nutlin-3a and Retinoblastoma

While mortality is low for intraocular retinoblastoma patients in the developed world who receive aggressive multimodal therapy, partial or full loss of vision occurs in approximately 50% of patients with advanced bilateral retinoblastoma. Therapies that preserve vision and reduce late effects are needed. Because clinical trials for retinoblastoma are difficult due to the young age of the patient population and relative rarity of the disease, robust preclinical testing of new therapies is critical. The last decade has seen advances towards identifying new therapies including the development of animal models of retinoblastoma for preclinical testing, progress in local drug delivery to reach intraocular targets, and improved understanding of the underlying biological mechanisms that give rise to retinoblastoma. This review discusses advances in these areas, with a focus on discovery and development of small molecules for the treatment of retinoblastoma, including novel targeted therapeutics such as inhibitors of the MDMX-p53 interaction (nutlin-3a), histone deacetylase (HDAC) inhibitors, and spleen tyrosine kinase (SYK) inhibitors.

Topics: Animals; Antineoplastic Agents; Drug Discovery; Histone Deacetylase Inhibitors; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Piperazines; Protein Interaction Maps; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-mdm2; Retina; Retinal Neoplasms; Retinoblastoma; Small Molecule Libraries; Syk Kinase; Tumor Suppressor Protein p53

Retinoblastoma is the most common intraocular tumor in children. Malfunctioning of many signaling pathways regulating cell survival or apoptosis, make the disease more vulnerable. Notably, resistance to chemotherapy mediated by MRP-1, lung-resistance protein (LRP) is the most challenging aspect to treat this disease. Presently, much attention has been given to the recently developed anticancer drug nutlin-3a because of its non-genotoxic nature and potency to activate tumor suppressor protein p53. However, being a substrate of multidrug resistance protein MRP1 and Pgp its application has become limited. Currently, research has step towards reversing Multi drug resistance (MDR) by using curcumin, however its clinical relevance is restricted by plasma instability and poor bioavailability. In the present investigation we tried to encapsulate nutlin-3a and curcumin in PLGA nanoparticle (NPs) surface functionalized with folate to enhance therapeutic potential of nutlin-3a by modulating MDR. We document that curcumin can inhibit the expression of MRP-1 and LRP gene/protein in a concentration dependent manner in Y79 cells. In vitro cellular cytotoxicity, cell cycle analysis and apoptosis studies were done to compare the effectiveness of native drugs (single or combined) and single or dual drug loaded nanoparticles (unconjugated/folate conjugated). The result demonstrated an augmented therapeutic efficacy of targeted dual drug loaded NPs (Fol-Nut-Cur-NPs) over other formulation. Enhanced expression or down regulation of proapoptotic/antiapoptotic proteins respectively and down-regulation of bcl2 and NFκB gene/protein by Fol-Nut-Cur-NPs substantiate the above findings. This is the first investigation exploring the role of curcumin as MDR modulator to enhance the therapeutic potentiality of nutlin-3a, which may opens new direction for targeting cancer with multidrug resistance phenotype.

Topics: Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Curcumin; Drug Carriers; Drug Resistance, Neoplasm; Folic Acid; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; Lactic Acid; Membrane Potential, Mitochondrial; Multidrug Resistance-Associated Proteins; Nanoparticles; NF-kappa B; Piperazines; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Proto-Oncogene Proteins c-bcl-2; Retinoblastoma; Tumor Suppressor Protein p53

Retinoblastoma is a rare childhood cancer of the retina that begins in utero and is diagnosed in the first years of life. The goals of retinoblastoma treatment are ocular salvage, vision preservation, and reduction of short- and long-term side effects without risking mortality because of tumor dissemination. To identify better chemotherapeutic combinations for the treatment of retinoblastoma, several groups have developed genetic mouse models and orthotopic xenograft models of human retinoblastoma for preclinical testing. Previous studies have implicated the MDMX protein in the suppression of the p53 pathway in retinoblastoma and shown that the MDM2/MDMX antagonist, Nutlin-3a, can efficiently induce p53-mediated cell death in retinoblastoma cell lines. However, Nutlin-3a cannot be administered systemically to treat retinoblastoma, because it has poor penetration across the blood-ocular barrier. Therefore, we developed an ocular formulation of Nutlin-3a, Nutlin-3a(OC), and tested the pharmacokinetics and efficacy of this new formulation in genetic and human retinoblastoma orthotopic xenograft models of retinoblastoma. Here, we show that Nutlin-3a(OC) specifically and efficiently targets the p53 pathway and that the combination of Nutlin-3a(OC) with systemic topotecan is a significantly better treatment for retinoblastoma than currently used chemotherapy in human orthotopic xenografts. Our studies provide a new standardized approach to evaluate and prioritize novel agents for incorporation into future clinical trials for retinoblastoma.

Topics: Animals; Conjunctiva; Disease Models, Animal; Humans; Imidazoles; Intraocular Pressure; Mice; Mice, Inbred C57BL; Mice, SCID; Piperazines; Proto-Oncogene Proteins c-mdm2; Retinal Neoplasms; Retinoblastoma; Solubility; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

In around 50% of all human cancers the tumor suppressor p53 is mutated. It is generally assumed that in the remaining tumors the wild-type p53 protein is functionally impaired. The two main inhibitors of p53, hMDM2 (MDM2) and hMDMX (MDMX/MDM4) are frequently overexpressed in wild-type p53 tumors. Whereas the main activity of hMDM2 is to degrade p53 protein, its close homolog hMDMX does not degrade p53, but it represses its transcriptional activity. Here we study the role of hMDMX in the neoplastic transformation of human fibroblasts and embryonic retinoblasts, since a high number of retinoblastomas contain elevated hMDMX levels.. We made use of an in vitro transformation model using a retroviral system of RNA interference and gene overexpression in primary human fibroblasts and embryonic retinoblasts. Consecutive knockdown of RB and p53, overexpression of SV40-small t, oncogenic HRasV12 and HA-hMDMX resulted in a number of stable cell lines representing different stages of the transformation process, enabling a comparison between loss of p53 and hMDMX overexpression. The cell lines were tested in various assays to assess their oncogenic potential.. Both p53-knockdown and hMDMX overexpression accelerated proliferation and prevented growth suppression induced by introduction of oncogenic Ras, which was required for anchorage-independent growth and the ability to form tumors in vivo. Furthermore, we found that hMDMX overexpression represses basal p53 activity to some extent. Transformed fibroblasts with very high levels of hMDMX became largely resistant to the p53 reactivating drug Nutlin-3. The Nutlin-3 response of hMDMX transformed retinoblasts was intact and resembled that of retinoblastoma cell lines.. Our studies show that hMDMX has the essential properties of an oncogene. Its constitutive expression contributes to the oncogenic phenotype of transformed human cells. Its main function appears to be p53 inactivation. Therefore, developing new drugs targeting hMDMX is a valid approach to obtain new treatments for a subset of human tumors expressing wild-type p53.

Topics: Animals; Cell Adhesion; Cell Cycle Proteins; Cell Proliferation; Cell Shape; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; Chorioallantoic Membrane; Fibroblasts; Gene Expression; Gene Expression Profiling; Humans; Imidazoles; Mice; Mice, Inbred BALB C; Mice, Nude; Mice, SCID; Neoplasm Transplantation; Nuclear Proteins; Oncogenes; Piperazines; Primary Cell Culture; Proto-Oncogene Proteins; Recombinant Proteins; Retina; Retinoblastoma; Tumor Suppressor Protein p53

The role of miR-34a, a p53-regulated microRNA, in retinoblastoma (RB) was investigated.. The expression of miR-34 family members in RB cells was determined by semiquantitative RT-PCR and real-time qPCR. Regulation of miR-34a expression by p53-activating compounds was determined by qPCR analysis. The tumor suppressor functions of miR-34a in RB cell lines were determined by tetrazolium-based cell growth assay and by caspase-3/7 and activated caspase-3 apoptotic activity assays. Additive growth inhibitory properties of miR-34a in combination with topotecan were determined by cell growth assay. miR-34a targets in RB cells were identified by real-time qPCR expression analysis of previously reported and GenMiR++-predicted mRNAs.. Differential miR-34a and miR-34b expression was observed in RB cell lines and tumor samples. miR-34a expression could be increased in Y79 cells, but not Weri-Rb1 cells, after p53 activation. This differential regulation was not caused by genomic alterations at the miR-34a p53 binding site or mature gene. Exogenous miR-34a inhibited Y79 and Weri-Rb1 cell growth and increased apoptotic activity in Y79 cells. Increased inhibition of Y79 and Weri-Rb1 cell growth was observed with combination miR-34a and topotecan treatment. mRNA expression changes were observed in 7 of 7 previously reported and 13 of 18 GenMiR++-predicted miR-34a targets after transfection of Y79 cells with miR-34a compared with negative control microRNA.. miR-34a functions as a tumor suppressor in RB cells and is a potential therapeutic target. Differential expression, regulation, and activity of miR-34a in RB cells may suggest further p53 pathway inactivation in RB.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Caspases; Cell Survival; Doxorubicin; Gene Expression Regulation, Neoplastic; Humans; Imidazoles; MicroRNAs; Piperazines; Retinal Neoplasms; Retinoblastoma; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Topotecan; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle Proteins; Cell Transformation, Neoplastic; Cellular Senescence; Cyclin D; Cyclin-Dependent Kinases; Cyclins; DNA Damage; Eye Neoplasms; Genes, p16; Genes, p53; Genes, Retinoblastoma; Humans; Imidazoles; Mice; Models, Biological; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; Retinoblastoma; Retinoblastoma Protein; Topotecan; Tumor Suppressor Protein p14ARF; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

To determine the efficacy of inducing p53-mediated cell death in retinoblastoma cells by Nutlin 3A, a small molecule HDM2 inhibitor.. Retinoblastoma cell lines WERI-RB-1 and Y79 were treated with Nutlin 3A. Cell viability assays, Western blot analyses, confocal microscopy, and flow cytometry were performed to measure cell survival, p53 protein levels, activation of downstream targets, and apoptosis. To determine whether the effects of Nutlin 3A were p53-dependent, cell viability assays were performed on Y79 cells expressing short interfering RNA (siRNA) against p53.. Nutlin 3A induced cell death in Y79 and WERI-RB-1 in the 5- to 10-microM dose range. Treated cells demonstrated increased protein levels of p53 and the p53 targets p21 and HDM2. Phosphorylation of p53-serine-15, a marker for activation of p53 via genotoxic mechanisms, was absent. Y79 cells expressing siRNA against p53 demonstrated resistance to Nutlin 3A.. Nutlin 3A induced p53-mediated apoptosis in a dose-dependent, nongenotoxic fashion in 2 retinoblastoma cell lines.. Nutlin 3A is effective against retinoblastoma cells in a nongenotoxic manner. Because the mutagenic effects of radiation and chemotherapy may increase risks of secondary tumor formation, targeted p53 activation may be a safer alternative treatment for retinoblastoma in the future.

Topics: Annexin A5; Apoptosis; Blotting, Western; Cell Survival; Dose-Response Relationship, Drug; Flow Cytometry; Fluorescent Antibody Technique, Indirect; Humans; Imidazoles; Microscopy, Confocal; Phosphorylation; Piperazines; Proto-Oncogene Proteins c-mdm2; Retinal Neoplasms; Retinoblastoma; RNA, Small Interfering; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Up-Regulation

Topics: Animals; Cell Cycle Proteins; Cell Division; Humans; Imidazoles; Mice; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins; Retinoblastoma; Retinoblastoma Protein; Tumor Suppressor Protein p53

Most human tumours have genetic mutations in their Rb and p53 pathways, but retinoblastoma is thought to be an exception. Studies suggest that retinoblastomas, which initiate with mutations in the gene retinoblastoma 1 (RB1), bypass the p53 pathway because they arise from intrinsically death-resistant cells during retinal development. In contrast to this prevailing theory, here we show that the tumour surveillance pathway mediated by Arf, MDM2, MDMX and p53 is activated after loss of RB1 during retinogenesis. RB1-deficient retinoblasts undergo p53-mediated apoptosis and exit the cell cycle. Subsequently, amplification of the MDMX gene and increased expression of MDMX protein are strongly selected for during tumour progression as a mechanism to suppress the p53 response in RB1-deficient retinal cells. Our data provide evidence that the p53 pathway is inactivated in retinoblastoma and that this cancer does not originate from intrinsically death-resistant cells as previously thought. In addition, they support the idea that MDMX is a specific chemotherapeutic target for treating retinoblastoma.

Topics: Animals; Cell Cycle Proteins; Cell Death; Cell Division; DNA Damage; Gene Amplification; Humans; Imidazoles; Mice; Mice, Inbred C57BL; Nuclear Proteins; Piperazines; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Retina; Retinoblastoma; Retinoblastoma Protein; Tumor Suppressor Protein p14ARF; Tumor Suppressor Protein p53