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Clinical Cancer Research Vol. 11, 6730-6737, September 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Enhancement of Cisplatin Sensitivity of Cisplatin-Resistant Human Cervical Carcinoma Cells by Bryostatin 1

Sanghamitra Mohanty, Jie Huang and Alakananda Basu

Authors' Affiliation: Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas

Requests for reprints: Alakananda Basu, Department of Molecular Biology and Immunology, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107. Phone: 817-735-2487; Fax: 817-735-2118; E-mail: abasu{at}hsc.unt.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
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 cisplatin-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-(4,5-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 PKC{delta} 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 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} down-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.

cis-Diamminedichloroplatinum(II) or cisplatin is one of the most effective anticancer drugs used in the treatment of solid tumors, including ovarian, testicular, cervical, and small cell lung cancers (1, 2). The acquisition of resistance by tumor cells to cisplatin is one of the major problems in cisplatin therapy. Although DNA is the primary target of cisplatin, other cellular factors can influence its cytotoxicity. We, and others, have shown that the protein kinase C (PKC) signaling pathway plays an important role in regulating cisplatin sensitivity (3–9).

PKC represents a family of at least 10 isozymes that have been categorized into three groups: conventional or cPKCs ({alpha}, ßI, ßII, and {gamma}), novel or nPKCs ({delta}, {varepsilon}, {eta}, and {theta}), and atypical or aPKCs ({zeta} and {lambda}/{iota}; refs. 10–13). Whereas cPKCs are dependent on Ca2+ and diacylglycerol for activity, nPKCs are Ca2+-independent. aPKCs are insensitive to both Ca2+ and diacylglycerol. Tumor-promoting phorbol esters are potent activators of PKCs and can substitute for diacylglycerol (10). Prolonged cellular exposure to phorbol esters can lead to depletion or down-regulation of conventional and novel PKCs (10, 13, 14).

PKC{delta} has been intimately associated with DNA damage–induced apoptosis (15, 16). There are, however, controversies regarding how PKC{delta} influences apoptosis. PKC{delta} is a substrate for caspase-3 and proteolytic activation of PKC{delta} has been linked to DNA damage–induced apoptosis (15, 16). Several studies suggested that activation of PKC{delta} was associated with cell death (17–20). However, PKC{delta} has been shown to promote cell survival and chemotherapeutic drug resistance in human non–small cell lung cancer cells (21). We have shown that prolonged cellular exposure to PKC activators that led to down-regulation of PKC{delta} enhanced cell death induced by the DNA-damaging agent cisplatin (5, 9). Paradoxically, rottlerin, a pharmacologic inhibitor of PKC{delta}, prevented cisplatin-induced apoptosis (8, 9).

Bryostatin 1, a macrocyclic lactone derived from the marine bryozoan Bugula neritina, belongs to a unique class of PKC activators (22, 23). It binds to and activates PKC, but it can also act as a partial agonist and often antagonizes its own effect or the effects of phorbol esters (23, 24). Unlike phorbol esters, bryostatin 1 lacks tumor-promoting activity and is an important candidate for anticancer therapy (24–26). Although bryostatin 1 does not affect proliferation of HeLa cells by itself, it enhances cellular sensitivity to cisplatin significantly at subnanomolar concentrations (6, 9). It, however, elicits a biphasic effect on cisplatin sensitization (6). We have shown that the biphasic concentration response of bryostatin 1 on cisplatin-induced cell death can be explained by PKC{delta} down-regulation and caspase activation (9).

We have recently shown that the regulation of PKC{delta} by phorbol 12,13-dibutyrate (PDBu) was affected in HeLa cells that acquired resistance to cisplatin (27). The level of PKC{delta} was elevated in cisplatin-resistant HeLa (HeLa/CP) cells and the ability of PDBu to induce down-regulation of PKC{delta} was compromised in HeLa/CP cells compared with drug-sensitive parental cells (27). Because the regulation of PKC{delta} by bryostatin 1 and PDBu is distinct and bryostatin 1 is already in clinical trials, we have examined how bryostatin 1 influences PKC{delta} regulation in cisplatin-resistant HeLa cells, and if bryostatin 1 could be used to circumvent cisplatin resistance.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Materials. 12-O-Tetradecanoylphorbol 13-acetate and PDBu were purchased from LC Service Corporation (Woburn, MA). Cisplatin and MTT were from Sigma (St. Louis, MO). Small interfering RNAs (siRNA) and polyclonal antibodies to PKC{delta} and PKC{varepsilon} were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal antibody to PKC{zeta}, and monoclonal antibody to PKC{alpha} were from Upstate Biotechnology, Inc. (Lake Placid, NY). Polyclonal antibody to caspase-3 and monoclonal antibody to poly(ADP-ribose) polymerase were from PharMingen (San Diego, CA). Annexin V conjugated to Alexa Fluor 488 and propidium iodide were purchased from Molecular Probes (Eugene, OR). Horseradish peroxidase–conjugated goat anti-mouse and donkey anti-rabbit antibodies were obtained from Jackson ImmunoResearch Lab., Inc. (West Grove, PA). Polyvinylidene difluoride membrane was from Millipore (Bedford, MA) and enhanced chemiluminescence detection kit was from Amersham (Arlington Heights, IL).

Cell culture and transfection. Human cervical carcinoma HeLa cells and its cisplatin-resistant variants (HeLa/CP) were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 2 mmol/L glutamine, and kept in a humidified incubator at 37°C with 95% air and 5% CO2. HeLa cells were transfected with full-length PKC{delta} cloned into pcDNA5 using LipofectAMINE (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol and selected using geneticin.

Assessment of cell viability by MTT Assay. Exponentially growing cells were plated in microtiter plates and incubated at 37°C in 5% CO2. The following day, cells were pretreated with or without PKC activators as indicated in the text and then with different concentrations of cisplatin. The number of viable cells was determined using the dye MTT as previously described (5).

Assessment of apoptosis by flow cytometric analysis. Cells were pretreated with PKC activators for the indicated periods of time and then treated with cisplatin. At the end of the incubation, cells were harvested and washed with PBS. Nuclei were isolated, stained with propidium iodide, and DNA content was analyzed using a flow cytometer (Coulter Epics, Miami, FL; ref. 28).

Annexin V/propidium iodide binding assay. Cells were treated with or without PKC activators and then with cisplatin for 3 hours. Cisplatin-containing media was removed and cells were incubated in drug-free media. At the end of the incubation, both detached cells and attached cells were collected and washed with PBS. Cells were then stained with Annexin V-Alexa 488 conjugate and propidium iodide according to the manufacturer's protocol and analyzed using a flow cytometer (Coulter Epics, Miami, FL).

Immunoblot analysis. Cell extracts containing equal amounts of proteins were electrophoresed by SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membrane. Immunoblot analyses were done as described before (9).

Knockdown of PKC isozymes. Control siRNA or siRNA targeted against PKC{alpha} or PKC{delta} were introduced into HeLa cells using LipofectAMINE 2000 (Invitrogen) and manufacturer's protocol. Briefly, cells were seeded 1 day before transfection. LipofectAMINE 2000 and siRNA diluted in Opti-MEM were mixed gently at a ratio of 300 ng LipofectAMINE 2,000:133 ng siRNA and incubated at room temperature for 15 to 20 minutes. Culture medium was replaced with Opti-MEM and 100 µL of siRNA/LipofectAMINE 2000 complexes were added to cells. After 4 to 6 hours, fresh culture medium was added to cells; 48 hours following siRNA transfection, cells were treated with cisplatin.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Differential effects of protein kinase C activators on protein kinase C{delta} down-regulation. We have recently shown that the ability of PDBu to down-regulate PKC{delta} was compromised in HeLa cells that acquired resistance to cisplatin (HeLa/CP; ref. 27). In the present study, we compared the effects of several PKC activators that differ structurally and functionally on the down-regulation of PKC{delta} in HeLa and HeLa/CP cells. Figure 1 shows that prolonged cellular exposure to PKC activators, including 12-O-tetradecanoylphorbol 13-acetate, PDBu, bryostatin 1, and indolactam V led to down-regulation of PKC{delta} in HeLa cells. In contrast, with the exception of bryostatin 1, PKC activators failed to induce substantial down-regulation of PKC{delta} in HeLa/CP cells. The ability of these PKC activators to induce down-regulation of PKC{alpha} and PKC{varepsilon} was comparable in HeLa and HeLa/CP cells, suggesting that there was no general alteration in the degradative pathway. None of the PKC activators had any effect on the down-regulation of phorbol ester–insensitive atypical PKC{zeta}. Thus, the level of PKC{zeta} also served as control for equal loading.



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  		Fig. 1. Effects of PKC activators on the down-regulation of PKC isozymes in HeLa and HeLa/CP cells. Cells were treated with or without 100 nmol/L 12-O-tetradecanoylphorbol 13-acetate, 1 µmol/L PDBu, 1 nmol/L bryostatin 1, or 10 µmol/L indolactam V for 24 hours. Western blot analyses were done with total cell lysates using antibodies specific to PKC isozymes. Results are representative of at least three independent experiments.

 
Because bryostatin 1 exhibits biphasic down-regulation of PKC{delta} in HeLa cells (9), we examined the effect of increasing concentrations of bryostatin 1 on PKC{delta} down-regulation in HeLa/CP cells. Figure 2 shows that bryostatin 1 induced biphasic down-regulation of PKC{delta} in both HeLa and HeLa/CP cells. The maximum down-regulation was achieved by 1 nmol/L bryostatin 1 and higher concentrations of bryostatin 1 were less effective than 1 nmol/L bryostatin 1 in inducing down-regulation of PKC{delta}. In contrast, down-regulation of PKC{alpha} by bryostatin 1 increased with increasing concentrations of bryostatin 1, such that 1 nmol/L bryostatin 1 had little effect on PKC{alpha} down-regulation, whereas 1 µmol/L bryostatin 1 caused complete down-regulation of PKC{alpha} in both HeLa and HeLa/CP cells.



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  		Fig. 2. Effects of different concentrations of bryostatin 1 on down-regulation of PKC isozymes in HeLa and HeLa/CP cells. Cells were treated with different concentrations of bryostatin 1 for 24 hours and Western blot analyses were done with total cell lysates. Results are representative of at least three independent experiments.

 
Comparison of the effects of phorbol 12,13-dibutyrate and bryostatin 1 on the sensitization of HeLa and HeLa/CP cells to cisplatin. We have previously shown that bryostatin 1 elicits a biphasic concentration response on cisplatin-induced cell death in HeLa cells (6). To examine if PKC{delta} down-regulation by bryostatin 1 was associated with cellular sensitization to cisplatin in HeLa/CP cells, we determined the concentration response of bryostatin on cellular sensitivity to cisplatin. The IC50 of cisplatin at different concentrations of bryostatin 1 was determined from the MTT assay. As shown in Fig. 3, the IC50 of cisplatin decreased with increasing concentrations of bryostatin 1 up to 1 nmol/L, and then the ability of bryostatin 1 to enhance cisplatin sensitivity decreased gradually such that 1 µmol/L bryostatin 1 was much less effective than 1 nmol/L bryostatin 1 in enhancing cellular sensitivity to cisplatin. We also assessed cell death by monitoring the appearance of a hypodiploid peak in a flow cytometer. As evident in Fig. 4, bryostatin 1 induced a similar biphasic response in enhancing cisplatin-induced cell death. Whereas treatment of HeLa/CP cells with 30 µmol/L cisplatin resulted in the appearance of 36% cells in the sub-G1 phase, pretreatment of HeLa/CP cells with 1 nmol/L bryostatin 1 prior to cisplatin treatment increased cell death to 56%. However, 1 µmol/L bryostatin 1 had little effect on cisplatin-induced cell death. Thus, cisplatin sensitization by different concentrations of bryostatin 1 correlated with PKC{delta} down-regulation.



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  		Fig. 3. Effects of different concentrations of bryostatin 1 on the sensitivity of HeLa/CP cells to cisplatin. Cells were pretreated with different concentrations of bryostatin 1 and then exposed to varying concentrations of cisplatin. Cell survival was determined by the MTT assay as described in Materials and Methods. IC50 values were determined from the cell survival curves.

 


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  		Fig. 4. Concentration-response of PDBu and bryostatin 1 on the sensitization of HeLa/CP cells to cisplatin. Cells were pretreated with or without different concentrations of PDBu or bryostatin 1 and then with 30 µmol/L cisplatin. Nuclei were stained with propidium iodide and DNA was analyzed using a flow cytometer.

 
Because PDBu failed to cause substantial down-regulation of PKC{delta} in HeLa/CP cells, we compared the ability of PDBu and bryostatin 1 to influence cisplatin sensitivity in HeLa and HeLa/CP cells. Figure 5 shows that PDBu decreased the IC50 of cisplatin from 6.4 to 1.9 µmol/L, whereas bryostatin 1 decreased the IC50 of cisplatin from 6.4 to 1.7 µmol/L in HeLa cells. The IC50 of CP for HeLa/CP cells was >30 µmol/L. PDBu and bryostatin 1 decreased the IC50 of HeLa/CP cells to 16.8 and 14 µmol/L, respectively. This difference was not statistically significant. The concentration response of PDBu shows that the maximum sensitization of HeLa/CP cells to cisplatin was achieved with 100 nmol/L PDBu (Fig. 4). Thus, PDBu was less potent than bryostatin 1. Furthermore, PDBu did not elicit a biphasic concentration response on cisplatin sensitization.



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  		Fig. 5. Comparison of the effects of PDBu and bryostatin 1 on the sensitivity of HeLa and HeLa/CP cells to cisplatin. Cells were treated without ({circ}) or with 1 µmol/L PDBu ({bullet}) or 1 nmol/L bryostatin 1 ({blacktriangleup}) and then treated with different concentrations of cisplatin. The cell survival was determined by the MTT assay as described in Materials and Methods. Results are representative of at least three independent experiments.

 
We also monitored apoptotic cell death by the Annexin V dye-binding assay. Cells were costained with the cell-impermeant dye propidium iodide to distinguish apoptotic cells from necrotic cells. When cells undergo apoptosis, phosphatidylserine is flipped from the inner to the outer leaflet of plasma membrane. During the early stage of apoptosis, Annexin V binds to phosphatidylserine on the cell surface. However, during the late stage of apoptosis, Annexin V can enter through the membrane and therefore late-stage apoptosis cannot be distinguished from necrosis. Viable cells are shown at the bottom left quadrant (negative for both Annexin V and propidium iodide). Cells stained with Annexin V conjugate alone (bottom right quadrant) represent apoptotic cells, whereas cells costained with Annexin V conjugate and propidium iodide (top right quadrant) represent late apoptotic and necrotic cells. We have indicated the total percentage of cell death in Fig. 6. Less than 10% cells underwent apoptosis in untreated HeLa and HeLa/CP cells. Because PKC activators enhance cisplatin-induced cell death, we treated HeLa cells with 5 µmol/L cisplatin that by itself had only a modest effect on cell death, and increased apoptotic cell death from 7.8% to 11.4%. PDBu and bryostatin 1 increased cisplatin-induced cell death in HeLa cells to 62% and 68%, respectively. In HeLa/CP cells, 15 µmol/L cisplatin had little effect on cell death and 45 µmol/L cisplatin increased cell death from 4.9% to 26.6%. Although the effect of bryostatin 1 seems to be less when cells were treated with 15 µmol/L cisplatin, both PDBu and bryostatin 1 were almost equally effective in enhancing cisplatin-induced apoptosis when HeLa/CP cells were treated with 45 µmol/L CP. These results suggest that PKC{delta} down-regulation was not essential for cisplatin sensitization by PDBu.



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  		Fig. 6. Comparison of the effects of PDBu and bryostatin 1 on cisplatin-induced apoptosis in HeLa and HeLa/CP cells. Cells were pretreated with 1 µmol/L PDBu or 1 nmol/L bryostatin 1 for 16 hours followed by treatment with cisplatin for 3 hours. Cells were then incubated in drug-free media for 20 hours. The results are representative of two independent experiments.

 
Effect of protein kinase C{delta} overexpression on cisplatin sensitization by bryostatin 1. Because down-regulation of PKC{delta} was associated with cisplatin sensitization by bryostatin 1, we examined the effect of PKC{delta} overexpression on the sensitization of HeLa cells to cisplatin by bryostatin 1. Figure 7A shows that 1 nmol/L of bryostatin 1 caused substantial down-regulation of PKC{delta} in HeLa cells but bryostatin 1 had little effect on PKC{delta} down-regulation in PKC{delta}-overexpressing HeLa cells (HeLa/PKC{delta}) cells. Ectopic expression of PKC{delta} alone decreased cisplatin-induced cell death from 19% to 11%. Pretreatment of HeLa cells with bryostatin 1 enhanced cisplatin-induced cell death in HeLa and HeLa/PKC{delta} cells to 30% and 19%, respectively. Thus, overexpression of PKC{delta} inhibited cisplatin-induced cell death.



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  		Fig. 7. Effect of PKC{delta} overexpression on cisplatin-induced cell death. HeLa cells transfected with or without full-length PKC{delta} were treated with or without 1 nmol/L bryostatin 1 and then with 3.3 µmol/L cisplatin for 24 hours. A, Western blot analysis of HeLa and PKC{delta} overexpressing HeLa cells (HeLa/{delta}) pretreated with or without 1 nmol/L bryostatin 1 for 24 hours. B, nuclei were stained with propidium iodide and DNA was analyzed using a flow cytometer.

 
Effect of protein kinase C{delta} and protein kinase C{alpha} knockdown on cisplatin sensitization. Because activation of PKC is a prerequisite for its down-regulation, it is difficult to distinguish between persistent activation from down-regulation because residual PKC remains in an active state. To further examine if depletion of PKC{delta} was necessary for cisplatin sensitization, we used siRNA targeted against PKC{delta} mRNA to knock down PKC{delta}. As shown in Fig. 8, we were unable to detect PKC{delta} in HeLa cells transfected with PKC{delta} siRNA. However, cisplatin-induced activation of caspase-3 as determined by the increase in processed caspase-3 and cleavage of poly(ADP-ribose) polymerase was reduced in HeLa cells transfected with PKC{delta} siRNA compared with cells transfected with control siRNA. In contrast, knockdown of PKC{alpha} enhanced activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase.



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  		Fig. 8. Effect of knock-down of PKC isozymes on caspase-3 activation in HeLa cells. HeLa cells were transfected with control siRNA or siRNA targeted against PKC{alpha} or PKC{delta}. Cells were then treated with or without 10 µmol/L cisplatin for 2 hours and then incubated in drug-free media for 24 hours. Western blot analyses were done with indicated antibodies. Only the processed forms of 17- and 12-kDa active caspase-3 are shown. Porin was used to control for loading. The results are representative of two independent experiments.

 
We also examined if knockdown of PKC{alpha} and PKC{delta} by siRNA influences cisplatin-induced cell death when HeLa cells are exposed to cisplatin continuously. We treated cells with 2 µmol/L cisplatin because continuous exposure to 10 µmol/L cisplatin caused extensive cell death. Figure 9 shows that 23% of cells underwent apoptosis when HeLa cells were treated with cisplatin. Knockdown of PKC{delta} decreased cells in the sub-G1 phase to 18%, whereas knockdown of PKC{alpha} enhanced the percentage of cells in the sub-G1 phase to 38%. These results show that although depletion of conventional PKC{alpha} was associated with cisplatin-induced cell death, depletion of novel PKC{delta} in fact inhibited cisplatin-induced cell death.



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  		Fig. 9. Effect of knock-down of PKC{alpha} and PKC{delta} on cisplatin-induced apoptosis in HeLa cells. HeLa cells were transfected with control siRNA or siRNA targeted against PKC{alpha} or PKC{delta}. Cells were then treated with or without 2 µmol/L cisplatin for 24 hours. Nuclei were stained with propidium iodide and DNA was analyzed using a flow cytometer.

 
Because the ability of PDBu to down-regulate PKC{delta} was compromised in cisplatin-resistant HeLa cells yet both PDBu and bryostatin 1 sensitized HeLa/CP cells to cisplatin, we examined the consequence of PKC{alpha} knockdown on cisplatin-induced cell death in HeLa/CP cells. Figure 10 shows that depletion of PKC{alpha} using siRNA, enhanced cisplatin-induced apoptosis as determined by the activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase but depletion of PKC{delta} inhibited cisplatin-induced apoptosis. These results suggest that the ability of PDBu to sensitize HeLa/CP cells to cisplatin could be explained by down-regulation of PKC{alpha}.



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  		Fig. 10. Effect of knock-down of PKC{alpha} and PKC{delta} on cisplatin-induced apoptosis in HeLa/CP cells. HeLa/CP cells transfected with control siRNA or siRNA targeted against PKC{alpha} or PKC{delta} were treated with or without 30 µmol/L cisplatin for 15 hours. Western blot analyses were done with the indicated antibodies.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The results of our present study show that PKC activators, such as PDBu and bryostatin 1 not only sensitize HeLa cells to cisplatin, they also enhance the sensitivity of cisplatin-resistant HeLa cells to cisplatin. However, the mechanism by which they regulate cisplatin sensitivity is distinct. Our results suggest that PKC{delta} can function as both pro- and antiapoptotic protein, and persistent activation or down-regulation of PKC{delta} by bryostatin 1 correlated with cellular sensitization to cisplatin. However, depletion of PKC{delta} was not sufficient to explain cisplatin sensitization by PDBu. We showed that PKC{alpha} acts as an antiapoptotic protein and depletion of PKC{alpha} was associated with cisplatin sensitization by PDBu.

We have previously shown that bryostatin 1 exhibits biphasic down-regulation of PKC{delta} (9) and down-regulation of PKC{delta} by bryostatin 1 correlated with sensitization of HeLa cells to cisplatin (6, 9). Paradoxically, PKC{delta} inhibitor rottlerin blocked cisplatin-induced cell death (8, 9). These observations raised an important question whether activation or down-regulation of PKC{delta} was associated with cell death. There are several potential mechanisms that regulate cisplatin-induced cell death, including cisplatin uptake, DNA damage and DNA repair. Although PKC activators caused a modest increase in cisplatin uptake, they had no effect on cisplatin efflux (5, 6, 29). Furthermore, bryostatin 1 reversed the increase in cisplatin uptake by PDBu but had no effect on the rate of cisplatin efflux (29). We have, however, shown that PKC acts upstream of caspases to regulate cisplatin-induced caspase activation and the biphasic concentration response of bryostatin 1 on cisplatin-induced cell death could be explained by its effect on cisplatin-induced caspase activation (9). Furthermore, PKC{delta} inhibitor rottlerin, which was shown to act at a step subsequent to DNA damage but prior to caspase activation, inhibited cisplatin-induced caspase activation (8, 30).

We have shown that down-regulation of PKC{delta} by bryostatin 1 also correlated with sensitization of cisplatin-resistant HeLa cells to cisplatin. However, although PDBu failed to induce down-regulation of PKC{delta} in HeLa/CP cells, both activators were equally effective in sensitizing HeLa/CP cells to cisplatin. These results were based on three independent assays—MTT assay, the appearance of a hypodiploid peak in a flow cytometer, and Annexin V dye binding assay. These results suggest that down-regulation of PKC{delta} was not sufficient to explain cisplatin sensitization by PKC activators.

There are differences in how phorbol esters and bryostatin 1 influence cisplatin sensitivity. First, bryostatin 1 is highly potent and it sensitizes cells at subnanomolar concentrations; the maximum sensitization was achieved with 1 nmol/L bryostatin 1. In contrast, PDBu was less potent than bryostatin 1 in enhancing cisplatin-induced cell death and the effects of 10 to 1,000 nmol/L PDBu on cisplatin-induced cell death were comparable. Second, unlike PDBu, bryostatin 1 is a partial agonist and it prevents its own effect or the effects of phorbol esters at higher concentrations (6).

The regulation of PKCs by PDBu and bryostatin 1 is also distinct. HeLa cells express several PKC isozymes, including PKC{alpha}, -{delta}, -{varepsilon} and -{zeta}. Unlike PDBu, bryostatin 1 induced biphasic down-regulation of PKC{delta} and maximum down-regulation of PKC{delta} was achieved at 1 nmol/L. However, bryostatin 1 did not induce biphasic down-regulation of PKC{alpha}. In addition, whereas 1 nmol/L bryostatin 1 was most effective in inducing PKC{delta} down-regulation, it had little effect on PKC{alpha} down-regulation. Thus, in HeLa cells, 1 nmol/L bryostatin 1 that caused maximum sensitization to cisplatin predominantly induced down-regulation of PKC{delta}, whereas 1 µmol/L PDBu caused down-regulation of both PKC{alpha} and PKC{delta}. In HeLa/CP cells, PDBu primarily caused down-regulation of PKC{alpha} because it failed to induce substantial down-regulation of PKC{delta}.

Because down-regulation of PKCs is a consequence of their activation, reversal of PKC{delta} down-regulation at higher concentrations of bryostatin 1 suggests that PKC{delta} that accumulates at higher concentrations of bryostatin 1 is inactive. In addition, because activation of PKC precedes its down-regulation, the residual PKC following down-regulation remains in an active state. The turnover rate of PKC{delta} is ~8 hours (31) and is greater than most PKCs. Thus, the consequence of PKC{delta} down-regulation could be distinct from the effect of PKC{delta} inhibition or depletion.

We therefore examined how knockdown of PKC{delta} influences cisplatin sensitivity. Our results show that depletion of PKC{delta} by siRNA targeted against PKC{delta} in fact reduced cellular sensitivity to cisplatin, suggesting that PKC{delta} was required for cisplatin-induced cell death. This is consistent with the proapoptotic function of PKC{delta}. The generation of PKC{delta} catalytic fragment has been associated with cell death by various apoptotic stimuli (15). Our results suggest that PKC{delta} may also function as antiapoptotic protein. First, overexpression of PKC{delta} was associated with cisplatin resistance (27). Second, down-regulation of PKC{delta} correlated with cisplatin sensitization by bryostatin 1. Finally, ectopic expression of PKC{delta} in HeLa cells inhibited cisplatin-induced cell death. We propose that whereas proteolytic activation of PKC{delta} during apoptosis may be associated with cell death, PKC{delta} holoenzyme inhibits cisplatin-induced cell death. Depletion of PKC{delta} by siRNA not only removes the full-length antiapoptotic PKC{delta} but it also prevents generation of cleaved fragments of PKC{delta} that act as proapoptotic proteins. This may be why the effect of siRNA depletion of PKC{delta} on cell death was modest. These results also explain why down-regulation of PKC{delta} correlates with cisplatin sensitization yet PKC{delta} inhibitor rottlerin prevents cell death by cisplatin. The biphasic response of PKC{delta} down-regulation and cisplatin sensitization by bryostatin 1 could be explained by the fact that low concentrations of bryostatin 1 caused activation of PKC{delta} followed by depletion of inactive PKC{delta} holoenzyme. Accumulation of inactive PKC{delta} at higher concentrations of bryostatin 1 may prevent cell death by cisplatin. This is consistent with the results that overexpression of PKC{delta} inhibited cisplatin-induced cell death. The inability of exogenously expressed PKC{delta} to obliterate responsiveness to bryostatin could be explained by the down-regulation of endogenous PKC{delta} by bryostatin 1. Knockdown of PKC{alpha}, however, enhanced cellular sensitivity to cisplatin. We also found that Gö 6976, which inhibits conventional PKC{alpha}, caused substantial increase in the sensitivity of HeLa/CP cells to cisplatin (data not shown), suggesting that PKC{alpha} acts as an antiapoptotic protein, and that inhibition of PKC{alpha} is associated with cisplatin sensitization. Thus, although PDBu fails to down-regulate PKC{delta} in HeLa/CP cells, depletion of PKC{alpha} by prolonged cellular exposure to PDBu may relieve its antiapoptotic function. Therefore, PKC{alpha} may provide a better target in producing cisplatin-induced cell death in HeLa/CP cells.

Bryostatin 1 is already in phase I and II clinical trials, either as a single agent or in combination with chemotherapeutic drugs. Although some studies showed encouraging results, others were not successful (32–35). The regulation of PKC by bryostatin 1 is complex and an understanding of how bryostatin 1 regulates cell death is critical to use it effectively in the clinic. Although phorbol esters cannot be used in the clinic, they provide an important pharmacologic tool to discern the function of PKC. Our results show that PKC activators could be used to enhance cellular sensitivity to cisplatin and shed light on the complex regulation of PKC by bryostatin 1.


   Acknowledgments
 
We thank Dr. Usha Sivaprasad for critical reading of the manuscript and Haidi Tu for technical assistance.


   Footnotes
 
Grant support: National Cancer Institute grant CA85682.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: S. Mohanty is currently at the Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas.

Received 2/28/05; revised 6/17/05; accepted 6/21/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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