Pharmacological modulation of protein kinase C for overcoming drug resistance in colorectal cancer. Preliminary results of in vitro studies.

Background: Protein kinase C (PKCs) is a family of protein kinase enzymes that regulate many important cellular processes. Some PKC isoforms, e.g. PKCbI and II, are involved in the progression of colorectal cancer (CRC) and confer resistance to apoptosis induced by anti-cancer drugs. The pharmacological inhibition of PKCs-mediated signal transduction with specific inhibitors may enhance the effects of chemotherapy and revert tumor drug resistance. Inhibitors of PKCb isoforms enzastaurin (E) and midostaurin (M) are able to induce apoptosis and inhibit tumor angiogenesis. Materials and methods: Human CRC cell lines sensitive to 5-fluorouracil (5-FU) (HCT-8, HT29) or SN-38 (HCT-8, H630), with acquired resistance to 5-FU (HCT-8/FUI/15R, HCT-8/FUB/2R) or SN38 (HCT-8/SN-38/C e HCT-8/SN-38/D) and with intrinsic resistance to 5-FU and SN-38 (SW620) were used. Cell lines were treated with E or M in combination with 5-FU or SN-38. Cell growth inhibition was determined by the SRB assay and synergistic effects of combined drug treatments by the method of Drewinko. PKCs mRNA expression levels were analyzed by RT-PCR in the above cell lines and in fresh-frozen human CRC explants. Results: In cell lines sensitive (HCT-8, HT29) or resistant to 5-FU (HCT-8/FUI/5R, HCT-8/FUB/2R, SW620) treated with E or M, ICs20 ranged from 1 to 2 mM and from 0.1 to 0.2 mM, respectively. Similar ICs20 were obtained in cell lines sensitive (HCT-8, H630) or resistant to SN-38 (HCT-8/SN-38/C and HCT-8/SN-38/D, SW620). Overall, an addictive effect between PKCb inhibitors and 5-FU or SN-38 was observed in cell lines with intrinsic or acquired resistance to 5-FU or SN-38. This effect occurred at all concentrations and exposure times tested. In particular, a synergistic effect was observed in HCT-8/FUI/15R cells exposed to E and 5-FU. mRNA expression levels of various PKC isoforms (a, bI, bII, g, d, e, q, i, z) showed high variability in all cell lines and CRC explants. PKC isoforms bI and bII were detectable only in SW620 cells. Conclusion: The results obtained so far demonstrate additive effects of PKCb inhibitors combined with 5-FU and SN-38 in cells with both intrinsic or acquired resistance. The mechanisms underlying the interactions between these drugs are being studied. The variability of basal expression levels of PKC isoforms in CRC cell lines and explants represents an initial observation useful for the characterization of PKCs role in sensitivity/resistance to chemotherapy for CRC. Supported by I.T.T.