Determinants of trimetrexate lethality in human colon cancer cells

Abstract

Summary We examined the cytotoxicity and biochemical effects of the lipophilic antifol trimetrexate (TMQ) in two human colon carcinoma cell lines, SNU-C4 and NCI-H630, with different inherent sensitivity to TMQ. While a 24 h exposure to 0.1 M TMQ inhibited cell growth by 50-60% in both cell lines, it did not reduce clonogenic survival. A 24 h exposure to 1 and 10 ILM TMQ produced 42% and 50% lethality in C4 cells, but did not affect H630 cells. Dihydrofolate reductase (DHFR) and thymidylate synthase were quantitatively and qualitatively similar in both lines. During drug exposure, DHFR catalytic activity was inhibited by > 85% in both cell lines; in addition, the reduction in apparent free DHFR binding capacity (,<20% of control), depletion of dTTP, ATP and GTP pools and inhibition of [6-3H]deoxyuridine incorporation into DNA were similar in C4 and H630 cells. TMQ produced a more striking alteration of the pH step alkaline elution profile of newly synthesised DNA in C4 cells compared with 630 cells, however, indicating greater interference with DNA chain elongation or more extensive DNA damage. When TMQ was removed after a 24 h exposure to 0.1 tM, recovery of DHFR catalytic activity and apparent free DHFR binding sites was evident over the next 24 -48 h in both cell lines. With 1 and 10 JAM, however, persistent inhibition of DHFR was evident in C4 cells, whereas DHFR recovered in H630 cells. These data suggest that, although DHFR inhibition during TMQ exposure produced growth inhibition, DHFR catalytic activity 48 h after drug removal was a more accurate predictor of lethality in these two cell lines. Several factors appeared to influence the duration of DHFR inhibition after drug removal, including initial TMQ concentration, declining cytosolic TMQ levels after drug removal, the ability to acutely increase total DHFR content and the extent of TMQ-mediated DNA damage. The greater sensitivity of C4 cells to TMQ-associated lethality may be attributed to the greater extent of TMQ-mediated DNA damage and more prolonged duration of DHFR inhibition after drug exposure.

From methotrexate to pemetrexed and beyond. A review of the pharmacodynamic and clinical properties of antifolates

“…Whilst this observation might support the contention that TMTX has an advantage over MTX, multidrug resistant cell lines are resistant to TMTX [86][87][88]. An alternative mechanism of resistance results from increased levels of DHFR occurring via a posttranslational mechanism [89,90].…”

Folate receptor 1 is necessary for neural plate cell apical constriction during Xenopus neural tube formation

“…Chemotherapeutic efficacy and DHFR inhibitory effect of the drugs have been demonstrated in vitro, in human colon and Chinese hamster ovary cancer cells and in mouse leukemia cells, and in vivo, in mice inoculated with leukemia cells and in rat intestine (Grem et al, 1994;Hook et al, 1986;Zimmerman et al, 1987). Results show that none of the lipophilic DHFR inhibitors, used at saturating doses, induces NTDs (Fig.…”

“…Hence, this model system provides the opportunity to determine novel mechanisms triggered by folate action. Indeed, we find that inhibiting one of the most important enzymes of folate metabolism using analogs of methotrexate that bypass the uptake systems due to their lipophilic nature (Grem et al, 1994;Hook et al, 1986;Zimmerman et al, 1987), does not induce NTDs, unlike methotrexate, suggesting that folate interaction with its receptor is relevant for the formation of the neural tube besides its action as a vitamin and growth-promoting factor.…”

“…We assessed the effect of three lipophilic methotrexate analogs that also inhibit dihydrofolate reductase (DHFR) but, unlike MTX, do not affect folate interaction with the folate receptor or carrier (Grem et al, 1994;Hook et al, 1986;Zimmerman et al, 1987). Chemotherapeutic efficacy and DHFR inhibitory effect of the drugs have been demonstrated in vitro, in human colon and Chinese hamster ovary cancer cells and in mouse leukemia cells, and in vivo, in mice inoculated with leukemia cells and in rat intestine (Grem et al, 1994;Hook et al, 1986;Zimmerman et al, 1987).…”

Synthetic Lethal Interactions Prediction Based on Multiple Similarity Measures Fusion

“…Researches show that Trimetrexate has potential anti-cancer activity and can be used to treat several types of cancer including colon cancer by inhibiting DHFR [41,42] .…”