Effect of the PKC-beta inhibitor, enzastaurin, in colorectal cancer and melanoma cell lines
Authors: Ana Margarida Abrantes, Ana Goncalves, Henriqueta Breda, Ana Bela Ribeiro, Filomena Botelho
Journal: Cancer Research (2007)

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA

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The important role of PKC in processes relevant to neoplastic transformation, carcinogenesis and tumour cell invasion renders it a potentially suitable target for anticancer therapy. The development of a selective inhibitor of PKC-beta agents, such as enzastaurin, with antitumoral activity both alone or in combination with other cytotoxic agents, remains a novel and interesting cancer therapeutic approach that will certainly be relevant in the future.

Our first approach is to evaluate the effect of the PKC-beta inhibitor, enzastaurin, in cell proliferation and death, in colorectal cancer and melanoma cell lines.

For this purpose we began to evaluate PKC beta-I and beta-II gene expression in the cancer cell lines by RT-PCR. Colon cancer cells, WiDR (adenocarcimona in situ ) and the melanoma cells, A375 and C32 (melanocytic and amelonocytic cells, respectively), are incubated in absence and presence of enzastaurin in different concentrations (1, 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300 and 400 µM), each 24 h during 96 h. We also incubate WiDR cells with conventional chemotherapeutic drugs, namely oxaliplatin and 5-Fluouracil and A375 and C32 cells with ATRA.

Cell growth and viability were evaluated by cell density and viability tests using Trypan blue exclusion (proliferative and viability curves), and MTT assay, each 24 h during 72-96 hours to establish a dose-response curve and the IC50 for each drug. Cell death analysis is performed by morphological studies using optic microscopy and by flow cytometry, using annexin-V, and/or propidium iodine incorporation.

In colon cancer and melanoma cells we didn’t detected any PKC gene expression. However this result may be related with a lower expression of PKC gene not detected by the conventional PCR used. We are trying other PCR techniques to confirm our results

However, our preliminary results suggest that enzastaurin induces a decrease in WiDR, A375 and C32 cells viability in a dose, time and cell line dependent manner, as single agent. The cytotoxic effect seems to be observed after 24h of treatment. On the other hand, the WiDR cells seem to be the more sensitive (ranging concentration of IC50 between 15-50 μM) cancer cell line and the C32 cells the less sensitive (ranging concentration of IC50 between 100-200 μM) cells. In fact we observe the some cytotoxic effect in colon cancer cells (WiDR) with lower concentration of enzastaurin than in the melanoma cell lines. However, we observe some differences between the melanoma cell lines, being the melanocytic one (A375) more sensitive to enzastaurin.

In spite of our preliminary results didn’t detected PKC-beta I and II gene expression, enzastaurin shows efficacy as single agent in melanoma and colon cancer cell lines with the tested doses which may suggest other mechanisms involved besides PKC inhibition and/or lower PKC gene expression.