Schedule-dependent cytotoxic synergism of pemetrexed and erlotinib in BXPC-3 and PANC-1 human pancreatic cancer cells

Wang,Lin; Zhu,Zhi-Xia; Zhang,Wen-Ying; Zhang,Wei-Min

doi:10.3892/etm.2011.293

Schedule-dependent cytotoxic synergism of pemetrexed and erlotinib in BXPC-3 and PANC-1 human pancreatic cancer cells

Authors:
- Lin Wang
- Zhi-Xia Zhu
- Wen-Ying Zhang
- Wei-Min Zhang
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Affiliations: Department of Oncology, Guangzhou General Hospital of Guangzhou Military Command (Guangzhou Liuhuaqiao Hospital), Guangzhou, Guangdong 510010, P.R. China
Published online on: June 22, 2011 https://doi.org/10.3892/etm.2011.293
Pages: 969-975

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Abstract

Previous studies have shown that both pemetrexed, a cytotoxic drug, and erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), inhibit the cell growth of pancreatic cancer cells. However, whether they exert a synergistic antitumor effect on pancreatic cancer cells remains unknown. The present study aimed to assess the synergistic effect of erlotinib in combination with pemetrexed using different sequential administration schedules on the proliferation of human pancreatic cancer BXPC-3 and PANC-1 cells and to probe its cellular mechanism. The EGFR and K-ras gene mutation status was examined by quantitative PCR high-resolution melting (qPCR‑HRM) analysis. BXPC-3 and PANC-1 cells were incubated with pemetrexed and erlotinib using different administration schedules. MTT assay was used to determine cytotoxicity, and cell cycle distribution was determined by flow cytometry. The expression and phosphorylation of EGFR, HER3, AKT and MET were determined using Western blotting. Both pemetrexed and erlotinib inhibited the proliferation of BXPC-3 and PANC-1 cells in a dose- and time-dependent manner in vitro. Synergistic effects on cell proliferation were observed when pemetrexed was used in combination with erlotinib. The degree of the synergistic effects depended on the administration sequence, which was most obvious when erlotinib was sequentially administered at 24-h interval following pemetrexed. Cell cycle studies revealed that pemetrexed induced S arrest and erlotinib induced G0/G1 arrest. The sequential administration of erlotinib following pemetrexed induced S arrest. Western blot analyses showed that pemetrexed increased and erlotinib decreased the phosphorylation of EGFR, HER3 and AKT, respectively. However, both pemetrexed and erlotinib exerted no significant effects on the phosphorylation of c-MET. The phosphorylation of EGFR, HER3 and AKT was significantly suppressed by scheduled incubation with pemetrexed followed by erlotinib, but not by concomitant or sequential incubation with erlotinib followed by pemetrexed. In summary, our results demonstrated that the combined use of erlotinib and pemetrexed exhibited a strong synergism in BXPC-3 and PANC-1 cells. The inhibitory effects were strongest after sequential administration of pemetrexed followed by erlotinib. The synergistic effects may be related to activation of the EGFR/HER3/AKT pathway induced by pemetrexed.

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1.	Michaud DS: Epidemiology of pancreatic cancer. Minerva Chir. 59:99–111. 2004.PubMed/NCBI
2.	Saidi RF, Remine SG and Jacobs MJ: Interferon receptor alpha/beta is associated with improved survival after adjuvant therapy in resected pancreatic cancer. HPB (Oxford). 9:289–294. 2007. View Article : Google Scholar : PubMed/NCBI
3.	Burris HA III, Moore MJ, Andersen J, et al: Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol. 15:2403–2413. 1997.PubMed/NCBI
4.	Moore MJ, Goldstein D, Hamm J, et al: erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol. 25:1960–1966. 2007. View Article : Google Scholar
5.	Taylor EC, Kuhnt D, Shih C, et al: A dideazatetrahydrofolate analog lacking a chiral center at C-6: N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo [2,3-d]pyrimidin-5yl)ethyl [benzoyl]-L-glutamic acid is an inhibitor of thymidylate synthase. J Med Chem. 35:4450–4454. 1992.PubMed/NCBI
6.	Chattopadhyay S, Moran RG and Goldman ID: pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications. Mol Cancer Ther. 6:404–417. 2007. View Article : Google Scholar : PubMed/NCBI
7.	Miller KD, Picus J, Blanke C, John W, Clark J, Shulman LN, Thornton D, Rowinsky E and Loehrer PJ Sr: Phase II study of the multitargeted antifolate LY231514 (ALIMTA, MTA, pemetrexed disodium) in patients with advanced pancreatic cancer. Ann Oncol. 11:101–103. 2000. View Article : Google Scholar : PubMed/NCBI
8.	Giovannetti E, Mey V, Danesi R, Mosca I and Del Tacca M: Synergistic cytotoxicity and pharmacogenetics of gemcitabine and pemetrexed combination in pancreatic cancer cell lines. Clin Cancer Res. 10:2936–2943. 2004. View Article : Google Scholar : PubMed/NCBI
9.	Li T, Ling YH, Goldman ID and Perez-Soler R: Schedule-dependent cytotoxic synergism of pemetrexed and erlotinib in human non-small cell lung cancer cells. Clin Cancer Res. 13:3413–3422. 2007. View Article : Google Scholar : PubMed/NCBI
10.	Solomon B and Bunn PA Jr: Clinical activity of pemetrexed: a multitargeted antifolate anticancer agent. Future Oncol. 1:733–746. 2005. View Article : Google Scholar : PubMed/NCBI
11.	Lu YY, Jing DD, Xu M, Wu K and Wang XP: Anti-tumor activity of erlotinib in the BxPC-3 pancreatic cancer cell line. World J Gastroenterol. 14:5403–5411. 2008. View Article : Google Scholar : PubMed/NCBI
12.	Giovannetti E, Lemos C, Tekle C, Smid K, Nannizzi S, Rodriguez JA, Ricciardi S, Danesi R, Giaccone G and Peters GJ: Mechanisms underlying the synergistic interaction of erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor, with the multitargeted antifolate pemetrexed in non-small-cell lung cancer cells. Mol Pharmacol. 73:1290–1300. 2008. View Article : Google Scholar
13.	Davies AM, Ho C, Lara PN Jr, Mack P, Gumerlock PH and Gandara DR: Pharmacodynamic separation of epidermal growth factor receptor tyrosine kinase inhibitors and chemotherapy in non-small-cell lung cancer. Clin Lung Cancer. 7:385–388. 2006. View Article : Google Scholar : PubMed/NCBI
14.	Mahaffey CM, Davies AM, Lara PN Jr, Pryde B, Holland W, Mack PC, Gumerlock PH and Gandara DR: Schedule-dependent apoptosis in K-ras mutant non-small-cell lung cancer cell lines treated with docetaxel and erlotinib: rationale for pharmacodynamic separation. Clin Lung Cancer. 8:548–553. 2007. View Article : Google Scholar
15.	Giovannetti E, Lemos C, Tekle C, et al: Molecular mechanisms underlying the synergistic interaction of erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor, with the multitargeted antifolate pemetrexed in non-small-cell lung cancer cells. Mol Pharmacol. 73:1290–1300. 2008. View Article : Google Scholar
16.	Li J, Li Y, Feng ZQ and Chen XG: Anti-tumor activity of a novel EGFR tyrosine kinase inhibitor against human NSCLC in vitro and in vivo. Cancer Lett. 279:213–220. 2009. View Article : Google Scholar : PubMed/NCBI
17.	Lu YY, Jing DD, Xu M, Wu K and Wang XP: Anti-tumor activity of erlotinib in the BxPC-3 pancreatic cancer cell line. World J Gastroenterol. 14:5403–5411. 2008. View Article : Google Scholar : PubMed/NCBI
18.	Sun SY, Rosenberg LM, Wang X, Zhou Z, Yue P, Fu H and Khuri FR: Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition. Cancer Res. 65:7052–7058. 2005. View Article : Google Scholar : PubMed/NCBI
19.	Roskoski R Jr: The ErbB/HER receptor protein-tyrosine kinases and cancer. Biochem Biophys Res Commun. 319:1–11. 2004. View Article : Google Scholar : PubMed/NCBI
20.	Tobita K, Kijima H, Dowaki S, et al: Epidermal growth factor receptor expression in human pancreatic cancer: Significance for liver metastasis. Int J Mol Med. 11:305–309. 2003.PubMed/NCBI
21.	Frolov A, Schuller K, Tzeng CW, Cannon EE, Ku BC, Howard JH, Vickers SM, Heslin MJ, Buchsbaum DJ and Arnoletti JP: ErbB3 expression and dimerization with EGFR influence pancreatic cancer cell sensitivity to erlotinib. Cancer Biol Ther. 6:548–554. 2007. View Article : Google Scholar : PubMed/NCBI
22.	Giovannetti E, Mey V, Nannizzi S, Pasqualetti G, Del Tacca M and Danesi R: Pharmacogenetics of anticancer drug sensitivity in pancreatic cancer. Mol Cancer Ther. 5:1387–1395. 2006. View Article : Google Scholar : PubMed/NCBI
23.	Liles JS, Arnoletti JP, Tzeng CW, Howard JH, Kossenkov AV, Kulesza P, Heslin MJ and Frolov A: ErbB3 expression promotes tumorigenesis in pancreatic adenocarcinoma. Cancer Biol Ther. 10:555–563. 2010. View Article : Google Scholar : PubMed/NCBI
24.	Yonezawa M, Wada K, Tatsuguchi A, Akamatsu T, Gudis K, Seo T, Mitsui K, Nagata K, Tanaka S, Fujimori S and Sakamoto C: Heregulin-induced VEGF expression via the ErbB3 signaling pathway in colon cancer. Digestion. 80:215–225. 2009. View Article : Google Scholar : PubMed/NCBI
25.	Miller TW, Pérez-Torres M, Narasanna A, et al: Loss of phosphatase and tensin homologue deleted on chromosome 10 engages ErbB3 and IGF-IR signaling to promote antiestrogen resistance in breast cancer. Cancer Res. 69:4192–4201. 2009. View Article : Google Scholar : PubMed/NCBI
26.	Chen K, Iribarren P, Gong W and Wang JM: The essential role of phosphoinositide 3-kinases (PI3Ks) in regulating pro-inflammatory responses and the progression of cancer. Cell Mol Immunol. 2:241–252. 2005.PubMed/NCBI
27.	Buck E, Eyzaguirre A, Haley JD, Gibson NW, Cagnoni P and Iwata KK: Inactivation of Akt by the epidermal growth factor receptor inhibitor erlotinib is mediated by HER-3 in pancreatic and colorectal tumor cell lines and contributes to erlotinib sensitivity. Mol Cancer Ther. 5:2051–2059. 2006. View Article : Google Scholar : PubMed/NCBI
28.	Engelman JA, Jänne PA, Mermel C, Pearlberg J, Mukohara T, Fleet C, Cichowski K, Johnson BE and Cantley LC: ErbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines. Proc Natl Acad Sci USA. 102:3788–3793. 2005. View Article : Google Scholar : PubMed/NCBI
29.	Engelman JA, Zejnullahu K, Mitsudomi T, et al: MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 316:1039–1043. 2007. View Article : Google Scholar : PubMed/NCBI
30.	Turke AB, Zejnullahu K, Wu YL, et al: Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC. Cancer Cell. 17:77–88. 2010. View Article : Google Scholar : PubMed/NCBI
31.	Arteaga CL: HER3 and mutant EGFR meet MET. Nat Med. 13:675–677. 2007. View Article : Google Scholar : PubMed/NCBI