Abstract
Survivin, a member of inhibitor of apoptosis family, is associated with both prostate cancer progression and drug resistance. Therefore, we hypothesized that survivin may play a potentially important role in hormone-refractory prostate cancer (HRPC) and bone metastatic disease; thus, targeting of survivin signaling could enhance therapeutic efficacy in prostate cancer. 3,3′-Diindolylmethane (DIM) has been known to have cancer chemoprevention activity. However, no information is available regarding the down-regulation of survivin by DIM, which could result in the chemosensitization of HRPC cells to Taxotere-induced killing. We investigated the effect of DIM alone or in combination with Taxotere using LNCaP and C4-2B prostate cancer cells. We observed that DIM enhanced Taxotere-induced apoptotic death in both cell lines. These enhancing effects were related to a decrease in survivin expression as well as androgen receptor and nuclear factor-κB (NF-κB) DNA-binding activity. We also found that knockdown of survivin expression by small interfering RNA transfection increased DIM-induced cell growth inhibition and apoptosis, whereas overexpression of survivin by cDNA transfection abrogated DIM-induced cell growth inhibition and apoptosis in both prostate cancer cells. Importantly, luciferase assays showed a significant reduction of survivin-Luc and NF-κB-Luc activity in prostate cancer cells exposed to DIM and Taxotere. Furthermore, combination treatment significantly inhibited C4-2B bone tumor growth, and the results were correlated with the down-regulation of survivin. From these results, we conclude that inactivation of survivin by DIM enhanced the therapeutic efficacy of Taxotere in prostate cancer in general, which could be useful for the treatment of HRPC and metastatic prostate cancer. [Cancer Res 2009;69(10):4468–75]
Introduction
Prostate cancer is one of the leading causes of death among men in the United States (1). Almost all prostate cancer patients treated with hormone ablation therapy develop hormone-refractory prostate cancer (HRPC) and bone metastatic disease with functional androgen receptor (AR; refs. 2–7). Progression of prostate cancer to androgen independence remains the primary barricade in improving patient survival due to complex mechanisms underlying the evolution to androgen independence, and at present, there is no curative treatment for HRPC and bone metastatic disease (3). Although the use of several different agents resulted in pain relief in some patients, there was little or no effect on survival. Therefore, an important challenge to develop treatments that are more effective depend on our understanding of the molecular mechanism(s) of prostate cancer progression, which will help to identify many potential therapeutic target genes that are involved in apoptosis, growth factor, and cell signaling.
It is known that regulation of apoptosis has a central role in the development of prostate cancer and its progression to an androgen-independent state, which is, in part, due to up-regulation of antiapoptotic genes after androgen deprivation (2, 3, 8–11). Survivin is a member of the inhibitor of apoptosis proteins (IAP) that is expressed at high levels in most human cancers and may facilitate evasion from apoptosis and progression of prostate cancer to an androgen-independent state (9, 12–15). Survivin is also highly articulated in all major tumor types and its role in metastasis remains unknown (9). Moreover, it is undetectable in most normal differentiated tissues. Since a correlation exists between high expression of survivin in tumors and poor survival among patients with various cancers (16–18), survivin is considered a novel target in various cancer therapies (19). In this study, we examined whether survivin could play a potentially important role in HRPC and bone metastatic disease and studied whether targeting the survivin pathway could enhance the therapeutic efficacy.
There is increasing evidence suggesting that survivin is associated with both progression of HRPC and resistance to chemotherapy (9, 12, 13, 20, 21). Therefore, suppressing survivin signaling may be a novel and effective therapeutic approach for HRPC. Currently, no information is available regarding the consequence of blocking survivin signaling, which may lower the antiapoptotic threshold in cancer cells, thus sensitizing prostate tumor cells to apoptosis. Several studies on survivin have revealed that several existing anticancer drugs show survivin-suppressive activity through various cell signaling pathways (22–24). However, the safety and toxic profile for potential use of chemotherapeutic agents, particularly for cancer chemotherapy, are major concerns for their efficacy. Thus, there is a dire need for the development of a novel and specific anti-survivin therapeutic strategy for the treatment of HRPC.
Previous studies have shown that 3,3′-diindolylmethane (DIM), a major in vivo acid-catalyzed condensation product of indole-3-carbinol, is thought to have protective effects against the development of human prostate cancer as well as breast cancer (11, 25–28). DIM is known to act as a potent inducer of apoptosis associated with down-regulation of Akt/nuclear factor-κB (NF-κB) and survivin in breast cancer cells (11, 26). It has also been indicated that HRPC is an aggressive and treatment-resistant disease and the pharmacologic therapeutic approach for this disease has previously shown limited efficacy (29, 30). Moreover, the function of DIM in HRPC therapy is unknown. In spite of the knowledge of DIM as a cancer chemopreventive agent, understanding of the molecular mechanism(s) of action by which DIM targets prostate cancer and its role in cancer therapy is lacking. Several molecular mechanism(s) that could be related to the multiple gene alterations observed in advanced prostate cancer are involved in the transition toward an androgen-independent state, including changes in cell growth and antiapoptotic factors such as Akt/NF-κB and survivin (31, 32). In HRPC, enhanced function of AR, together with the activation of Akt/NF-κB pathways, also promotes cancer cells to become resistant to androgen deprivation therapy (31, 32). In addition, previous studies have shown that androgen could induce oxidative stress resulting in the production of reactive oxygen species, which in turn could activate NF-κB and contribute to the induction of cell growth during the development or progression of prostate cancer (33). However, the down-regulation of survivin via inactivation of AR and NF-κB signaling by DIM could be an important strategy for the treatment of prostate cancer, especially for HRPC. The present options for treating metastatic prostate cancer are limited to surgery, chemotherapy, and radiation therapy or combined modality therapy. Commonly used anticancer agents for the treatment of HRPC do not result in clinically meaningful responses in most prostate cancer patients, suggesting the need for a combination therapy (34). In this study, we examined whether DIM increases apoptotic cell death, thereby inactivating survivin leading to chemosensitization of HRPC cells to Taxotere-induced killing using both LNCaP and C4-2B prostate cancer cells, which is more similar to human HRPC. Here, we report the mechanistic role of a nontoxic dietary chemopreventive agent, DIM, for the treatment as well as enhancement of the therapeutic efficacy of Taxotere for prostate cancer in general but most importantly for HRPC and bone metastatic disease. We believe that targeting survivin by DIM could be a novel approach for the treatment of HRPC and bone metastatic disease.
Materials and Methods
Cell culture and experimental reagents. The human prostate cancer cell lines LNCaP (hormone-sensitive and AR-positive) and C4-2B (hormone-insensitive and AR-positive) were used in this study. DIM was generously provided by Dr. Michael Zeligs (BioResponse) and dissolved in DMSO to make a 50 mmol/L stock solution. Anti-AR (Santa Cruz Biotechnology), anti-prostate-specific antigen (Lab Vision), anti-survivin (R&D Systems), anti-FoxM1 (Santa Cruz Biotechnology), anti-caspase-3 (Cell Signaling), anti-human poly(ADP-ribose) polymerase (PARP) antibody (Biomol), anti-NF-κB p65 (Upstate), and anti-β-actin (Sigma) primary antibodies were used for Western blot analysis. All secondary antibodies were obtained from Pierce. Taxotere (Aventis Pharmaceuticals) was dissolved in DMSO to make a 4 μmol/L stock solution.
Cell proliferation inhibition studies by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. LNCaP and C4-2B prostate cancer cells were seeded in 96-well plates. After 24 h, the cells were treated with 10, 30, and 40 μmol/L DIM followed by treatment of Taxotere with 0.5, 1.0, and 1.5 nmol/L for 24 to 72 h. After treatment, the cell growth studies were done by MTT as described earlier (26, 27).
Histone/DNA ELISA for detection of apoptosis. The cytoplasmic histone/DNA fragments from prostate cancer cells treated with 10 or 30 μmol/L DIM followed by treatment of Taxotere with 1.0 or 1.5 nmol/L for 24, 48, or 72 h were extracted and the Cell Death Detection ELISA Kit (Roche) was used to detect apoptosis in prostate cancer cells as described earlier (11, 26, 27).
Western blot analysis. Prostate cancer cells were treated with DIM at various concentrations for different times followed by treatment with and without Taxotere for 72 h. Cells were lysed in lysis buffer and protein concentrations were then measured using the Bio-Rad assay system (Bio-Rad). Total proteins were fractionated using SDS-PAGE and transferred to nitrocellulose membrane for Western blotting as described earlier (11, 26, 27).
NF-κB DNA-binding activity measurement. Prostate cancer cells were plated at a density of 1 × 106 in 100 mm dishes and cultured for 72 h. Subsequently, the cells were treated as described above. The total proteins and the nuclear proteins were extracted and subjected to Western blot analysis for survivin, AR, and NF-κB p65 and electrophoretic mobility shift assay (EMSA) for NF-κB DNA-binding activity by Odyssey Infrared Imaging System using NF-κB IRDye-labeled oligonucleotide (L1-COR, Inc.) as described earlier (26, 27, 35). Using frozen tumor tissues, nuclear proteins were also extracted as described previously (26, 27, 35).
Plasmids and transfection. Prostate cancer cells were transfected with survivin small interfering RNA (siRNA; Santa Cruz Biotechnology) and survivin cDNA, respectively, using Lipofectamine 2000 (11). The transfected cells were treated with 30 μmol/L DIM for 48 or 72 h. The cell growth and apoptotic cell death of transfected cells with and without treatments were measured using MTT assay and ELISA, respectively.
Luciferase assay. NF-κB-Luc (Stratagene) and survivin-Luc (plasmid containing 1,430 bp survivin promoter with luciferase coding sequence; Health Research) were transfected into C4-2B cells using Lipofectamine (Invitrogen) for 24 h followed by treatment with DIM (30 μmol/L), Taxotere (1.5 nmol/L), and DIM plus Taxotere for 24 h. Luciferase activities in the samples were measured following the procedures as described earlier (36). CMV-β-gal reporter construct transfection was used for normalization of transfection efficiency.
Animal studies. The severe combined immunodeficiency (SCID)-human prostate cancer and breast cancer model of experimental bone metastasis was used for our study as described earlier (37–39). Briefly, suspensions of C4-2B cells were injected intraosseously by insertion of a 27-gauge needle through the mouse (Taconic Farms) skin directly into the marrow surface of the previously implanted bone (38, 39). The mice were divided into four groups of 6 to 8 animals in each group. Group I was assigned as control and group II mice were given DIM (3.5 mg/d/animal; refs. 11, 26–28). In addition, the mice from group III received three doses of Taxotere (5 mg/kg body weight given intravenously) every 72 h after 24 h DIM gavaging (39, 40). Group IV was exposed to DIM and treated with Taxotere as shown for groups II and III. The mice in the intervention groups were given DIM by oral gavage every day for 5 weeks. The volume of the bone tumor in each group was determined by weekly caliper measurements according to the formula: ab2/2, where a is length and b is cross-sectional diameter. The mice were sacrificed 10 to 11 weeks after cell injection.
Histology and immunohistochemistry. Tissue collection, fixation, and H&E and immunohistochemical staining were done according to the methods as described earlier (26, 38, 39).
Statistical analysis. Data are represented as mean ± SD for the absolute values or percent of controls as indicated in the vertical axis legend of Figs. 1 to 3. The statistical significance of differential findings between experimental groups and control was determined by Student's t test as implemented by Excel 2000 (Microsoft) and GraphPad StatMate software (GraphPad Software). P < 0.05 was used to indicate statistical significance.
Effect of DIM and Taxotere on prostate cancer cell growth. For single-agent treatment, prostate cancer cells were treated with DIM (10, 30, or 40 μmol/L) or Taxotere (0.5, 1.0, or 1.5 nmol/L) alone for 72 h (A and B, left and middle). For combination, prostate cancer cells were treated with 30 μmol/L DIM and then exposed to 1.5 nmol/L Taxotere for 72 h (A and B, right). DIM in combination with Taxotere significantly inhibited cell proliferation in LNCaP (A, right) and C4-2B (B, right) prostate cancer cells as measured by MTT assay. Data from three individual experiments. *, P < 0.05; **, P < 0.01.
Effect of DIM and Taxotere on prostate cancer cell growth. For single-agent treatment, prostate cancer cells were treated with DIM (10, 30, or 40 μmol/L) or Taxotere (0.5, 1.0, or 1.5 nmol/L) alone for 72 h (A and B, left and middle). For combination, prostate cancer cells were treated with 30 μmol/L DIM and then exposed to 1.5 nmol/L Taxotere for 72 h (A and B, right). DIM in combination with Taxotere significantly inhibited cell proliferation in LNCaP (A, right) and C4-2B (B, right) prostate cancer cells as measured by MTT assay. Data from three individual experiments. *, P < 0.05; **, P < 0.01.
Results
DIM sensitizes prostate cancer cells to Taxotere-induced growth inhibition. We found that treatment of prostate cancer cells by DIM and Taxotere caused 45% to 65% growth inhibition (Fig. 1A, and B, left and middle). However, DIM in combination with a lower dose of Taxotere resulted in 85% to 90% growth inhibition in both prostate cancer cells, suggesting the greater inhibitory effect of combination treatment (Fig. 1A and B, right). These results show that combination of DIM along with a lower dose of Taxotere elicits significantly greater inhibition of cancer cell growth compared with either agent alone. The lower dose of Taxotere in inhibiting cell growth when combined with a nontoxic dietary chemopreventive agent (DIM) will have significant ramification for extending our studies for human prostate cancer treatment.
DIM sensitizes prostate cancer cells to Taxotere-induced apoptosis. By apoptotic cell death ELISA analysis, we found that 30 μmol/L DIM combined with lower dose of Taxotere induced greater apoptosis in prostate cancer cells compared with single-agent treatment (Fig. 2A). We also observed that DIM and Taxotere combination treatment in vitro produced stronger PARP cleavages compared with monotreatment (Fig. 2B), suggesting that the combination treatment could induce greater apoptosis in prostate cancer cells. Using Western blot analysis, we found that DIM alone or in combination with chemotherapeutic agents down-regulated the expression of survivin, AR, prostate-specific antigen, FoxM1, and NF-κB p65 in both prostate cancer cells (Fig. 2B). The combined treatment of DIM and Taxotere also resulted in an increase of caspase-3 activation in both prostate cancer cells (Fig. 2B). These results are consistent with the cell growth inhibition observed by MTT assay, suggesting that greater cell growth inhibition resulting from the combination treatment is partly mediated through the increased induction of apoptosis in prostate cancer cells.
Increased apoptotic response was evident in the combination treatment group relative to untreated control or single-agent–treated group as measured by ELISA (A). Western blot analysis showed that DIM in combination with Taxotere (Tax) significantly decreased the expression of survivin, poly(ADP-ribose) polymerase (PARP), AR, prostate-specific antigen (PSA), FoxM1, caspase-3, and NF-κB p65 (B). DIM and Taxotere inhibited the activity of NF-κB in prostate cancer cells as measured by EMSA (C). Retinoblastoma (Rb) protein level served as nuclear protein loading control. *, P < 0.05; **, P < 0.01.
Increased apoptotic response was evident in the combination treatment group relative to untreated control or single-agent–treated group as measured by ELISA (A). Western blot analysis showed that DIM in combination with Taxotere (Tax) significantly decreased the expression of survivin, poly(ADP-ribose) polymerase (PARP), AR, prostate-specific antigen (PSA), FoxM1, caspase-3, and NF-κB p65 (B). DIM and Taxotere inhibited the activity of NF-κB in prostate cancer cells as measured by EMSA (C). Retinoblastoma (Rb) protein level served as nuclear protein loading control. *, P < 0.05; **, P < 0.01.
DIM inhibits activation of NF-κB. Treatment with DIM resulted in a decreased NF-κB activity in both prostate cancer cells as shown in Fig. 2C (left and right). Interestingly, the combination of DIM and Taxotere showed inactivation NF-κB in both prostate cancer cell lines (Fig. 2C). These results show that DIM not only down-regulates the NF-κB activity but also inhibits NF-κB even more in the presence of a lower concentration of Taxotere, which could be responsible for better cell killing by combination treatment.
Down-regulation of survivin expression by siRNA transfection promotes DIM-induced cell growth inhibition and apoptosis. We found that treatment of cells with DIM or survivin siRNA alone for 72 h generally caused 60% to 70% of growth inhibition in prostate cancer cells compared with control. However, DIM plus survivin siRNA resulted in ∼90% growth inhibition compared with control (Fig. 3A,, left). Survivin siRNA-transfected prostate cancer cells were significantly more sensitive to spontaneous and DIM-induced apoptosis (Fig. 3A , right). These results suggest that DIM plus survivin siRNA promotes cell growth inhibition and apoptosis to a greater degree compared with either agent alone.
A, down-regulation of survivin expression by siRNA promotes DIM-induced cell growth inhibition and apoptosis in prostate cancer cells. B, overexpression of survivin by cDNA transfection abrogated DIM-induced cell growth inhibition and apoptosis in prostate cancer. C, transcriptional down-regulation of survivin by treatment with DIM. The results evidently show that luciferase activity from cell lysate of survivin-Luc (SurLuc)-transfected or NF-κB-Luc–transfected cells were decreased significantly in the presence of DIM and Taxotere. *, P < 0.05; **, P < 0.01.
A, down-regulation of survivin expression by siRNA promotes DIM-induced cell growth inhibition and apoptosis in prostate cancer cells. B, overexpression of survivin by cDNA transfection abrogated DIM-induced cell growth inhibition and apoptosis in prostate cancer. C, transcriptional down-regulation of survivin by treatment with DIM. The results evidently show that luciferase activity from cell lysate of survivin-Luc (SurLuc)-transfected or NF-κB-Luc–transfected cells were decreased significantly in the presence of DIM and Taxotere. *, P < 0.05; **, P < 0.01.
Overexpression of survivin by cDNA transfection reduces DIM-induced cell growth inhibition and apoptosis. Overexpression of survivin by cDNA transfection rescued DIM-induced cell growth inhibition and abrogated DIM-induced apoptosis to a certain degree (Fig. 3B,, left and right). We found that treatment of cells with survivin cDNA for 48 h promotes 80% to 90% of cell growth in prostate cancer cells compared with control. However, DIM plus survivin cDNA resulted in 35% to 50% growth inhibition compared with cDNA alone (Fig. 3B , left). These results provide evidence for a potential role of survivin during DIM-induced cell growth inhibition and apoptosis in prostate cancer cells.
DIM suppresses survivin gene transcription mediated by inhibition of NF-κB activity. Luciferase assays showed an increase in activity of prostate cancer cells transfected with survivin-Luc or NF-κB-Luc. In contrast, our results (Fig. 3C) clearly show that luciferase activity in survivin-Luc–transfected or NF-κB-Luc–transfected cells were significantly decreased in combination treatment. These results further showed that the down-regulation of survivin expression by DIM is a transcriptional event.
DIM enhances in vivo therapeutic efficacy of Taxotere. We found that DIM as well as Taxotere alone inhibited the C4-2B tumor growth within the bone environment to some extent. Interestingly, the combination of DIM and Taxotere showed 80% inhibition in tumor growth relative to control, showing the enhanced inhibitory effect of DIM and Taxotere combination in our model (Fig. 4A). We measured the DNA-binding activity of NF-κB and two of its target genes, such as survivin, AR, and NF-κB p65, within tumor tissues. Our results clearly show that survivin and its effector genes, such as AR, NF-κB p65, and NF-κB activity, were significantly down-regulated in specimens obtained from the combination group (Fig. 4B and C). These in vivo results were similar to our in vitro findings, suggesting that the inactivation of survivin is at least one of the molecular events by which the drug combination potentiates antitumor activity in our experimental model.
A, under the experimental conditions, combined treatment (DIM and Taxotere) caused an 80% reduction in tumor volume compared with control group (A, left). Comparison of the tumor volumes in each group on the day when all mice were sacrificed (A, right). *, P < 0.05; **, P < 0.01. The total proteins and the nuclear proteins were extracted from randomly selected frozen tumor tissues obtained from each treatment group of animals and subjected to Western blot and EMSA, respectively (B and C). These results showed that DIM and Taxotere were effective in down-regulating survivin, AR, NF-κB p65, and NF-κB in tumors from treated animals relative to tumors from control animals (B and C).
A, under the experimental conditions, combined treatment (DIM and Taxotere) caused an 80% reduction in tumor volume compared with control group (A, left). Comparison of the tumor volumes in each group on the day when all mice were sacrificed (A, right). *, P < 0.05; **, P < 0.01. The total proteins and the nuclear proteins were extracted from randomly selected frozen tumor tissues obtained from each treatment group of animals and subjected to Western blot and EMSA, respectively (B and C). These results showed that DIM and Taxotere were effective in down-regulating survivin, AR, NF-κB p65, and NF-κB in tumors from treated animals relative to tumors from control animals (B and C).
Tumor histology. The histology of the tumor in all sections was similar, each consisting of a high-grade carcinoma with only focal areas indicative of adenocarcinoma differentiation (Fig. 5A). Our findings suggest reduction in the percentage of viable tumor mass from the control to the treatment group, particularly for the combination treatment (Table 1). Also, extensive bone destruction with areas of bone remodeling/new bone formation was particularly evident in the control group (Fig. 5A) but not as apparent in the treatment groups. All of the treatment groups, especially in the combination treatment group (Fig. 5A; Table 1), showed a higher percentage of bony tissue compared with the control group in which the bony elements were almost completely destroyed/absent (Fig. 5A; Table 1).
Inhibition of C4-2B tumor growth by DIM and Taxotere using a SCID-hu model of prostate cancer bone metastasis (A). Mostly viable high-grade carcinoma with extensive bone destruction and only rare bony elements (control). Partially viable tumor with areas of acute hemorrhagic necrosis, fibrosis, and bony elements (DIM). Similar to DIM, the section shows partially viable tumor with a combination of acute hemorrhagic necrosis, fibrosis, and bony elements (Taxotere). The combined group showed a relatively lower percentage of viable tumor, more bony elements, and predominantly organizing necrosis/fibrosis (DIM+Tax). Immunohistochemical staining for AR (B) and survivin (C) in DIM plus Taxotere-treated and untreated animal tumors done on randomly selected tumor tissues. Tumor cells in untreated control group show intensive staining of AR (B) and survivin (C). In contrast, tumor cells in DIM plus Taxotere-treated group showing weaker staining of AR (B) and survivin (C).
Inhibition of C4-2B tumor growth by DIM and Taxotere using a SCID-hu model of prostate cancer bone metastasis (A). Mostly viable high-grade carcinoma with extensive bone destruction and only rare bony elements (control). Partially viable tumor with areas of acute hemorrhagic necrosis, fibrosis, and bony elements (DIM). Similar to DIM, the section shows partially viable tumor with a combination of acute hemorrhagic necrosis, fibrosis, and bony elements (Taxotere). The combined group showed a relatively lower percentage of viable tumor, more bony elements, and predominantly organizing necrosis/fibrosis (DIM+Tax). Immunohistochemical staining for AR (B) and survivin (C) in DIM plus Taxotere-treated and untreated animal tumors done on randomly selected tumor tissues. Tumor cells in untreated control group show intensive staining of AR (B) and survivin (C). In contrast, tumor cells in DIM plus Taxotere-treated group showing weaker staining of AR (B) and survivin (C).
Morphologic characteristics: microscopic evaluation of H&E
. | Average % viable tumor . | Average % acute necrosis or hemorrhage . | Average % fibrosis and/or fatty connective tissue . | Average % intact bony tissue . |
---|---|---|---|---|
Control | 62 | 22 | 13 | 4 |
DIM | 53 | 28 | 12 | 5 |
Taxotere | 51 | 32 | 8 | 8 |
DIM + Taxotere | 23 | 10 | 43 | 22 |
. | Average % viable tumor . | Average % acute necrosis or hemorrhage . | Average % fibrosis and/or fatty connective tissue . | Average % intact bony tissue . |
---|---|---|---|---|
Control | 62 | 22 | 13 | 4 |
DIM | 53 | 28 | 12 | 5 |
Taxotere | 51 | 32 | 8 | 8 |
DIM + Taxotere | 23 | 10 | 43 | 22 |
NOTE: All microscopic evaluations were done using randomly selected tumor tissues from control and treatment groups.
Analysis of survivin and AR expression by immunohistochemistry. The expression of AR (Fig. 5B) and survivin (Fig. 5C) was significantly decreased in C4-2B bone tumors in SCID-hu mice receiving the DIM compared with control. Importantly, the combination of DIM and Taxotere showed greater degree of down-regulation of AR (Fig. 5B) and survivin (Fig. 5C). Overall, our results suggest that the DIM and in combination with Taxotere may indeed reduce the levels of AR and survivin, which is mediated by the inactivation of NF-κB in the tumor microenvironment resulting in antitumor activity in our experimental model of prostate cancer bone metastasis.
Discussion
There is collective evidence suggesting that survivin is a strong apoptosis inhibitor, the expression of which is increased in aggressive prostate cancer compared with normal tissue (9, 12, 13, 21). Therefore, down-regulation of survivin mediated by the inactivation of NF-κB could be an important approach for devising novel therapeutic strategies for HRPC. Our data showed that DIM treatment in combination with Taxotere caused significant induction of apoptosis by down-regulation of survivin in prostate cancer cells. Several studies have suggested that the activation of NF-κB, a transcription factor, regulates hundreds of genes, such as survivin, which is critically involved in apoptosis (41). In this study, we also observed a drastic reduction of survivin and NF-κB DNA-binding activity in prostate cancer cells exposed to DIM and Taxotere. These results suggest that DIM may down-regulate survivin through NF-κB-mediated transcriptional inactivation, thereby inactivating AR, leading to apoptotic cell death and chemosensitization of HRPC cells to Taxotere.
Apoptosis is one of the most vital pathways through which chemopreventive agents inhibit the overall growth of cancer cells. Thus, it is important to investigate whether inhibition of cell proliferation and induction of apoptosis by any chemopreventive agents are associated with the down-regulation of antiapoptotic genes, such as survivin, which plays a critical role in prostate cancer cell progression (12, 13). It has also been shown that expression of survivin is associated with cancer cell viability and chemoresistance, which could be eliminated by chemopreventive agents (22, 42, 43). Naturally occurring dietary compounds found in cruciferous vegetables such as DIM have gained considerable attention as cancer chemopreventive agents. Previously, our laboratory as well others have shown a novel function of DIM, which induced apoptosis concomitant with down-regulation of survivin that could be due to the inactivation of Akt/NF-κB signaling in breast and prostate cancer cells (11, 25–28). In the present study, our results showed that DIM and Taxotere, as single agents, did induce apoptosis in prostate cancer cells as documented by ELISA and poly(ADP-ribose) polymerase cleavage assay, in conjunction with a more significant inhibition of cell growth as observed by MTT assay. However, treatment of cells with DIM in combination with a lower dose of Taxotere resulted in a significantly greater induction of apoptosis associated with the down-regulation of survivin in prostate cancer cells. Western blot analysis showed that the protein levels of AR, prostate-specific antigen, FoxM1, caspase-3, and NF-κB p65 were also down-regulated after DIM treatment. These results suggest that more significant inhibition of prostate cancer cell growth was caused by a low dose of combination treatment, which is mediated by the increased induction of apoptosis, acting through the inactivation of the protein survivin.
Recent evidence also indicates that the overexpression of survivin in tumors is associated with poor prognosis and increased tumor recurrence because it may overcome the G1-S checkpoint imposing progression of cells through cell cycle conferring proliferative advantage (9, 12–15, 19). In addition to its role in inhibiting apoptosis, survivin also interacts with the mitotic spindle (19, 44), which has been implicated as being essential for its antiapoptotic function (19). DIM per se was effective in down-regulating survivin in prostate cancer cells as well as inducing G1-S cell cycle arrest (45, 46). Based on our findings, one may speculate and argue that DIM in combination with Taxotere treatment may be effective in blocking cell cycle progression in the G1-S phase resulting from DIM-induced inhibition of survivin that leads the cells to undergo apoptosis (19, 44, 46). This highlights yet another important molecular mechanism of action of DIM in enhancing the therapeutic efficacy of cytotoxic agents because G1-S cell cycle arrest has emerged as an attractive therapeutic target for cancer therapy (19, 45, 46).
As documented previously, NF-κB found in prostate cancer cells may cause survival by inhibiting apoptosis. NF-κB also regulates the expression of a large number of genes that play critical roles in apoptosis, viral replication, tumorigenesis, various autoimmune diseases, and inflammation (47). Several studies have suggested that the activation of NF-κB up-regulates survivin gene expression, which protects cells from apoptosis (41, 48). Consistent with existing reports in the literature documenting that NF-κB induces survivin gene expression, we evaluated NF-κB activity to determine the mechanism(s) of increased apoptosis induced by DIM and Taxotere. Our results showed that DIM inhibited the expression of NF-κB activity, whereas Taxotere showed a lesser degree of apoptosis, supporting the observation that NF-κB causes inhibition of apoptosis, which is associated with up-regulation of survivin. It has been indicated that the survivin promoter region contains binding sites of several transcription factors such as NF-κB (49, 50). We hypothesized that DIM may inhibit the binding reaction of NF-κB in the survivin promoter region and inhibits transcription of the survivin gene. However, to further explore the inhibitory effects of DIM between survivin and NF-κB, we conducted transfection with a luciferase construct. Luciferase assays showed a significant reduction of survivin-Luc and NF-κB-Luc activity in prostate cancer cells exposed to DIM and Taxotere compared with control transfections where cells were transfected with empty vectors. In this study, we also found that down-regulation of survivin by survivin siRNA together with DIM treatment caused cell growth inhibition and apoptosis to a greater degree in prostate cancer cells compared with either agent alone. On the other hand, overexpression of survivin by survivin cDNA transfection abrogated DIM-induced apoptosis to a certain degree. Therefore, we strongly believe that down-regulation of survivin mediated by the inhibition of NF-κB is mechanistically linked with DIM and Taxotere-induced cell growth inhibition and apoptosis.
To test whether DIM has any antitumor effects in vivo and whether survivin is inactivated and correlated with down-regulation of NF-κB and induction of apoptotic cell death, we conducted an animal experiment using a xenograft model of experimental prostate cancer bone metastasis. We found that treatment of C4-2B prostate cancer cells with DIM sensitizes these cells to Taxotere, which could be mechanistically related to down-regulation of survivin and inactivation NF-κB DNA-binding activity in tumor tissues. Our present observation also showed that the maximum degree of necrosis occurred in DIM plus Taxotere treatment group. These results further support the conclusion that inhibition of survivin activation promotes Taxotere-induced apoptosis in vivo. In our immunohistochemical analysis, the expression of survivin and AR, two important molecules of tumor cell survival and metastasis, were significantly decreased in C4-2B bone tumors in SCID-hu mice receiving the DIM and Taxotere compared with control. In general, our in vitro and in vivo results showed that the utilization of a dietary agent DIM alone or in combination with other chemotherapeutic agents could be useful for the treatment of prostate cancer in general but most significantly for the treatment of hormone-refractory and metastatic prostate cancer. Importantly, our observations from preclinical animal model and in vitro studies revealed down-regulation of survivin as well as AR and NF-κB in sensitization experiments, which may be effective in contributing to the potentiation of Taxotere cytotoxicity by DIM. These observations on alterations in the expression status of survivin by DIM are of considerable importance, which strongly suggests that DIM sensitizes the prostate cancer cells to the cytotoxic effect of chemotherapeutic drugs.
In conclusion, the combination treatment significantly inhibited tumor cell growth, induced apoptosis, and inhibited the growth of experimental C4-2B cells in the bone microenvironment in our SCID-hu model of human prostate cancer bone metastasis. Our present findings suggest that DIM may function as an inhibitor of survivin signaling directly or indirectly due to the inactivation of AR and NF-κB, ultimately causing cell growth inhibition and induction of apoptosis, which may lead to chemosensitization of HRPC cells to Taxotere. Therefore, we believe that down-regulation of survivin and NF-κB signaling, thereby inactivating AR by DIM, could be a novel approach for sensitizing prostate cancer cells to Taxotere. Taken together, the results from our in vitro as well as in vivo experiments clearly suggest that the combination of DIM and Taxotere could be a potential regimen for the treatment of patients diagnosed with prostate cancer, especially HRPC and bone metastatic disease in the future. Further in-depth testing of combinations of various drugs with DIM are needed in support of designing mechanism-based anticancer therapies, particularly for bone metastatic prostate cancer for which there is no curative therapy.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Grant support: Department of Defense grant W81XWH-07-01-0145 (KM W. Rahman).
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.
We thank Kristin Dominiak for editorial assistance.