奥利司他联合EZH2抑制剂GSK126对胰腺癌细胞脂质代谢重编程的影响

doi:10.13481/j.1671-587X.20230108

摘要/Abstract

摘要：

目的阐明果蝇zeste基因增强子的人类同源物2（EZH2）抑制剂抗胰腺癌疗效不理想的可能机制，为探索新的胰腺癌治疗方案提供依据。方法采用不同浓度（0~50 μmol·L^－1）EZH2抑制剂GSK126分别处理胰腺癌PANC-1细胞、CFPAC-1细胞和胰腺转移癌SW1990细胞，CCK-8法检测各组细胞存活率。将3种细胞各分为对照组和GSK126组，GSK126孵育48 h后采用流式细胞术检测各组细胞凋亡率，油红O染色观察各组细胞中脂滴形成情况，检测各组细胞中甘油三酯（TG）水平，实时荧光定量PCR（RT-qPCR）法检测各组细胞中凋亡基因含半胱氨酸的天冬氨酸蛋白水解酶1、3、7、9（Caspase-1、Caspase-3、Caspase-7、Caspase-9）和血管内皮生长因子A（VEGFA）、E-钙黏蛋白（E-cadherin）及干性标志基因CD133、CD24和CD44 mRNA表达水平，以及脂代谢相关基因脂肪酸合成酶（FASN）、乙酰辅酶A羧化酶a （ACACA）、柠檬酸合成酶（CS）、ATP柠檬酸裂解酶（ACLY）、硬脂酰辅酶A去饱和酶（SCD）和酰基辅酶A合成酶短链家族成员2 （ACSS2）mRNA表达水平。将3种细胞各分为对照组、GSK126组、FASN抑制剂奥利司他（Orlistat）组和GSK126+Orlistat组，细胞孵育48 h后，CCK-8法检测各组细胞存活率，计算两药联合指数（CI），油红O染色观察各组细胞中脂滴形成情况，检测各组细胞中TG水平，流式细胞术检测各组细胞凋亡率，RT-qPCR法检测各组细胞中凋亡基因Caspase-1、Caspase-3、Caspase-7和Caspase-9及干性基因CD133、CD24和CD44 mRNA表达水平。结果 GSK126呈浓度依赖性抑制胰腺癌细胞增殖。与对照组比较，GSK126组3种细胞凋亡率和凋亡基因mRNA表达水平差异无统计学意义（P>0.05），GSK126组SW1990细胞中VEGFA和PANC-1细胞中E-cadherin mRNA表达水平明显降低（P<0.05），3种细胞中CD133及CFPAC-1和SW1990细胞中CD24 mRNA表达水平均明显升高（P<0.01）。与对照组比较，GSK126组3种细胞中脂滴数量明显增加，TG水平和FASN mRNA表达水平均明显升高（P<0.05或 P<0.01），其他脂代谢相关基因也有不同程度升高。与对照组、GSK126组和Orlistat组比较，GSK126 +Orlistat组细胞存活率明显降低（P<0.05或P<0.01），经计算两药CI均<1，具有协同作用。与对照组、GSK126组和Orlistat组比较，GSK126+Orlistat组细胞中脂滴数量明显减少；与对照组和GSK126组比较，GSK126+Orlistat组细胞中TG水平明显降低（P<0.01），凋亡基因和干性基因mRNA表达水平明显升高（P<0.05或 P<0.01）；与对照组、GSK126组和Orlistat组比较，GSK126+ Orlistat组细胞凋亡率明显升高（P<0.01）。结论 GSK126在抑制胰腺癌细胞增殖的同时可诱发细胞内脂代谢重编程，削弱了其抗癌效果，联合使用FASN抑制剂Orlistat可部分逆转该效应，协同抑制细胞增殖，明显增强细胞凋亡并降低干性基因表达。

关键词: 胰腺肿瘤, 化学疗法, EZH2抑制剂, GSK126, 脂代谢重编程, 脂肪酸合成酶

Abstract:

Objective：To clarify the possible mechanism of suboptimal efficacy of enhancer of zeste homolog 2 （EZH2） inhibitors in pancreatic cancer， and to provide basis for exploring a new treatment plan for pancreatic cancer. Methods The pancreatic cancer PANC-1 cells， CFPAC-1 cells and pancreatic metastasis cancer SW1990 cells were treated with different concentrations（0－50 μmol·L^－1）of EZH2 inhibitor GSK126. CCK8 method was used to detect the survival rates of the cells in various groups. Then the cells were divided into control group and GSK126 group. After 48 h of GSK126 incubation， the apoptotic rates of the cells in various groups were detected by flow cytometry，the lipid droplet formation in the cells in various groups were observed with oil red O staining，and the levels of triglyceride（TG） in the cells in various groups were detected.The expression levels of apoptosis genes cysteine-aspartic acid protease（1，3，7 and 9）（Caspase-1， Caspase-3， Caspase-7， Caspase-9）， vascular endothelial growth factor A （VEGFA）， E-cadherin and stemness markers CD133， CD24 and CD44 mRNA and the expression levels of fat acid synthase （FASN）， acetyl CoA carboxylase alpha （ACACA）， citrate synthase （CS）， ATP citrate lyase （ACLY）， stearoyl-CoA desaturase （SCD） and acyl-CoA synthetase short chain family member 2（ACSS2） mRNA were detected by real-time fluorescence quantitative PCR（RT-qPCR） method. The three kinds of cells were divided into control group， GSK126 group， FASN inhibitor Orlistat （Orlistat） group and GSK126+Orlistat group，respectively. After incubated for 48 h， the survival rates of cells in various groups were detected by CCK-8 method， and the combination index （CI） value of the two drugs was calculated.The lipid droplet formation in the cells in various groups were observed with oil red O staining，and the levels of TG in the cells in various groups were detected； the apoptotic rates of cells in various groups were detected by flow cytometry，and the expression levels of Caspase-1， Caspase-3， Caspase-7 and Caspase-9 and CD133， CD24 and CD44 mRNA in the cells in various groups were detected by RT-qPCR method. Results GSK126 inhibited the proliferation of pancreatic cancer cells in a concentration dependent manner. There were no significant differences in the apoptotic rates and apoptosis gene mRNA expression levels in the three kinds of cells between control group and GSK126 group（P>0.05）. Compared with control group， the expression levels of VEGFA mRNA in the SW1900 cells and E-cadherin mRNA in the PANC-1 cells in GSK126 group were significantly decreased （P<0.05）；the expression levels of CD133 in three kinds of cells and the expression levels of CD24 mRNA in the CFPAC-1 cells and SW1990 cells were signicantly increased （P<0.05）.Compared with control group， the number of lipid droplets in three kinds of cells in GSK126 group were significantly increased，the TG levels and the expression levels of FASN mRNA were significantly increased （P<0.05 or P<0.01）， and the expression levels of other lipid metabolism-related genes were also increased to varying degrees.Compared with control group， GSK126 group and Orlistat group， the survival rates of the cells in GSK126+Orlistat group were significantly decreased（P<0.05 or P<0.01）； the CI of two drugs was less than 1， which meaned synergistic effect of GSK126 and Orlistat.Compared with control group，GSK126 group and the number of lipid droplets in the cells in GSK126+Orlistat group was signifieantly decreased；compared with control group and GSK126 group，the TG level in the cells in GSK126+Orlistat group was significantly decreased（P<0.01），and the expression levels of apoptosis-related gene and stemness gene mRNA were significantly increased（P<0.05 or P<0.01）；compared with control group，GSK126 group and Orlistat group，the apoptotic rates of the cells in GSK126+Orlistat group was significantly increased（P<0.01）. Conclusion GSK126 inhibits the proliferation of pancreatic cancer cells and induces reprogramming of lipid metabolism， which weakens its anticancer effect. The combined use of GSK126 and FASN inhibitor Orlistat can partially reverse this effect， synergistically inhibit the cell proliferation， enhance apoptosis and reduce the expression of stemness genes.

Key words: Pancreas neoplasms, Chemotherapy, EZH2 inhibitor, GSK126, Lipid metabolism reprogramming, Fatty acid synthase

中图分类号:

R735.9

武晓慧,杨帆,郭新茹,袁轲,马综艺,廉婷,JHA Rajiv Kumar. 奥利司他联合EZH2抑制剂GSK126对胰腺癌细胞脂质代谢重编程的影响[J]. 吉林大学学报(医学版), 2023, 49(1): 55-66.

Xiaohui WU,Fan YANG,Xinru GUO,Ke YUAN,Zongyi MA,Ting LIAN,Rajiv Kumar JHA. Effect of Orlistat combined with EZH2 inhibitor GSK126 on lipid metabolism reprogramming in pancreatic cancer cells[J]. Journal of Jilin University(Medicine Edition), 2023, 49(1): 55-66.

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参考文献 23

1	ZENG S Y， PÖTTLER M， LAN B， et al. Chemoresistance in pancreatic cancer［J］. Int J Mol Sci， 2019， 20（18）： 4504.
2	SUNG H， FERLAY J， SIEGEL R L， et al. Global cancer statistics 2020： GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries［J］.CA Cancer J Clin，2021，71（3）：209-249.
3	SUNAMI Y， BÖKER V， KLEEFF J. Targeting and reprograming cancer-associated fibroblasts and the tumor microenvironment in pancreatic cancer［J］. Cancers， 2021， 13（4）： 697.
4	GARDNER K P， TSAI S， ALDAKKAK M， et al. CXCR4 expression in tumor associated cells in blood is prognostic for progression and survival in pancreatic cancer［J］. PLoS One， 2022， 17（3）： e0264763.
5	OUGOLKOV A V， BILIM V N， BILLADEAU D D. Regulation of pancreatic tumor cell proliferation and chemoresistance by the histone methyltransferase enhancer of zeste homologue 2［J］. Clin Cancer Res， 2008， 14（21）： 6790-6796.
6	HAN T， JIAO F， HU H， et al. EZH2 promotes cell migration and invasion but not alters cell proliferation by suppressing E-cadherin， partly through association with MALAT-1 in pancreatic cancer［J］. Oncotarget， 2016， 7（10）： 11194-11207.
7	JIN X， YANG C， FAN P， et al. CDK5/FBW7-dependent ubiquitination and degradation of EZH2 inhibits pancreatic cancer cell migration and invasion［J］. J Biol Chem， 2017， 292（15）： 6269-6280.
8	KURMASHEVA R T， SAMMONS M， FAVOURS E，et al. Initial testing （stage 1） of tazemetostat （EPZ-6438）， a novel EZH2 inhibitor， by the Pediatric Preclinical Testing Program［J］. Pediatr Blood Cancer， 2017， 64（3）.DOI： 10.1002/pbc.26218 . doi: 10.1002/pbc.26218
9	LEE W， TECKIE S， WIESNER T， et al. PRC2 is recurrently inactivated through EED or SUZ12 loss in malignant peripheral nerve sheath tumors［J］. Nat Genet， 2014， 46（11）： 1227-1232.
10	DUAN R， DU W F， GUO W J. EZH2： a novel target for cancer treatment［J］.J Hematol Oncol，2020，13（1）： 104.
11	XU H， ZHANG L S， QIAN X H， et al. GSK343 induces autophagy and downregulates the AKT/mTOR signaling pathway in pancreatic cancer cells［J］. Exp Ther Med， 2019， 18（4）： 2608-2616.
12	HUANG X， YAN J， ZHANG M， et al. Targeting epigenetic crosstalk as a therapeutic strategy for EZH2-aberrant solid tumors［J］. Cell， 2018， 175（1）： 186-199.
13	CHOU T C. Theoretical basis， experimental design， and computerized simulation of synergism and antagonism in drug combination studies［J］. Pharmacol Rev， 2006， 58（3）： 621-681.
14	WANG Y Y， WANG M， WEI W J， et al. Disruption of the EZH2/miRNA/β-catenin signaling suppresses aerobic glycolysis in glioma［J］. Oncotarget， 2016， 7（31）： 49450-49458.
15	AVAN A， CREA F， PAOLICCHI E， et al. Molecular mechanisms involved in the synergistic interaction of the EZH2 inhibitor 3-deazaneplanocin A with gemcitabine in pancreatic cancer cells［J］. Mol Cancer Ther， 2012， 11（8）： 1735-1746.
16	SUBRAMANIAM D， RAMALINGAM S， HOUCHEN C W， et al. Cancer stem cells： a novel paradigm for cancer prevention and treatment［J］. Mini Rev Med Chem， 2010， 10（5）： 359-371.
17	SWIERCZYNSKI J， HEBANOWSKA A， SLEDZINSKI T. Role of abnormal lipid metabolism in development， progression， diagnosis and therapy of pancreatic cancer［J］. World J Gastroenterol， 2014， 20（9）： 2279-2303.
18	TADROS S， SHUKLA S K， KING R J， et al. De novo lipid synthesis facilitates gemcitabine resistance through endoplasmic reticulum stress in pancreatic cancer［J］. Cancer Res， 2017， 77（20）： 5503-5517.
19	HERMS A， BOSCH M， ARIOTTI N， et al. Cell-to-cell heterogeneity in lipid droplets suggests a mechanism to reduce lipotoxicity［J］. Curr Biol， 2013， 23（15）： 1489-1496.
20	PETAN T， JARC E， JUSOVIĆ M. Lipid droplets in cancer： guardians of fat in a stressful world［J］. Molecules， 2018， 23（8）： E1941.
21	CHA J Y， LEE H J. Targeting lipid metabolic reprogramming as anticancer therapeutics［J］. J Cancer Prev， 2016， 21（4）： 209-215.
22	OLZMANN J A， CARVALHO P. Dynamics and functions of lipid droplets［J］. Nat Rev Mol Cell Biol， 2019， 20（3）： 137-155.
23	FAZLI H R， MORADZADEH M， MEHRBAKHSH Z， et al. Diagnostic significance of serum fatty acid synthase in patients with pancreatic cancer［J］. Middle East J Dig Dis， 2021， 13（2）： 115-120.

Gene	Forward (5'—3')	Reverse (5'—3')
Caspase-1	GCCTGTTCCTGTGATGTGGAG	TGCCCACAGACATTCATACAGTTTC
Caspase-3	GACTCTGGAATATCCCTGGACAACA	AGGTTTGCTGCATCGACATCTG
Caspase-7	ATGTCACCATGCGATCCATCA	TCCGTTTCGAACGCCCATA
Caspase-9	CTTGAGACACTCGCTCAGCTTC	CTTGAGACACTCGCTCAGCTTC
CD133	AGTGGCATCGTGCAAACCTG	CTCCGAATCCATTCGACGATA
CD24	CTCCTACCCACGCAGATTTATT	GTGGTGGCATTAGTTGGATTTG
CD44	GAAATGGCACCACTGCTTATG	CTACTAGGAGTTGCCTGGATTG
VEGFA	AGGGCAGAATCATCACGAAGT	AGGG TCTCGATTGGATGGCA
E-cadherin	ATCAAAGGTATCACGGCAAACG	CGGAGAGCTCGTCCACGTAT
FASN	CTAGGTTTGATGCCTCCTTCTT	GATGGCTTCATAGGTGACTTCC
ACACA	CTTGCTAGGGCAGAAGGTATTC	CCCAGGCCACATGAAACATA
CS	GAGCAGGGTAAAGCCAAGAA	CCAAACAGGACCGTGTAGTAAT
ACLY	GTCTCATCGGAGTCGCATTT	CTCCTTCCCAGCACAAAGAT
SCD	GCACATCAACTTCACCACATTC	GTAGTTTCCATCTCCGGTTCTT
ACSS2	ACGCTTTGAGACAACCTACTT	CCTGCCAGTGATCCAGTAATAG
β-actin	ATTGCCGACAGGATGCAGA	GAGTACTTGCGC TCAGGAGGA

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