Jennifer Sigmond

Induction of resistance to the multitargeted antifolate Pemetrexed (ALIMTA) in WiDr human colon cancer cells is associated with thymidylate synthase overexpression

Pemetrexed (ALIMTA, MTA) is a novel thymidylate synthase (TS) inhibitor and has shown activity against colon cancer, mesothelioma and nonsmall-cell lung cancer. We induced resistance to Pemetrexed in the human colon cancer cell line WiDr by using a continuous exposure to stepwise increasing Pemetrexed concentrations (up to 20 microM) as well as a more clinically relevant schedule with intermittent exposure (up to 50 microM) for 4 hr every 7 days, resulting in WiDr variants WiDr-cPEM and WiDr-4PEM, respectively. However, using the same conditions, it was not possible to induce resistance in the WiDr/F cell line, a variant adapted to growth under low folate conditions. Mechanisms of resistance to Pemetrexed were determined at the level of TS, folylpolyglutamate synthetase (FPGS) and reduced folate carrier (RFC). WiDr-4PEM and WiDr-cPEM showed cross-resistance to the polyglutamatable TS inhibitor Raltitrexed (6- and 19-fold, respectively) and the nonpolyglutamatable TS-inhibitor Thymitaq (6- and 42-fold, respectively) but not to 5-fluorouracil. The ratios of TS mRNA:beta actin mRNA in WiDr-4PEM and WiDr-cPEM were 5-fold (P=0.01) and 18-fold (P=0.04) higher, respectively, compared to WiDr (ratio: 0.012). In addition, TS protein expression in the resistant WiDr variants was elevated 3-fold compared to WiDr, while the catalytic activity of TS with 1 microM dUMP increased from 30 pmol/hr/10(6) cells in WiDr cells to 2201 and 7663 pmol/hr/10(6) cells in WiDr-4PEM and WiDr-cPEM, respectively. The activity of FPGS was moderately decreased, but not significantly different in all WiDr variants. Finally, no evidence was found that decreased catalytic activity of RFC was responsible for the obtained Pemetrexed resistance. Altogether, these results indicate that resistance to Pemetrexed in the colon cancer cell line WiDr was solely due to upregulation of TS of which all related parameters (mRNA and protein expression and TS activity) were increased, rather than alterations in FPGS or RFC activity.

Pyrimidine and Purine Analogues, Effects on Cell Cycle Regulation and the Role of Cell Cycle Inhibitors to Enhance Their Cytotoxicity

In anti-cancer treatment, deoxynucleoside analogues are widely used in combination chemotherapy. Improvement can be achieved by rational design of novel combinations with cell cycle inhibitors. These compounds inhibit protein kinases, preventing the cell cycle from continuing when affected by deoxynucleoside analogs. The efficacy is dependent on the site of cell cycle inhibition, whether multiple cyclin-dependent kinases are inhibited and whether the inhibitors should be given before or after the deoxynucleoside analogs. The action of cell cycle inhibition in vivo may be limited by unfavorable pharmacokinetics. Preclinical and clinical studies will be discussed, aiming to design improved future strategies.

Staurosporine increases toxicity of gemcitabine in non-small cell lung cancer cells: role of protein kinase C, deoxycytidine kinase and ribonucleotide reductase

Gemcitabine, a deoxycytidine analog, active against non-small cell lung cancer, is phosphorylated by deoxycytidine kinase (dCK) to active nucleotides. Earlier, we found increased sensitivity to gemcitabine in P-glycoprotein (SW-2R160) and multidrug resistance-associated protein (SW-2R120), overexpressing variants of the human SW1573 non-small cell lung cancer cells. This was related to increased dCK activity. As protein kinase C (PKC) is higher in 2R120 and 2R160 cells and may control the dCK activity, we investigated whether gemcitabine sensitivity was affected by the protein kinase C inhibitor, staurosporine, which also modulates the cell cycle. Ten nmol/l staurosporine enhanced the sensitivity of SW1573, 2R120 and 2R160 cells 10-fold, 50-fold and 270-fold, respectively. Staurosporine increased dCK activity about two-fold and the activity of thymidine kinase 2, which may also activate gemcitabine. Staurosporine also directly increased dCK in cell free extracts. Staurosporine decreased expression of the free transcription factor E2F and of ribonucleotide reductase (RNR), a target for gemcitabine inhibition. In conclusion, staurosporine may potentiate gemcitabine by increasing dCK and decreasing E2F and RNR, which will lead to a more pronounced RNR inhibition.

Pharmacological Aspects of the Enzastaurin-Pemetrexed Combination in Non-Small Cell Lung Cancer (NSCLC)

Conventional regimens have limited impact against NSCLC. Current research is focusing on multiple pathways as potential targets, and this review describes pharmacological aspects underlying the combination of the PKCbeta-inhibitor enzastaurin with the multitargeted antifolate pemetrexed. Pemetrexed is commonly used, alone or combined with platinum compounds, in NSCLC treatment, and ongoing studies are evaluating its target, thymidylate synthase (TS), as predictor of drug activity. Enzastaurin is a biological targeted agent being actively investigated against different tumors as single agent or in combination. All the downstream events following PKCbeta inhibition by enzastaurin are not completely known, and assays to evaluate possible biomarkers, such as expression of PKC, VEGF and GSK3beta, in tissues and/or in blood samples, are being developed. Enzastaurin-pemetrexed combination was synergistic in preclinical models, including NSCLC cells, where enzastaurin reduced phosphoCdc25C, resulting in G2/M-checkpoint abrogation, and Akt and GSK3beta; phosphorylation, favoring apoptosis induction in pemetrexed-damaged cells. Enzastaurin also significantly reduced VEGF secretion and pemetrexed-induced upregulation of TS expression, possibly via E2F-1 reduction, while the combination decreased TS activity. Similarly, the accumulation of deoxyuridine (a marker of TS inhibition) and the reduction of GSK3beta phosphorylation were detectable in clinical samples from a phase-Ib trial of pemetrexed-enzastaurin combination. In conclusion, the favorable toxicity profile and the multiple effects of enzastaurin on signaling pathways involved in cell cycle control, apoptosis and angiogenesis, as well as on proteins involved in pemetrexed activity, provide experimental basis for future studies on enzastaurin-pemetrexed combination and their possible pharmacodynamic markers in NSCLC patients.

Combinations of 5-fluorouracil with UCN-01 or staurosporine.

The action of 5‐Fluorouracil (5‐FU) is mediated by inhibition of thymidylate synthase (TS), which is regulated by cell cycle proteins controlled by protein phosphorylation. We studied the effects of staurosporine and its analogue UCN‐01, inhibitors of protein kinase C (PKC) on 5‐FU cytotoxicity in Lovo colon cancer cells. Each drug contributes equally to the cell cycle effects of the 5‐FU combinations. In sequential drug administration, the cell cycle distribution was determined by the first drug. Simultaneous 5‐FU combinations induced additive effects in induction of apoptosis. When staurosporine was used as the second drug, induction of apoptosis was 2‐fold higher than the sum of both drugs alone. Based on induction of apoptosis 5‐FU addition prior to the PKC inhibitors seemed preferable.

Enhanced Activity of Deoxycytidine Kinase After Pulsed Low Dose Rate and Single Dose Gamma Irradiation

In both pulsed low dose rate (LDR) and single high dose radiation schedules, gemcitabine pretreatment sensitizes tumor cells to radiation. These radiosensitizing effects could be the result of decreased DNA repair. In this study, the effect of irradiation on the deoxycytidine kinase (dCK) needed for DNA repair was investigated. The activity of dCK, a deoxynucleoside analogue-activating enzyme was increased upon irradiation in both schedules. No change in dCK protein expression was observed that indicates a post-translational regulation. The benefit of this increased activity induced by irradiation should be further investigated in combination with deoxynucleoside analogues activated by this enzyme.

Quantitative real time PCR of deoxycytidine kinase mRNA by Light Cycler PCR; in relation to enzyme activity.

Deoxycytidine kinase (dCK) is essential for the phosphorylation of several deoxyribonucleosides and various analogues such as gemcitabine (2′,2′‐difluorodeoxycytidine). We developed and optimized a sensitive real time Light Cycler (LC) PCR assay for dCK with SYBR green detection. The enzymatic activity measured in the same human xenografts of dCK correlated excellently with dCK mRNA expression levels measured by the LC. This assay can be used for evaluation of dCK expression in tumors.

Induction of Resistance to the Multi-Targeted Antifolate MTA (LY231514) in Widr Human Colon Cancer Cells

The enzyme thymidylate synthase (TS) catalyses the methylation of 2′-deoxyuridine-5′monophosphate (dUMP) to 2′-deoxythymidine-5′-monophosphate (dTMP), an essential precursor during DNA synthesis (1). 5,10-Methylene tetrahydrofolate (CH2-THF) is the limiting methyl donor during this reaction. TS, is usually elevated in tumors (2) and is therefore an interesting target for anticancer agents such as the antifolate multi-targeted antifolate (MTA, ALIMTA, PEMETREXED)(3,4), which inhibits activity of TS by competition with the binding site of CH2-THF of TS. MTA is currently been developed as an anticancer agent for treatment of colorectal, non-small-cell lung cancer and mesothelioma (4). Resistance to this agent might develop similarly to other antifolates, and might be due to elevated activity or mutations of the target enzyme TS, impaired polyglutamation of antifolates, decreased transport into the cells, but also apoptosis regulating proteins may be involved (5,6). For this purpose we induced resistance to the antifolate MTA in the colon cancer cell line WiDr by exposure to 50 mM MTA for 4 hours every week, resulting in the WiDr-4LY cell line, and by continuous exposure for 72 hr to 20 mM MTA every week, resulting in WiDr-cLY. The resistant WiDr variants were studied on the level of TS and folyl polyglutamate synthetase (FPGS).