Enhancement of cisplatin sensitivity of cisplatin-resistant human cervical carcinoma cells by bryostatin 1

Purpose: Bryostatin 1, a unique protein kinase C (PKC) activator, is already in the clinical trials. An understanding of complex regulation of PKC by bryostatin 1 is essential for effective use of bryostatin 1 in the clinic. We have previously shown that the ability of bryostatin 1 to enhance cisplatin sensitivity correlated with its ability to down-regulate PKC delta in HeLa cells. We have investigated how bryostatin 1 influences PKC delta regulation in cisplatiln-resistant HeLa (HeLa/CP) cells, and if bryostatin 1 could be used to reverse cisplatin resistance. Experimental Design: Phorbol 12,13-dibutyrate (PDBu); bryostatin 1, and small, interfering RNA were used to manipulate PKC level/activation status. Cell death was monitored by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Annexin V dye-binding assay, and analysis of hypodiploid peak in a flow cytometer. Results: Bryostatin 1 elicited a biphasic concentration response on PKC delta down-regulation and cisplatin-induced cell cleathin HeLa/CP cells; the maximum effect was achieved with 1 nmol/L bryostatin 1. Down-regulation of PKC alpha increased with increasing concentrations of bryostatin 1. PDBu induced down-regulation of PKC alpha in HeLa and HeLa/CP cells but it had little effect on PKC delta clown-regulation in HeLa/CP cells. However, both PDBu And bryostatin 1 enhanced the sensitivity of HeLa/CP cells to cisplatin. Knockdown of PKC delta by small interfering RNA inhibited cisplatin-induced apoptosis but knockdown of PKC alpha enhanced cisplatin-induced cell death. Conclusions: These results suggest that although PKC delta acts as a proapoptotic protein, full-length PKC delta may inhibit cisplatin-induced cell death. Thus, persistent activation/down-regulation of PKC delta by bryostatin 1 was-associated with cisplatin sensitization. Furthermore, PKC alpha acts as an antiapoptotic protein and down-regulation of PKC alpha by, PDBu was associated with cellular sensitization to cisplatin.