after intervention with the optimal concentration were estimated and compared.SKBR3 cells in the log-phase of growth were randomly divided into blank control group(without intervention),LAP group(intervened with 1.00 μmol/L LAP),PDT group(intervened with 1.00 mmol/L 5-ALA,followed by laser irradiation),LAP+PDT group(intervened with 1.00 μmol/L LAP,then 1.00 mmol/L 5-ALA,followed by laser irradiation).Flow cytometry was used to detect the apoptotic status of each group.In addition,fluorescence intensity of green fluorescent protein and mitochondrial membrane potential were detected by Rhodamine 123,and the expression of HER2 was detected by Western blotting.Results The IC50 of LAP was 1.65 μmol/L,and 1.00 μmol/L(the growth inhibition rate of LAP group was 40.12%)was chosen as the administration concentration of LAP and LAP+PDT groups.The IC50 of 5-ALA was 1.57 mmol/L.The administration concentration of 5-ALA in PDT and LAP+PDT groups was determined as 1.00 mmol/L(the growth inhibition rate of PDT group was 41.23%).When LAP was 1.00 mmol/L and 5-ALA was 1.00 μmol/L,the growth inhibition rate of LAP+PDT group was 79.71%.Combined treatment Q ≈ 1.23.The growth inhibition rates of LAP,PDT and LAP+PDT groups at 24 and 48 hours were higher than those of blank control group(P<0.05);the growth inhibition rates of PDT group at 48 hours were higher than those of LAP group(P<0.05);the growth inhibition rates of LAP+PDT group at 24 and 48 hours were higher than those of LAP group and PDT group(P<0.05).Compared with the blank control group,the cells in LAP and PDT groups mainly manifested as apoptosis,while those in LAP + PDT group showed obvious apoptosis and necrosis,and the proportion of early apoptosis was very low.The fluorescence intensity of green fluorescent protein in LAP and PDT groups decreased,and that of LAP + PDT group decreased most obviously.Mitochondrial membrane potential in LAP,PDT and LAP + PDT groups was lower than that in blank control group(P<0.05);mitochondrial membrane potential in PDT group and LAP + PDT group was lower than that in LAP group(P<0.05);mitochondrial membrane potential in LAP + PDT group was lower than that in PDT group(P<0.05).The expression of HER2 in blank control group was higher than that of other groups(P<0.05),and it in LAP group was higher than that of PDT and LAP + PDT groups,too(P<0.05),and similar status was found when compared HER2 in PDT group with that of LAP + PDT group(P<0.05).Conclusion 5-ALA had obvious inhibitory effect on SKBR3 cells,and the inhibitory effect became stronger when PDT was used in combination with LAP.The mechanism is related to the reduction of mitochondrial membrane potential and the expression of HER2 in cancer cells.Therefore,PDT combined with LAP is expected to improve the quality of life and survival rate of HER2-positive breast cancer patients,and has a good application prospect.