Cellular and Pharmacogenetics Foundation of Synergistic Interaction of Pemetrexed and Gemcitabine in Human Non–Small-Cell Lung Cancer Cells

Abstract

Gemcitabine and pemetrexed are effective agents in the treatment of non-small-cell lung cancer (NSCLC), and the present study investigates cellular and genetic aspects of their interaction against A549, Calu-1, and Calu-6 cells. Cells were treated with pemetrexed and gemcitabine, and their interaction was assessed using the combination index. The role of drug metabolism in gemcitabine cytotoxicity was examined with inhibitors of deoxycytidine kinase (dCK), 5Ј-nucleotidase, and cytidine deaminase, whereas the role of pemetrexed targets, thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) in drug chemosensitivity was analyzed in cytotoxicity rescue studies. The effect of gemcitabine and pemetrexed on Akt phosphorylation was investigated with enzyme-linked immunosorbent assay, whereas quantitative polymerase chain reaction (PCR) was used to study target gene-expression profiles and its modulation by each drug. Synergistic cytotoxicity was demonstrated, and pemetrexed significantly decreased the amount of phosphorylated Akt, enhanced apoptosis, and increased the expression of dCK in A549 and Calu-6 cells, as well as the expression of the human nucleoside equilibrative transporter 1 (hENT1) in all cell lines. PCR demonstrated a correlation between dCK expression and gemcitabine sensitivity, whereas expression of TS, DHFR, and GARFT was predictive of pemetrexed chemosensitivity. These data demonstrated that 1) gemcitabine and pemetrexed synergistically interact against NSCLC cells through the suppression of Akt phosphorylation and induction of apoptosis; 2) the gene expression profile of critical genes may predict for drug chemosensitivity; and 3) pemetrexed enhances dCK and hENT1 expression, thus suggesting the role of gene-expression modulation for rational development of chemotherapy combinations.Despite recent advances in early diagnosis and treatment, non-small-cell lung cancer (NSCLC) is a disease with a grim prognosis. Extensive clinical studies demonstrated that chemotherapy increases survival in the adjuvant setting (Arriagada et al., 2004) and in patients with advanced disease (Reck and Gatzemeier, 2004). Nonetheless, response rates remain lower than 15%, and median survival is less than 6 months, thus emphasizing the need for new effective drugs and combination regimens (Rosell and Crinò, 2002). However, the rationale for chemotherapy combinations has remained mostly empirical, determined from the antitumor activity of each agent and the lack of overlapping toxicities, despite many attempts to discover preclinical models for rational selection of drug interactions.Gemcitabine (2Ј,2Ј-difluorodeoxycytidine) is a deoxycytidine analog with a broad spectrum of anticancer activity against several solid tumors in preclinical models, and it is M.D.T. was supported by unrestricted research grants from Eli Lilly (Florence, Italy). R. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.doi:10.1124/...

In vitro synergistic cytotoxicity of gemcitabine and pemetrexed and pharmacogenetic evaluation of response to gemcitabine in bladder cancer patients

“…P-Ser473 Akt and Akt total concentrations were calculated from standard curves and values of P-Ser473 Akt were normalised for total Akt and protein content, which was measured with the Lowry reagent (Sigma), as described previously (Giovannetti et al, 2005).…”

“…Forward and reverse primers and probes for dCK (NM_000788), cN-II 5 0 -NT (NM_012229), CDA (NM_001785), TS (NM_0010711), DHFR (NM_000791) and GARFT (NM_000819) were designed on the basis of the gene sequence obtained from the GeneBank database with the Primer Express software (Applied Biosystems, V.2.0), as previously described (Giovannetti et al, 2005); while primers and probes for hENT1 (NM_004955), hCNT1 (NM_004213) and for the regulatory M1 (NM_001033), and catalytic M2 (NM_001034) subunits of RR were obtained from Applied Biosystems Assay-on-Demand s products (Hs00168784, Hs00188418, Hs0035724 and Hs00191940).…”

“…Cells plated and treated with gemcitabine alone or in combination with pemetrexed (0.3 nM -33.3 mM) as described in 'Assay of Cytotoxicity', were simultaneously exposed to 2 0 -deoxycytidine (10 mM), to inhibit drug activation by phosphorylation (dCK), to the specific hENT1 inhibitor nitrobenzylthioinosine (NBMPR, 100 nM) or to dipyridamole, a non-specific inhibitor of nucleoside transporters, used at concentration of 10 mM, as previously described (Giovannetti et al, 2005).…”

“…Modulation of enzymes involved in gemcitabine uptake and metabolism may influence drug activity against human tumour cell lines and, among chemotherapeutic drugs, pemetrexed appears to be a potential candidate because of its ability to deplete cellular nucleotide pools by inhibiting nucleotide synthesis (Giovannetti et al, 2005). Indeed, pemetrexed and its polyglutamates are potent, tight-binding inhibitors of folate-dependent enzymes, including thymidylate synthase (TS), dihydrofolate reductase (DHFR), and glycinamide ribonucleotide formyltransferase (GARFT) and show activity against a wide variety of solid tumours, including bladder cancer (Calvert, 2004).…”

Expression of gemcitabine- and cisplatin-related genes in non-small-cell lung cancer

“…37 On the contrary, patients with adenocarcinoma had better outcome in the regimen containing pemetrexed in association with cisplatin, possibly because of the lower expression levels of the main target of pemetrexed, thymidylate synthase in adenocarcinoma than in squamous-cell carcinoma. 39,40 The predictive role for histology for pemetrexed was supported by phase II studies and by a retrospective review of two large, randomized, phase III trial. 38 A recent review of 408 publications reported that 11 single-arm or pooled population studies showed …”