Mechanism of Triptolide-Induced Autophagic Death of Drug-Resistant Cervical Cancer Cells Based on JAK2/STAT3 Signaling Pathway
LIU Xiu-ling1, WANG Ru-na1, FU Xiao-chun1, ZHANG Hai-long2, WANG Pei-pei2, ZHAO Guang-cai3*
1. Department of Gynecological Oncology, Zhumadian Central Hospital, Zhumadian 463000, China; 2. Department of Pharmacy, Zhumadian Central Hospital, Zhumadian 463000, China; 3. Department of Obstetrics, Qilu Hospital of Shandong University, Qingdao 266003, China
Abstract：OBJECTIVE To investigate the effect and mechanism of triptolide (TPL)-induced autophagic death of cisplatin (DDP)-resistant cervical cancer cells. METHODS DDP-resistant cervical cancer cell lines HeLa/DR was cultured in vitro, and divided into blank group, DDP (10 μg·mL-1) group, TPL (50 nmol·L-1) group, DDP+TPL group for pharmacodynamic research and into TPL group, N-acetyl-L-cysteine(NAC, 5 mmol·L-1) group, TPL+NAC group, inhibitor of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) AG490 (8 μg·mL-1) group, TPL+activator of JAK2/STAT3(RO8191, 20 μmol·L-1) group for mechanism research. CCK-8 method was used to detect the cell proliferation, the IC50 and drug sensitivity index (SI) were calculated, and the combined drug use index (CI) of DDP and apatinib was calculated by median pharmacodynamics (chou-talalay) method. The apoptosis and cell cycle were detected by Flow cytometry. The number of lysosomes and phagocytic function were observed by Mitotracker Green and DQ BSA red staining, and the number of mitochondrial autophagosomes was observed by GFP-LC3 and Mitrotracker co-localization staining. Western blot was used to detect the expression of the related proteins. RESULTS When the dosage of TPL was less than 200 nmol·L-1, the CI was <1, indicating that TPL had synergistic effect with DDP, and when the dosage of TPL was 50 nmol·L-1, the synergistic effect of TPL and DDP was most significant. Besides, 50 nmol·L-1of TPL could increase the sensitivity of HeLa/DR cells to DDP, 5-fluorouracil, and paclitaxel (SI=8.05±0.28,3.17±0.14, 4.05±0.12). Compared with DDP group, the apoptosis rate and the ratio of G0/G1 phase cells were significantly increased in DDP+TPL group (P<0.05). Besides, although co-localization of LC3-Mitro green fluorescence and LC3-II/LC3-I ratio and Beclin1 expression in total mitochondrial proteins were increased (P<0.05), the fluorescence ratio of BSA DQ red/Lysotracker green decreased (P<0.05). Compared with the blank group, the DCF fluorescence intensity of HeLa/DR cells in TPL group was significantly increased, and the addition of NAC could significantly reduce the amount of ROS production, while the relative expression of Beclin1 protein and the ratio of LC3-II/LC3-I protein were decreased. Network pharmacology revealed the molecular interaction among TPL-cervical cancer-JAK2/STAT3 pathways. Western blot showed that the protein expression ratio of p-JAK2/JAK2 and p-STAT3/STAT3 in the TPL group and AG490 group was significantly reduced compared with the blank group, but the expression ratio of p-JAK2/JAK2 and p-STAT3/STAT3 protein in the TPL+RO8191 group and TPL+NAC group were higher than those in the TPL group (P<0.001). CONCLUSION TPL can inhibit the JAK2/STAT3 signaling pathway by inducing ROS production, promote mitochondrial damage, and destroy the mitochondrial-lysosome interaction established by the action of DDP, thereby inducing the autophagic death of HeLa/DR cells cooperated with DDP.
刘秀玲, 王茹娜, 符小春, 张海龙, 王培培, 赵广彩. 基于JAK2/STAT3信号通路探讨雷公藤甲素诱导宫颈癌耐药细胞自噬性死亡的机制[J]. 中国药学杂志, 2022, 57(24): 2099-2107.
LIU Xiu-ling, WANG Ru-na, FU Xiao-chun, ZHANG Hai-long, WANG Pei-pei, ZHAO Guang-cai. Mechanism of Triptolide-Induced Autophagic Death of Drug-Resistant Cervical Cancer Cells Based on JAK2/STAT3 Signaling Pathway. Chinese Pharmaceutical Journal, 2022, 57(24): 2099-2107.
BRAY F, FERLAY J, SOERJOMATARAM I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality world wide for 36 cancers in 185 countries [J]. CA Cancer J Clin, 2018, 68(6):394-424.
[2]
JU Y Y, ZHANG F F, WANG X H, et al. Current status and progress of treatment of recurrent cervical cancer[J]. J Int Obstet Gynecol, 2021, 48(1):56-60, 70.
[3]
TONG L, ZHAO Q F, DATAN E, et al. Triptolide: reflections on two decades of research and prospects for the future[J]. Nat Prod Rep, 2021, 38(4):843-860.
[4]
WEI Y M, WANG Y H, XUE H Q, et al. Triptolide, a potential autophagy modulator[J]. Chin J Integr Med(中国结合医学杂志), 2019, 25(3):233-240.
[5]
KOCATURK N M, AKKOC Y, KIG C, et al. Autophagy as a molecular target for cancer treatment[J]. Eur J Pharm Sci, 2019, 134:116-137.
[6]
WANG T, LIU Q, DUAN L. MBNL1 regulates resistance of HeLa cells to cisplatin via Nrf2[J]. Biochem Biophys Res Commun, 2020, 522(3):763-769.
[7]
WANG H, LIU G, LI S W, et al. The role of HMGB1/STAT3/Nanog signal pathway in cisplatin chemoresistance during the treatment of cervical cancer[J]. J Hubei Univ Med(湖北医药学院学报), 2018, 37(5):410-415.
[8]
HE M, CHEN L, ZENG S, et al. Research progress on regulation of autophagy of tumor cells by traditional Chinese medicine[J]. Chin Tradit Herb Drugs(中草药), 2021, 52(10):3142-3150.
[9]
CHENG X, FENG H, WU H, et al. Targeting autophagy enhances apatinib-induced apoptosis via endoplasmic reticulum stress for human colorectal cancer[J]. Cancer Lett, 2018, 431(9):105-114.
[10]
WEI Y M, LUAN Z H, LIU B W, et al. Autophagy in triptolide-mediated cytotoxicity in hepatic cells[J]. Int J Toxicol, 2019, 38(5):436-444.
[11]
LUO Z, XU X, SHO T, et al. ROS-induced autophagy regulates porcine trophectoderm cell apoptosis, proliferation, and differentiation[J]. Am J Physiol Cell Physiol, 2019, 316(2):C198-C209.
[12]
CHUANG K C, CHANG C R, CHANG S H, et al. Imiquimod-induced ROS production disrupts the balance of mitochondrial dynamics and increases mitophagy in skin cancer cells[J]. J Dermatol Sci, 2020, 98(3):152-162.
[13]
DAI C, CICCOTOSTO G D, CAPPAI R, et al. Rapamycin confers neuroprotection against colistin-induced oxidative stress, mitochondria dysfunction, and apoptosis through the activation of autophagy and mTOR/Akt/CREB signaling pathway[J]. ACS Chem Neurosci, 2018, 9(4):824-837.
[14]
OJHA R, SINGH S K, BHATTACHARYYA S.JAK-mediated autophagy regulates stemness and cell survival in cisplatin resistant bladder cancer cells[J]. Biochim Biophys Acta, 2016, 1860(11 Pt A):2484-2497.
[15]
CHEN B, LI D, LI M, et al. Induction of mitochondria-mediated apoptosis and PI3K/Akt/ mTOR-mediated autophagy by aflatoxin B2 in hepatocytes of broilers[J]. Oncotarget, 2016, 7(51):84989-84998.
[16]
XU Z, HAN X, OU D, et al. Targeting PI3K/AKT/mTOR-mediated autophagy for tumor therapy[J]. Appl Microbiol Biotechnol, 2020, 104(2):575-587.
[17]
ZHANG L, LIU H, WANG X M, et al. Study on the mechanism of Hela cell apoptosis mediated by vorinolta and paclitaxel enhancing ROS level[J]. Prog Obstet Gynecol, 2020, 104(2):575-587.
[18]
PALMIERI M, PAL R, SARDIELLO M, et al. AKT modulates the autophagy-lysosome pathway via TFEB[J]. Cell Cycle, 2017, 16(13):1237-1238.
[19]
CAO Y, WANG J, TIAN H, et al. Mitochondrial ROS accumulation inhibiting JAK2/STAT3 pathway is a critical modulator of CYT997-induced autophagy and apoptosis in gastric cancer[J]. J Exp Clin Cancer Res, 2020, 39(1):119. Doi: 10.1186/s13046-020-01621-y.
2022, Vol. 57 
