Adenovirus-mediated inhibition of survivin expression sensitizes human prostate cancer cells to paclitaxel in vitro and in vivo

BACKGROUND: Improvements in the response rates to chemotherapy would represent an important advancement in the care of patients with metastatic prostate cancer. There is accumulating evidence that Survivin, a member of the inhibitor of apoptosis (IAP) family, is associated with both cancer progression and drug resistance. The purpose of this study is to investigate the role of Survivin in paclitaxel-resistance and whether the targeting of Survivin sensitizes prostate cancer cells to paclitaxel. METHODS: Human prostate cell lines PC-3, DU-145, and LNCaP were infected with replication-deficient adenoviruses encoding either wild-type Survivin [pAd-S(WT)], to examine Survivin overexpression effects, or a phosphorylation-defective Survivin Thr34 --> Ala dominant negative mutant [pAd-S(T34A)], to examine Survivin inactivation effects. The effects of wild-type or mutant Survivin on spontaneous and paclitaxel-induced apoptosis were investigated both in vitro and in vivo. RESULTS: Forced overexpression of wild-type Survivin with pAd-S(WT) increased resistance to paclitaxel in all cell lines, both in vitro and in vivo. Inhibition of Survivin using pAd-S(T34A) resulted in a significant increase in the rate of spontaneous and paclitaxel-induced apoptosis in all cell lines, both in vitro and in vivo. This effect was abolished by co-treatment with VAD-CHO (Calbiochem, San Diego, CA), a pan-caspase inhibitor, indicating that Survivin normally mediates resistance to paclitaxel through suppression of caspase-mediated apoptosis. CONCLUSIONS: Survivin mediates paclitaxel-resistance in prostate cancer cells. The inhibition of Survivin sensitizes prostate cancer cells to paclitaxel-induced apoptosis through a caspase-dependant mechanism in vitro and in vivo.     

Ionizing radiation enhances the therapeutic potential of TRAIL in prostate cancer in vitro and in vivo: Intracellular mechanisms

BACKGROUND: We assessed the influence of sequential treatment of ionizing radiation followed by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) on intracellular mechanisms of apoptosis of prostate tumor cells in vitro and in vivo. METHODS: Prostate normal and cancer cells were exposed to irradiation and TRAIL. Four- to 6-week-old athymic nude mice were injected s.c. with PC-3 tumor cells. Tumor bearing mice were exposed to irradiation and TRAIL, either alone or in combination (TRAIL after 24 hr of irradiation), and tumor growth, apoptosis, and survival of mice were examined. Expressions of death receptors, Bcl-2 family members, and caspase were measured by Western blotting, ELISA, and ribonuclease protection assay; tumor cellularity was assessed by H&E staining; inhibition of p53 was performed by RNA interference (RNAi) technology, and apoptosis was measured by annexin V/propidium iodide staining, and terminal deoxynucleotidyltransferase-mediated nick end labeling assay. RESULTS: Irradiation significantly augmented TRAIL-induced apoptosis in prostate cancer cells through upregulation of DR5, Bax, and Bak, and induction of caspase activation. Dominant negative FADD and p53 siRNA inhibited the synergistic interaction between irradiation and TRAIL. The pretreatment of cells with irradiation followed by TRAIL significantly enhanced more apoptosis than single agent alone or concurrent treatment. Furthermore, irradiation sensitized TRAIL-resistant LNCaP cells to undergo apoptosis. The sequential treatment of xenografted mice with irradiation followed by TRAIL-induced apoptosis through activation of caspase-3, induction of Bax and Bak, and inhibition of Bcl-2, and completely eradicated the established tumors with enhanced survival of nude mice. CONCLUSION: The sequential treatment with irradiation followed by TRAIL can be used as a viable option to enhance the therapeutic potential of TRAIL in prostate cancer.     

Decreased gene expression of steroid 5 alpha-reductase 2 in human prostate cancer: implications for finasteride therapy of prostate carcinoma

BACKGROUND: Steroid 5alpha-reductase 2 (SRD5A2) catalyzes the conversion of testosterone to the more potent androgen, DHT, in the prostate. The therapeutic influence of SRD5A2 inhibitor finasteride on prostate cancer is currently unknown. The direction and extent of changes in SRD5A2 expression in disease tissues is a relevant issue in this regard. METHODS: The expression differences of SRD5A2 in tissues representative of normal, benign, and malignant growth in the human prostate were examined in parallel by comparative analysis of relevant microarray gene expression data. Semiquantitative RT-PCR was used to further verify the gene expression differences of SRD5A2. RESULTS: Consistently decreased expression of SRD5A2 was observed in 25 prostate cancer samples when compared to 25 matched normal samples and nine BPH samples. Expression differences among these samples for six other genes were presented in parallel as indicators of the direction and extent of expression changes. These additional genes include SRD5A1, Hepsin (overexpressed in prostate cancer), AMACR (overexpressed in prostate cancer), Keratin 8 (epithelial marker), smooth muscle actin (stromal marker), Nell2 (overexpressed in BPH). Semiquantitative RT-PCR verified the expression differences for SRD5A2 in six normal, six BPH, and six prostate cancer samples. CONCLUSIONS: Results from this study, combined with those from previous studies, indicate an association of prostate cancer with reduced 5alpha-reductase enzymatic activity as a result of remarkably decreased expression of the SRD5A2 gene. The implications of this study for finasteride therapy of prostate cancer are discussed.     

Antisense MDM2 enhances the response of androgen insensitive human prostate cancer cells to androgen deprivation in vitro and in vivo

BACKGROUND: Antisense MDM2 oligonucleotide (AS-MDM2) sensitizes androgen sensitive LNCaP cells to androgen deprivation (AD) in vitro and in vivo. In this study, we investigated the effects of AS-MDM2 combined with AD on androgen resistant LNCaP (LNCaP-Res) and moderately androgen resistant bcl-2 overexpressing LNCaP (LNCaP-BST) cells. METHODS: The LNCaP-Res cell line was generated by culturing LNCaP cells in medium containing charcoal-stripped serum for more than 1 year. Apoptosis was quantified in vitro by Annexin V staining and caspase 3 + 7 activity. For the in vivo studies, orthotopic tumor growth was monitored by magnetic resonance imaging (MRI). AS-MDM2 and the mismatch control were given by i.p. injection at doses of 25 mg/kg per day, 5 days/week for 15 days. RESULTS: LNCaP-Res cells expressed high levels of androgen receptor (AR) and bcl-2, and displayed no growth inhibition to AD. AS-MDM2 caused significant reductions in MDM2 and AR expression, and increases in p53 and p21 expression in both cell lines. AS-MDM2 + AD resulted in the highest levels of apoptosis in vitro and tumor growth inhibition in vivo in both cell lines; although, these effects were less pronounced in LNCaP-BST cells. CONCLUSIONS: AS-MDM2 + AD enhanced apoptotic cell death in vitro and tumor growth inhibition in vivo in androgen resistant cell lines. The action of AS-MDM2 + AD was influenced somewhat by bcl-2 expression as an isolated change (LNCaP-BST cells), but not when accompanied by other molecular changes associated with androgen insensitivity (LNCaP-Res cells). MDM2 knockdown has promise for the treatment of men with early hormone refractory disease.     

Effects of sequential treatments with chemotherapeutic drugs followed by TRAIL on prostate cancer in vitro and in vivo

BACKGROUND: Tumor necrosis factor related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo-2L) is a novel anticancer agent, capable of inducing apoptosis preferentially in tumor and transformed cells. TRAIL-R1/death receptor (DR)4 and TRAIL-R2/DR5 are members of the tumor necrosis factor (TNF) receptor family, and can be activated by the TRAIL. We examined the clinical potential of chemotherapeutic drugs and TRAIL for the treatment of prostate cancer. METHODS: Prostate and bladder cancer cells were exposed to chemotherapeutic drugs (paclitaxel, vincristine, vinblastine, etoposide, doxorubicin, and camptothecin) and TRAIL. Cell viability was measured by sodium 3'[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) assay; expressions of death receptors and Bcl-2 family members were measured by Western blotting, ELISA and ribonuclease protection assay. PC-3 tumor cells xenografted athymic nude mice were exposed to chemotherapeutic drugs and TRAIL, either alone or in combination, to measure tumor growth and survival of mice. Apoptosis was measured by annexin V-FITC/propidium iodide staining, and terminal deoxynucleotidyltransferase-mediated nick end labeling assay. Caspase-3 activity was measured by the Western blotting and immunohistochemistry. RESULTS: TRAIL induced apoptosis with varying sensitivity. Chemotherapeutic drugs (paclitaxel, vincristine, vinblastine, etoposide, doxorubicin, and camptothecin) significantly augmented TRAIL-induced apoptosis in cancer cells through up-regulation of DR4, DR5, Bax, and Bak, and induction of caspase activation. Mitochondrial pathway enhanced the synergistic interactions between drugs and TRAIL. The sequential treatment of mice with chemotherapeutic drugs followed by TRAIL induced caspase-3 activity, and apoptosis, inhibited angiogenesis, completely eradicated the established tumors, and enhanced survival of mice. CONCLUSIONS: Chemotherapeutic drugs can be used to enhance the therapeutic potential of TRAIL in prostate cancer.     

Adenoviral infection of survivin antisense sensitizes prostate cancer cells to etoposide in vivo

BACKGROUND: We aimed to investigate whether use of a survivin antisense fragment carried by an adenovirus vector (Ad.survivin-AS) could enhance the therapeutic efficacy of chemotherapy for androgen-independent prostate cancer. METHODS: We used Ad.survivin-AS to promote apoptosis through inhibition of survivin expression. Recombinant adenoviruses alone or in combination with chemotherapeutic agents were tested for anti-cancer activity both in vitro and in vivo. RESULTS: Infection with Ad.survivin-AS strongly inhibited survivin expression in a dose- and time-dependent manner, resulting in significant antitumor activity in vitro and in vivo. Downregulation of survivin expression potentiated induction of apoptosis by the chemotherapeutic agents docetaxel and etoposide in DU145 cells. In particular, the combination of etoposide and Ad.survivin-AS demonstrated dramatic growth inhibition with no tumor regrowth being observed during the experimental period. CONCLUSIONS: The prominent synergy of this combination may provide a basis for clinical application of Ad.survivin-AS as a chemosensitizer of etoposide.     

BM18: A novel androgen-dependent human prostate cancer xenograft model derived from a bone metastasis

BACKGROUND: Androgen-dependent prostate cancer (PrCa) xenograft models are required to study PrCa biology in the clinically relevant in vivo environment. METHODS: Human PrCa tissue from a femoral bone metastasis biopsy (BM18) was grown and passaged subcutaneously through male severe combined immune-deficient (SCID) mice. Human mitochondria (hMt), prostate specific antigen (PSA), androgen receptor (AR), cytokeratin-18 (CK-18), pan-cytokeratin, and high molecular weight-cytokeratin (HMW-CK) were assessed using immunohistochemistry (IHC). Surgical castration was performed to examine androgen dependence. Serum was collected pre- and post-castration for monitoring of PSA levels. RESULTS: BM18 stained positively for hMt, PSA, AR, CK-18, pan keratin, and negatively for HMW-CK, consistent with the staining observed in the original patient material. Androgen-deprivation induced tumor regression in 10/10 castrated male SCID mice. Serum PSA levels positively correlated with BM18 tumor size. CONCLUSIONS: BM18 expresses PSA and AR, and rapidly regresses in response to androgen withdrawal. This provides a new clinically significant PrCa model for the study of androgen-dependent growth.     

Weekly paclitaxel plus estramustine combination therapy in hormone-refractory prostate cancer: A pilot study

BACKGROUND: Paclitaxel used in combination with estramustine has been shown to exert synergistic cytotoxicity in patients with hormone-refractory prostate cancer (HRPC). There have been few reports of this therapy in an Asian male population. METHODS: Nine patients with progressive metastatic HRPC completed at least one cycle of combination therapy employing weekly paclitaxel plus estramustine. Paclitaxel was given weekly for 3 weeks as a 2-h intravenous infusion at a dose of 100 mg/infusion. The cycle was repeated every 4 weeks. A dose of 280 mg of oral estramustine was administrated twice daily for 21 days from the first day of each cycle. Both efficacy and toxicity were recorded. RESULTS: Grade 1 sensory neuropathy was seen in three patients (33%) and grade 4 thrombopenia/anemia was seen in one patient (11%). Performance status improved in three of seven patients (43%), while six patients (67%) showed a 50% or greater decline in prostate-specific antigen levels. Two of these patients experienced significant improvement in bone pain. One patient died of cardiac infarction during this trial and another died of disseminated intravascular coagulopathy subsequent to gastrointestinal bleeding. An additional patient suffered non-fatal pulmonary infarction. The one-year median survival rate was 22.2% and the overall survival period was 36 weeks. CONCLUSION: Although weekly paclitaxel plus estramustine may pose a significant risk, this combination may have a beneficial effect on the quality of life HRPC patients. A well-designed phase I-II trial in an Asian male population is highly recommended.     

Androgen receptor-dependent regulation of Bcl-xL expression: Implication in prostate cancer progression

BACKGROUND: Recently we reported that silencing the androgen receptor (AR) gene reduced Bcl-xL expression that was associated with a profound apoptotic cell death in prostate cancer cells. In this study we further investigated AR-regulated Bcl-xL expression. METHODS: Prostate cancer cell line LNCaP and its sublines, LNCaP/PURO and LNCaP/Bclxl, were used for cell proliferation assay and xenograft experiments in nude mice. Luciferase gene reporters driven by mouse or human bcl-x gene promoter were used to determine androgen regulation of Bcl-xL expression. RT-PCR and Western blot assays were conducted to assess Bcl-xL gene expression. Chromatin immunoprecipitation assay was performed to determine AR interaction with Bcl-xL promoter. Bcl-xL-induced alteration of gene expression was examined using cDNA microarray assay. RESULTS: In cultured prostate cancer LNCaP cells, androgen treatment significantly increased Bcl-xL expression at mRNA and protein levels via an AR-dependent mechanism. Promoter analyses demonstrated that the AR mediated androgen-stimulated bcl-x promoter activation and that the AR interacted with bcl-x promoter. Enforced expression of Bcl-xL gene dramatically increased cell proliferation in vitro and promoted xenograft tumor growth in vivo. Genome-wide gene profiling analysis revealed that Bcl-xL expression was significantly higher in metastatic and castration-resistant diseases compared to normal prostate tissues or primary cancers. Bcl-xL overexpression significantly increased the expression of cyclin D2, which might be responsible for Bcl-xL-induced cell proliferation and tumor growth. CONCLUSIONS: Taken together, our data strongly suggest that androgen stimulates Bcl-xL expression via the AR and that increased Bcl-xL expression plays a versatile role in castration-resistant progression of prostate cancer.     

Detection of human papillomavirus DNA and p53 gene mutations in human prostate cancer

The relationship between integration with human papillomavirus (HPV) and p53 gene mutations in tissues of prostate cancer was examined. Tissue samples analyzed were obtained by total prostatectomy (29 stage B cancer cases) and from autopsy (22 endocrine therapy-resistant metastatic disease cases). HPV DNA was detected in 8 of 51 (16%, 5 in stage B and 3 in autopsy cases) by polymerase chain reaction (PCR) using consensus primers on L1 region. Genotypes of HPV were entirely type 16. Structural abnormalities of p53 gene were detected in 7 of the 22 autopsy cases (32%) by PCR-single-strand conformation polymorphism analysis and direct sequencing. No p53 gene mutation was found in stage B cancer cases. Analysis of mutation spectra revealed clear differences between Japanese and Westerners. There was a significant difference in the mutation frequency between stage B and autopsy cases (P < 0.01, Fisher's exact test). One case showed both integration of HPV and p53 gene mutation in different cancer foci. However, the other cases revealed an inverse correlation between the presence of HPV DNA and p53 gene mutations. These data show that p53 genetic alteration is correlated with the progression of prostate cancer, in contrast to the integration of HPV that may occur in a relatively early stage. In conclusion, this study may indicate that either p53 gene mutation or the presence of HPV's oncogenic protein E6 is involved in the development of prostate cancer. © 1996 Wiley-Liss, Inc.     

反义MDM2联合紫杉醇对胶质瘤细胞U251的作用及其机制

目的探讨MDM2反义寡核苷酸联合紫杉醇对U251人胶质瘤细胞株的作用。方法以Lipofectamine介导的MDM2反义寡核苷酸转染U251人胶质瘤细胞株，免疫组织化学法检测胶质瘤细胞株MDM2蛋白的改变，噻唑蓝（MTT）检测细胞增殖，流式细胞仪检测凋亡，Western blot检测野型生p53（wild typ p53，wtp53）的表达，构建荷胶质瘤裸鼠模型，采用瘤内注射和腹腔给药方式，以MDM2反义寡核苷酸联合紫杉醇治疗，研究其联合抗肿瘤作用。结果脂质体介导反义MDM2 ODN转染U251人胶质瘤细胞株后，MDM2表达减少，与紫杉醇联合使用可增加细胞的凋亡率，野型生p53的表达量增加，裸鼠动物试验联合治疗组瘤重显著小于单独用药组及对照组。结论MDM2反义寡核苷酸可显著降低MDM2在U25I中的表达，引起细胞凋亡，MDM2反义寡核苷酸与紫杉醇联合作用U251人胶质瘤细胞株具有协同作用。     

Complex functions of mutant p53 alleles from human prostate cancer

BACKGROUND: Few studies have used multiple assays to examine the functionality of mutant p53 in prostate cancer (CaP). We employed seven functional assays to study 16 representative mutant p53 alleles, six from localized and ten from metastatic CaP. METHODS: Yeast assays were employed to determine loss of function (LOF), partial function (PF), and dominant-negative status. Assays using p53-null Saos2 cells were used to determine whether mammalian cells transfected with mutant p53 could up-regulate the MDR-1 or PCNA promoters, alter IL-6 expression or confer the ability to grow in soft agar. As a further test of gain of function (GOF), p53-null PC3 cells stably transfected with these mutant p53 alleles were examined for cell cycle distributions. RESULTS: All 16 mutant p53 alleles demonstrated either total or partial LOF. All but one allele also had at least one gain of function; however, the pattern of GOF was different for each mutant allele. Alleles derived from both localized and metastatic CaP had similar GOF characteristics; however, only alleles from metastatic disease had significantly increased S-phase fractions. CONCLUSIONS: Different mutant p53 alleles from CaP had different, complex functional profiles. The lack of predictable patterns for these alleles suggest that each mutation may uniquely affect p53 function.     

Concentration of enzymatically active prostate-specific antigen (PSA) in the extracellular fluid of primary human prostate cancers and human prostate cancer xenograft models

BACKGROUND: Prostate-specific antigen (PSA) targeted prodrugs are under development in our laboratory. Concentrations of total PSA and enzymatically active PSA produced by various human prostate cancer xenograft models have not been well characterized. METHODS: The concentration of PSA secreted into the extracellular fluid (ECF) in normal human prostate tissue, primary prostate cancers obtained directly from patients, and serially passageable human prostate cancer xenografts (PC-82, LNCaP, LAPC-4) were determined using Tandem assays. Percent enzymatically active PSA in the ECF and in conditioned media was also determined using a previously validated assay employing a monoclonal antibody to the PSA catalytic site. In addition, the concentration and activity of PSA within sera from men with and without prostate cancer, as well as from tumor-bearing animals, was likewise assayed. RESULTS: Normal human prostate tissue and primary human prostate cancers have high concentrations of PSA in the ECF (i.e., 1600-2100 nM). The majority of this PSA is enzymatically active (i.e., 80-90%). Human PC-82 prostate cancer xenografts also have high concentrations of PSA in the ECF (624 +/- 360 nM), and the majority of this PSA is also enzymatically active (i.e., 66 +/- 4%). In contrast, much lower concentrations of PSA are found in the ECF from LNCaP (45 +/- 9 nM) and LAPC-4 (7.3 +/- 0.6 nM). Only a small portion of the total PSA isolated from DHT-containing, serum-free, conditioned media from these cell lines is enzymatically active (i.e., approximately 18%). While PSA was detected in all serum samples regardless of the type of host, no enzymatically active PSA was detected in any of these serum samples. CONCLUSIONS: Prostate cancers obtained directly from patients produce and secrete large amounts of PSA, the majority of which is highly enzymatically active. In contrast, while PSA was detected in the sera, none of this PSA was enzymatically active. This is also the case for the human PC-82 prostate cancer xenografts. In contrast, LNCaP and LAPC-4 human prostate cancer xenograft models secrete approximately 70-300-fold less PSA in the ECF than prostate cancers from patients and the majority of this PSA is enzymatically inactive. Also, the serum from these animals had detectable PSA, but none of this PSA was enzymatically active. Thus, these latter two prostate cancer models define the least and the PC-82, the most, optimized xenograft model for screening PSA targeted prodrugs.