Combination treatment with triptolide and hydroxycamptothecin synergistically enhances apoptosis in A549 lung adenocarcinoma cells through PP2A-regulated ERK, p38 MAPKs and Akt signaling pathways

Meng,Guanmin; Wang,Wei; Chai,Kequn; Yang,Suwen; Li,Fangqiong; Jiang,Kai

doi:10.3892/ijo.2015.2814

Combination treatment with triptolide and hydroxycamptothecin synergistically enhances apoptosis in A549 lung adenocarcinoma cells through PP2A-regulated ERK, p38 MAPKs and Akt signaling pathways

Authors:
- Guanmin Meng
- Wei Wang
- Kequn Chai
- Suwen Yang
- Fangqiong Li
- Kai Jiang
View Affiliations

Affiliations: Department of Clinical Laboratory, Tongde Hospital of Zhejiang Province, Hangzhou 310012, P.R. China, Department of Clinical Laboratory, Sir Run Run Shaw Hospital, Medical College, Zhejiang University, Hangzhou 310016, P.R. China
Published online on: January 7, 2015 https://doi.org/10.3892/ijo.2015.2814
Pages: 1007-1017
Copyright: © Meng et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Lung cancer is the leading cause of cancer death worldwide. Recently, two plant-derived drugs triptolide (TP) and hydroxycamptothecin (HCPT) both have shown broad-spectrum anticancer activities. Our previous study documented that combination treatment with these two drugs acted more effectively than mono-therapy, however, the molecular basis underlying the synergistic cytotoxicity remains poorly understood. In this study, we aimed to clarify the molecular mechanism of TP/HCPT anticancer effect in A549 lung adenocarcinoma cells, by investigating the involvement of phosphatase 2A (PP2A) and PP2A-regulated mitogen-activated protein kinases (MAPKs) and Akt signaling pathways. The results showed that TP and HCPT synergistically exerted cytotoxicity in the growth of A549 cells. Combinatorial TP/HCPT treatment significantly enhanced the activation of caspase-3 and -9, Bax/Bcl-2 ratio, release of cytochrome c from mitochondrial and subsequent apoptosis. While the Akt survival pathway was inhibited, ERK and p38 MAPKs were dramatically activated. Furthermore, the activity of PP2A was significantly augmented. Regulation of p38, ERK and Akt by PP2A was demonstrated, by using a specific PP2A inhibitor okadaic acid (OA). Finally, pharmacological inhibitors OA, SB203580, SP600125 and PD98059 confirm the role of PP2A and its substrates ERK, p38 MAPK and Akt in mediating TP/HCPT-induced apoptosis. Taken together, this study provides the first evidence for a synergistic TP/HCPT anticancer activity in A549 cells and also supports a critical role of PP2A and PP2A-regulated signaling pathways, providing new insight into the mode of action of TP/HCPT in cancer therapy.

View Figures

View References

1	He YY, Zhang XC, Yang JJ, et al: Prognostic significance of genotype and number of metastatic sites in advanced non-small-cell lung cancer. Clin Lung Cancer. 15:441–447. 2014. View Article : Google Scholar : PubMed/NCBI
2	Rajeswaran A, Trojan A, Burnand B and Giannelli M: Efficacy and side effects of cisplatin- and carboplatin-based doublet chemotherapeutic regimens versus non-platinum-based doublet chemotherapeutic regimens as first line treatment of metastatic non-small cell lung carcinoma: a systematic review of randomized controlled trials. Lung Cancer. 59:1–11. 2008. View Article : Google Scholar
3	Chen Y, Zhu J and Zhang W: Antitumor effect of traditional Chinese herbal medicines against lung cancer. Anticancer Drugs. 25:983–989. 2014. View Article : Google Scholar : PubMed/NCBI
4	Wang S, Wu X, Tan M, et al: Fighting fire with fire: poisonous Chinese herbal medicine for cancer therapy. J Ethnopharmacol. 140:33–45. 2012. View Article : Google Scholar : PubMed/NCBI
5	Kupchan SM, Court WA, Dailey RG, Gilmore CJ and Bryan RF: Triptolide and tripdiolide, novel antileukemic diterpenoid triepoxides from Tripterygium wilfordii. J Am Chem Soc. 94:7194–7195. 1972. View Article : Google Scholar : PubMed/NCBI
6	Corson TW and Crews CM: Molecular understanding and modern application of traditional medicines: triumphs and trials. Cell. 130:769–774. 2007. View Article : Google Scholar : PubMed/NCBI
7	Liu Y, Xiao E, Yuan L and Li G: Triptolide synergistically enhances antitumor activity of oxaliplatin in colon carcinoma in vitro and in vivo. DNA Cell Biol. 33:418–425. 2014. View Article : Google Scholar : PubMed/NCBI
8	Zhao H, Yang Z, Wang X, et al: Triptolide inhibits ovarian cancer cell invasion by repression of matrix metalloproteinase 7 and 19 and upregulation of E-cadherin. Exp Mol Med. 44:633–641. 2012. View Article : Google Scholar : PubMed/NCBI
9	Nakazato T, Sagawa M and Kizaki M: Triptolide induces apoptotic cell death of multiple myeloma cells via transcriptional repression of Mcl-1. Int J Oncol. 44:1131–1138. 2014.PubMed/NCBI
10	Chen F, Liu Y, Wang S, et al: Triptolide, a Chinese herbal extract, enhances drug sensitivity of resistant myeloid leukemia cell lines through downregulation of HIF-1α and Nrf2. Pharmacogenomics. 14:1305–1317. 2013. View Article : Google Scholar : PubMed/NCBI
11	Wu PP, Liu KC, Huang WW, et al: Triptolide induces apoptosis in human adrenal cancer NCI-H295 cells through a mitochondrial-dependent pathway. Oncol Rep. 25:551–557. 2011.
12	Mujumdar N, Banerjee S, Chen Z, et al: Triptolide activates unfolded protein response leading to chronic ER stress in pancreatic cancer cells. Am J Physiol Gastrointest Liver Physiol. 306:G1011–G1020. 2014. View Article : Google Scholar : PubMed/NCBI
13	Li J, Liu R, Yang Y, Huang Y, Li X and Shen X: Triptolide-induced in vitro and in vivo cytotoxicity in human breast cancer stem cells and primary breast cancer cells. Oncol Rep. 31:2181–2186. 2014.PubMed/NCBI
14	Hsiang YH, Hertzberg R, Hecht S and Liu LF: Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem. 260:14873–14878. 1985.PubMed/NCBI
15	Zaki NM: Augmented cytotoxicity of hydroxycamptothecin-loaded nanoparticles in lung and colon cancer cells by chemosensitizing pharmaceutical excipients. Drug Deliv. 21:265–275. 2014. View Article : Google Scholar
16	Hu W, Zhang C, Fang Y and Lou C: Anticancer properties of 10-hydroxycamptothecin in a murine melanoma pulmonary metastasis model in vitro and in vivo. Toxicol In Vitro. 25:513–520. 2011. View Article : Google Scholar
17	Fu YR, Yi ZJ, Yan YR and Qiu ZY: Hydroxycamptothecin-induced apoptosis in hepatoma SMMC-7721 cells and the role of mitochondrial pathway. Mitochondrion. 6:211–217. 2006. View Article : Google Scholar : PubMed/NCBI
18	Kotoh S, Naito S, Yokomizo A, et al: Increased expression of DNA topoisomerase I gene and collateral sensitivity to camptothecin in human cisplatin-resistant bladder cancer cells. Cancer Res. 54:3248–3252. 1994.PubMed/NCBI
19	Liu Y, Chen F, Wang S, et al: Low-dose triptolide in combination with idarubicin induces apoptosis in AML leukemic stem-like KG1a cell line by modulation of the intrinsic and extrinsic factors. Cell Death Dis. 4:e9482013. View Article : Google Scholar : PubMed/NCBI
20	Alsaied OA, Sangwan V, Banerjee S, et al: Sorafenib and triptolide as combination therapy for hepatocellular carcinoma. Surgery. 156:270–279. 2014. View Article : Google Scholar : PubMed/NCBI
21	Tang XY, Zhu YQ, Tao WH, Wei B and Lin XL: Synergistic effect of triptolide combined with 5-fluorouracil on colon carcinoma. Postgrad Med J. 83:338–343. 2007. View Article : Google Scholar : PubMed/NCBI
22	Pigneux A, Mahon FX, Uhalde M, et al: Triptolide cooperates with chemotherapy to induce apoptosis in acute myeloid leukemia cells. Exp Hematol. 36:1648–1659. 2008. View Article : Google Scholar : PubMed/NCBI
23	Chen Z, Sangwan V, Banerjee S, et al: Triptolide sensitizes pancreatic cancer cells to TRAIL-induced activation of the death receptor pathway. Cancer Lett. 348:156–166. 2014. View Article : Google Scholar : PubMed/NCBI
24	Wang W, Yang S, Su Y, et al: Enhanced antitumor effect of combined triptolide and ionizing radiation. Clin Cancer Res. 13:4891–4899. 2007. View Article : Google Scholar : PubMed/NCBI
25	Yang SW, Wang W, Xie XY, Zhu WP and Li FQ: In vitro synergistic cytotoxic effect of triptolide combined with hydroxycamptothecin on pancreatic cancer cells. Am J Chin Med. 39:121–134. 2011. View Article : Google Scholar : PubMed/NCBI
26	Chou TC and Talalay P: Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 22:27–55. 1984. View Article : Google Scholar : PubMed/NCBI
27	Chou TC: Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev. 58:621–681. 2006. View Article : Google Scholar : PubMed/NCBI
28	Meng G, Sun Y, Fu W, Guo Z and Xu L: Microcystin-LR induces cytoskeleton system reorganization through hyperphosphorylation of tau and HSP27 via PP2A inhibition and subsequent activation of the p38 MAPK signaling pathway in neuroendocrine (PC12) cells. Toxicology. 290:218–229. 2011. View Article : Google Scholar : PubMed/NCBI
29	Zhao S, Xiong Z, Mao X, et al: Atmospheric pressure room temperature plasma jets facilitate oxidative and nitrative stress and lead to endoplasmic reticulum stress dependent apoptosis in HepG2 cells. PLoS One. 8:e736652013. View Article : Google Scholar : PubMed/NCBI
30	Han J, Back SH, Hur J, et al: ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nat Cell Biol. 15:481–490. 2013. View Article : Google Scholar : PubMed/NCBI
31	Nakagawa T, Zhu H, Morishima N, et al: Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature. 403:98–103. 2000. View Article : Google Scholar : PubMed/NCBI
32	Komatsu M, Furukawa T, Ikeda R, et al: Involvement of mitogen-activated protein kinase signaling pathways in microcystin-LR-induced apoptosis after its selective uptake mediated by OATP1B1 and OATP1B3. Toxicol Sci. 97:407–416. 2007. View Article : Google Scholar : PubMed/NCBI
33	Cowan KJ and Storey KB: Mitogen-activated protein kinases: new signaling pathways functioning in cellular responses to environmental stress. J Exp Biol. 206:1107–1115. 2003. View Article : Google Scholar : PubMed/NCBI
34	Downward J: Mechanisms and consequences of activation of protein kinase B/Akt. Curr Opin Cell Biol. 10:262–267. 1998. View Article : Google Scholar : PubMed/NCBI
35	Sontag E: Protein phosphatase 2A: the Trojan Horse of cellular signaling. Cell Signal. 13:7–16. 2001. View Article : Google Scholar : PubMed/NCBI
36	Chen W, Wang Z, Jiang C and Ding Y: PP2A-mediated anti-cancer therapy. Gastroenterol Res Pract. 2013:6754292013. View Article : Google Scholar
37	Perrotti D and Neviani P: Protein phosphatase 2A: a target for anticancer therapy. Lancet Oncol. 14:e229–e238. 2013. View Article : Google Scholar : PubMed/NCBI
38	Janssens V and Rebollo A: The role and therapeutic potential of Ser/Thr phosphatase PP2A in apoptotic signalling networks in human cancer cells. Curr Mol Med. 12:268–287. 2012. View Article : Google Scholar : PubMed/NCBI
39	Chen J, Martin BL and Brautigan DL: Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science. 257:1261–1264. 1992. View Article : Google Scholar : PubMed/NCBI
40	Kuo YC, Huang KY, Yang CH, Yang YS, Lee WY and Chiang CW: Regulation of phosphorylation of Thr-308 of Akt, cell proliferation, and survival by the B55alpha regulatory subunit targeting of the protein phosphatase 2A holoenzyme to Akt. J Biol Chem. 283:1882–1892. 2008. View Article : Google Scholar
41	Lee ER, Kim JH, Choi HY, Jeon K and Cho SG: Cytoprotective effect of eriodictyol in UV-irradiated keratinocytes via phosphatase-dependent modulation of both the p38 MAPK and Akt signaling pathways. Cell Physiol Biochem. 27:513–524. 2011. View Article : Google Scholar : PubMed/NCBI
42	Chen L, Liu L, Yin J, Luo Y and Huang S: Hydrogen peroxide-induced neuronal apoptosis is associated with inhibition of protein phosphatase 2A and 5, leading to activation of MAPK pathway. Int J Biochem Cell Biol. 41:1284–1295. 2009. View Article : Google Scholar
43	Li XJ, Jiang ZZ and Zhang LY: Triptolide progress on research in pharmacodynamics and toxicology. J Ethnopharmacol. 155:67–79. 2014. View Article : Google Scholar : PubMed/NCBI
44	Tan W, Lu J, Huang M, et al: Anti-cancer natural products isolated from chinese medicinal herbs. Chin Med. 6:272011. View Article : Google Scholar : PubMed/NCBI
45	Hengartner MO: The biochemistry of apoptosis. Nature. 407:770–776. 2000. View Article : Google Scholar : PubMed/NCBI
46	Chen YW, Huang CF, Yang CY, Yen CC, Tsai KS and Liu SH: Inorganic mercury causes pancreatic beta-cell death via the oxidative stress-induced apoptotic and necrotic pathways. Toxicol Appl Pharmacol. 243:323–331. 2010. View Article : Google Scholar
47	Carter BZ, Mak DH, Schober WD, et al: Triptolide induces caspase-dependent cell death mediated via the mitochondrial pathway in leukemic cells. Blood. 108:630–637. 2006. View Article : Google Scholar : PubMed/NCBI
48	Wang Q, Du H, Geng G, et al: Matrine inhibits proliferation and induces apoptosis via BID-mediated mitochondrial pathway in esophageal cancer cells. Mol Biol Rep. 41:3009–3020. 2014. View Article : Google Scholar : PubMed/NCBI
49	Li Y, Zhang S, Geng JX and Hu XY: Curcumin inhibits human non-small cell lung cancer A549 cell proliferation through regulation of Bcl-2/Bax and cytochrome C. Asian Pac J Cancer Prev. 14:4599–4602. 2013. View Article : Google Scholar : PubMed/NCBI
50	Qin R, Shen H, Cao Y, et al: Tetrandrine induces mitochondria-mediated apoptosis in human gastric cancer BGC-823 cells. PLoS One. 8:e764862013. View Article : Google Scholar : PubMed/NCBI
51	Yip KW and Reed JC: Bcl-2 family proteins and cancer. Oncogene. 27:6398–6406. 2008. View Article : Google Scholar : PubMed/NCBI
52	Giorgi C, De Stefani D, Bononi A, Rizzuto R and Pinton P: Structural and functional link between the mitochondrial network and the endoplasmic reticulum. Int J Biochem Cell Biol. 41:1817–1827. 2009. View Article : Google Scholar : PubMed/NCBI
53	Deniaud A, Sharaf el dein O, Maillier E, et al: Endoplasmic reticulum stress induces calcium-dependent permeability transition, mitochondrial outer membrane permeabilization and apoptosis. Oncogene. 27:285–299. 2008. View Article : Google Scholar
54	Pinton P, Giorgi C, Siviero R, Zecchini E and Rizzuto R: Calcium and apoptosis: ER-mitochondria Ca²⁺ transfer in the control of apoptosis. Oncogene. 27:6407–6418. 2008. View Article : Google Scholar : PubMed/NCBI
55	Shao H, Ma J, Guo T and Hu R: Triptolide induces apoptosis of breast cancer cells via a mechanism associated with the Wnt/β-catenin signaling pathway. Exp Ther Med. 8:505–508. 2014.PubMed/NCBI
56	Whitmarsh AJ and Davis RJ: Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med (Berl). 74:589–607. 1996. View Article : Google Scholar
57	Xia Z, Dickens M, Raingeaud J, Davis RJ and Greenberg ME: Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 270:1326–1331. 1995. View Article : Google Scholar : PubMed/NCBI
58	Lee ER, Kang YJ, Kim JH, Lee HT and Cho SG: Modulation of apoptosis in HaCaT keratinocytes via differential regulation of ERK signaling pathway by flavonoids. J Biol Chem. 280:31498–31507. 2005. View Article : Google Scholar : PubMed/NCBI
59	Leung HW, Hour MJ, Chang WT, et al: P38-associated pathway involvement in apoptosis induced by photodynamic therapy with Lonicera japonica in human lung squamous carcinoma CH27 cells. Food Chem Toxicol. 46:3389–3400. 2008. View Article : Google Scholar : PubMed/NCBI
60	Turowski P, Favre B, Campbell KS, Lamb NJ and Hemmings BA: Modulation of the enzymatic properties of protein phosphatase 2A catalytic subunit by the recombinant 65-kDa regulatory subunit PR65alpha. Eur J Biochem. 248:200–208. 1997. View Article : Google Scholar : PubMed/NCBI
61	Muslin AJ: MAPK signalling in cardiovascular health and disease: molecular mechanisms and therapeutic targets. Clin Sci (Lond). 115:203–218. 2008. View Article : Google Scholar