nolatrexed and Colorectal-Neoplasms

Topics: Antineoplastic Agents; Clinical Trials as Topic; Colorectal Neoplasms; Enzyme Inhibitors; Folic Acid; Glutamates; Guanine; Humans; Pemetrexed; Quinazolines; Thiophenes; Thymidylate Synthase

Thymidylate synthase (TS) is a critical enzyme for DNA replication and cell growth because it is the only de novo source of thymine nucleotide precursors for DNA synthesis. TS is the primary target of 5-fluorouracil (5-FU), which has been used for cancer treatment for more than 40 years. However, dissatisfaction with the overall activity of 5-FU against the major cancers, and the recognition that TS still remains an attractive target for anticancer drugs because of its central position in the pathway of DNA synthesis, led to a search for new inhibitors of TS structurally analogous to 5,10-methylenetetrahydrofolate, the second substrate of TS. TS inhibitory antifolates developed to date that are in various stages of clinical evaluation are ZD 1694 and ZD9331 (Astra-Zeneca, London, UK), (Eli Lilly, Indianapolis, IN), LY231514 (BW1843U89 (Glaxo-Wellcome, Research Triangle Park, NC), and AG337 and AG331 (Agouron, La Jolla, CA). Although each of these compounds has TS as its major intracellular site of action, they differ in propensity for polyglutamylation and for transport by the reduced folate carrier. LY231514 also has secondary target enzymes. As a result, each compound is likely to have a different spectrum of antitumor activity and toxicity. This review will summarize the development and properties of this new class of TS inhibitors.

Topics: Animals; Antimetabolites, Antineoplastic; Colorectal Neoplasms; Enzyme Inhibitors; Folic Acid; Folic Acid Antagonists; Glutamates; Guanine; Humans; Indoles; Isoindoles; Pemetrexed; Quinazolines; Thiophenes; Thymidylate Synthase

To investigate the effect of the RNAi and the chemotherapy drugs nolatrxed on the expression of thymidylate synthase(TS) and the growth of the colorectal carcinoma LOVO cells.. The siRNA was constructed targeting the human TS gene, and then transfected into the human colorectal cancer LOVO cells. RT-PCR and Western blot technique were used to observe the TS gene and protein expression levels, and MTT was used to detect cell proliferation after silencing the TS gene. In addition, siRNA and nolatrxed were applied to the LOVO cells to observe the TS protein expression and cell growth.. TS siRNA significantly reduced the expression of TS gene and protein in LOVO cells, and inhibited cell growth. The IC50 value of LOVO cells was (1.46±0.25) μmol/L in TS siRNA combined with nolatrexed group, (6.81±0.31) μmol/L in the negative control group, and (6.47±0.43) μmol/L in the single nolatrexed group. After treatment of TS siRNA combined with nolatrexed on LOVO cells for 36 hours, the apoptosis index was higher than that in single TS siRNA and nolatrexed[(62.12±0.89)% vs.(21.56±0.67)% and(40.51±0.83)%, both P<0.05].. TS siRNA can partly suppress the expression of TS gene in LOVO cells, inhibit cell proliferation, promote cell apoptosis and enhance cell sensitivity to apoptosis induced by nolatrexd.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Humans; Quinazolines; RNA Interference; Thymidylate Synthase

The serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A) are key enzymes in regulating entry into the cell cycle, mitosis and apoptosis. Inhibition of PP1 and PP2A is associated with enhanced S-phase entry culminating in G(2)/M arrest and apoptotic cell death. Thymidylate synthase (TS) is a key regulatory enzyme in DNA synthesis, inhibition of which is often a first-line treatment for colorectal carcinoma. In this study the effect of combining PP inhibition with TS inhibition in two colorectal cell lines was examined.. Cantharidin and nolatrexed were used to inhibit PP and TS activity, respectively. The MTT cytotoxicity assay and cell cycle analysis were performed following single-drug treatment of HT29 and HCT116 colorectal cell lines. The median effect method was used to determine a combination index (CI), where drug antagonism was indicated by a CI>1.1, additivity by a CI between 0.9 and 1.1, and synergism by a CI<0.9.. Both cell lines were equally sensitive to cantharidin alone (GI(50) values 5.4 and 7.3 micro M), which induced a significant increase in the S-phase population of both cell lines within 6 h with a concomitant increase in DNA synthesis. This response culminated in G(2)/M cell cycle arrest within 24 h and subsequent cell death. In response to nolatrexed alone, HT29 cells were more sensitive than HCT116 cells (GI(50) 1.9 micro M vs 9.8 micro M), with G(1)/S-phase cell cycle arrest occurring within 24 h in both cell lines. In HT29 cells, this was followed by cell death, whereas in HCT116 cells, a proportion of cells died following arrest but the predominant event was re-entry into the cell cycle. The simultaneous exposure of HT29 cells to the combination of nolatrexed and cantharidin in drug molar ratios of 1:1 and 1:2.5 for 72 h was synergistic producing composite CIs of 0.88 and 0.87, respectively. The sequence of nolatrexed followed by cantharidin 24 h later resulted in greater synergism (CI values of 0.75, 0.52, 0.55, 0.68 for molar ratios of 10:1, 1:1, 1:2.5, 1:10), whereas the reverse sequence was antagonistic, suggesting that the point of interaction is downstream of TS inhibition. In HCT116 cells only additive and antagonistic interactions were observed for any of the treatment combinations. The lack of synergism in these cells may be caused by the reduced sensitivity of these cells to nolatrexed as a single agent.. The effect of TS inhibition can be enhanced by the inhibition of serine/threonine protein phosphatases.

Topics: Antimetabolites, Antineoplastic; Cantharidin; Carcinoma; Cell Cycle; Cell Division; Cell Line, Tumor; Colorectal Neoplasms; DNA, Neoplasm; Drug Synergism; Enzyme Inhibitors; HT29 Cells; Humans; Phosphoprotein Phosphatases; Quinazolines; Thymidylate Synthase

To study the dynamic changes in thymidylate synthase (TS) expression in different cell lines treated with nolatrexed.. For studying the time course of the action of the drug, LoVo and Lo2 cells were treated separately with nolatrexed at the concentrations of 10 and 75 micromol/L (approximately their respective IC(90) value) for 0 to 60 h, while in the dose response study, treatment with nolatrexed for 36 h was performed with the dose range of 0 to 40 micromol/L for LoVo cells and 0 to 125 micromol/L for Lo2 cell. TS expression was detected by flow cytometry and Western blotting.. The dynamic changes in TS expression with respect of the time course and dose response differed significantly in the two cell lines after nolatrexed treatment. In the time course study, TS expression increased continuously in LoVo cells, as compared with that in the control cells, whereas the TS expression initially decreased and then began to increase gradually 24 h after the treatment, remaining still below the control level after 60 h. In the dose response study, TS expression in LoVo cells gave rise to a peak in the curve, with the peak level approaching the IC(90) value. In Lo2 cells, when the concentration of nolatrexed was blow the IC(90) value, the decrement in TS expression were similar, but as the concentration exceeded the IC(90) value, TS expression decreased dose-dependently.. Nolatrexed may elicit TS induction in the cells, and the difference in the dynamic changes of TS expression between different cell lines after nolatrexed treatment contributes to different profiles of drug resistance.

Topics: Cell Line; Colorectal Neoplasms; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Hepatocytes; Humans; Quinazolines; Thymidylate Synthase; Tumor Cells, Cultured