Dual effects of glutathione-S-transferase pi on As2O3 action in prostate cancer cells: enhancement of growth inhibition and inhibition of apoptosis

To determine the effects of glutathione-S-transferase pi (GSTpi) on the actions of As2O3, As2O3-induced growth inhibition and apoptosis was studied in three prostate cancer cell lines: DU-145, PC-3 and LNCaP cells. As2O3 inhibited cell proliferation of DU-145 and PC-3 cells (both cells express GSTpi), but not of LNCaP cells (which lack GSTpi expression) at concentrations below 1 microM. LNCaP cells stably transfected and expressed GSTpi (LNCaP/GSTpi) became sensitive to As2O3 growth inhibition. As2O3 arrested cell growth of DU-145, PC-3 and LNCaP/GSTpi cells in the G2/M phase of the cell cycle at low concentrations (<2 microM), but did not induce apoptosis. At higher concentrations (10-20 microM), As2O3 induced apoptosis in LNCaP cells, but not in DU-145 or PC-3 cells. The apoptosis induction due to As2O3 treatment of LNCaP cell correlated with the activation of JNK and p38 and induction of p53 protein. LNCaP/GSTpi cells became insensitive to As2O3-induced apoptosis with reduced JNK activition. These data indicate that GSTpi increases growth inhibition due to As2O3 treatment and prevents As2O3-induced apoptosis in prostate cancer cells. Therefore, it appears that As2O3 inhibits cell growth and induces apoptosis through different mechanisms.     

Cryptophycin-induced hyperphosphorylation of Bcl-2, cell cycle arrest and growth inhibition in human H460 NSCLC cells

Bcl-2 has been described as a factor that can protect from apoptosis. The protective effect of Bcl-2 may be lost if the protein is phosphorylated. Bcl-2 phosphorylation can be induced by agents that affect microtubule depolymerization or prevent microtubule assembly. In 13 human tumor cell lines there was a high degree of heterogeneity in Bcl-2 protein expression. Human H460 non-small-cell lung carcinoma (NSCLC) cells expressed high levels of Bcl-2 and were selected for study. Western blot analysis for Bcl-2 phosphorylation was carried out after 4 h and 24 h of exposure to cryptophycin 52, cryptophycin 55, paclitaxel or vinblastine. Cryptophycin 52 and cryptophycin 55 were very potent inducers of Bcl-2 phosphorylation. After 4 h of exposure, Bcl-2 phosphorylation was evident with 0.05 nM cryptophycin 52, 0.25 nM cryptophycin 55, 5 nM vinblastine and 50 nM paclitaxel. The hyperphosphorylated form of Bcl-2 was evident after 24 h exposure of H460 cells to 0.25 nM cryptophycin 52 or cryptophycin 55 and 50 nM vinblastine or paclitaxel. The effects of the compounds on the cell cycle paralleled those on Bcl-2 phosphorylation. In H460 cells 90% cell killing was obtained with 0.13 nM cryptophycin 52, 0.2 nM cryptophycin 55, 20 nM paclitaxel and > 100 nM vinblastine after 24 h of exposure as determined by colony formation. In Bcl-2-negative Calu-6 NSCLC cells, 90% cell killing was obtained with 0.03 nM cryptophycin 52, 0.1 nM cryptophycin 55, 11 nM paclitaxel and 0.5 nM vinblastine using the same experimental design. Thus, cryptophycins are potent inducers of Bcl-2 phosphorylation. The cryptophycins were more potent cytotoxic agents in Bcl-2-negative Calu-6 cells than in Bcl-2-positive H460 cells indicating that pathways triggered by Bcl-2 phosphorylation are involved in cryptophycin-induced lethality.     

Bcl-2 and Bcl-x(L) differentially protect human prostate cancer cells from induction of apoptosis by melanoma differentiation associated gene-7, mda-7/IL-24

Subtraction hybridization identified melanoma differentiation associated gene-7, mda-7, in the context of terminally differentiated human melanoma cells. Based on its structure, cytokine-like properties and proposed mode of action, mda-7 has now been classified as IL-24. When expressed by means of a replication-incompetent adenovirus, Ad.mda-7 induces apoptosis in a broad range of cancer cells, without inducing harmful effects in normal fibroblast or epithelial cells. These unique properties of mda-7/IL-24 suggest that this gene will prove beneficial for cancer gene therapy. We now demonstrate that Ad.mda-7 decreases viability by induction of apoptosis in hormone-responsive (LNCaP) and hormone-independent (DU-145 and PC-3) human prostate carcinomas, without altering growth or survival in early-passage normal human prostate epithelial cells (HuPEC). Ad.mda-7 causes G(2)/M arrest and apoptosis in LNCaP (p53-wildtype), DU-145 (p53 mutant, Bax-negative) and PC-3 (p53-negative) prostate carcinomas, but not in HuPEC. Apoptosis induction correlated with changes in the ratio of pro- to antiapoptotic Bcl-2 protein family members. A potential functional role for changes in bcl-2 family gene expression in Ad.mda-7-induced apoptosis was suggested by the finding that forced overexpression of bcl-x(L) or bcl-2 differentially diminished the apoptotic effect of Ad.mda-7 in prostate carcinomas. These results confirm that induction of apoptosis by the mda-7/IL-24 gene in prostate cancer cells is Bax- and p53-independent and is mediated by mitochondrial pathways involving bcl-2 family gene members. The mda-7/IL-24 gene represents a new class of cancer-specific apoptosis-inducing genes with obvious potential for the targeted gene-based therapy of human prostate cancer.     

Profiling of signaling molecules in four different human prostate carcinoma cell lines before and after induction of apoptosis

We have treated four prostate tumor cell lines, DU-145, PC-3, LNCaP and 22RV1 with various concentrations of cisplatin in order to check for influence on viability and for onset of apoptosis induction. At a cisplatin concentration of 20 microM, 22RV1 and DU-145 cells showed approximately 22% and 18% and PC-3 and LNCaP cells showed approximately 4 and 10% dead cells, respectively. When checking for apoptosis induction, the differences among the cell lines became even more evident. DU-145 and 22RV1 cells showed apoptosis induction at 5- and 2-microM cisplatin, whereas in the case of LNCaP and PC-3 cells comparable apoptosis induction was observed at 100-microM cisplatin; hence, the difference between the two groups of cell lines with respect to apoptosis induction is 20- and 50-fold, respectively. We used 37 antibodies to screen the expression levels of key signaling molecules and their phosphorylation status where appropriate. DU-145 and PC-3 cells are androgen-receptor negative and harbor non-functional p53, whereas LNCaP and 22RV1 cells are androgen-receptor positive and harbor wild-type p53. The results of the profiling of DU-145 and PC-3 support the notion that an intact PTEN/AKT pathway (as found in DU-145 and 22RV1 cells) and the presence of active p38 are responsible for the high sensitivity to apoptosis induction and that neither the androgen receptor nor the p53 status is of primary importance for the differences observed with respect to apoptosis induction.     

Molecular determinants of cell death induction following adenovirus-mediated gene transfer of wild-type p53 in prostate cancer cells

Adenoviral vectors expressing wild-type p53 (Ad-p53) induce apoptosis in different types of cancer cells. The therapeutic utility of Ad-p53 is now being evaluated in prostate-cancer patients. Bcl-2 is frequently expressed by prostate-cancer cells and has previously been shown to inhibit p53-mediated cell death following genotoxic stress. We studied the impact of bcl-2 on Ad-p53-induced cell death in human prostate-cancer cells. Human prostate-cancer cell lines LNCaP (p53 wt) and PC3 (p53 mut) were stably transfected with bcl-2. After p53 transduction, cell viability, apoptosis induction and modulation of specific apoptosis-regulatory proteins were assessed. LNCaP vector control and bcl-2-expressing cells underwent similar decreases in viability associated with apoptosis induction following Ad-p53 infection. Increased bcl-2 expression provided significant protection to PC3 cells transduced with Ad-p53. These findings are correlated with modulations in bax, bcl-2, bcl-x(L) and p21 protein levels. These data suggest that Ad-p53 may be useful in the treatment of some prostate cancers.     

Tumor growth inhibition by arsenic trioxide (As2O3) in the orthotopic metastasis model of androgen-independent prostate cancer.

Arsenic trioxide (As2O3) induces clinical remission of patients with acute promyelocytic leukemia. As a novel anticancer agent for treatment of solid cancers, As2O3 is promising, but no in vivo experimental investigations of its efficacy on solid cancers have been done at clinically obtained concentrations. In addition, the cell death mechanism of As2O3 has yet to be clarified, especially in solid cancers. In this study, human androgen-independent prostate cancer cell lines, PC-3, DU-145, and TSU-PR1 were examined as cellular models for As2O3 treatment, and As2O3-induced cell death and inhibition of cell growth and colony formation were evaluated. The involvement of p38, c-Jun NH2-terminal kinase (JNK), caspase-3, and reactive oxygen species (ROS) were investigated in As2O3-induced cell death. Finally, As2O3 was administered to severe combined immunodeficient mice inoculated orthotopically with PC-3 cells to estimate in vivo efficacy. In all three of the cell lines, at high concentrations, As2O3 induced apoptosis and, at low concentrations, growth inhibition. As2O3 activated p38, JNK, and caspase-3 dose dependently. Treatment with the p38 inhibitor and over-expression of dominant-negative JNK did not guard against As2O3-induced cell death. In contrast with partial protection by the caspase-3 inhibitor, the antioxidant N-acetyl-L-cysteine gave marked protection from As2O3-induced apoptosis and eliminated the activation of p38, JNK, and caspase-3, and the generation of ROS. The orthotopic murine metastasis model showed in vivo tumor growth inhibition in orthotopic and metastatic lesions with no signs of toxicity. This study establishes that As2O3 provides a novel, safe approach for treatment of androgen-independent prostate cancer. Generation of ROS as a therapeutic target for the potentiation of As2O3-induced apoptosis also was shown.     

Methylseleninic acid sensitizes prostate cancer cells to TRAIL-mediated apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic agent that preferentially induces apoptosis in a variety of human cancer cells. Unfortunately, some tumor cells remain resistant to TRAIL. Therefore, agents that sensitize malignant cells to TRAIL-mediated cell death might be of particular importance for the development of novel antitumor therapeutic regimens. Recent studies establish a critical role of selenium in prostate cancer prevention in vitro and in vivo. Here, we demonstrate that concomitant administration of TRAIL and methylseleninic acid (MSA) produces synergistic effects on the induction of apoptosis in androgen-dependent LNCaP and androgen-independent DU-145 prostate cancer cells. MSA rapidly and specifically downregulates expression of the cellular FLICE inhibitory protein, a negative regulator of death receptor signaling. In addition, we demonstrate that the synergistic effects of MSA and TRAIL result from the activation of the mitochondrial pathway-mediated amplification loop. Addition of MSA effectively blocked TRAIL-mediated BAD phosphorylation at Ser112 and Ser136 in DU-145 cells and was accompanied by induction of the mitochondrial permeability transition and release of apoptogenic cytochrome c and Smac/DIABLO proteins from the mitochondria and into the cytosol. These results suggest that selenium-based dietary compounds may help to overcome resistance to TRAIL-mediated apoptosis in prostate cancer cells.     

Angiogenic potential of prostate carcinoma cells overexpressing bcl-2

BACKGROUND: Tumors commonly outgrow their blood supply, thereby creating hypoxic conditions, which induce apoptosis and increase expression of angiogenic growth factors. The bcl-2 oncogene inhibits apoptosis induced by a variety of stimuli, including hypoxia. On the basis of bcl-2's role in regulating apoptosis in response to hypoxia, we hypothesized that this oncogene might affect other responses to hypoxia, such as the expression of angiogenic growth factors. METHODS: Three prostate carcinoma cell lines, PC3, LNCaP, and DU-145, were stably transfected with a bcl-2 complementary DNA (cDNA), and transfectants were analyzed in vitro for the expression of angiogenic factors after exposure to either normoxic (19% O(2)) or hypoxic (1% O(2)) conditions. The in vivo angiogenic potential of the transfected cells was determined by analyzing vessel density in xenografts derived from them and by measuring the ability of these xenografts to induce neovascularization when implanted in mouse corneal micropockets. Statistical tests were two-sided. RESULTS: When exposed to hypoxic conditions, prostate carcinoma cells overexpressing bcl-2 expressed statistically significantly higher levels of vascular endothelial growth factor (VEGF), an angiogenic factor, than control-transfected cells (P = .001 for PC3, P = .04 for DU-145 after 48 hours). This effect of bcl-2 was independent of its antiapoptotic activity because increased expression of VEGF was detected in PC3 cells overexpressing bcl-2 even though PC3 cells are inherently resistant to hypoxia-induced apoptosis. In vivo, xenograft tumors derived from the bcl-2-overexpressing prostate carcinoma cell lines displayed increased angiogenic potential and grew more aggressively than tumors derived from the control cell lines (P =.03 for PC3). Treatment of bcl-2-overexpressing and control tumors with the antiangiogenic drug TNP-470 neutralized the aggressive angiogenesis in bcl-2-overexpressing tumors (P = .04 for PC3, P = .004 for DU-145) and the moderate angiogenesis in control tumors (P = .01 for PC3, P = .05 for DU-145), resulting in similar growth rates for both tumors. CONCLUSIONS: bcl-2 may play a dual role in tumorigenesis by suppressing apoptosis and by stimulating angiogenesis.     

Interruption of nuclear factor kappaB signaling by the androgen receptor facilitates 12-O-tetradecanoylphorbolacetate-induced apoptosis in androgen-sensitive prostate cancer LNCaP cells

12-O-tetradecanoylphorbolacetate (TPA) influences proliferation, differentiation, and apoptosis in a variety of cells including prostate cancer cells. Here, we show that androgen treatment potentiates TPA-induced apoptosis in androgen-sensitive prostate cancer LNCaP cells but not in androgen-independent prostate cancer cell lines DU145 and PC-3. The use of the antiandrogen bicalutamide (Casodex) rescued LNCaP cells from 5-alpha-dihydrotestosterone (DHT)/TPA-induced apoptosis, suggesting that DHT/TPA-induced apoptosis is mediated by androgen/androgen receptor (AR). In addition, a caspase-3 inhibitor (Ac-DEVD-CHO) reduced the level of apoptosis, suggesting that DHT/TPA-mediated apoptosis occurs through a caspase-3-dependent pathway. A functional reporter assay using nuclear factor (NF) kappaB-luciferase and an electromobility gel shift assay showed that DHT suppressed NFkappaB activity. In addition, apoptosis mediated by combined DHT/TPA treatment was abrogated by overexpression of the NFkappaB subunit p65 in LNCaP-p65 cells, suggesting that NFkappaB may play an important role in regulating the effects of androgen/AR and TPA on apoptosis. Furthermore, use of the c-Jun N-terminal kinase (JNK) inhibitor SB202190 showed that the combination of DHT/TPA increased JNK activation in LNCaP cells but not in LNCaP-p65 cells, demonstrating that NFkappaB may be able to suppress JNK activity. These results indicate that androgen/AR facilitates TPA-induced apoptosis by interruption of the NFkappaB signaling pathway, leading to activation of JNK in LNCaP cells. These data describe a signaling pathway that could potentially be useful in proposed therapeutic treatment strategies exploiting combinations of different agents that control apoptosis in prostate tumors.     

C-Jun N-terminal kinase is required for phorbol ester- and thapsigargin-induced apoptosis in the androgen responsive prostate cancer cell line LNCaP

In early, androgen dependent stages of prostate cancer, androgen withdrawal, the major course of therapy in prostate cancer, leads to a rapid regression of the tumor as a result of apoptosis. However, prostate cancer invariably progresses to an androgen independent and apoptosis resistant stage for which no curative treatment is available. The molecular details of regression upon androgen withdrawal and progression to a resistant state are largely unknown. Here we show that c-Jun N-terminal Kinase (JNK) is activated strongly and in a sustained fashion by 12-O-tetradecanoylphorbol 13-acetate (TPA) and thapsigargin (TG), two agents which were previously shown to lead to apoptosis in the androgen responsive prostate cancer cell line LNCaP. The time course of JNK induction by both compounds correlated very well with the onset and progression of apoptosis in LNCaP cells. Inhibition of either ERK or p38 pathways did not affect TPA-induced cell death. In the androgen-independent prostate cancer cell lines DU-145 and PC-3, and in the cervical carcinoma cell line HeLaS3, TPA did not lead to apoptosis and there were no significant changes in JNK activity upon TPA treatment. The failure of TPA to induce JNK activity in PC-3, DU-145, and HelaS3 cells was not due to a general defect in JNK signaling since ultraviolet (UV) irradiation dramatically increased JNK activity in all four cell lines. Specific inhibition of JNK by expression of the JNK Inhibitory Protein (JIP) dramatically inhibited both TPA- and TG-induced apoptosis. Furthermore, apoptosis induced by both agents was completely blocked by ectopic expression of the baculovirus caspase-inhibitor P35. Surprisingly, ZVAD-fmk, a cell-permeable fluoromethylketone inhibitor of caspases, had no effect on TPA-induced apoptosis, whereas it completely inhibited TG-induced cell death; JNK activity was not affected in either case. This indicates that ZVAD-fmk does not inhibit some of the caspases involved in TPA-induced apoptosis, and that despite the common requirement of JNK activation, TPA- and TG-induced cell death are mechanistically different. Furthermore, it also suggests that JNK is either upstream or independent of caspases in LNCaP cells. Collectively, these results indicate that apoptosis in LNCaP cells requires a sustained increase in JNK activity and caspase activation; components of these signaling pathways may be defective in the androgen independent prostate cancer cell lines.     

A recombinant defective adenoviral agent expressing anti-bcl-2 ribozyme promotes apoptosis of bcl-2-expressing human prostate cancer cells.

Over-expression of bcl-2, a potent anti-apoptosis protein, is likely to be one of the genetic mechanisms through which human prostate cancer cells develop resistance to hormonal and other forms of therapy. To develop a therapeutic agent for hormone-resistant prostate cancer based on suppression of bcl-2 expression, we had previously designed and synthesized a dual-hammerhead ribozyme capable of recognizing and specifically cleaving human bcl-2 mRNA in vitro as well as in vivo. To increase the efficiency by which the anti-bcl-2 ribozyme can be delivered to target cells, we have created a recombinant replication-deficient (defective) adenoviral agent capable of expressing the anti-bcl-2 ribozyme upon infection. This viral agent effectively reduces intracellular levels of bcl-2 mRNA and protein in cultured LNCaP prostate cancer cells following standard infection procedures. Likewise, the defective adenovirus-anti-bcl-2 ribozyme induces extensive apoptosis in several androgen-sensitive (LNCaP) and androgen-insensitive (LNCaP/bcl-2 and PC-3) human prostate cancer cell lines that express differing amounts of bcl-2 protein. One androgen-insensitive prostate cancer cell line, DU-145, lacking in bcl-2 expression, was found to be completely refractory to the effects of the virus ribozyme, supporting the concept that the cytotoxic effects of the ribozyme are based solely on its effects on bcl-2 expression. Our results support further development of this adenovirus/anti-bcl-2 ribozyme for potential gene therapeutic purposes in certain forms of hormone-resistant prostate cancer where over-expression of bcl-2 proto-oncogene is indicated.     

Interleukin-17 receptor-like gene is a novel antiapoptotic gene highly expressed in androgen-independent prostate cancer

We have recently identified a new gene, interleukin-17 receptor-like (IL-17RL), which is expressed in normal prostate and prostate cancer. This investigation is focused on the role of IL-17RL in prostate cancer. We found that IL-17RL was expressed at significantly higher levels in several androgen-independent prostate cancer cell lines (PC3, DU145, cds1, cds2, and cds3) and tumors compared with the androgen-dependent cell lines (LNCaP and MLC-SV40) and tumors. In an in vivo model of human prostate tumor growth in nude mice (CWR22 xenograft model), IL-17RL expression in tumors was induced by androgen deprivation. The relapsed androgen-independent tumors expressed higher levels of IL-17RL compared with the androgen-dependent tumors. Overexpression of IL-17RL in tumor necrosis factor alpha (TNFalpha)-sensitive LNCaP cells inhibited TNFalpha-induced apoptosis by blocking activation of caspase-3 downstream to caspase-2 and caspase-8. Reciprocally, knocking down IL-17RL expression by small interfering RNA induced apoptosis in all the prostate cancer cell lines studied. Taken together, these results show that IL-17RL is a novel antiapoptotic gene, which may confer partially the property of androgen-independent growth of prostate cancer by promoting cell survival. Thus, IL-17RL is a potential therapeutic target in the treatment of prostate cancer.     

In vitro pharmacology of cryptophycin 52 (LY355703) in human tumor cell lines

Purpose: Cryptophycin 52 (LY355703) is a new member of the cryptophycin family of antitumor agents that is currently undergoing clinical evaluation for cancer chemotherapy. The mechanism of action of the cryptophycin class of compounds is associated with an action on microtubules. This report details the pharmacological profile of this new clinical compound in a panel of human tumor cell lines. Methods: Antiproliferative effects of cryptophycin 52 were measured indirectly by detection of the metabolic reduction of alamarBlue^®. Cytoxicity was assessed by enzymatic dye activation (calcein AM) combined with dye exclusion (ethidium homodimer) and by clonogenicity assay. Cell cycle effects were evaluated using flow cytometry and fluorescence microscopy. Results: Both antiproliferative and cytotoxic effects of cryptophycin 52 were concentration- and time-dependent. IC₅₀ values for antiproliferative activity in both solid and hematologic tumor cell lines were in the low picomolar range, and without exception, were significantly below values for the antimitotic agents paclitaxel and vinblastine. Flow cytometry and microscopic examination of tumor cells treated with cryptophycin 52 indicated that they accumulated in the mitotic phase of the cell cycle. Cryptophycin 52 was tested for its sensitivity to multidrug-resistance in several paired cell lines in which a sensitive parental line was matched with a multidrug-resistant derivative line. The resistant lines have been shown to over express Pgp and/or MRP multidrug-resistance transport factors. Compared to other antimitotic agents (paclitaxel, vinblastine, vincristine), the potency of cryptophycin 52 was shown to be minimally affected in multidrug-resistant cells compared to their sensitive parental lines. Conclusion: Cryptophycin 52 has potent antimitotic, antiproliferative and cytotoxic activity in in vitro human tumor cell models. It is significantly more potent and less sensitive to multidrug resistance mechanisms than other antimitotic antitumor agents currently used in cancer therapy. These characteristics may translate into therapeutic advantages for the clinical use of cryptophycin 52 in cancer chemotherapy.     

Mechanisms controlling cell cycle arrest and induction of apoptosis after 12-lipoxygenase inhibition in prostate cancer cells

Extensive studies have implicated the role of dietary fatty acids in prostatecancer progression. Platelet-type 12-Lipoxygenase (12-LOX) has beenshown to regulate growth, metastasis, and angiogenesis of prostate cancer. The effect of two 12-LOX inhibitors, Baicalein and N-benzyl-N-hydroxy-5-phenylpentamide (BHPP), on the mechanisms controlling cell cycle progression and apoptosis were examined in two prostate cancer cell lines, PC3 and DU-145. Treatment with Baicalein or BHPP resulted in a dose-dependent decrease in cell proliferation, as measured by BrdUrd incorporation. This growth arrest was shown to be because of cell cycle inhibition at G0/G1, and was associated with suppression of cyclin D1 and D3 protein levels. PC3 cells also showed a strong decrease in phosphorylated retinoblastoma (pRB) protein, whereas the other retinoblastoma-associated proteins, p107 and p130, were inhibited in DU-145 cells. Treatment with 12-hydroxyeicosatetraenoic acid in the presence of Baicalein blocked loss of pRB, whereas 12(S)-HETE alone induced pRB expression. Treatment with either Baicalein or BHPP resulted in significant apoptosis in both cell lines as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. DU-145 cells underwent apoptosis more rapidly than PC-3 cells. The mechanisms involved were decreased phosphorylation of Akt, loss of survivin and subsequent activation of caspase-3 and caspase-7 in each cell line, decreased Bcl-2 and Bcl-X(L) expression in DU-145, and a shift in Bcl-2/Bax levels favoring apoptosis in PC-3 cells. Addition of 12(S)-HETE protected both cell lines from Baicalein-induced apoptosis, whereas other LOX metabolites, 5(S)-HETE, or 15(S)-HETE did not. These results show that the 12-LOX pathway is a critical regulator of prostate cancer progression and apoptosis, by affecting various proteins regulating these processes. Therefore, inhibition of 12-LOX is a potential therapeutic agent in the treatment of prostate cancer.     

Progression to androgen-independent LNCaP human prostate tumors: cellular and molecular alterations

Lethal phenotypes of human prostate cancer are characterized by progression to androgen-independence and metastasis. For want of a clinically relevant animal model, mechanisms behind this progression remain unclear. Our study used an in vivo model of androgen-sensitive LNCaP human prostate cancer cell xenografts in male SCID mice to study the cellular and molecular biology of tumor progression. Primary tumors were established orthotopically, and the mice were then surgically castrated to withdraw androgens. Five generations of androgen-independent tumors were developed using castrated host mice. Tumor samples were used to determine expressions of cellular and molecular markers. Androgen-independent tumors had increased proliferation and decreased apoptosis compared to androgen-sensitive tumors, outcomes associated with elevated expression of p53, p21/waf1, bcl-2, bax and the bcl-2/bax ratio. Blood vessel growth in androgen-independent tumor was associated with increased expression of vascular endothelial growth factor. Overexpression of androgen receptor mRNA and reduced expression of androgen receptor protein in androgen-independent tumors suggest that the androgen receptor signaling pathway may play an important role in the progression of human prostate cancer to androgen-independence. The in vivo orthotopic LNCaP tumor model described in our study mimics the clinical course of human prostate cancer progression. As such, it can be used as a model for defining the molecular mechanisms of prostate cancer progression to androgen-independence and for evaluating the effect of preventive or therapeutic regimens for androgen-independent human prostate cancer.     

Raloxifene,a selective estrogen receptor modulator,induces apoptosis in androgen-responsive human prostate cancer cell line LNCaP through an androgen-independent pathway

Raloxifene, a selective estrogen receptor (ER) modulator, is a mixed estrogen agonist/antagonist that has been shown to prevent osteoporosis and breast cancer in women. Because the prostate contains a high level of ER-beta, the present study investigated the effect of raloxifene in the androgen-sensitive human prostate cancer cell line LNCaP. Previously, it has been demonstrated that LNCaP cells express ER-beta but not ER-alpha and that tamoxifene induces apoptosis in these cells. After treatment with raloxifene, a dramatic increase in cell death occurred in a dose-dependent manner (10(-9) to 10(-6) M range). Using the terminal deoxynucleotidyl transferase-mediated nick end labeling apoptotic assay, we demonstrated that the nuclear fragmentation was due to apoptosis. The dramatic change in cellular morphology after treatment with raloxifene was no longer observed when cells were pretreated with a pan-caspase inhibitor, Z-VAD-FMK, and a specific caspase-9 inhibitor, Z-LEHD-FMK. Furthermore, immunoblot demonstrated an activation of caspase-9 in LNCaP cells. Because LNCaP cells contain a mutated androgen receptor that allows cellular proliferation in the presence of antiandrogens, prostate-specific antigen assay and transfection with a reporter construct containing luciferase gene under the control of androgen response element (pARE) were carried out. The results demonstrated that raloxifene does not significantly alter androgen receptor activity in LNCaP cells. Taken together, these results demonstrate that raloxifene, a selective ER modulator, induces apoptosis in the androgen-sensitive human prostate cancer cell line LNCaP through an androgen-independent pathway.     

Nitroxide tempo, a small molecule, induces apoptosis in prostate carcinoma cells and suppresses tumor growth in athymic mice

BACKGROUND: In previous studies, nitroxide tempo (2, 2, 6, 6-tetramethyl-piperidine-1-oxyl), a small molecule, induced cell death in cancer cells. The current study examined the antineoplastic properties of tempo in the human hormone-dependent/hormone-independent prostate carcinoma models (LNCaP, DU-145, and PC-3). METHODS: The apoptotic effects of tempo were examined by the flow cytometric analysis of cells labeled with fluorescein isothiocyanate-conjugated annexin-V, and by electron microscopy. Enzymatic assays were performed to measure the activities of 2 cysteine proteases, i.e., caspase-9 and caspase-3, in tempo-treated cells. The effects of tempo on cell proliferation and on cell cycle distribution profiles were measured by the flow cytometric assay using immunofluorescent staining of incorporated 5'-bromo-2'-deoxyuridine (BrdU) coupled with 7-amino-actinomycin D (7-AAD) staining of total DNA. The number of proliferating cells was also determined independently by enzyme-linked immunosorbent assay using chemiluminescent detection of incorporated BrdU. Subcutaneous growth of human prostate carcinoma in athymic mice was monitored after intratumoral administration of tempo into tumor-bearing mice. In addition, cell viability assays were performed to compare the cytotoxic effect of a combination of doxorubicin or mitoxantrone and tempo with single agents. RESULTS: Tempo treatment of prostate carcinoma cells caused a significant increase in the number of apoptotic cells compared with control groups (tempo, 2.5 mM, 24 hours: DU-145, approximately 3.4-fold; PC-3, approximately 6-7-fold; tempo 1 mM, 24 hours: LNCaP, approximately 12-fold). Tempo-induced loss of cell viability was blocked partially or completely after pretreatment of cells with actinomycin-D or cycloheximide, suggesting a de novo macromolecule synthesis-dependent mechanism of cell death. Electron microscopy revealed aggregation and marginalization of chromatin in the nuclei of a large number of tempo-treated LNCaP cells. Tempo treatment of LNCaP cells resulted in enhanced activities of caspase-9 (tempo, 5 mM, 15 hours: approximately 2-fold) and caspase-3 (tempo, 2.5 mM, 24 hours: approximately 12-fold). Tempo treatment also led to an enhanced number of cells in G2/M phase of the cell cycle (tempo, 5.0 mM, 24 hours: DU-145, approximately 1.6-fold; PC-3, approximately 1.5-fold; LNCaP, approximately 5.3-fold), and decreased BrdU incorporation indicative of a decline in the number of proliferating cells (tempo, 2.5 mM, 24 or 48 hours; DU-145, approximately 2-3-fold; PC-3, approximately 1.2-fold; LNCaP, approximately 5-10-fold). Administration of tempo into LNCaP tumor-bearing mice resulted in a significant inhibition of tumor growth (percent initial tumor volume [Day 30, n = 4]: vehicle, 845.35 +/- 272.83; tempo, 9.72 +/- 9.72; tempo vs. vehicle, P < 0.02). In hormone-refractory prostate carcinoma cells, a combination of relatively low doses of tempo and doxorubicin or mitoxantrone caused enhanced cytotoxicity as compared with single agents. CONCLUSIONS: These data demonstrated that nitroxide tempo induced apoptosis and activated a caspase-mediated signaling pathway in prostate carcinoma cells. Tempo treatment also caused cell cycle arrest in G2/M phase and decreased the number of proliferating cells (S phase). Tempo treatment of tumor-bearing mice led to inhibition of tumor growth, suggesting that tempo is a novel member of the small-molecule family of antineoplastic agents.     

Implication of multiple mechanisms in apoptosis induced by the synthetic retinoid CD437 in human prostate carcinoma cells

The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) induces apoptosis in several types of cancer cell. CD437 inhibited the growth of both androgen-dependent and -independent human prostate carcinoma (HPC) cells in a concentration-dependent manner by rapid induction of apoptosis. CD437 was more effective in killing androgen-independent HPC cells such as DU145 and PC-3 than the androgen-dependent LNCaP cells. The caspase inhibitors Z-VAD-FMK and Z-DEVD-FMK blocked apoptosis induced by CD437 in DU145 and LNCaP cells, in which increased caspase-3 activity and PARP cleavage were observed, but not in PC-3 cells, in which CD437 did not induce caspase-3 activation and PARP cleavage. Thus, CD437 can induce either caspase-dependent or caspase-independent apoptosis in HPC cells. CD437 increased the expression of c-Myc, c-Jun, c-Fos, and death receptors DR4, DR5 and Fas. CD437's potency in apoptosis induction in the different cell lines was correlated with its effects on the expression of oncogenes and death receptors, thus implicating these genes in CD437-induced apoptosis in HPC cells. However, the importance and contribution of each of these genes in different HPC cell lines may vary. Because CD437 induced the expression of DR4, DR5 and Fas, we examined the effects of combining CD437 and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Fas ligand, respectively, in HPC cells. We found synergistic induction of apoptosis, highlighting the importance of the modulation of these death receptors in CD437-induced apoptosis in HPC cells. This result also suggests a potential strategy of using CD437 with TRAIL for treatment of HPC. Oncogene (2000) 19, 4513 - 4522.