Antisense Bcl-2 sensitizes prostate cancer cells to radiation

BACKGROUND: Bcl-2 is anti-apoptotic and overexpression is associated with prostate tumor aggressiveness. We hypothesized that Bcl-2 has a role in prostate cancer radiation (RT) response. The relationship of Bcl-2 expression in four prostate cancer cell lines, and the effect of modulating expression with a Bcl-2 antisense oligonucleotide (G3139, Genasense, oblimersen sodium, Genta Incorporated), to RT was examined. METHODS: The four cell lines studied were LNCaP (wild type-p53), PC3 (p53 null), Bcl-2 stably transfected LNCaP (LNCaP-BST), and Bcl-2 stably transfected PC3 (PC3-BST) cells. Cells were treated with antisense (AS) Bcl-2 alone or with RT (2-6 Gy). Following RT, cells were processed at 3-6 hr for Western blots, 18 hr for Annexin V staining and flow cytometric analysis, 24 hr for caspases 3+7 quantification by fluorometric assay, and immediately for clonogenic survival. RESULTS: AS caused a significant reduction in Bcl-2 expression in all cell lines. P53 expression was elevated following RT treatment in LNCaP and LNCaP-BST cells. P21 was increased by RT treatment in all cell lines. AS caused a significant increase in caspase 3+7 activity over the mismatch (MM) controls in all cell lines. When AS was combined with RT, caspase 3+7 activity was further increased significantly over all other groups in all cell lines. Moreover, AS+RT resulted in significantly reduced clonogenic survival over MM+RT, which was dampened in the Bcl-2 overexpressing lines. CONCLUSIONS: To our knowledge, these data demonstrate for the first time that a Bcl-2 specific AS oligonucleotide sensitizes prostate cancer cells to RT. p53 is not required for this effect.     

Overexpression of the cytoprotective protein clusterin decreases radiosensitivity in the human LNCaP prostate tumour model

OBJECTIVE: To evaluate the effect of clusterin overexpression on radiation-induced tumour growth rates and apoptosis in human prostate LNCaP cells, as prostate cancer cells are relatively resistant to radiation-induced apoptosis and local recurrences are common, but overexpression of the anti-apoptotic protein clusterin can accelerate progression to androgen-independence and to confer a chemoresistant phenotype in various prostate cancer models. MATERIALS AND METHODS: Western blot analysis and immunohistochemistry were used to compare clusterin expression levels in parental (P) and clusterin-transfected (T) LNCaP cells in vitro and in vivo. The effects of radiation on clusterin-expression in both parental LNCaP/P and clusterin-transfected LNCaP/T tumours were analysed by Northern blot analysis. The cellular response to radiation was determined up to 3 weeks after irradiation using tetrazolium and re-growth assays, and cell-cycle analysis by flow cytometry. RESULTS: Clusterin mRNA expression increased from undetectable to low levels in LNCaP/P tumours after radiation and more than three-fold in LNCaP/T tumours. Clusterin overexpression decreased the radiosensitivity in a time-dependent manner, reducing the extent of growth arrest and apoptosis by up to 54%. Re-growth assays showed that the improved survival rates of LNCaP/T cells after radiation did not change after 3 days, remaining constant over 3 weeks. CONCLUSIONS: These results identify clusterin as a promoter of cell survival that may help mediate resistance to radiation-induced apoptosis. Furthermore, clusterin overexpression seems to provide an extended protection against radiation-induced cell cycle arrest and apoptosis.     

Novel therapeutic strategy for advanced prostate cancer using antisense oligodeoxynucleotides targeting anti-apoptotic genes upregulated after androgen withdrawal to delay androgen-independent progression and enhance chemosensitivity

Progression to androgen-independence remains the main obstacle to improving survival for patients with advanced prostate cancer. In this review, findings are summarized that have recently been demonstrated to establish novel therapeutic strategy targeting several genes playing functionally important roles after androgen withdrawal and during androgen-independent progression. The authors initially characterized changes in gene expression after androgen withdrawal in the androgen-dependent Shionogi and LNCaP tumor models using cDNA arrays. Based on these results, they focused on genes highly upregulated after androgen ablation (i.e. bcl-2, bcl-xL, TR.PM-2, IGFBP-5), which have anti-apoptotic or mitogenic activities, and thereby confer a resistance to androgen withdrawal as well as cytotoxic chemotherapy. The authors further demonstrated the efficacy of an antisense oligodeoxynucleotide (ODN) strategy for patients with advanced prostate cancer through the inhibition of target gene expression, resulting in a delay in the progression to androgen-independence by enhancing apoptotic cell death induced by androgen ablation and chemotherapy. The authors also showed the effectiveness of combined antisense ODN therapy and cytotoxic chemotherapy by achieving additive or synergistic effects. These findings provide a basic significance for the design of clinical studies using antisense ODN either alone or in combination with chemotherapeutic agents in patients with advanced prostate cancer.     

Livin靶向ASODN对前列腺癌PC3细胞生物学特性的影响

目的：合成Livin靶向的反义核苷酸（ASODN）,观察其对前列腺癌PC3细胞凋亡和增殖等生物学特性的影响。方法：合成全硫代磷酸化修饰的LivinASODN并通过脂质体转染前列腺癌PC3细胞。采用MTT法检测其对细胞增殖的影响,采用RT—PCR检测转染后Livin基因mRNA的表达情况,采用流式细胞仪检测转染后细胞的凋亡效应,采用体内成瘤实验比较细胞在裸鼠体内成瘤性的变化,采用激酶法测定Caspase-3活性的变化。结果：LivinASODN转染PC3细胞后,与对照组相比,实验组细胞内LivinmRNA的表达水平下调（P〈O．01）;细胞的增殖受到显著抑制（P〈O．01）;细胞凋亡率明显增高（P〈O．01）;肿瘤细胞在荷瘤鼠体内的成瘤体积小于对照组（P〈O．05）;Caspase-3活性明显增加（P〈0．05）。结论：靶向Livin基因的ASODN干扰了前列腺癌PC3细胞中Livin基因的表达,抑制了细胞的增殖并诱导其凋亡,延缓了肿瘤细胞的生长。     

Clusterin expression is significantly enhanced in prostate cancer cells following androgen withdrawal therapy

INTRODUCTION AND OBJECTIVES: Progression of prostate cancer to androgen independence (AI) results in part from the upregulation of anti-apoptotic genes following androgen withdrawal, and androgen-independent disease remains the primary obstacle to improved survival. Testosterone-repressed prostate message-2 (TRPM-2) encodes the anti-apoptotic protein clusterin, which is upregulated in response to cellular compromise as observed in normal and malignant tissues undergoing apoptosis. Systemic administration of antisense clusterin oligonucleotides in prostate cancer xenograft models delays progression to AI and enhances chemosensitivity. The objective of this study was to define changes in clusterin expression following neoadjuvant hormone therapy (NHT) in prostate cancer patients. MATERIALS AND METHODS: Archival radical prostatectomy (RP) specimens were obtained for 128 patients who received either no NHT or treatment for 2-8 weeks, 3 months, or 8 months. Paired needle biopsy specimens were acquired for 30 patients and all tissues were subjected to clusterin immunohistochemistry. Western blot analysis was performed on frozen tissue from 5 untreated and 5 treated patients. RESULTS: Clusterin expression in malignant prostatic tissue was significantly greater in patients who underwent preoperative NHT (P < 0.001). Needle biopsies obtained prior to NHT consistently demonstrated lower staining intensity than corresponding RP specimens (P < 0.001). Western blot analysis confirmed clusterin levels increased 17-fold beginning within 4 weeks after androgen withdrawal. CONCLUSIONS: Upregulation of clusterin levels following androgen ablation therapy may represent an adaptive cell survival response following apoptotic signals like androgen withdrawal. These findings support clusterin as a valid therapeutic target in strategies employing novel multimodality therapy for advanced prostate cancer.     

The leader sequence triggers and enhances several functions of clusterin and is instrumental in the progression of human prostate cancer in vivo and in vitro

OBJECTIVE: To investigate the role of the leader sequence (which during clusterin biosynthesis facilitates its proper post-translational processing and secretion) in the functional activities of clusterin, a ubiquitous secretory glycoprotein with many biological functions, reported to be pro-apoptotic and anti-apoptotic in target cells, but for which the dual mechanism remains unclear. MATERIALS AND METHODS: We designed an expression vector starting from the second in-frame ATG on the full-length human clusterin cDNA that was capable of driving the expression of both the full-length and the truncated isoforms of clusterin. We established stable expression clones of the androgen-dependent prostate cancer line LNCaP expressing clusterin with and without the leader sequence. This induced expression provided an opportunity to evaluate both the in vivo and in vitro actions of clusterin expression. RESULTS: The LNCaP cells expressing clusterin with the leader sequence resisted apoptosis induced by tumour necrosis factor (TNF)-alpha, but clones with no leader sequence were highly susceptible to TNF-alpha-induced apoptosis. Furthermore, in the absence of the leader sequence, the expressed clusterin had a molecular weight consistent with that of the predicted holoprotein (40 kDa), suggesting a compromised post-translational processing with diffuse distribution throughout the cytoplasm. However, cells transfected with the full-length vector expressed clusterin of 60 and 35 kDa variants, and located exclusively in the Golgi apparatus. In vivo, only the overexpression of the full-length clusterin is anti-apoptotic and stimulates the proliferation of tumour. CONCLUSION: The leader sequence is important in determining the functions of clusterin, which include anti-apoptotic and anti-necrotic properties. The lack of the leader sequence allowed the incompletely processed clusterin to induce apoptosis in target cells; without the leader sequence, clusterin functions differently. Thus, the leader sequence is a trigger for many functions of clusterin in the progression of human prostate cancer cells.     

Chemosensitization of gemcitabine‐resistant human bladder cancer cell line both in vitro and in vivo using antisense oligonucleotide targeting the anti‐apoptotic gene,clusterin

OBJECTIVE

To characterize changes in clusterin (sCLU‐2) expression in bladder cancer cells after continuous treatment with gemcitabine and to determine whether knockdown of sCLU‐2 can re‐introduce sensitivity of gemcitabine‐resistant cells to treatment with gemcitabine.

MATERIALS AND METHODS

A human bladder cancer cell line, UM‐UC‐3, was continuously exposed to increasing doses of gemcitabine in vitro, and a gemcitabine‐resistant cell line UM‐UC‐3R was developed. The role of sCLU‐2 in chemoresistant phenotype acquired in both in vitro and in vivo was then analysed using antisense oligonucleotide targeting the sCLU‐2 gene (OGX‐011).

RESULTS

Treatment of parental UM‐UC‐3 cells (UM‐UC‐3P) with gemcitabine induced transient up‐regulation of sCLU‐2 protein. There was a sustained increase in sCLU‐2 expression levels in UM‐UC‐3R compared with UM‐UC‐3P cells (6.4‐fold). Treatment of UM‐UC‐3R cells with OGX‐011 resulted in a dose‐dependent and sequence‐ specific inhibition in sCLU‐2 expression. Furthermore, OGX‐011 chemo‐sensitized UM‐UC‐3R cells to gemcitabine in vitro with a reduction in the concentration that reduces the effect by 50% (IC₅₀) from 100 nm to 10 nm . Tumour volume and the incidence of metastasis in nude mice injected with UM‐UC‐3R cells was significantly greater than those of nude mice injected with UM‐UC‐3P cells; however, systemic administration of OGX‐011 plus a low dose of gemcitabine significantly suppressed tumour volume and the incidence of metastasis in both groups.

CONCLUSION

These findings suggest that sCLU‐2 plays a significant role in the acquisition of chemoresistant phenotype in bladder cancer cells and the knockdown of sCLU‐2 using OGX‐011 combined with a chemotherapeutic agent could be an attractive approach for advanced bladder cancer through the enhancement of chemosensitivity.     

An antisense oligonucleotide to cIAP-1 sensitizes prostate cancer cells to fas and TNFalpha mediated apoptosis

BACKGROUND: The inhibitors of apoptosis (IAP) proteins are a family of structurally homologous caspase inhibitors. We synthesized an antisense oligonucleotide (AO) to target a region within the BIR domain of cIAP-1 and examined its ability to facilitate apoptosis in prostate cancer cells. METHODS: We transfected the IAP AO into PC3 and DU145 cells and determined alterations in IAP expression using Western blotting. Apoptosis and viability were assessed using propidium iodide (PI) DNA incorporation with flow cytometry. Pacitaxel, caffeic acid phenethyl ester (CAPE), Fas antibody, and TNFalpha were used as 'second hit' agents in association with the AO. RESULTS: Western blotting showed a down-regulation in cIAP-1 expression and higher levels of spontaneous apoptosis in both cell types with no alteration in overall cell viability. AO sensitized PC3 cells, to Fas antibody and TNFalpha-mediated apoptosis, but not to apoptosis mediated by paclitaxel or CAPE. CONCLUSIONS: cIAP-1 down-regulation increased spontaneous apoptosis in prostate cancer cells and sensitized PC3 cells to receptor-mediated apoptosis.     

Response to a lethal dose of heat shock by a transient up-regulation of clusterin expression followed by down-regulation and apoptosis in prostate and bladder cancer cells

BACKGROUND: Clusterin is a ubiquitous, secretory glycoprotein with a wide array of functions. Recent studies have implicated that clusterin functioned as heat shock response proteins. The objective of the present study was to determine the fate of clusterin expression in cancer cells, which were subjected to a lethal dose of heat shock, in an attempt to shed light on mechanisms of action of clusterin. METHODS: A prostate cancer line, PC-3, and a bladder cancer line, TSU-Pr1, were selected owing to their aggressive growth behaviors. Apoptosis was assessed by enzymatic activities of terminal deoxynucleotidyl transferase as well as by activities of caspase-3. Cells were exposed to 45 degrees C for a period of 60 min. RESULTS: Both cell lines underwent a transient up-regulation of clusterin expression followed by down-regulation and apoptosis. TSU-Prl cells produced higher levels of clusterin and displayed a greater resistance to apoptosis than did PC-3 cells. The addition of exogenous clusterin to the cultures of PC-3 cells protected apoptosis. Treatment of oligonucleotide antisense to clusterin to the cultures of TSU-Pr1 cells enhanced apoptosis mediated by heat shock. CONCLUSION: Clusterin offers a protection to PC-3 and TSU-Prl cells against heat shock and plays an important role in the cascade of events initiated by heat shock. Prostate 53: 277-285, 2002.     

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.     

Protection of androgen-dependent human prostate cancer cells from oxidative stress-induced DNA damage by overexpression of clusterin and its modulation by androgen

BACKGROUND: Recent studies reported that oxidative stress is one of the major factors associated with the progression of prostate cancer through the accumulation of DNA damage. In the present study, we investigated the effect of oxidative stress on cell injury using androgen-dependent human prostate cancer LNCaP cells overexpressing clusterin, which has been shown to play crucial roles in the acquisition of resistance to several apoptotic stimuli. METHODS: We introduced clusterin cDNA into LNCaP cells which do not express a detectable level of clusterin expression, and generated a clusterin-overexpressing cell line (LNCaP/Cl) and a control vector only-transfected cell line (LNCaP/Co). The effects of hydrogen peroxide (H2O2) treatment on the LNCaP sublines with and without the addition of dihydrotestosterone (DHT) were analyzed using the in vitro mitogenic assay and lipid peroxidation assay, and morphological changes in the LNCaP sublines after H2O2 treatment were examined by staining with Hoechst 33258. The degrees of DNA damage induced by H2O2 into the LNCaP sublines were evaluated by the measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG) level. RESULTS: H2O2-induced apoptosis in LNCaP/Cl was significantly suppressed compared with that in LNCaP/Co through the inhibition of membrane damage; however, the measurement of 8-OHdG level demonstrated that DNA damage was more intensively accumulated in LNCaP/Cl cells than LNCaP/Co cells. Furthermore, DHT suppressed the incidence of apoptotic cell death and enhanced the formation of 8-OHdG in both LNCaP/Cl and LNCaP/Co cells after H2O2 treatment in a dose-dependent manner. CONCLUSIONS: These findings suggest that clusterin may contribute to conferring resistance to oxidative stress-mediated cellular injury on prostate cancer cells, especially in the presence of androgen.     

Clinical experience of hormone therapy to bone metastatic prostate cancer

BACKGROUND: A novel hormone therapy was instituted against prostate cancer with bone metastases and its therapeutic efficacy was investigated. METHODS: A total of 35 patients who had been pathologically diagnosed with carcinoma of the prostate between December [corrected] 1994 and December 2003 were entered into the present study. Patients aged over 80 years were excluded from the study. As for the treatment methodology, diethylstilbestrol diphosphate (DES-P) at 500 mg/day was intravenously injected for 20-40 days, followed by monotherapy with an analog of luteinizing hormone-releasing hormone (LHRH). In all subjects, surgical castration was not conducted. The survival rate was analysed according to the method of Kaplan-Meier. RESULTS: One of the 35 patients was excluded from the study as this patient did not meet the inclusion criteria. There were four patients who dropped out of the study. On histology, 17 patients had moderately differentiated adenocarcinomas and 17 patients had poorly differentiated adenocarcinomas. As for the extent of disease (EOD), the patients were classified as with a score of 1 in 10 patients, 2 in 13 patients, 3 in 7 patients and 4 in 4 patients. The 5-year progression-free survival rate and overall survival rate were 24.3% and 60.6%, respectively. CONCLUSION: Our new hormone therapy in the management of prostate cancer metastatic to the bone has demonstrated markedly superior therapeutic results compared to those so far obtained.     

前列腺雄激素调节基因:一个新的雄激素非依赖性前列腺癌治疗的潜在靶点(英文)

目的:初步研究前列腺雄激素调节基因(PAR)与雄激素—雄激素受体信号转导通路的关系及其在前列腺癌细胞恶性转化过程中的作用,探讨通过抑制 PAR 基因治疗雄激素非依赖性前列腺癌的可能性。方法:用 RT-PCR 检测 LNCaP、PC3细胞中 PAR 基因 mRNA 表达水平的差异。分别用 RT-PCR 检测双氢睾酮对LNCaP、PC3及稳定转染了 pcDNA3-AR 的 PC3细胞株 PC3-AR 的 PAR 基因 mRNA 表达的调节作用,并观察这一调节作用是否可被雄激素受体拮抗剂氟他胺阻断。进一步用 RNA 干扰技术下调 PC3细胞 PAR 的表达,用细胞计数、软琼脂克隆形成实验、流式细胞术研究 PAR 基囚表达下调对 PC3细胞生长的抑制作用。结果:PC3细胞 PAR 基因 mRNA 的表达是 LNCaP 细胞的3倍;双氢睾酮可调节 LNCaP 和 PC3-AR 细胞株PAR 基因 mRNA 表达水平,此种对 PAR 表达的调节作用可被氟他胺阻断:双氢睾酮对 PC3细胞 PAR 基因mRNA 表达无明显影响。RNA 干扰可抑制 PC3细胞 PAR 基因表达,使细胞增殖受抑制,细胞周期阻滞于G_2-M 期,凋亡增加。结论:PAR 可能是雄激素—雄激素受体信号转导通路下游的与雄激素非依赖性前列腺癌恶性表型密切相关的癌基因,有望成为其基冈和药物治疗的靶点。