Top

Published in:

Open Access 01-08-2010 | Original Article

Cellular and molecular mechanisms for the synergistic cytotoxicity elicited by oxaliplatin and pemetrexed in colon cancer cell lines

Authors: Sara Nannizzi, Gareth J. Veal, Elisa Giovannetti, Valentina Mey, Simona Ricciardi, Christopher J. Ottley, Mario Del Tacca, Romano Danesi

Published in: Cancer Chemotherapy and Pharmacology | Issue 3/2010

Abstract

Purpose

Oxaliplatin effect in the treatment of colorectal cancer is improved upon combination with thymidylate synthase (TS) inhibitors. Pemetrexed is polyglutamated by the folylpolyglutamate synthase (FPGS) and blocks folate metabolism and DNA synthesis by inhibiting TS, dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). The present study evaluates the pharmacological interaction between oxaliplatin and pemetrexed in colorectal cancer cells.

Methods

Human HT29, WiDr, SW620 and LS174T cells were treated with oxaliplatin and pemetrexed. Drug interaction was studied using the combination index method, while cell cycle was investigated with flow cytometry. The effects of drugs on Akt phosphorylation and apoptosis were studied with ELISA and fluorescence microscopy, respectively. RT-PCR analysis was performed to assess whether drugs modulated the expression of pemetrexed targets and of genes involved in DNA repair (ERCC1 and ERCC2). Finally, platinum–DNA adduct levels were detected by ultra-sensitive multi-collector inductively coupled plasma mass spectrometry (ICP-MS).

Results

A dose-dependent inhibition of cell growth was observed after drug exposure, while a synergistic interaction was detected preferentially with sequential combinations. Oxaliplatin enhanced cellular population in the S-phase. Drug combinations increased apoptotic indices with respect to single agents, and both drugs inhibited Akt phosphorylation. RT-PCR analysis showed a correlation between the FPGS/(TS × DHFR × GARFT) ratio and pemetrexed sensitivity, as well as a downregulation of ERCC1, ERCC2, TS, DHFR and GARFT after drug exposure. In addition, pretreatment with pemetrexed resulted in an increase of oxaliplatin–DNA adducts.

Conclusion

These data demonstrate that oxaliplatin and pemetrexed synergistically interact against colon cancer cells, through modulation of cell cycle, inhibition of Akt phosphorylation, induction of apoptosis and modulation of gene expression.

Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ (2009) Cancer statistics, 2009. CA Cancer J Clin 59:225–249CrossRefPubMed

Goldberg RM (2005) Advances in the treatment of metastatic colorectal cancer. Oncologist 10:40–48CrossRefPubMed

Meyerhardt JA, Mayer RJ (2005) Systemic therapy for colorectal cancer. N Engl J Med 352:476–487CrossRefPubMed

Raymond E, Faivre S, Chaney S, Woynarowski J, Cvitkovic E (2002) Cellular and molecular pharmacology of oxaliplatin. Mol Cancer Ther 1:227–235PubMed

Kweekel DM, Gelderblom H, Guchelaar HJ (2005) Pharmacology of oxaliplatin and the use of pharmacogenomics to individualize therapy. Cancer Treat Rev 31:90–105CrossRefPubMed

Reardon JT, Vaisman A, Chaney SG, Sancar A (1999) Efficient nucleotide excision repair of cisplatin, oxaliplatin, and Bis-aceto-ammine-dichloro-cyclohexylamine-platinum(IV) (JM216) platinum intrastrand DNA diadducts. Cancer Res 59:3968–3971PubMed

Arnould S, Hennebelle I, Canal P, Bugat R, Guichard S (2003) Cellular determinants of oxaliplatin sensitivity in colon cancer cell lines. Eur J Cancer 39:112–119CrossRefPubMed

Chang IY, Kim MH, Kim HB, Lee DY, Kim SH, Kim HY, You HJ (2005) Small interfering RNA-induced suppression of ERCC1 enhances sensitivity of human cancer cells to cisplatin. Biochem Biophys Res Commun 327:225–233CrossRefPubMed

Shirota Y, Stoehlmacher J, Brabender J, Xiong YP, Uetake H, Danenberg KD, Groshen S, Tsao-Wei DD, Danenberg PV, Lenz HJ (2001) ERCC1 and thymidylate synthase mRNA levels predict survival for colorectal cancer patients receiving combination oxaliplatin and fluorouracil chemotherapy. J Clin Oncol 19:4298–4304PubMed

10.

Xu Z, Chen ZP, Malapetsa A, Alaoui-Jamali M, Bergeron J, Monks A, Myers TG, Mohr G, Sausville EA, Scudiero DA, Aloyz R, Panasci LC (2002) DNA repair protein levels vis-a-vis anticancer drug resistance in the human tumor cell lines of the National Cancer Institute drug screening program. Anticancer Drugs 13:511–519CrossRefPubMed

11.

Peters GJ, Backus HH, Freemantle S, van Triest B, Codacci-Pisanelli G, van der Wilt CL, Smid K, Lunec J, Calvert AH, Marsh S, McLeod HL, Bloemena E, Meijer S, Jansen G, van Groeningen CJ, Pinedo HM (2002) Induction of thymidylate synthase as a 5-fluorouracil resistance mechanism. Biochim Biophys Acta 1587:194–205PubMed

12.

Adjei AA (1999) A review of the pharmacology and clinical activity of new chemotherapy agents for the treatment of colorectal cancer. Br J Clin Pharmacol 48:265–277CrossRefPubMed

13.

Bocci G, Danesi R, Allegrini G, Innocenti F, Di Paolo A, Falcone A, Conte PF, Del Tacca M (2002) Severe 5-fluorouracil toxicity associated with a marked alteration of pharmacokinetics of 5-fluorouracil and its catabolite 5-fluoro-5, 6-dihydrouracil: a case report. Eur J Clin Pharmacol 58:593–595CrossRefPubMed

14.

Shih C, Chen VJ, Gossett LS, Gates SB, MacKellar WC, Habeck LL, Shackelford KA, Mendelsohn LG, Soose DJ, Patel VF, Andis SL, Bewley JR, Rayl EA, Moroson BA, Beardsley GP, Kohler W, Ratnam M, Schultz RM (1997) LY231514, a pyrrolo[2, 3-d]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes. Cancer Res 57:1116–1123PubMed

15.

Chattopadhyay S, Moran RG, Goldman ID (2007) Pemetrexed: biochemical and cellular pharmacology, mechanisms, and clinical applications. Mol Cancer Ther 6:404–417CrossRefPubMed

16.

Giovannetti E, Mey V, Danesi R, Mosca I, Del Tacca M (2004) Synergistic cytotoxicity and pharmacogenetics of gemcitabine and pemetrexed combination in pancreatic cancer cell lines. Clin Cancer Res 10:2936–2943CrossRefPubMed

17.

Scagliotti GV, Parikh P, von Pawel J, Biesma B, Vansteenkiste J, Manegold C, Serwatowski P, Gatzemeier U, Digumarti R, Zukin M, Lee JS, Mellemgaard A, Park K, Patil S, Rolski J, Goksel T, de Marinis F, Simms L, Sugarman KP, Gandara D (2008) Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer. J Clin Oncol 26:3543–3551CrossRefPubMed

18.

Atkins JN, Jacobs SA, Wieand HS, Smith RE, John WJ, Colangelo LH, Vogel VG, Kuebler JP, Cescon TP, Miller BJ, Geyer CE Jr, Wolmark N (2005) Pemetrexed/oxaliplatin for first-line treatment of patients with advanced colorectal cancer: a phase II trial of the National Surgical Adjuvant Breast and Bowel Project Foundation Research Program. Clin Colorectal Cancer 5:181–187CrossRefPubMed

19.

Giovannetti E, Lemos C, Tekle C, Smid K, Nannizzi S, Rodriguez JA, Ricciardi S, Danesi R, Giaccone G, Peters GJ (2008) 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 73:1290–1300CrossRefPubMed

20.

Giovannetti E, Mey V, Nannizzi S, Pasqualetti G, Marini L, Del Tacca M, Danesi R (2005) Cellular and pharmacogenetics foundation of synergistic interaction of pemetrexed and gemcitabine in human non-small-cell lung cancer cells. Mol Pharmacol 68:110–118PubMed

21.

Cooper B, Veal GJ, Radivoyevitch T, Tilby MJ, Meyerson HJ, Lazarus HM, Koc ON, Creger RJ, Pearson G, Nowell G, Gosky D, Ingalls ST, Hoppel CL, Gerson SL (2004) A Phase I and pharmacodynamic study of fludarabine, carboplatin and topotecan in patients with relapsed, refractory, or high-risk acute leukemia. Clin Cancer Res 10:6830–6839CrossRefPubMed

22.

Veal GJ, Errington J, Tilby MJ, Pearson ADJ, Foot ABM, McDowell H, Ellershaw C, Pizer B, Nowell GM, Pearson DG, Boddy AV (2007) Adaptive dosing and platinum–DNA adduct formation in children receiving high dose carboplatin for the treatment of solid tumours. Br J Cancer 96:725–731CrossRefPubMed

23.

Raymond E, Louvet C, Tournigand C, Coudray AM, Faivre S, De Gramont A, Gespach C (2002) Pemetrexed disodium combined with oxaliplatin, SN38, or 5-fluorouracil, based on the quantitation of drug interactions in human HT29 colon cancer cells. Int J Oncol 21:361–367PubMed

24.

Celio L, Sternberg CN, Labianca R, La Torre I, Amoroso V, Barone C, Pinotti G, Cascinu S, Di Costanzo F, Cetto GL, Bajetta E (2009) Pemetrexed in combination with oxaliplatin as a first-line therapy for advanced gastric cancer: a multi-institutional phase II study. Ann Oncol 20:1062–1067CrossRefPubMed

25.

Arnould S, Guichard S, Hennebelle I, Cassar G, Bugat R, Canal P (2002) Contribution of apoptosis in the cytotoxicity of the oxaliplatin-irinotecan combination in the HT29 human colon adenocarcinoma cell line. Biochem Pharmacol 64:1215–1226CrossRefPubMed

26.

Faivre S, Chan D, Salinas R, Woynarowska B, Woynarowski JM (2003) DNA strand breaks and apoptosis induced by oxaliplatin in cancer cells. Biochem Pharmacol 66:225–237CrossRefPubMed

27.

Troiani T, Lockerbie O, Morrow M, Ciardiello F, Eckhardt SG (2006) Sequence-dependent inhibition of human colon cancer cell growth and of prosurvival pathways by oxaliplatin in combination with ZD6474 (Zactima), an inhibitor of VEGFR and EGFR tyrosine kinases. Mol Cancer Ther 5:1883–1894CrossRefPubMed

28.

Temmink OH, Hoebe EK, van der Born K, Ackland SP, Fukushima M, Peters GJ (2007) Mechanism of trifluorothymidine potentiation of oxaliplatin-induced cytotoxicity to colorectal cancer cells. Br J Cancer 96:231–240CrossRefPubMed

29.

Mey V, Giovannetti E, De Braud F, Nannizzi S, Curigliano G, Verweij F, De Cobelli O, Pece S, Del Tacca M, Danesi R (2006) In vitro synergistic cytotoxicity of gemcitabine and pemetrexed and pharmacogenetic evaluation of response to gemcitabine in bladder cancer patients. Br J Cancer 95:289–297CrossRefPubMed

30.

Tonkinson JL, Worzalla JF, Teng CH, Mendelsohn LG (1999) Cell cycle modulation by a multitargeted antifolate, LY231514, increases the cytotoxicity and antitumor activity of gemcitabine in HT29 colon carcinoma. Cancer Res 59:3671–3676PubMed

31.

Fresno Vara JA, Casado E, de Castro J, Cejas P, Belda-Iniesta C, Gonzalez-Baron M (2004) PI3K/Akt signalling pathway and cancer. Cancer Treat Rev 30:193–204CrossRefPubMed

32.

Nemoto S, Nakamura M, Osawa Y, Kono S, Itoh Y, Okano Y, Murate T, Hara A, Ueda H, Nozawa Y, Banno Y (2009) Sphingosine kinase isoforms regulate oxaliplatin sensitivity of human colon cancer cells through ceramide accumulation and Akt activation. J Biol Chem 284:10422–10432CrossRefPubMed

33.

Arango D, Wilson AJ, Shi Q, Corner GA, Aranes MJ, Nicholas C, Lesser M, Mariadason JM, Augenlicht LH (2004) Molecular mechanisms of action and prediction of response to oxaliplatin in colorectal cancer cells. Br J Cancer 91:1931–1946CrossRefPubMed

34.

Hanada M, Feng J, Hemmings BA (2004) Structure, regulation and function of PKB/AKT—a major therapeutic target. Biochim Biophys Acta 1697:3–16PubMed

35.

Kim JH, Lee KW, Jung Y, Kim TY, Ham HS, Jong HS, Jung KH, Im SA, Kim TY, Kim NK, Bang YJ (2005) Cytotoxic effects of pemetrexed in gastric cancer cells. Cancer Sci 96:365–371CrossRefPubMed

36.

Rots MG, Willey JC, Jansen G, Van Zantwijk CH, Noordhuis P, DeMuth JP, Kuiper E, Veerman AJ, Pieters R, Peters GJ (2000) mRNA expression levels of methotrexate resistance-related proteins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method. Leukemia 14:2166–2175CrossRefPubMed

37.

Matsumoto S, Igishi T, Hashimoto K, Kodani M, Shigeoka Y, Nakanishi H, Touge H, Kurai J, Makino H, Takeda K, Yasuda K, Hitsuda Y, Shimizu E (2004) Schedule-dependent synergism of vinorelbine and 5-fluorouracil/UFT against non-small cell lung cancer. Int J Oncol 25:1311–1318PubMed

38.

van der Wilt CL, van Laar JA, Gyergyay F, Smid K, Peters GJ (1992) Biochemical modification of the toxicity and the anti-tumour effect of 5-fluorouracil and cis-platinum by WR-2721 in mice. Eur J Cancer 28A:2017–2024CrossRefPubMed

39.

Guichard S, Hennebelle I, Bugat R, Canal P (1998) Cellular interactions of 5-fluorouracil and the camptothecin analogue CPT-11 (irinotecan) in a human colorectal carcinoma cell line. Biochem Pharmacol 55:667–676CrossRefPubMed

40.

Yeh KH, Cheng AL, Wan JP, Lin CS, Liu CC (2004) Down-regulation of thymidylate synthase expression and its steady-state mRNA by oxaliplatin in colon cancer cells. Anticancer Drugs 15:371–376CrossRefPubMed

41.

Novakovic P, Stempak JM, Sohn KJ, Kim YI (2006) Effects of folate deficiency on gene expression in the apoptosis and cancer pathways in colon cancer cells. Carcinogenesis 27:916–924CrossRefPubMed

42.

Fujishima H, Nakano S, Masumoto N, Esaki T, Tatsumoto T, Kondo T, Niho Y (1997) Inhibition by 5-fluorouracil of ERCC1 and gamma-glutamylcysteine synthetase messenger RNA expression in a cisplatin-resistant HST-1 human squamous carcinoma cell line. Oncol Res 9:167–172PubMed

43.

Ojima E, Inoue Y, Watanabe H, Hiro J, Toiyama Y, Miki C, Kusunoki M (2006) The optimal schedule for 5-fluorouracil radiosensitization in colon cancer cell lines. Oncol Rep 16:1085–1091PubMed

44.

Balin-Gauthier D, Delord JP, Pillaire MJ, Rochaix P, Hoffman JS, Bugat R, Cazaux C, Canal P, Allal BC (2008) Cetuximab potentiates oxaliplatin cytotoxic effect through a defect in NER and DNA replication initiation. Br J Cancer 98:120–128CrossRefPubMed

45.

Kopetz S, Lesslie DP, Dallas NA, Park SI, Johnson M, Parikh NU, Kim MP, Abbruzzese JL, Ellis LM, Chandra J, Gallick GE (2009) Synergistic activity of the Src family kinase inhibitor dasatinib and oxaliplatin in colon carcinoma cells is mediated by oxidative stress. Cancer Res 69:3842–3849CrossRefPubMed

Title: Cellular and molecular mechanisms for the synergistic cytotoxicity elicited by oxaliplatin and pemetrexed in colon cancer cell lines
Authors: Sara Nannizzi
Gareth J. Veal
Elisa Giovannetti
Valentina Mey
Simona Ricciardi
Christopher J. Ottley
Mario Del Tacca
Romano Danesi
Publication date: 01-08-2010
Publisher: Springer-Verlag
Published in: Cancer Chemotherapy and Pharmacology / Issue 3/2010
Print ISSN: 0344-5704
Electronic ISSN: 1432-0843
DOI: https://doi.org/10.1007/s00280-009-1195-2

Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras

Prof. Fabrice Barlesi

Developed by: Springer Medicine

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Purpose

Methods

Results

Conclusion

Please log in to get access to this content

Other articles of this Issue 3/2010

Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce apoptosis in multi-drug-resistant cancer cells

GCS induces multidrug resistance by regulating apoptosis-related genes in K562/AO2 cell line

Bendamustine: something old, something new

Patterns of relapse and metastatic spread in HER2-overexpressing breast cancer according to estrogen receptor status

Anemia response and safety to epoetin-beta treatment in patients with neoadjuvant therapy prior to primary digestive tract tumor surgery

Differential apoptotic response of human cancer cells to organoselenium compounds

Keynote webinar | Spotlight on antibody–drug conjugates in cancer