Combined antitumoral effects of pretubulysin and methotrexate

Abstract

Pretubulysin (PT), a potent tubulin‐binding antitumoral drug, and the well‐established antimetabolite methotrexate (MTX) were tested separately or in combination (PT+MTX) for antitumoral activity in L1210 leukemia cells or KB cervix carcinoma cells in vitro and in vivo in NMRI‐nu/nu tumor mouse models. In cultured L1210 cells, treatment with PT or MTX displays strong antitumoral effects in vitro, and the combination PT+MTX exceeds the effect of single drugs. PT also potently kills the MTX resistant KB cell line, without significant MTX combination effect. Cell cycle analysis reveals the expected arrest in G1/S by MTX and in G2/M by PT. In both cell lines, the PT+MTX combination induces a G2/M arrest which is stronger than the PT‐triggered G2/M arrest. PT+MTX does not change rates of apoptotic L1210 or KB cells as compared to single drug applications. Confocal laser scanning microscopy images show the microtubule disruption and nuclear fragmentation induced by PT treatment of L1210 and KB cells. MTX changes the architecture of the F‐actin skeleton. PT+MTX combines the toxic effects of both drugs. In the in vivo setting, the antitumoral activity of drugs differs from their in vitro cytotoxicity, but their combination effects are more pronounced. MTX on its own does not display significant antitumoral activity, whereas PT reduces tumor growth in both L1210 and KB in vivo models. Consistent with the cell cycle effects, MTX combined at moderate dose boosts the antitumoral effect of PT in both in vivo tumor models. Therefore, the PT+MTX combination may present a promising therapeutic approach for different types of cancer.

Simultaneous Inhibition of Protein Kinase CK2 and Dihydrofolate Reductase Results in Synergistic Effect on Acute Lymphoblastic Leukemia Cells

“…Additionally, we observed CCRF-CEM cell accumulation in both S-and G 2 /M phases of the cell cycle after the combined treatment, which seems to have been an additive effect, since methotrexate and CX-4945 used individually led to G 2 /M- phase arrest and S-phase arrest, respectively. Interestingly, our results demonstrating G 2 /M-phase arrest after methotrexate treatment are contrary to previous results obtained for L1210 mouse leukemia suggesting that methotrexate cytotoxicity is related to irreversible S-phase and G 1 /S-phase arrest (9,24). S-Phase arrest occurring after CK2 inhibition in the CCRF-CEM line is consistent with our previous data demonstrating the effect of another CK2 inhibitor, 4,5,6,7-tetrabromo-2-methyl-1H-benzimidazol-1-yl)acetonitrile, on cell-cycle progression in this cell line (17).…”

“…Other studies have shown that the combination of methotrexate and suberoylanilide hydroxamic acid (a histone deacetylase inhibitor) has a synergistic effect in leukemia cells with combination index values in the range of 0.64-0.84, depending on the cell line used (for CCRF-CEM with line: CI=0.84) (8). The most recent research carried out on L1210 mouse lymphocytic leukemia cell line showed that combined treatment of pretubulysin, a potent tubulin-binding antitumoral drug, with methotrexate displayed strong anticancer effects in both in vitro and in vivo models (9). DHFR, with two other enzymes, namely thymidylate synthase (TYMS), and serine hydroxymethyltransferase (SHMT), constitute the thymidylate synthesis cycle, providing the substrate required for DNA synthesis and repair (10).…”

Overview of Dual-Acting Drug Methotrexate in Different Neurological Diseases, Autoimmune Pathologies and Cancers

“…The IC 50 value of the formulation was significantly lower than those of free drugs. The use of MTX and a potent tubulin-binding anti-tumoural drug pretubulysin, alone and in the form of a combined regimen, is described by Kern et al [224] The authors demonstrated the benefits of using the drug regimen and indicated that it was a promising therapeutic approach for various types of cancer, among other in lung cancer therapy. Sekimura et al presented a case of primary lung cancer with MTX-associated lymphoproliferative disorder [271] which occurred in a patient receiving oral MTX and prednisolone for RA for about 15 years.…”

Reactive oxygen species, not Ca2+, mediates methotrexate‐induced autophagy and apoptosis in spermatocyte cell line

“…Apoptosis is a severe toxic phenomenon marked by cell shrinkage, chromatin condensation, nuclear DNA fragmentation, a decrease in the mitochondrial membrane potential (MMP) and caspase activation . MTX exerts its antitumour effects via the induction of apoptosis in many cancers . Unfortunately, it can also induce apoptosis in healthy tissues, including the testes, and the underlying molecular mechanism is poorly characterized.…”

“…9 MTX exerts its antitumour effects via the induction of apoptosis in many cancers. [10][11][12] Unfortunately, it can also induce apoptosis in healthy tissues, including the testes, 13,14 and the underlying molecular mechanism is poorly characterized. Autophagy is an intracellular system that degrades damaged cytoplasmic components and proteins to eliminate old and unhealthy cells.…”