Enhancement of cisplatin sensitivity of cisplatin-resistant human cervical carcinoma cells by bryostatin 1.
OBJECTIVE
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 PKCdelta in HeLa cells. We have investigated how bryostatin 1 influences PKCdelta regulation in cisplatin-resistant HeLa (HeLa/CP) cells, and if bryostatin 1 could be used to reverse cisplatin resistance.


METHODS
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-(4,5-dimethylthiazol-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 PKCdelta down-regulation and cisplatin-induced cell death in HeLa/CP cells; the maximum effect was achieved with 1 nmol/L bryostatin 1. Down-regulation of PKCalpha increased with increasing concentrations of bryostatin 1. PDBu induced down-regulation of PKCalpha in HeLa and HeLa/CP cells but it had little effect on PKCdelta down-regulation in HeLa/CP cells. However, both PDBu and bryostatin 1 enhanced the sensitivity of HeLa/CP cells to cisplatin. Knockdown of PKCdelta by small interfering RNA inhibited cisplatin-induced apoptosis but knockdown of PKCalpha enhanced cisplatin-induced cell death.


CONCLUSIONS
These results suggest that although PKCdelta acts as a proapoptotic protein, full-length PKCdelta may inhibit cisplatin-induced cell death. Thus, persistent activation/down-regulation of PKCdelta by bryostatin 1 was associated with cisplatin sensitization. Furthermore, PKCalpha acts as an antiapoptotic protein and down-regulation of PKCalpha by PDBu was associated with cellular sensitization to cisplatin.
Mohanty
Sanghamitra
S
Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.
Huang
Jie
J
Basu
Alakananda
A
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D054713:Bryostatins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007769:Lactams / Q000494:pharmacology; D018942:Macrolides / Q000494:pharmacology*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D002583:Uterine Cervical Neoplasms / Q000201:enzymology / Q000235:genetics / Q000473:pathology
D000970:Antineoplastic Agents; D054713:Bryostatins; D007211:Indoles; D007769:Lactams; D018942:Macrolides; D034741:RNA, Small Interfering; D015240:Phorbol 12,13-Dibutyrate; C046785:bryostatin 1; C043969:indolactam V; D011065:Poly(ADP-ribose) Polymerases; C496628:PRKCA protein, human; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D013755:Tetradecanoylphorbol Acetate; D002945:Cisplatin