不同pH值条件下多柔比星对胃癌BGC823细胞增殖、凋亡的影响及机制研究
|
摘要
目的探讨在不同pH值培养条件下多柔比星(doxorubicin,ADM)对胃癌BGC823细胞增殖、凋亡能力的影响及初步机制研究。方法采用细胞活性测定试剂盒(CCK-8)法检测不同pH值培养条件下多柔比星对胃癌细胞BGC823增殖活性的影响;应用Annexin V/PI双染法,在荧光显微镜下观察凋亡细胞的形态,用流式细胞仪检测胃癌细胞凋亡率;采用qRT-PCR法检测p-AKT、BCL-2和NF-κB基因在胃癌细胞中的表达。结果①ADM对胃癌BGC823细胞的半数抑制浓度(IC_(50))值为5.277μg/mL,考虑化疗药物毒性及后续实验,ADM剂量选择该细胞株IC_(50)值1/4的浓度(即1.319μg/mL)用于进行后续实验;②随着pH值升高,ADM对BGC823细胞增殖活性的抑制作用增强,当pH达到7.4时,抑制增殖作用最明显;③随着pH值升高,ADM对BGC823细胞凋亡水平明显增加,当pH达到7.4时,促凋亡作用最明显,再提高pH浓度,促凋亡作用能力不明显;④不同pH(pH 6.8～7.6)环境下,ADM处理后,随着pH升高,BCL-2、NF-κB和p-AKT基因表达水平明显降低。结论升高细胞外pH值能明显提高ADM对BGC823细胞抑制细胞增殖、促进细胞凋亡的能力,这种与pH值相关的ADM抗肿瘤效果可能是通过抑制AKT的磷酸化,阻止NF-κB、Bcl-2等抗凋亡基因表达来实现的。
Objective To investigate the effects of doxorubicin on the proliferation and apoptosis of gastric cancer BGC823 cells under different pH conditions and the preliminary mechanism. Methods The effect of doxorubicin(ADM) cultured under different pH conditions on the proliferation of gastric cancer cell line BGC823 was detected by cell viability assay kit(CCK-8); Morphology of apoptotic cells were observed under fluorescence microscope; The apoptosis of BGC823 was assessed with Annexin V-PI; The expression of p-AKT, BCL-2 and NF-κB genes and proteins in gastric cancer cells were detected by qRT-PCR. Results ①The half-inhibitory concentration(IC_(50)) of ADM on gastric cancer BGC823 cells was 5.277 μg/mL. Considering the toxicity of chemotherapy drugs and subsequent experiments, the ADM dose was selected to be 1/4 times the IC_(50) value of the cell line(1.319 μg/mL) for subsequent experiments; ②With the increase of pH, the inhibitory effect of ADM on the proliferation of BGC823 cells was enhanced. When the pH reached 7.4, the inhibition of proliferation was the most obvious. ③With the increase of pH, the apoptosis of BGC823 cells increased significantly with ADM. When the pH reached 7.4, the pro-apoptotic effect was the most obvious, and the pH concentration was further increased, and the ability to promote apoptosis was not obvious; ④Under different pH(pH 6.8-7.6) environment, the expression levels of BCL-2, NF-κB and p-AKT genes decreased significantly with the increase of pH after ADM treatment. Conclusion Increasing the extracellular pH can significantly increase the ability of ADM to inhibit cell proliferation and promote apoptosis in BGC823 cells. The anti-tumor effect of this pH-related ADM may be achieved by inhibiting the phosphorylation of AKT and preventing the expression of anti-apoptotic proteins such as NF-κB and Bcl-2.
Objective To investigate the effects of doxorubicin on the proliferation and apoptosis of gastric cancer BGC823 cells under different pH conditions and the preliminary mechanism. Methods The effect of doxorubicin(ADM) cultured under different pH conditions on the proliferation of gastric cancer cell line BGC823 was detected by cell viability assay kit(CCK-8); Morphology of apoptotic cells were observed under fluorescence microscope; The apoptosis of BGC823 was assessed with Annexin V-PI; The expression of p-AKT, BCL-2 and NF-κB genes and proteins in gastric cancer cells were detected by qRT-PCR. Results ①The half-inhibitory concentration(IC_(50)) of ADM on gastric cancer BGC823 cells was 5.277 μg/mL. Considering the toxicity of chemotherapy drugs and subsequent experiments, the ADM dose was selected to be 1/4 times the IC_(50) value of the cell line(1.319 μg/mL) for subsequent experiments; ②With the increase of pH, the inhibitory effect of ADM on the proliferation of BGC823 cells was enhanced. When the pH reached 7.4, the inhibition of proliferation was the most obvious. ③With the increase of pH, the apoptosis of BGC823 cells increased significantly with ADM. When the pH reached 7.4, the pro-apoptotic effect was the most obvious, and the pH concentration was further increased, and the ability to promote apoptosis was not obvious; ④Under different pH(pH 6.8-7.6) environment, the expression levels of BCL-2, NF-κB and p-AKT genes decreased significantly with the increase of pH after ADM treatment. Conclusion Increasing the extracellular pH can significantly increase the ability of ADM to inhibit cell proliferation and promote apoptosis in BGC823 cells. The anti-tumor effect of this pH-related ADM may be achieved by inhibiting the phosphorylation of AKT and preventing the expression of anti-apoptotic proteins such as NF-κB and Bcl-2.
引文
[1] CHEN W,ZHENG R,BAADE P D,et al.Cancer statistics in China,2015[J].CA Cancer J Clin,2016,66(2):115-132.
[2] WANG M,ZHAO J,ZHANG L,et al.Role of tumor microenvironment in tumorigenesis[J].J Cancer,2017,8(5):761-773.
[3] GOSWAMI K K,GHOSH T,GHOSH S,et al.Tumor promoting role of anti-tumor macrophages in tumor microenvironment[J].Cell Immunol,2017,316(2):1-10.
[4] AMITH S R,FONG S,BAKSH S,et al.Na+/H+ exchange in the tumour microenvironment:does NHE1 drive breast cancer carcinogenesis?[J].Int J Dev Biol,2015,59(7-9):367-377.
[5] KHAWAR I A,KIM J H,KUH H J.Improving drug delivery to solid tumors:Priming the tumor microenvironment[J].J Control Release,2015,201(3):78-89.
[6] LORZ A,LORENZI T,CLAIRAMBAULT J,et al.Modeling the effects of space structure and combination therapies on phenotypic heterogeneity and drug resistance in solid tumors[J].Bull Math Biol,2015,77(1):1-22.
[7] FELIPE A V,OLIVEIRA J,MORAES A A,et al.Reversal of multidrug resistance in an epirubicin-resistant gastric cancer cell subline[J].Asian Pac J Cancer Prev,2018,19(5):1237-1242.
[8] FENG C,RUI M,SHEN H,et al.Tumor-specific delivery of doxorubicin through conjugation of pH-responsive peptide for overcoming drug resistance in cancer[J].Int J Pharm,2017,528(1-2):322-333.
[9] 范平生,朱德发,陈飞虎,等.pH值对LAK细胞增殖的影响及与rIL-2联合抗恶性肿瘤的研究[J].微循环学杂志,2002,12(2):1-3,57.
[10] 范平生,朱德发,胡冰,等.[H+]对LAK细胞、rIL-2、IFN-α2b或联合ADM抗恶性肿瘤的影响[J].免疫学杂志,2003,19(3):21-24.
[11] FAN P,WANG S,XIU L,et al.Basic and clinical study of increased effect of partial anti-tumor agents by infusing sodium bicarbonate through target artery[J].CG J Clin,2007,6(1):13-17.
[12] 范平生,谢钊,吴志丽,等.经靶动脉灌注碳酸氢钠提高部分抗肿瘤药物疗效[J].临床肿瘤学杂志,2005,10(1):52-54.
[13] 段巧虹,张腾跃,黄海良,等.曲妥珠单抗对不同pH培养HER2阳性乳腺癌细胞增殖的抑制及促凋亡作用[J].微循环学杂志,2013,23(4):1-3.
[14] 王涛,郑永光,范康武,等.胃癌与肿瘤微环境的研究进展[J].实用肿瘤学杂志,2014,28(6):561-564.
[15] PEPPICELLI S,ANDREUCCI E,RUZZOLINI J,et al.The acidic microenvironment as a possible niche of dormant tumor cells[J].Cell Mol Life Sci,2017,74(15):2761-2771.
[16] TAYLOR S,SPUGNINI E P,ASSARAF Y G,et al.Microenvironment acidity as a major determinant of tumor chemoresistance:Proton pump inhibitors (PPIs) as a novel therapeutic approach[J].Drug Resist Updat,2015,72(23):69-78.
[17] ALFAROUR K O.Tumor metabolism,cancer cell transporters,and microenvironmental resistance[J].J Enzyme Inhib Med Chem,2016,31(6):859-866.
[18] 杨梦迪,王智煜,赵晖.肿瘤相关成纤维细胞对肿瘤微环境及肿瘤免疫代谢影响的研究进展[J].癌症进展,2018,16(3):268-271.
[19] CHEN F,ZHUANG X,LIN L,et al.New horizons in tumor microenvironment biology:challenges and opportunities[J].BMC Med,2015,13(1):45.
[20] 崔立文,徐金升,白亚玲,等.细胞外pH值及其波动对高磷诱导的大鼠血管平滑肌细胞凋亡的作用及机制研究[J].中国全科医学,2015,18(33):4090-4095.
[21] PHATAK N R,STANKOWSKA D L,KRISHNAMOORTHY R R.Bcl-2,Bcl-xL,and p-AKT are involved in neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma[J].Mol Vis,2016,22:1048-1061.
[22] YOKOYAMA T,KOHN E C,BRILL E,et al.Apoptosis is augmented in high-grade serous ovarian cancer by the combined inhibition of Bcl-2/Bcl-xL and PARP[J].Int J Oncol,2017,12(50):1064-1074.
[23] 陈浩洋,郑永绵,刘明辉.Gab2基因在胃癌中的表达及对胃癌细胞增殖、凋亡、迁移及AKT、p-AKT、Bax、Bcl-2表达的影响研究[J].癌症进展,2017,15(3):262-266.
[24] 劳海黎,李霞,李延海,等.PI3K/Akt信号通路抑制剂对人胃癌细胞增殖及相关基因表达的影响[J].国际医药卫生导报,2016,22(14):2030-2033.
[25] 斯庆图娜拉,刘磊.抑制自噬增加PI3K/AKT/mTOR信号通路抑制剂引起的胃癌细胞死亡[J].中国实验诊断学,2015,19(9):1457-1460.
该文章发表作者为新乡医学院临床医学专业型在读硕士,中国科学技术大学附属第一医院西区(安徽省肿瘤医院)为新乡医学院临床医学培养单位,导师范平生即通信作者为中国科学技术大学附属第一医院西区(安徽省肿瘤医院)肿瘤内二科主任,拥有该单位项目基金。
[2] WANG M,ZHAO J,ZHANG L,et al.Role of tumor microenvironment in tumorigenesis[J].J Cancer,2017,8(5):761-773.
[3] GOSWAMI K K,GHOSH T,GHOSH S,et al.Tumor promoting role of anti-tumor macrophages in tumor microenvironment[J].Cell Immunol,2017,316(2):1-10.
[4] AMITH S R,FONG S,BAKSH S,et al.Na+/H+ exchange in the tumour microenvironment:does NHE1 drive breast cancer carcinogenesis?[J].Int J Dev Biol,2015,59(7-9):367-377.
[5] KHAWAR I A,KIM J H,KUH H J.Improving drug delivery to solid tumors:Priming the tumor microenvironment[J].J Control Release,2015,201(3):78-89.
[6] LORZ A,LORENZI T,CLAIRAMBAULT J,et al.Modeling the effects of space structure and combination therapies on phenotypic heterogeneity and drug resistance in solid tumors[J].Bull Math Biol,2015,77(1):1-22.
[7] FELIPE A V,OLIVEIRA J,MORAES A A,et al.Reversal of multidrug resistance in an epirubicin-resistant gastric cancer cell subline[J].Asian Pac J Cancer Prev,2018,19(5):1237-1242.
[8] FENG C,RUI M,SHEN H,et al.Tumor-specific delivery of doxorubicin through conjugation of pH-responsive peptide for overcoming drug resistance in cancer[J].Int J Pharm,2017,528(1-2):322-333.
[9] 范平生,朱德发,陈飞虎,等.pH值对LAK细胞增殖的影响及与rIL-2联合抗恶性肿瘤的研究[J].微循环学杂志,2002,12(2):1-3,57.
[10] 范平生,朱德发,胡冰,等.[H+]对LAK细胞、rIL-2、IFN-α2b或联合ADM抗恶性肿瘤的影响[J].免疫学杂志,2003,19(3):21-24.
[11] FAN P,WANG S,XIU L,et al.Basic and clinical study of increased effect of partial anti-tumor agents by infusing sodium bicarbonate through target artery[J].CG J Clin,2007,6(1):13-17.
[12] 范平生,谢钊,吴志丽,等.经靶动脉灌注碳酸氢钠提高部分抗肿瘤药物疗效[J].临床肿瘤学杂志,2005,10(1):52-54.
[13] 段巧虹,张腾跃,黄海良,等.曲妥珠单抗对不同pH培养HER2阳性乳腺癌细胞增殖的抑制及促凋亡作用[J].微循环学杂志,2013,23(4):1-3.
[14] 王涛,郑永光,范康武,等.胃癌与肿瘤微环境的研究进展[J].实用肿瘤学杂志,2014,28(6):561-564.
[15] PEPPICELLI S,ANDREUCCI E,RUZZOLINI J,et al.The acidic microenvironment as a possible niche of dormant tumor cells[J].Cell Mol Life Sci,2017,74(15):2761-2771.
[16] TAYLOR S,SPUGNINI E P,ASSARAF Y G,et al.Microenvironment acidity as a major determinant of tumor chemoresistance:Proton pump inhibitors (PPIs) as a novel therapeutic approach[J].Drug Resist Updat,2015,72(23):69-78.
[17] ALFAROUR K O.Tumor metabolism,cancer cell transporters,and microenvironmental resistance[J].J Enzyme Inhib Med Chem,2016,31(6):859-866.
[18] 杨梦迪,王智煜,赵晖.肿瘤相关成纤维细胞对肿瘤微环境及肿瘤免疫代谢影响的研究进展[J].癌症进展,2018,16(3):268-271.
[19] CHEN F,ZHUANG X,LIN L,et al.New horizons in tumor microenvironment biology:challenges and opportunities[J].BMC Med,2015,13(1):45.
[20] 崔立文,徐金升,白亚玲,等.细胞外pH值及其波动对高磷诱导的大鼠血管平滑肌细胞凋亡的作用及机制研究[J].中国全科医学,2015,18(33):4090-4095.
[21] PHATAK N R,STANKOWSKA D L,KRISHNAMOORTHY R R.Bcl-2,Bcl-xL,and p-AKT are involved in neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma[J].Mol Vis,2016,22:1048-1061.
[22] YOKOYAMA T,KOHN E C,BRILL E,et al.Apoptosis is augmented in high-grade serous ovarian cancer by the combined inhibition of Bcl-2/Bcl-xL and PARP[J].Int J Oncol,2017,12(50):1064-1074.
[23] 陈浩洋,郑永绵,刘明辉.Gab2基因在胃癌中的表达及对胃癌细胞增殖、凋亡、迁移及AKT、p-AKT、Bax、Bcl-2表达的影响研究[J].癌症进展,2017,15(3):262-266.
[24] 劳海黎,李霞,李延海,等.PI3K/Akt信号通路抑制剂对人胃癌细胞增殖及相关基因表达的影响[J].国际医药卫生导报,2016,22(14):2030-2033.
[25] 斯庆图娜拉,刘磊.抑制自噬增加PI3K/AKT/mTOR信号通路抑制剂引起的胃癌细胞死亡[J].中国实验诊断学,2015,19(9):1457-1460.
该文章发表作者为新乡医学院临床医学专业型在读硕士,中国科学技术大学附属第一医院西区(安徽省肿瘤医院)为新乡医学院临床医学培养单位,导师范平生即通信作者为中国科学技术大学附属第一医院西区(安徽省肿瘤医院)肿瘤内二科主任,拥有该单位项目基金。