Hideaki Miyake

Use of antisense oligonucleotides targeting the cytoprotective gene, clusterin, to enhance androgen- and chemo-sensitivity in prostate cancer

The discovery and targeting of genes mediating androgen-independence may lead to the development of novel therapies that delay progression of hormone refractory prostate cancer (HRPC). Clusterin is a stress-associated cell survival gene that increases after androgen ablation. Here, we review clusterin’s functional role in apoptosis and the use of antisense oligonucleotides (ASOs) against clusterin to enhance apoptosis in prostate cancer models. Immunostaining of tissue microarrays constructed from untreated and post-hormone treated radical prostatectomy specimens confirm that clusterin is highly expressed in virtually all HRPC cells, 80% of prostate cancer cells after neoadjuvant hormone therapy, but is low or absent (<20%) in untreated specimens. Overexpression of clusterin in LNCaP cells confers resistance to both androgen ablation and chemotherapy. Clusterin ASOs reduced clusterin levels in a dose-dependent and sequence-specific manner. Adjuvant treatment with murine clusterin ASOs after castration of mice bearing Shionogi tumors decreased clusterin levels, accelerated apoptotic tumor regression, and significantly delayed the recurrence of androgen-independent tumors. A human clusterin ASO targeting the translation initiation site and incorporating MOE-gapmer backbone (OGX-011) synergistically enhanced the cytotoxic effects of paclitaxel in human xenografts of prostate, renal cell, bladder, and lung cancer. Clusterin, is an anti-apoptosis protein upregulated in an adaptive cell survival manner by androgen ablation and chemotherapy that confers resistance to various cell death triggers. Suppression of clusterin levels using ASOs enhances cell death following treatment with androgen ablation, radiation, and chemotherapy.

Stress protein GRP78 prevents apoptosis induced by calcium ionophore, ionomycin, but not by glycosylation inhibitor, tunicamycin, in human prostate cancer cells

GRP78 induction has recently been shown to play a critical role in maintaining cell viability against several kinds of stress, including depletion of endoplasmic reticulum Ca2+ and accumulation of unglycosylated proteins, under specific experimental conditions. However, the functional significance of GRP78 induction after stressful treatment has not been well defined. This article characterizes the different biological features associated with GRP78 induction by two kinds of stress agents, calcium ionophore, ionomycin (IM), and glycosylation inhibitor, tunicamycin (TM), focusing on the association with apoptosis in human prostate cancer cells. Both IM and TM treatment resulted in marked induction of GRP78 transcription in androgen‐dependent prostate cancer LNCaP cells maintained in medium without androgen, but not in medium containing androgen, as measured by Northern blotting and nuclear run‐off assays. After pretreatment with tumor necrosis factor‐α, which has potent cytotoxic effects on LNCaP cells, both IM and TM could induce substantial increases in GRP78 transcription in LNCaP cells, even in medium containing androgen. Under both experimental conditions described, DNA fragmentation assays showed a direct correlation between the onset of apoptosis in LNCaP cells after IM treatment and the initiation of GRP78 transcript induction, while induction of GRP78 expression preceded TM‐induced apoptosis. To elucidate the functional differences of GRP78 induction by IM and TM, an antisense oligodeoxynucleotide (ODN) targeted against the grp78 gene was designed to reduce GRP78 expression in a sequence‐specific and dose‐dependent manner. Antisense GRP78 ODN treatment substantially enhanced apoptosis of LNCaP cells induced by IM compared with mismatch control ODN treatment, whereas no marked differences were observed in apoptotic features induced by TM with antisense GRP78 and mismatch control ODN treatment. Studies of additional androgen‐independent prostate cancer PC3 cells also demonstrated a correlation between GRP78 induction and resistance to apoptosis after IM treatment, but not after TM treatment. These findings suggest that there are at least two GRP78 signaling pathways, which play different roles in resistance against stress‐induced apoptosis. J. Cell. Biochem. 77:396–408, 2000.

Polymeric micellar paclitaxel phosphorylates Bcl-2 and induces apoptotic regression of androgen-independent LNCaP prostate tumors

Paclitaxel has been difficult to evaluate in preclinical tumor model systems because its poor solubility requires a Cremophor EL formulation, which results in lethal anaphylaxis. We tested the effectiveness of a novel polymeric micellar paclitaxel on androgen‐independent tumor growth in the LNCaP tumor model.

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

The paper by Zellweger et al. builds on the continuing story of clusterin (TRPM‐2) in the development and progression of prostate cancer. This group have published a series of papers on this protein, showing that it correlates with progression to androgen‐independence and resistance to apoptosis. One of their recent papers has shown that ‘knocking out’ clusterin increases radiation sensitivity in prostate cancer cells. The current paper reports that increasing the expression of clusterin in LNCaP cells increases the cells resistance to radiation‐induced apoptosis. Manipulating identified survival proteins has important implications in preventing androgen‐independent progression. Clusterin is such a survival protein and represents an important drug target in the near future.

Synergistic chemosensitization and inhibition of tumor growth and metastasis by adenovirus-mediated P53 gene transfer in human bladder cancer model.

OBJECTIVESnTo determine whether an adenovirus-mediated p53 gene (Ad5CMV-p53) transfer enhances cisplatin cytotoxicity in vitro and whether Ad5CMV-p53 and cisplatin synergistically inhibit growth and metastasis in vivo using human bladder cancer KoTCC-1 cells.nnnMETHODSnMTT assays and DNA fragmentation assays were used to examine the effects of treatment with Ad5CMV-p53 and/or cisplatin on growth inhibition and induction of apoptosis, respectively, in KoTCC-1 cells. The efficacies of combined Ad5CMV-p53 and/or cisplatin therapy against growth and metastasis of KoTCC-1 tumors were assessed using subcutaneous and orthotopic tumor cell injection models.nnnRESULTSnAd5CMV-p53 substantially enhanced cisplatin chemosensitivity in a dose-dependent manner, reducing the median IC(50) by more than 50%. Characteristic apoptotic DNA laddering was induced by the combination of sublethal doses of Ad5CMV-p53 and cisplatin, but not by either agent alone. Furthermore, combined Ad5CMV-p53 and cisplatin therapy synergistically inhibited growth of subcutaneous KoTCC-1 tumors and the incidence of metastasis after orthotopic injection.nnnCONCLUSIONSnThese findings illustrate that combined treatment with Ad5CMV-p53 and cisplatin could be an attractive strategy for inhibiting progression of bladder cancer through effective induction of apoptosis.

Beyond simple castration: targeting the molecular basis of treatment resistance in advanced prostate cancer

Over the past 20xa0years, research on hormonal treatments for prostate cancer focused on maximizing androgen ablation through combination therapy. Unfortunately, maximal androgen ablation increases treatment-related side effects and expense and has not significantly prolonged time to androgen-independent (AI) progression. Intermittent androgen suppression (IAS) is based on the hypothesis that if tumor cells surviving androgen withdrawal can be forced along a normal pathway of differentiation by androgen replacement, then apoptotic potential might be restored, androgen dependence may be prolonged and progression to androgen independence may be delayed. Observations from animal model studies suggest that progression to androgen independence is delayed by IAS and this strategy is now being evaluated in phase III trials. Another strategy for improving therapies in advanced prostate cancer involves targeting genes that are activated by either androgen withdrawal or chemotherapy to delay or prevent the emergence of the resistant AI phenotype. Targeted inhibition of stress-associated increases in gene expression precipitated by androgen withdrawal or chemotherapy may enhance treatment-induced apoptosis and delay progression to AI disease. Proteins fulfilling these criteria include antiapoptotic members of the Bcl-2 protein family, clusterin, Hsp27, and IGFBP-2 and IGFBP-5. The purpose of this paper is to review the rationale and progress in using targeted gene therapies to enhance tumor cell death after androgen withdrawal or taxane chemotherapy. Antisense oligonucleotides offer one approach to target genes involved in cancer progression, especially those not amenable to small molecule or antibody inhibition. The current status and future direction of several antisense oligonucleotides that have potential clinical use in cancer are also reviewed.

CALCIUM IONOPHORE, IONOMYCIN INHIBITS GROWTH OF HUMAN BLADDER CANCER CELLS BOTH IN VITRO AND IN VIVO WITH ALTERATION OF BCL-2 AND BAX EXPRESSION LEVELS

PURPOSEnThe objective of this study was to characterize the antiproliferative effects of the calcium ionophore, ionomycin on the human bladder cancer cell line HT1376 both in vitro and in vivo.nnnMATERIALS AND METHODSnThe in vitro growth rate of HT1376 after treatment with ionomycin was measured by using the MTT assay. The apoptotic features in HT1376 after ionomycin treatment were evaluated by flow cytometric analysis and DNA degradation assay. Bcl-2 and Bax expression levels in HT1376 after ionomycin treatment were examined by Northern and Western blot analyses. The effects of intratumoral injection of ionomycin on the growth of subcutaneous HT1376 tumors established in athymic nude mice were then tested. The efficacy of combined treatment with ionomycin and cisplatin against HT1376 growth was also examined.nnnRESULTSnThe in vitro growth rate of HT1376 was suppressed by ionomycin in a dose- and time-dependent manner, and characteristic apoptotic DNA degradation was observed in HT1376. Ionomycin treatment caused a marked decrease in the ratios of Bcl-2 to Bax mRNA and protein in HT1376 cells. Intratumoral injection of ionomycin into subcutaneous HT1376 tumors reduced the tumorigenicity in nude mice. Furthermore, these in vivo growth-inhibitory effects of ionomycin were significantly enhanced by pretreatment with cisplatin.nnnCONCLUSIONSnThese findings suggest that ionomycin-based therapy could be used as a novel therapeutic strategy for advanced bladder cancer through the effective induction of apoptosis by decreasing the ratio of Bcl-2 to Bax.

Overexpression of Bcl-2 regulates sodium butyrate- and/or docetaxel-induced apoptosis in human bladder cancer cells both in vitro and in vivo.

Sodium butyrate (NaBt), a physiologically occurring short‐chain fatty acid, induces differentiation as well as apoptosis in numerous cell types, and this induction is partially regulated by Bcl‐2 expression. The objectives of our study were to characterize the in vitro effects of NaBt and/or docetaxel on the growth, cell cycle and apoptosis of human bladder cancer cells, and to determine whether tumor growth in vivo is inhibited by isobutyramide, an orally bioavailable Bt analogue, and/or docetaxel by using Bcl‐2‐transfected human bladder cancer cell line KoTCC‐1/BH and control vector only‐transfected cell line KoTCC‐1/C. NaBt caused a decrease in growth of both KoTCC‐1/C and KoTCC‐1/BH cells, however, its growth inhibitory effect was significantly greater in KoTCC‐1/C cells. One mM NaBt resulted in G1 cell cycle arrest, accompanied by up‐regulation of p21 (waf1/cip1) and down‐regulation of cyclin D1 in KoTCC‐1/C cells, whereas KoTCC‐1/BH showed resistance to G1 cell cycle arrest. An amount of 5 mM NaBt induced apoptosis, accompanied by up‐regulation of Bak in KoTCC‐1/C cells but failed to induce apoptosis in KoTCC‐1/BH cells despite substantial down‐regulation of Bcl‐2. Oral administration of isobutyramide significantly reduced the KoTCC‐1/C tumor volume compared with the KoTCC‐1/BH tumor volume in nude mice. Furthermore, docetaxel induced Bcl‐2 phosphorylation in KoTCC‐1/BH cells and combined treatment with isobutyramide and docetaxel synergistically inhibited the growth of KoTCC‐1/BH cells both in vitro and in vivo. These findings suggest that isobutyramide therapy could be a novel therapeutic strategy for patients with bladder cancer if docetaxel is combined according to the Bcl‐2 expression status.

Differential involvement of the Fas receptor/ligand system in p53-dependent apoptosis in human prostate cancer cells.

The objective of this study was to characterize the involvement of the Fas receptor/ligand system in p53‐dependent apoptosis in human prostate cancer cells.

Targeting Cell Survival Genes Using Antisense Oligonucleotides to Improve Chemo- and Hormonal-Therapies in Prostate Cancer

Abstract Androgen withdrawal remains the most effective form of systemic therapy for men with advanced prostate cancer, with symptomatic and/or objective response rates in 80% of patients. Unfortunately, progression to androgen-independent (AI) disease occurs within a few years and is the main obstacle to improving survival and quality of life in men with advanced prostate cancer. Novel therapeutic strategies targeting the molecular basis of androgen- and chemotherapy-resistance of prostate cancer are needed because hormone-refractory prostate cancer (HRPC) historically has responded poorly to cytotoxic chemotherapy. Results of recent phase II studies using taxane-based combinations have demonstrated high objective response rates in patients with measurable disease, as well as high rates of prostate-specific antigen (PSA) decline. Phase III trials are underway to confirm these results. Another strategy being investigated to improve outcomes in advanced prostate cancer involves targeting genes, which are activated by either androgen withdrawal or chemotherapy, to delay or prevent the emergence of androgen independence. Targeting genes that are upregulated after chemotherapy or androgen withdrawal (e.g. Bcl-2, clusterin, cathepsins, Bcl-xL), which function to prevent castration-induced apoptosis or activate alternative growth factor pathways, may enhance tumor cell death, prolong time to overt recurrence and ultimately prolong survival.