Serine/threonine protein phosphatase inhibition enhances the effect of thymidylate synthase inhibition.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
The effect of TS inhibition can be enhanced by the inhibition of serine/threonine protein phosphatases.
Sakoff
Jennette A
JA
Department of Medical Oncology, Newcastle Mater Misericordiae Hospital, Edith Street, 2298 Waratah, NSW, Australia. jennette.sakoff@newcastle.edu.au
Howitt
Ian J
IJ
Ackland
Stephen P
SP
McCluskey
Adam
A
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology* / Q000627:therapeutic use; D002193:Cantharidin / Q000494:pharmacology* / Q000627:therapeutic use; D002277:Carcinoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D004273:DNA, Neoplasm / Q000096:biosynthesis; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D019073:HT29 Cells; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011799:Quinazolines / Q000494:pharmacology* / Q000627:therapeutic use; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*
D000964:Antimetabolites, Antineoplastic; D004273:DNA, Neoplasm; D004791:Enzyme Inhibitors; D011799:Quinazolines; D013940:Thymidylate Synthase; D010749:Phosphoprotein Phosphatases; D002193:Cantharidin; C099178:nolatrexed