nutlin-3a and Prostatic-Neoplasms

Prostate cancer is the most common carcinoma of the male urinary system in developed countries. Androgen deprivation therapy has been commonly used in the treatment of prostate cancer for decades, but most patients will inevitably develop into more aggressive castration-resistant prostate cancer. Therefore, novel strategies are urgent to address this resistance mechanism. In this review, we discussed some new strategies for targeting androgen receptors through degradation pathways as potential treatments for prostate cancer.

Topics: Androgen Receptor Antagonists; Animals; Antineoplastic Agents; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Heat-Shock Proteins; Humans; Male; Prostatic Neoplasms; Proteolysis; Receptors, Androgen; Signal Transduction; Thiohydantoins; Ubiquitination

Topics: Adenocarcinoma; Apoptosis; Aurora Kinase B; CDC2 Protein Kinase; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Humans; Imidazoles; Male; Piperazines; Prostatic Neoplasms; Proto-Oncogene Proteins c-mdm2

Escape from apoptosis, one of the characteristic features of cancer cells, is a case that reduces the therapeutic efficacy of apoptosis-inducing molecules used in the cancer treatment. Stabilization of the P53 protein, which is responsible for the regulation of apoptosis mechanism in the cell, is therefore an important therapeutic goal. Nutlin3a inhibits the degradation of the P53 protein, triggers P53-mediated apoptosis in cancer cells and enhances the effectiveness of chemotherapeutics. However, its low aqueous solubility is the major disadvantage when it comes to in vivo administration. In order to facilitate an aqueous formulation of Nutlin3a and to enhance its apoptotic activity on cancer cells, Nutlin3a was encapsulated in solid lipid nanoparticles (SLNs) prepared by Ouzo method. Physicochemical characterization was performed and activity of apoptosis induction on wild-type P53 expressing LNCaP prostate cancer cell line was evaluated. Nutlin3a-loaded cationic solid lipid nanoparticles were found to stabilize functional P53 at protein level. In addition, induction rate of apoptosis by nanoparticles was higher than Nutlin3a solution in DMSO, proving this nanoparticle formulation is a promising candidate for increasing the efficiency of Nutlin3a for P53(+) cancer cases. Thus, it is anticipated that the results will contribute to evaluate the use of lipid-based nanocarriers to enhance the therapeutic potential of small molecules that are important in cancer cure.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Drug Delivery Systems; Humans; Imidazoles; Male; Nanoparticles; Piperazines; Prostatic Neoplasms; Tumor Suppressor Protein p53

Inhibition of androgen receptor (AR) signalling represents the conventional medical management of prostate cancer. Ultimately this treatment fails because tumors develop an incurable, castrate resistant phenotype, resulting in an unmet need for new treatments in prostate cancer. The AR remains a viable therapeutic target in castrate resistant disease, such that novel ways of downregulating AR activities are attractive as potential treatments. Here we describe a mechanism by which the AR can be downregulated by the MDM2 antagonist Nutlin-3, resulting in loss of pro-survival c-FLIP gene expression and apoptosis. We additionally show that loss of c-FLIP sensitises prostate cancer cells to Nutlin-3. Finally, we demonstrate that the unrelated MDM2 antagonist Mi-63 also impinges upon AR signalling, supporting the concept of future treatment of prostate cancer with MDM2 antagonists.

Topics: Apoptosis; Biomarkers; CASP8 and FADD-Like Apoptosis Regulating Protein; Cell Cycle Checkpoints; Cell Line, Tumor; Drug Resistance, Neoplasm; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Imidazoles; Male; Piperazines; Prostatic Neoplasms; Protein Binding; Proto-Oncogene Proteins c-mdm2; Receptors, Androgen; Ubiquitination

Prostate cancer is the second most commonly diagnosed cancer in men, and approximately one-third of those diagnosed succumb to the disease. The development of prostate cancer from small regions of hyperplasia to invasive tumors requires genetic and epigenetic alterations of critical cellular components to aid in the development of cells more adapted for aberrant growth. The p53 transcription factor is a critical element in the cell's ability to regulate the cell cycle and its response to DNA damage. Mutations within the DNA-binding domain of p53 are common and allow the formation of tetramers; however, these alterations prevent this protein complex from associating with target gene promoters. In the present study, we examined the effects of p53 functionality in prostate cancer cells that harbored wild-type (WT) or mutant forms of the protein in response to commonly used chemotherapeutic drugs. The androgen receptor positive 22Rv-1 and LNCaP prostate cancer cell lines carry WT p53 and were demonstrated to have a decrease in chemotherapeutic drug sensitivity when transfected with a dominant-negative (DN) p53. Conversely, expression of the WT p53 in the p53-mutated and more advanced DU145 prostate cancer cell line significantly increased its overall sensitivity to anti-neoplastic drugs. Furthermore, analysis of colony formation in soft agar revealed that the functional status of p53 in each cell line altered the cell's ability to proliferate in an anchorage-independent fashion. Prostate cancer colony growth was more prevalent when p53 transcriptional activity was decreased, whereas growth was more limited in the presence of functional p53. These results demonstrate that the functional status of the tumor suppressor p53 is important in the progression of prostate cancer and dictates the overall effectiveness a given drug would have on disease treatment.

Topics: Antineoplastic Agents; Cell Line, Tumor; Humans; Imidazoles; Male; Piperazines; Prostatic Neoplasms; Proto-Oncogene Proteins c-mdm2; Tumor Suppressor Protein p53

Hormone therapy is the standard of care for newly diagnosed or recurrent prostate cancers. It uses anti-androgen agents, castration, or both to eliminate cancer promoting effect of testicular androgen. The p53 tumor suppressor controls a major pathway that can block cell proliferation or induce apoptosis in response to diverse forms of oncogenic stress. Activation of the p53 pathway in cancer cells expressing wild-type p53 has been proposed as a novel therapeutic strategy and recently developed MDM2 antagonists, the nutlins, have validated this in preclinical models of cancer. The crosstalk between p53 and androgen receptor (AR) signaling suggest that p53 activation could augment antitumor outcome of androgen ablation in prostate cancer. Here, we test this hypothesis in vitro and in vivo using the MDM2 antagonist, nutlin-3 and the p53 wild-type prostate cancer cell line, LNCaP.. Using charcoal-stripped serum as a cellular model of androgen deprivation, we show an increased apoptotic effect of p53 activation by nutlin-3a in the androgen-dependent LNCaP cells and to a lesser extent in androgen-independent but responsive 22Rv1 cell line. This effect is due, at least in part, to an enhanced downregulation of AR expression by activated p53. In vivo, androgen deprivation followed by two weeks of nutlin administration in LNCaP-bearing nude mice led to a greater tumor regression and dramatically increased survival.. Since majority of prostate tumors express wild-type p53, its activation by MDM2 antagonists in combination with androgen depletion may offer an efficacious new approach to prostate cancer therapy.

Topics: Androgens; Animals; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cell Survival; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Male; Mice; Mice, Nude; MicroRNAs; Mutation; Piperazines; Prostatic Neoplasms; Proto-Oncogene Proteins c-mdm2; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Renal cell cancers (RCC) are notoriously resistant to chemotherapy and radiotherapy. While mutations of the p53 tumor suppressor gene frequently contribute to therapy resistance in other epithelial cancers, p53 mutations are relatively rare in RCC. To date, there is conflicting evidence as to whether p53 signaling and function are otherwise proficient or defective in tumors with wild-type p53. In this study, we assayed p53 function in a series of RCC cell lines and normal proximal epithelial tubule cells using two different MDM-2 antagonists, Nutlin-3a and MI-219. Most cell lines with wild-type p53 responded to MDM-2 antagonists as evidenced by induction of p53 and its target gene p21. RCC cell lines treated with MDM-2 antagonists consistently accumulated in the G2/M phase of the cell cycle and this event was associated with inhibition of proliferation in RCC cell lines but not in normal proximal epithelial tubule cells. MDM-2 antagonists did not induce significant cell death in RCC cell lines, even with induction of p53-dependent pro-apoptotic genes. In contrast, MDM-2 antagonists caused significant cell death in LNCaP prostate adenocarcinoma cells. RCC cell lines with reduced p53, either by mutation or through ectopic expression of p53 shRNA, demonstrated enhanced sensitivity to cell death following sequential treatment with DNA damage and G2/M checkpoint abrogation. Our results suggest that wild-type p53 RCC cell lines are proficient in p53-dependent cell cycle arrest but defective in p53-dependent cell death.

Topics: Apoptosis; Carcinoma, Renal Cell; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Doxorubicin; Gene Expression Regulation, Neoplastic; Genes, p53; Humans; Imidazoles; Kidney Tubules, Proximal; Male; Mutation; Phosphorylation; Piperazines; Polymerase Chain Reaction; Prostatic Neoplasms; Proto-Oncogene Proteins c-mdm2; RNA, Small Interfering; Signal Transduction; Tumor Suppressor Protein p53

Nutlin-3 is a small-molecule inhibitor that acts to inhibit MDM2 binding to p53 and subsequent p53-dependent DNA damage signaling. Whether Nutlin-3 alters cell toxicity following DNA damage under oxic versus hypoxic conditions has not been studied. The potential radiosensitization (0-10 Gy) properties of Nutlin-3 (dose range, 2-10 micromol/L for up to 24 h) were investigated in vitro using three prostate cancer cell lines, 22RV1 [wild-type p53 (WTp53)], DU145 (mutated p53), and PC-3 (p53-null) under oxic (21% O(2)), hypoxic (0.2% O(2)), and anoxic (0% O(2)) conditions. As a single agent, Nutlin-3 (2-10 micromol/L) stabilized p53 and p21(WAF) levels and was toxic to WTp53-22RV1 cells (IC(50), 4.3 micromol/L) but had minimal toxicity toward p53-deficient cells (IC(50), >10 micromol/L). When combined with radiation under oxic conditions, Nutlin-3 decreased clonogenic survival in all three cell lines: 22RV1 [sensitizing enhancement ratio (SER), 1.24], DU145 (SER, 1.27), and PC-3 (SER, 1.12). Anoxia induced p53 protein expression in 22RV1 cells and this was augmented by Nutlin-3 treatment. Furthermore, Nutlin-3 was more effective as a radiosensitizer under hypoxic conditions particularly in WTp53-expressing cells: 22RV1 (SER, 1.78), DU145 (SER, 1.31), and PC-3 (SER, 1.28). The decrease in clonogenic survival with Nutlin-3 was not correlated to altered levels of radiation-induced apoptosis within the three cell lines. Our results indicate that Nutlin-3 can act as a radiosensitizer via p53-independent mechanisms under low O(2) levels. Nutlin-3 may be a useful adjunct to improve the therapeutic ratio using precision radiotherapy targeted to hypoxic cells and warrants further study in vivo.

Topics: Apoptosis; Blotting, Western; Caspases; Cell Cycle; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Imidazoles; Male; Piperazines; Prostatic Neoplasms; Proto-Oncogene Proteins c-mdm2; Radiation-Sensitizing Agents; Stereoisomerism; Tumor Cells, Cultured; Tumor Stem Cell Assay; Tumor Suppressor Protein p53

Small molecule MDM2 antagonists including nutlin-3 have been shown to be effective against a range of cancer cell types and nutlin-3 can inhibit growth of LNCaP xenografts. We compared the efficacy of nutlin-3 in three prostate cancer cell types and provide an insight into the mechanism of nutlin-3.. Nutlin-3 efficacy was measured using proliferation assays, cell cycle analysis, apoptosis assays, quantitative RT-PCR, and immunoblotting. Chromatin immunoprecipitation (ChIP) assays were also performed.. Nutlin-3 can specifically inhibit proliferation of LNCaP cells through cell cycle arrest and apoptosis. This coincides with increased levels of the p53-responsive transcripts p21, PUMA, gadd45, and Mdm2 and recruitment of p53 to chromatin. Nutlin-3 also reduces androgen receptor levels, resulting in altered receptor recruitment to chromatin.. Our study demonstrates that small molecule MDM2 antagonists might be useful in the treatment of human prostate cancers that retain functional p53 and androgen receptor signaling.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Plant; Genes, p53; Humans; Imidazoles; Male; Nuclear Proteins; Piperazines; Prostate-Specific Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Receptors, Androgen; Signal Transduction