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Introduction

Prostate cancer (PCa) is the most common non-cutaneous cancer and the second leading cause of cancer death in American males (1). Despite the initial regression/stabilization brought by androgen deprivation therapy, a large number of PCa eventually relapses and progresses to castration-resistant prostate cancer (CRPC), with a median survival of only 7–12 months (2), for which, better understanding and newer management is urgently needed. Chemotherapy with new agents such as taxol analogs, and new combinatorial regimens has recently made some progress (3). However, the overall outcome of current chemotherapeutic strategies is still unsatisfactory (4).

Camptothecin (CPT) and its analogs are a promising class of anticancer drugs, which have advanced to the forefront of chemotherapy when used alone or in combination with others (5). CPT inhibits DNA topoisomerase I (Topo I) by blocking the rejoining step of the cleavage/religation reaction, resulting in accumulation of the covalent reaction intermediate, the cleavable complex, which leads to apoptosis and cell cycle arrest (6). The first two CPT derivatives, irinotecan (Camptosar) and topotecan (Hycamtin), were approved by US Food and Drug Administration (FDA) in 1996 for the treatment of colon/rectum cancer, and lung cancer (7). Other analogs, such as exatecan (8), have also shown very promising results in clinical trials. However, the effects of CPT analogs on PCa are controversial. Most preclinical studies have shown that CPTs are effective for both androgen-dependent and androgen-independent PCa (9,10). Unfortunately, a couple of clinical trials (phase I/II) with different CPT analogs have only shown very limited benefits on CRPC patients (11–13). Recently, we have demonstrated that NSC606985, a highly water-soluble CPT analog that has rarely been studied for its anticancer activity, produces a significant dose-dependent induction of cell apoptosis at nanomolar concentrations in DU145, LNCaP and PC3 PCa cells (14).

Previous studies have shown that CPT, and apoptotic effects of its analogs may involve protein kinase Cδ (PKCδ) signaling pathway (14–16). PKCδ is a member of the serine/threonine-specific PKC family, which has been found to be involved in diverse signaling pathways. From an oncology standpoint, PKCδ is generally considered as an anti-proliferative and pro-apoptotic protein kinase (17). Previous studies have shown that PKCδ activates both intrinsic and extrinsic apoptotic pathways in PCa cells under phorbol 12-myristate 13-acetate (PMA) stimulation (18,19). However, recent studies have gradually revealed that PKCδ can also function as an anti-apoptotic protein and it is critical for the survival of several cancer cells (20,21). It has been proposed that these apparent opposite actions of PKCδ are tightly regulated by epigenetic mechanisms including post-translational modifications and cellular compartmentalization (22).

In the present study, we aimed to investigate the effects of CPT analogs, mainly NSC, in LAPC4 cells, elucidate its potential molecular mechanism, and reveal its impact on future CRPC management.

Materials and methods

Materials

NSC, and topotecan were kindly provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutic Program, National Cancer Institute (Bethesda, MD, USA). Tissue culture medium, rottlerin, protease inhibitors, propidium iodide (PI), and anti-β-actin primary antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), L-glutamine, penicillin and streptomycin were from Gemini Bio-Products (Calabasas, CA, USA). Z-VAD-fluoromethylketone (FMK), RNase A, protease K, and M-MLV reverse transcriptase were from Promega (Madison WI, USA). Antibodies against cyclin A, and PKCδ were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The cytochrome c antibody (clone 7H8.2C12) was obtained from BD Pharmingen (San Diego, CA, USA). The SYBR-Green Real-Time PCR master mix was from Life Technologies (Grand Island, NY, USA). TriPure Isolation reagent was from Roche Applied Science (Mannheim, Germany).

Cell culture

LAPC4 cells (kindly provided by Dr C. Sawyer) were grown in Iscove's modified Dulbecco's medium supplemented with 15% FBS, 2 mM L-glutamine, 1 nM R1881, 50 U/ml of penicillin, and 50 µg/ml of streptomycin. R1881 was withdrawn 48 h before cell passage to conduct experiments. PC3, LNCaP and DU145 cells (ATCC, Rockville, MD, USA) were grown in RPMI-1640 and Dulbecco's modified Eagle's medium, respectively, supplemented with 10% FBS, 2 mM L-glutamine, 50 U/ml of penicillin and 50 µg/ml streptomycin. Cells were maintained in a 5% CO2 - 95% air humidified atmosphere at 37°C, and cultured in phenol red-free medium with 5% stripped FBS for 24 h before each experiment. The expression of androgen receptor (AR) was verified with real-time PCR whenever a new vial of LAPC4 was thawed. Treatment with 10 nM dihydrotestosterone was used as a positive control for stimulation of LAPC4 cell growth.

Cell proliferation assays

Cells were seeded in 96-well plates at ~30% density, and treated with various regimens as indicated in each experiment at 24 h after plating. The viable cell number was determined using the CellTiter AQueous One Solution Cell Proliferation assay kit from Promega, following the manufacturer's instructions. DNA biosynthesis was determined using the BrdU Cell Proliferation assay kit obtained from Calbiochem (San Diego, CA, USA) following the manufacturer's instructions.

Flow cytometry

Approximately 1×106 LAPC4 cells were plated in 60-mm plates and treated with vehicle control or various regimens as indicated in each experiment. At the end of the treatment, cells were collected, washed with phosphate-buffered saline (PBS) and fixed with 70% ethanol. For DNA content, cells were stained with PI (50 µg/ml) plus RNase A (20 µg/ml) in PBS, and analyzed using the FACScan Flow Cytometer (Becton-Dickinson, Heidelberg, Germany). The data from 1.0×104 cells/sample were collected and analyzed using the CellQuest software (Becton-Dickinson). For cell apoptosis analysis, cells were stained with Annexin V in combination with PI using the Annexin V-FITC apoptosis detection kit I from BD Pharmingen according to the manufacturer's instructions.

Determination of DNA fragmentation

NSC treated and untreated cells were harvested and incubated in a lysis buffer [50 mM Tris-HCl (pH 8.0), 20 mM ethylenediaminetetraacetic acid (EDTA), 10 mM NaCl, 1% sodium dodecyl sulfate (SDS)] for 20 min on ice. Samples were then centrifuged and treated with DNase-free RNase A and proteinase K. Following phenol and chloroform extraction, DNA was precipitated by ethanol and dissolved in 1X Tris-EDTA buffer. The DNA samples were then electrophoresed in a 2% agarose gel, visualized by ethidium bromide staining under ultraviolet light.

Western blot analysis

LAPC4 cells were harvested and total cellular proteins were extracted using a lysis buffer (62.5 mM Tris-HCl pH 6.8, 100 mM dithiothreitol, 2% SDS, 10% glycerol). The cytosolic, nucleus and mitochondrial protein extracts were prepared as described (14). The protein concentrations were determined using the Bio-Rad Protein assay (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer's instructions. Protein (25 µg) was loaded in each lane, electrophoresed on a 15% SDS-PAGE, and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA). The blots were blocked with TBST buffer [500 mM NaCl, 20 mM Tris-HCl (pH 7.4), and 0.1% Tween-20] containing 5% nonfat dry milk (Bio-Rad Laboratories) and then incubated with specific primary antibody in TBST buffer containing 1% nonfat dry milk at 4°C overnight. Following secondary antibody incubation for 1 h at room temperature, bands were visualized using a Super Signal Chemiluminescence kit (Millipore, Billerica, MA, USA), and exposed to Kodak X-Max film. β-actin was used as the internal control after stripping off the original membrane. The images were scanned and relative band intensities were calculated using the NIH ImageJ software.

PKCδ Steath™ RNAi transfections

Based upon the sequence of human PRKCD (GenBank™ accession no. NM_006254), custom steath™ RNAi oligos (Invitrogen, Carlsbad, CA, USA) at 25-base-pair in length, 5′-CCACUACAUCAAGAACCAUGAGUUU-3′ was designed. A non-specific Steath™ RNAi (NS-RNAi) control, 5′-CCAUGGCGCCAAUUCCAAACAGUUU-3′ was also synthesized. All RNAi transfections at doses from 25 to 200 nM were performed using Lipofectamine 2000 (Life Technologies) following the manufacturer's instructions. For the analysis of PKCδ knockdown following RNAi transfection, LAPC4 cells (5×105 cells/well) were seeded in 6-well plates and transfected with various concentrations of RNAi at 24 h after plating. The cells were harvested at 96 h of transfection. Total cellular proteins were extracted and subjected to western blot analysis as described above. For cell proliferation and flow cytometric analysis, LAPC4 cells were seeded in 96-well and 60 mm plates, respectively as described above. Cells were treated with various regimens for 72 h after 24-h RNAi transfection (100 nM) as indicated in each experiment. Cell proliferation was determined by BrdU assay and cell apoptosis and cell cycle were analyzed by flow cytometry as described above.

Real-Time PCR

Approximately 5×105 LAPC4 cells were seeded in 6-well plates and treated with different regiments, and harvested at different time-points as indicated in each experiment. Total cellular RNA was extracted with TriPure Isolation reagent following the manufacture's instructions. One microgram of RNA per sample was used for reverse transcription using M-MLV reverse transcriptase with RNase inhibitor. Real-time PCR was done with an Eco Real-Time PCR system (Illumina, San Diego, CA, USA). CCNA2 expression level was detected using 2X SYBR™-Green Real-Time PCR master mix. The expression level was normalized with human ACTB using ΔΔCt method. The primers used were as follows: for CCNA2 forward, 5′-TGGACCTTCACCAGACCTAC-3′ and reverse, 5′-GGTTGAGGAGAGAAACACCA-3′; for ACTB forward, 5′-CTAGAAGCATTTGCGGTGGACGATG-3′ and reverse, 5′-TCATGAAGTGTGACGTGGACATCCG-3′. The final concentration of forward and reverse primer in each reaction system was 200 nM, respectively, and the final concentration of total RNA templates was 40 ng/well. The cycling conditions are listed in Table I.

Table I Real-time PCR conditions.

Table I

Real-time PCR conditions.

Cycle no.	Step	Temperature (°C)	Time (sec)
1X	UDG incubation	50	120
1X	Polymerase activation	95	600
40X	Denature	95	15
	Anneal/extend	60	60
1X	Melt curve analysis	95	15
		55	15
		95	15

[i] UDG, uracil DNA glycosylase.

Statistics

The data are presented as mean ± standard error of the mean. One-way analysis of variance (ANOVA) following post-hoc Student-Newman-Keuls test was used to determine the differences among multiple groups. A p-value <0.05 was considered statistically significant. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (23). All analyses were performed using SigmaPlot 12.0 from Systat Software (Chicago, IL, USA).

Results

CPT and its analogs produce a dose- and time-dependent dual action on cell growth and death in LAPC4 cells

LAPC4 cells were treated with various doses of CPT and NSC for 24, 48 or 72 h. As shown in Fig. 1A, at 72 h, NSC at doses ranging from 10 to 100 nM produced a dose-dependent increase in viable cell number (1.7-folds of control at 100 nM, p<0.01); while at doses ranging from 500 nM to 5 µM, NSC produced a dose-dependent decrease in viable cell number (0.48-fold of control at 5 µM, p<0.01). This biphasic effect of NSC was also time-dependent (Fig. 1B). An increase in viable cell number was observed at 24 h and more obvious at 48 and 72 h with 50 nM NSC treatment, while a time-dependent decrease in viable cell number was observed when cells were treated with 1 µM NSC. Similar dose-dependent biphasic effect on viable cell number was observed after 72 h treatment with CPT (Fig. 1A) and topotecan (Fig. 1C) in LAPC4 cells, although the stimulation by CPT and topotecan was relatively weaker compared to NSC. Like NSC and CPT (14), topotecan at a low-dose (50 nM) did not stimulate cell growth, while significantly decreased viable cell numbers at a high-dose (1 µM) in DU145 and LNCaP cells (p<0.001) (Fig. 1C).

Figure 1 CPT and its analogs (NSC and topotecan) produce biphasic effect on cell growth and death in PCa cells. (A) Dose-dependent dual action of NSC and CPT on viable cell number in LAPC4 cells after 72 h treatment. The data are shown as mean ± SEM, n=6–12 for NSC, n=3–6 for CPT. (B) Time- and dose-dependent dual action of NSC on viable cell number in LAPC4 cells. The data are shown as mean ± SEM, (n=3). (C) Effects of topotecan treatment for 72 h on cell viability in different PCa cell lines. The data are shown as mean ± SEM, n=6–9. (D) Morphological changes of LAPC4 cells after NSC treatment (72 h). *p<0.05 and **p<0.001 compared to corresponding controls. CPT, camptothecin.

Morphological analysis showed that there were more viable cells with higher cell density in NSC 50 nM group than in control group after 72 h treatment (Fig. 1D). In contrast, 1 µM NSC induced significant decrease in cell number with apoptotic characteristics such as shrunken cells and apoptotic bodies (Fig. 1D).

NSC induces apoptosis in LAPC4 cells

To investigate whether the NSC-induced decrease in viable cell number is associated with cell apoptosis, Annexin V/PI staining was performed to determine the apoptotic and/or necrotic cells after NSC treatment in LAPC4. Treatment with NSC produced a time- and dose-dependent increase in early (Annexin V+/PI−), late apoptotic (Annexin V+/PI+) and necrotic cells (Annexin V−/PI+) as shown in Fig. 2A. The fractions of Annexin V+ cells were significantly increased at 48 and 72 h of NSC treatment, and the changes were much greater at 1 µM than 50 nM dose. The pro-apoptosis effect was further confirmed by demonstrating that NSC produced a time- and dose-dependent induction of DNA fragmentation (Fig. 2B), and cytosolic cytochrome c release (Fig. 2C).

Figure 2 NSC induces cell apoptosis in LAPC4 cells. (A) Time- and dose-dependent apoptosis of LAPC4 cells after NSC treatment. The data are shown as mean ± SEM, n=6. *p<0.05 and **p<0.01 compared to control. (B) NSC induced DNA fragmentation in LAPC4 cells. (C) NSC induced cytochrome c release from mitochondria to cytosol in LAPC4 cells.

The dual action of NSC in LAPC4 cells involves PKCδ

To investigate whether the NSC-caused dual action involves PKCδ activation, rottlerin was used to inhibit PKCδ activity. As shown in Fig. 3A, at 1 µM, rottlerin completely blocked the low-dose (50 nM) NSC-induced cell growth and significantly rescued the high-dose (1 µM) NSC-caused decrease in viable cell number. On the other hand, FMK, a pan-caspase inhibitor, significantly rescued the high-dose NSC-induced decrease in viable cell number without any effect on the low-dose NSC-induced cell growth. Moreover, PKCδ knockdown by specific RNAi (100 nM) blocked NSC dual action in LAPC4 cells as shown in Fig. 3B. In contract, transfection of an NS-RNAi failed to alter NSC effect on DNA biosynthesis (Fig. 3B). The knockdown of PKCδ by RNAi transfection was confirmed using western blot analysis (Fig. 3C and D).

Figure 3 The dual action of NSC involves PKCδ in LAPC4 cells. (A) Rottlerin (ROT) blocked NSC-induced biphasic effect in LAPC4 cells. (B) NSC-induced biphasic effect on DNA biosynthesis was blocked by rottlerin and knockdown of PKCδ in LAPC4 cells. The data in (A and B) are shown as mean ± SEM, n=6–9. *p<0.05 and **p<0.01 compared to control; ##p<0.01 compared to corresponding NSC treatment. (C and D) Confirmation of the knockdown of PKCδ by RNAi transfection with or without NSC treatment. ROT, rottlerin; FMK, Z-VAD-fluoromethylketone; NS RNAi, non-specific RNAi; PKCδ, protein kinase Cδ.

In flow cytometry analysis, as shown in Table II, co-administration of rottlerin at 1 µM for 72 h, which did not have any significant impact on cell apoptosis per se, markedly inhibited the NSC-induced apoptosis. PKCδ-knockdown significantly attenuated the 1 µM NSC-induced late (Annexin V+/PI+) and early (Annexin V+/PI−) apoptotic cells from 19.93 and 3.32% to 7.81 and 1.68%, respectively (Table II). In contrast, transfection of the NS-RNAi did not significantly affect NSC-induced apoptosis.

Table II Blockade of NSC-induced cell apoptosis by rottlerin and specific PKCδ RNAi using flow cytometry analysis (72 h).

Table II

Blockade of NSC-induced cell apoptosis by rottlerin and specific PKCδ RNAi using flow cytometry analysis (72 h).

Cell types	Early apoptotic cells Late apoptotic cells (Annexin V+/PI−, % of total cells)	(Annexin V+/PI+, % of total cells)
Treatment
Control	0.66±0.12	2.4±0.31
NSC 50 nM	3.31±0.64a	6.92±1.16b
NSC 1 µM	3.32±0.61a	19.93±4.16b
Rottlerin 1 µM	0.66±0.18	3.91±0.75
Rottlerin+NSC 50 nM	0.13±0.06	3.94±0.87c
Rottlerin+NSC 1 µM	5.11±1.28b	6.81±1.72b,d
PKCδ RNAi	2.59±0.81	4.60±0.81
PKCδ RNAi+NSC 50 nM	2.97±0.88	5.91±0.11
PKCδ RNAi+NSC 1 µM	1.68±0.43d	7.81±1.42a,c
NS RNAi	4.16±0.77	5.25±1.22
NS RNAi+NSC 50 nM	5.58±1.03	9.4±1.92a
NS RNAi+NSC 1 µM	14.85±3.89b	19.71±4.50b

{ label (or @symbol) needed for fn[@id='tfn2-ijo-51-05-1601'] } The data are shown as mean ± SEM of three independent experiments.

a p<0.05 and

b p<0.01 compared to control;

c p<0.05 and

d p<0.01 compared to the corresponding NSC treatment. PKCδ, protein kinase Cδ.

Consistent with the inhibition of NSC-induced cell apoptosis, NSC-induced cytochrome c release from mitochondria to cytosol was also greatly attenuated by the co-administration of 1 µM rottlerin (Fig. 4A) and the transfection of 100 nM PKCδ RNAi (Fig. 4B). However, the transfection of an NS-RNAi did not change the NSC-induced cytochrome c release as shown in Fig. 4B.

Figure 4 NSC-induced cytochrome c release from mitochondria to cytosol is blocked by rottlerin and knockdown of PKCδ in LAPC4 cells (72 h). (A) Cytochrome c release after 72 h NSC treatment with or without co-treatment of rottlerin (1 µM). (B) Cytochrome c release after 72 h NSC treatment with 24 h pre-transfection of either NS RNAi (100 nM), or PKCδ RNAi (100 nM). NS RNAi, non-specific RNAi. PKCδ, protein kinase Cδ.

NSC produces a dose-dependent differential PKCδ cleavage in subcellular compartments

To explore the potential mechanism of NSC dual action on cell growth and apoptosis, the proteolytic cleavage of PKCδ in various subcellular compartments were analyzed by western blot analysis. The total PKCδ expression level was not altered with NSC treatment, but a slight increase of PKCδ cleavage was observed in total cellular protein after NSC treatment as shown in Fig. 5A. Most interestingly, NSC treatment resulted in a dose- and time-dependent differentiated change of PKCδ proteolytic cleavage in different subcellular compartments as shown in Fig. 5B–D. Treatment with a high-dose (1 µM) NSC resulted in a more rapid and robust PKCδ cleavage in the membrane/mitochondrial fraction than those treated with a low-dose (50 nM) NSC (Fig. 5B). The level of mitochondrial PKCδ cleavage was elevated >4-fold at 24 h of 1 µM NSC treatment and sustained for at least 72 h. Similar time-dependent PKCδ cleavage was observed at either a low or a high-dose NSC treatment in the cytosol (Fig. 5C). Whereas in the nuclear compartment, NSC-induced increase in PKCδ cleavage was more rapid, intense and sustainable at low-dose (>4-fold) compared to high-dose treatment (~2-fold) (Fig. 5D). Moreover, the addition of 1 µM rottlerin greatly reduced both the low-dose and high-dose NSC-induced proteolytic cleavage of PKCδ in LAPC4 cells (Fig. 5E). Taken together, these data indicate that NSC produced a dose-dependent differential PKCδ cleavage in the subcellular compartments of LAPC4 cells.

Figure 5 NSC induces proteolytic cleavage of PKCδ in a subcellular compartment-specific manner in LAPC4 cells. (A) Total cellular PKCδ cleavage in LAPC4 cells after NSC treatment. (B) Mitochondrial PKCδ cleavage in LAPC4 cells after NSC treatment. (C) Cytosolic PKCδ cleavage in LAPC4 cells after NSC treatment. (D) Nuclear PKCδ cleavage in LAPC4 cells after NSC treatment. (E) Differential subsellular PKCδ cleavage in LAPC4 cells after NSC treatment with or without rottlerin (1 µM) co-treatment (72 h). The quantitative changes in PKCδ cleavage are indicated at the bottom of the PKCδ western blot analyses (A–D) expressed as folds of control. PKCδ, protein kinase Cδ.

NSC produces a dose-dependent alteration in cell cycle in LAPC4 cells

Analysis of nuclear DNA distribution showed that NSC produced a time- and dose-dependent increase in hypoploid cells (sub-G1 cells) as shown in Table III, in agreement with the Annexin V/PI staining analysis as described above (Fig. 2A and Table II). The fraction of sub-G1 cells, an important indicator of cell apoptosis, was markedly increased in cells treated with 1 µM NSC for 72 h, while it only had slight increase in cells treated with 50 nM NSC as shown in Table III. NSC at 50 nM produced a significant elevation in the fraction of G2/M cells. Consistent with the concept that NSC-induced cell apoptosis involves PKCδ activation, the addition of rottlerin and PKCδ RNAi transfection both markedly inhibited the high-dose (1 µM) NSC-induced sub-G1 cells (p<0.01) without significant alterations in NSC-induced other cell cycle changes. Whereas, a non-specific RNAi had no effect on NSC-induced cell cycle changes (Table III).

Table III NSC-induced cell cycle changes and the blockade by rottlerin and specific PKCδ RNAi using flow cytometry analysis.

Table III

NSC-induced cell cycle changes and the blockade by rottlerin and specific PKCδ RNAi using flow cytometry analysis.

Cell cycle	Sub-G1 (% of total cells)	G1 (% of total cells)	S (% of total cells)	G2/M (% of total cells)
Treatment
Control	2.54±0.35	58.74±2.27	11.07±0.60	28.63±1.61
NSC 50 nM	4.18±0.43	45.13±2.11b	10.24±1.16	38.96±1.88a
NSC 1 µM	16.44±1.63b	42.28±2.10b	17.25±1.67	23.18±2.02
Rottlerin 1 µM	4.60±0.29	54.78±2.03	10.31±1.34	30.62±1.65
Rottlerin+NSC 50 nM	4.87±0.45	47.28±1.98b	10.02±1.14	37.64±1.67a
Rottlerin+NSC 1 µM	7.14±0.72a,d	41.16±2.34b	18.31±1.35	33.77±1.79
PKCδ RNAi	3.76±0.53	53.82±2.02	11.94±1.31	32.68±1.87
PKCδ RNAi+NSC 50 nM	4.58±0.45	42.17±2.01b	12.73±1.20	39.54±1.74a
PKCδ RNAi+NSC 1 µM	6.42±0.74a,d	41.5±2.02b	18.39±1.55	32.85±2.07
NS RNAi	4.09±0.48	54.16±2.06	10.59±1.32	31.75±1.61
NS RNAi+NSC 50 nM	4.60±0.57	42.36±2.12b	13.19±1.22	38.82±2.02a
NS RNAi+NSC 1 µM	17.35±1.47b	42.57±2.36b	17.12±1.46	22.64±1.52

{ label (or @symbol) needed for fn[@id='tfn7-ijo-51-05-1601'] } LAPC4 cells were treated as indicated for 72 h and cell cycle was determined by flow cytometry as described in section Materials and methods. The data are shown as mean ± SEM of three independent experiments. NS RNAi, non-specific RNAi.

a p<0.05 and

b p<0.01 compared to control;

c p<0.05 and

d p<0.01 compared to the corresponding NSC treatment.

NSC-induced cell proliferation is associated with an induction of cyclin A expression and cyclin-dependent kinase activity

The activity of cyclin/cyclin-dependent kinase (CDK) complexes that regulate the progression of cell cycle is controlled by the synthesis of appropriate cyclins during a specific phase of the cell cycle. Western blot analysis demonstrated that treatment with NSC at either a low (50 nM) or a high-dose (1 µM) dramatically increased cyclin A expression in LAPC4, but not in DU145 cells (Fig. 6A, B and D). The NSC-induced cyclin A expression was greatly attenuated by rottlerin, but not by FMK (Fig. 6A). RT-PCR analysis confirmed that the levels of CCNA2 mRNA, the somatic type of cyclin A, were significantly elevated at 72 h of NSC treatment as shown in Fig. 6C. Co-administration of roscovitine (ROS), a selective CDKs inhibitor, at doses ranging from 0.01 to 1 µM completely blocked the low-dose NSC (50 nM) inducing cell growth in LAPC4 cells (Fig. 6E).

Figure 6 NSC-induced cell growth involves upregulation of cyclin A expression in LAPC4 cells. (A and B) Cyclin A expression in LAPC4 and DU145 cells after 72 h treatment of various regiments as indicated. (C and D) CCNA2 mRNA and cyclin A protein expression in LAPC4 cells after 72 h NSC treatment, respectively. The data are shown as mean ± SEM, n=5–9. (E) Effects of roscovitine (Ros) on cell growth in NSC-treated (50 nM) LAPC4 cells (72 h). The data are shown as mean ± SEM, n=6. *p<0.05 and **p<0.01 compared to control; ##p<0.01 compared to NSC treatment alone. ROT, rottlerin; FMK, Z-VAD-fluoromethylketone; Ros, roscovitine.

Discussion

CPT, a unique pentacyclic quinoline alkaloid isolated from Camptotheca acuminate (24), a native tree from Tibet, China, is one of the prominent leading compounds in anticancer drug development (6). Unfortunately, CPT analogs have only shown limited benefits in the treatment of CRPC (11–13). Previous studies indicate that NSC, a synthetic CPT analogue, induces cell apoptosis and growth inhibition in PCa cells (14), leukemia cells (16), and ovarian cancer cells (25) through an activation of the intrinsic apoptotic pathway and induction of cell cycle arrest. In the present study, we demonstrated, for the first time, that both CPT and its analogs such as NSC and topotecan produced a dose-dependent dual action on cell growth and cell apoptosis in LAPC4 PCa cells (Fig. 1). At low-doses (10–100 nM), both CPT and its analogs produced an overall favorable effect on cell proliferation, whereas, at high-doses (500 nM-5 µM), CPT and its analogs predominately caused cell apoptosis. Using NSC as an example, we further demonstrated that this dual action was mediated, at least in part, via PKCδ activation on both ends. This conclusion is supported by the results that co-administration of rottlerin, or transfection of PKCδ RNAi, both significantly inhibited the proliferative effect induced by low-dose NSC (Fig. 3A–B), and blocked the apoptotic effect induced by high-dose NSC (Figs. 3A and B and 4 and Table II).

PKCδ is abundantly expressed in mammalian tissues, and has been proved to be involved in the regulation of both intrinsic and extrinsic apoptosis for a wide range of stimuli through various cell-type specific pathways (18,26). It can be activated mainly by four mechanisms, one of which is proteolytic cleavage (22,27). Generally, when the intrinsic/mitochondrial apoptosis pathway is induced by cell stress such as DNA damage by UV, cisplatin and CPT analogs, PKCδ can be proteolytically cleaved by caspase-3 into a 38 kDa regulatory fragment, and a 41 kDa constantly activated catalytic fragment which translocates to mitochondria, leading to an amplification of the apoptosis cascade in various cell types (27–30). Furthermore, PKCδ also acts as a 'gatekeeper' to inhibit cell cycle progression through G1/S and G2/M checkpoints by mediating p21CIP1, a G1 CDK inhibitor, and decreasing the expression of cyclins (31). Our previous studies in leukemic cells have demonstrated that NSC rapidly induces the proteolytic activation of PKCδ with a loss of mitochondrial transmembrane potential (Δψm) and caspase-3 activation, which is completely blocked by co-administration of rottlerin (16). In the present study, we have demonstrated that NSC produced a time- and dose-dependent induction of cell apoptosis (Fig. 2) and PKCδ proteolytic activation (Fig. 5) in LAPC4 cells. These NSC-induced changes were blocked by co-administration of rottlerin or knockdown of PKCδ with a specific RNAi (Figs. 3, 4 and 5 and Tables II and III). Our results suggest that NSC induced DNA damage and PKCδ proteolytic activation, leading to an activation of the intrinsic apoptosis pathway in LAPC4 cells.

Most surprisingly, for the very first time, we found that both CPT and CPT analogs (NSC and topotecan) induced cell growth in LAPC4 cells at nanomolar doses. Unlike the NSC-induced apoptotic effect, this proliferative effect of NSC was involved in PKCδ, but not caspase activation (Figs. 3 and 6). Although PKCδ has long been proposed as a pro-apoptotic gene and a tumor suppressor, growing evidence suggests that it is also involved in cell proliferation and survival pathways (21,32,33). PKCδ activation has been reported to induce the insulin-like growth I factor (IGF-I) proliferative signaling in renal carcinoma cells (34), increase the anchorage-independent growth of metastatic breast cancer cells (35), and be correlated with reduced overall breast cancer patient survival (36). In contrast, downregulation of PKCδ in breast cancer cells results in impairment in cell survival and a potentiation of chemotherapeutic agent-induced apoptosis (37). Although not fully understood, PKCδ has been reported to regulate multiple molecular signal pathways including NF-κB, Akt-PI3K, mTOR, and Ras/Raf/MEK/MAPK mitogenic signal transduction pathways (20,21,38), which may lead to cell survival and proliferation. In the present study, we demonstrated that NSC greatly induced cyclin A expression in LAPC4 cells (Fig. 6), a well-known factor in accelerating cell proliferation via interacting with CDK2 (39). The NSC-induced cyclin A expression was associated with NSC-induced cell growth since roscovitine, a specific CDK inhibitor, completely blocked NSC-induced cell growth (Fig. 6E). Moreover, rottlerin, but not FMK, completely blocked NSC-induced cyclin A expression and cell growth, indicating that PKCδ activation is upstream of cyclin A expression (Fig. 6A). The NSC-induced cyclin A upregulation was not observed in DU145 cells, consistent with our precious study (14), in which we revealed a potent apoptotic but not proliferative effect of NSC in DU145 cells, indicating a cell-dependent differential effect of NSC in PCa cells. Taken together, our data strongly indicate that administration of NSC in LAPC4 cells produced a proteolytic activation of PKCδ leading to an upregulation of cyclin A, resulting in a promotion of cell survival and proliferation (Fig. 7). We therefore, for the first time, demonstrated that PKCδ activation is associated with both cell proliferation and apoptosis in the same cell line.

Figure 7 A schematic representation of NSC-induced dual action on cell proliferation and apoptosis in LAPC4 cells. Solid lines indicate defined actions and dashed lines possible actions. Top I, topoisomerase I; Rot, rottlerin; Ros, roscovitine; FMK, Z-VAD-fluoromethylketone; CDKs, cyclin dependent kinases.

It is intriguing why PKCδ activation by NSC results in this dose-dependent biphasic effect. This unconventional dose-response effect has traditionally been attributed to a functional antagonism model, which proposed that the drug interacts with two independent receptor-effector systems, causing effects that counteract each other in the same system (40–42). The overall outcome would therefore be governed by the sum of the two independent dose-response effects. However, unlike the two independent systems predicted in the model, we observed that the stimulation of both cell proliferation and apoptosis involve PKCδ activation. We have therefore hypothesized that this NSC biphasic effect may be mediated through a differential activation of PKCδ in subcellular compartments. This hypothesis is supported by our demonstration that a low-dose (50 nM) of NSC predominately increased the proteolytic PKCδ cleavage in nuclei, while a high-dose (1 µM) of NSC mainly induced PKCδ cleavage in mitochondria (Fig. 5). Although the functional significance of this subcellular differential activation of PKCδ remains to be elucidated, this is in agreement with previous demonstrations that PKCδ activation in mitochondria causes cell apoptosis (28,30), while its activation in nuclei may induce cell proliferation (43). Studies have shown that PKCδ nucleic trans-location is a central step mediating IGF-I-induced mitogenic and proliferative signaling in primary human skeletal cells (43), and in hypoxia-induced cell proliferation and differentiation in human lung epithelial cells (44). Based on previous studies and our present data, we propose that the dose-dependent biphasic effect of NSC on LAPC4 cells is an integration of cell proliferation and cell apoptosis induced by the differential subcellular activation of PKCδ as illustrated in Fig. 7. At low-doses of NSC, PKCδ activity is predominately elevated in the nucleus, presumably due to a relatively low-level DNA damage, leading to a more prominent upregulation of pro-proliferation genes such as cyclin A and consequently cell growth. On the other hand, PKCδ activation is dramatically increased in the mitochondrion at high NSC doses, presumably due to a higher level DNA damage, which leads to a more prominent activation of the intrinsic apoptotic pathway, outweighs its proliferative effect, and eventually causes cell death.

Finally, the biological and clinical significance of differential response to CPT analogs among PCa cells should not be underestimated. Among the cell lines tested, we have observed that the order of cell sensitivity to NSC-induced cell death is DU145 > PC3 > LNCaP > LAPC4 (14). DU145 cells are the most sensitive PCa cells to NSC with a dramatic apoptosis and cell death at nanomolar concentrations. However, at the same concentration, NSC and topotecan significantly induced cell viability in LAPC4 cells (Fig. 1). Moreover, unlike the molecular alterations observed in LAPC4 cells, NSC neither activates PKCδ (14), nor alters cyclin A expression (Fig. 6B) in DU145 cells. This cell-differential effect suggests that the response of individual patients to CPT chemotherapy is variable, which may account, at least in part, for the failure of CPT-related clinical trials in PCa patients (11–13).

In conclusion, the present study demonstrates that CPT and its analogs, such as NSC and topotecan, produce a dose-dependent biphasic effect on cell growth and apoptosis in LAPC4 PCa cells, which may be mediated through a differential subcellular activation of PKCδ. This atypical biphasic effect of CPT analogs has clear cell specificity. The differential response of different prostate cancer cells to CPT analogs underscores the importance of identifying specific biomarker(s) associated with drug sensitivity, which will guide clinical trials and patient management, leading to an individualized chemotherapy for PCa patients.

Acknowledgments

We are very grateful to Dr Charles Sawyer (Memorial Sloan-Kettering Cancer Center) for the LAPC4 cells. This study is supported in part by grants from the National Institute of Health (no. UL1-TR000457), the Argenbright Research Fund and the Cohen Research Fund.

References