小檗碱对高糖环境下HCCLM3细胞凋亡与自噬的影响
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摘要
目的:探讨小檗碱(berberine,BBR)对高糖环境下人肝癌HCCLM3细胞增殖、凋亡及自噬相关基因表达的影响,并探究其可能的抗肿瘤分子机制。方法:把HCCLM3细胞加入低、中、高质量浓度葡萄糖中,通过细胞增殖实验(cell counting kit-8,CCK-8)检测发现高质量浓度葡萄糖对HCCLM3细胞增殖的作用最明显。基于此,选择高糖培养HCCLM3细胞,加入BBR(5,10,20,30,40,50μmol·L~(-1))进行体外干预24 h,研究各组药物对HCCLM3细胞增殖的抑制效果,并设顺铂组对照,通过流式细胞仪观察细胞凋亡率的变化,实时荧光定量PCR(Real-time PCR)检测B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)和自噬相关基因Atg5,Beclin1 mRNA表达水平。结果:与空白组比较,随着葡萄糖浓度的增加,HCCLM3细胞在高糖组中增殖能力最强(P <0. 05); BBR对高糖培养下HCCLM3增殖的抑制作用与BBR浓度有相关性,BBR(5,10,20,30,40,50μmol·L~(-1))组抑制率依次为33. 86%,40. 75%(P <0. 05),49. 22%,55. 1%,60. 12%,61. 42%(P <0. 01);细胞凋亡率随BBR浓度增加有显著上升(P <0. 01);与单纯高糖组比较,各干预组中凋亡基因Bcl-2与自噬基因Atg5,Beclin1 mRNA表达上调(P <0. 05,P <0. 01)。结论:BBR对高糖环境下HCCLM3细胞增殖具有抑制作用,可能是通过调控Bcl-2的表达,诱导该细胞凋亡,同时又上调Beclin1,Atg5 mRNA表达水平,促进自噬发挥抗肿瘤作用。
Objective: To explore the anti-tumor molecular mechanism of berberine( BBR) by observing and analyzing its effect on proliferation,apoptosis and autophagy-related gene expression for HCCLM3 cells under high glucose condition. Method: HCCLM3 cells were added into low,medium or high-concentration groups of glucose. It was found in cell counting kit-8( CCK-8) that the high concentration of glucose had the most obvious effect on HCCLM3 cells proliferation. Based on the above experimental result,HCCLM3 cells treated with high concentration of glucose was selected and then different concentrations of berberine( 5,10,20,30,40,50 μmol·L~(-1)) was added for in vitro intervention for 24 h. Then the effect of each drug group on the proliferation of HCCLM3 cells were studied. At the same time,the control group of metformin was arranged. After that,the changes of apoptosis rate were observed by flow cytometry,and the expression levels of B-cell lymphoma-2( Bcl-2)and autophagy genes Atg5,Beclin1 were detced by Real-time polymerase chain reaction( Real-time PCR).Result: With the increase of glucose concentration,HCCLM3 cell had the strongest migration and proliferation ability in high glucose group( P < 0. 05). The inhibitory effect of BBR on the proliferation of HCCLM3 in high glucose culture was correlated with the concentration of BBR( 5,10,20,30,40,50 μmol·L~(-1)),and the inhibition rate was 33. 86%,40. 75%( P < 0. 05),49. 22%,55. 1%,60. 12%,and 61. 42% respectively from low concentration to high concentration( P < 0. 01). The apoptosis rate was significantly increased with the increase of BBR concentration( P < 0. 01). The expression levels of Bcl-2,Atg5 and Beclin1 gene were significantly upregulated in all intervening groups as compared with those in single high glucose group( P < 0. 05,P < 0. 01).Conclusion: BBR could inhibit the proliferation of HCCLM3 cells in high glucose environment. Its inhibition effect for HCCLM3 cells might be achieved by inducing apoptosis of the cells,regulating Bcl-2 and up-regulating the expression levels of autophagy gene Beclin1 and Atg5. Thus BBR plays an anti-tumor role through promoting autophagy in high glucose environment.
Objective: To explore the anti-tumor molecular mechanism of berberine( BBR) by observing and analyzing its effect on proliferation,apoptosis and autophagy-related gene expression for HCCLM3 cells under high glucose condition. Method: HCCLM3 cells were added into low,medium or high-concentration groups of glucose. It was found in cell counting kit-8( CCK-8) that the high concentration of glucose had the most obvious effect on HCCLM3 cells proliferation. Based on the above experimental result,HCCLM3 cells treated with high concentration of glucose was selected and then different concentrations of berberine( 5,10,20,30,40,50 μmol·L~(-1)) was added for in vitro intervention for 24 h. Then the effect of each drug group on the proliferation of HCCLM3 cells were studied. At the same time,the control group of metformin was arranged. After that,the changes of apoptosis rate were observed by flow cytometry,and the expression levels of B-cell lymphoma-2( Bcl-2)and autophagy genes Atg5,Beclin1 were detced by Real-time polymerase chain reaction( Real-time PCR).Result: With the increase of glucose concentration,HCCLM3 cell had the strongest migration and proliferation ability in high glucose group( P < 0. 05). The inhibitory effect of BBR on the proliferation of HCCLM3 in high glucose culture was correlated with the concentration of BBR( 5,10,20,30,40,50 μmol·L~(-1)),and the inhibition rate was 33. 86%,40. 75%( P < 0. 05),49. 22%,55. 1%,60. 12%,and 61. 42% respectively from low concentration to high concentration( P < 0. 01). The apoptosis rate was significantly increased with the increase of BBR concentration( P < 0. 01). The expression levels of Bcl-2,Atg5 and Beclin1 gene were significantly upregulated in all intervening groups as compared with those in single high glucose group( P < 0. 05,P < 0. 01).Conclusion: BBR could inhibit the proliferation of HCCLM3 cells in high glucose environment. Its inhibition effect for HCCLM3 cells might be achieved by inducing apoptosis of the cells,regulating Bcl-2 and up-regulating the expression levels of autophagy gene Beclin1 and Atg5. Thus BBR plays an anti-tumor role through promoting autophagy in high glucose environment.
引文
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[2] HUA F,YU J J,HU Z W. Diabetes and cancer,common threads and missing links[J]. Cancer Lett,2016,374(1):54-61.
[3] Onitilo A A,Engel J M,Glurich I,et al. Diabetes and cancer I:risk,survival,and implications for screening[J]. Cancer Causes Control,2012,23(6):967-981.
[4] WANG P,KANG D,CAO W,et al. Diabetes mellitus and risk of hepatocellular carcinoma:a systematic review and Meta-analysis[J]. Diabetes Metab Res Rev,2012,28(2):109-122.
[5] Arase Y,Kobayashi M,Suzuki F,et al. Effect of type2 diabetes on risk for malignancies includes hepatocellular carcinoma in chronic hepatitis C[J].Hepatology,2013,57(3):964-973.
[6] CHEN R,YI Y,LI B. High prevalence of diabetes mellitus and impaired glucose tolerance in liver cancer patients:a hospital based study of 4 610 patients with benign tumors or specific cancers[J]. F1000Res,2016,5:1397-1407.
[7] Wojciechowska J,Krajewski W,Bolanowski M,et al.Diabetes and cancer:a review of current knowledge[J].Exp Clin Endocrinol Diabetes, 2016, 124(5):263-275.
[8] Giovannucci E,Harlan D M,Archer M C,et al. Diabetes and cancer:a consensus report[J]. CA Cancer J Clin,2010,60(4):207-221.
[9]黄玲玲,傅缨,资晓飞,等.红花黄色素对人肝癌细胞凋亡及相关蛋白表达的影响[J].中国中医基础医学杂志,2016,22(8):1061-1064.
[10] Raff E J,Kakati D,Bloomer J R,et al. Diabetes mellitus predicts occurrence of cirrhosis and hepatocellular cancer in alcoholic liver and non-alcoholic fatty liver diseases[J]. J Clin Transl Hepatol,2015,3(1):9-16.
[11] Barone B B,Yeh H C,Snyder C F,et al. Long-term all-cause mortality in cancer patients with preexisting diabetes mellitus:a systematic review and Meta-analysis[J]. JAMA,2008,300(23):2754-2764.
[12] LI T C,CHEN H J,LI C I,et al. Hyperglycemia and chronic liver diseases on risk of hepatocellular carcinoma in Chinese patients with type 2 diabetes-national cohort of Taiwan diabetes study[J]. Int J Cancer,2015,136(11):2668-2679.
[13] Rolo A P,Palmeira C M. Diabetes and mitochondrial function:role of hyperglycemia and oxidative stress[J].Toxicol Appl Pharmacol,2006,212(2):167-178.
[14] Kellenberger L D,Petrik J. Hyperglycemia promotes insulin-independent ovarian tumor growth[J]. Gynecol Oncol,2018,18(30):122-127.
[15] Maiuri M C,Le T G,Criollo A,et al. Functional and physical interaction between Bcl-X(L)and a BH3-like domain in Beclin-1[J]. Embo J,2007,26(10):2527-2539.
[16] CUI Q, Tashiro S, Onodera S, et al. Autophagy preceded apoptosis in oridonin-treated human breast cancer MCF-7 cells[J]. Biol Pharm Bull,2007,30(5):859-864.
[17] Besson A,Dowdy S F,Roberts J M. CDK inhibitors:cell cycle regulators and beyond[J]. Developmental Cell,2008,14(2):159-169.
[18] Levine B,Sinha S,Kroemer G. Bcl-2 family members:dual regulators of apoptosis and autophagy[J].Autophagy,2008,4(5):600-606.
[19] HUANG S,Sinicrope F A. Celecoxib-induced apoptosis is enhanced by ABT-737 and by inhibition of autophagy in human colorectal cancer cells[J]. Autophagy,2010,6(2):256-269.
[20] Matsushita M,Suzuki N N,Obara K,et al. Structure of Atg5. Atg16,a complex essential for autophagy[J]. J Biol Chem,2007,282(9):6763-6772.
[21] LIANG L Z,MA B,LIANG Y J,et al. Obatoclax induces Beclin 1-and Atg5-dependent apoptosis and autophagy in adenoid cystic carcinoma cells[J]. Oral Dis,2015,21(4):470-477.
[22] Yousefi S, Perozzo R, Schmid I, et al. Calpainmediated cleavage of Atg5 switches autophagy to apoptosis[J]. Nat Cell Biol,2006,8(10):1124-1132.
[23] LIU D,YANG Y,LIU Q,et al. Inhibition of autophagy by 3-MA potentiates cisplatin-induced apoptosis in esophageal squamous cell carcinoma cells[J]. Med Oncol,2011,28(1):105-111.