S. S. Singhal

RLIP76 Inhibition: A Promising Developmental Therapy for Neuroblastoma

Refractory and relapsed neuroblastoma (NB) present with significant challenges in clinical management. Though primary NBs largely with wild-type p53 respond well to interventions, dysfunctional signaling in the p53 pathways in a MYCN oncogene driven background is found in a number of children with NB. The p53-mutant NB is largely unresponsive to available therapies and p53-independent targeted therapeutics represents a vital need in pediatric oncology. We analyzed the findings on mercapturic acid pathway (MAP) transporter RLIP76, which has broad and critical effects on multiple pathways as essential for carcinogenesis, oxidative stress and drug-resistance, is over-expressed in NB. RLIP76 inhibition by antibodies or depletion by antisense causes apoptosis and sensitization to chemo-radiotherapy in many cancers. In addition, recent studies indicate that the interactions between p53, MYCN, and WNT regulate apoptosis resistance and protein ubiquitination. RLIP76 and p53 interact with each other and colocalize in NB cells. Targeted depletion/inhibition of RLIP76 causes apoptosis and tumor regression in NB irrespective of p53 status. In the present review, we discuss the mechanisms and the role of RLIP76 in oxidative stress, drug-resistance and clathrin-dependent endocytosis (CDE), and analyze the molecular basis for the role of RLIP76 targeted approaches in the context principal drivers of NB pathogenesis, progression and drug-resistance. The evidence from RLIP76 studies in other cancers, when taken in the context of our recent RLIP76 focused mechanistic studies in NB, provides strong basis for further characterization and development of RLIP76 targeted therapies for NB.

Didymin: an orally active citrus flavonoid for targeting neuroblastoma

Neuroblastoma, a rapidly growing yet treatment responsive cancer, is the third most common cancer of children and the most common solid tumor in infants. Unfortunately, neuroblastoma that has lost p53 function often has a highly treatment-resistant phenotype leading to tragic outcomes. In the context of neuroblastoma, the functions of p53 and MYCN (which is amplified in ~25% of neuroblastomas) are integrally linked because they are mutually transcriptionally regulated, and because they together regulate the catalytic activity of RNA polymerases. Didymin is a citrus-derived natural compound that kills p53 wild-type as well as drug-resistant p53-mutant neuroblastoma cells in culture. In addition, orally administered didymin causes regression of neuroblastoma xenografts in mouse models, without toxicity to non-malignant cells, neural tissues, or neural stem cells. RKIP is a Raf-inhibitory protein that regulates MYCN activation, is transcriptionally upregulated by didymin, and appears to play a key role in the anti-neuroblastoma actions of didymin. In this review, we discuss how didymin overcomes drug-resistance in p53-mutant neuroblastoma through RKIP-mediated inhibition of MYCN and its effects on GRK2, PKCs, Let-7 micro-RNA, and clathrin-dependent endocytosis by Raf-dependent and -independent mechanisms. In addition, we will discuss studies supporting potential clinical impact and translation of didymin as a low cost, safe, and effective oral agent that could change the current treatment paradigm for refractory neuroblastoma.

The impact of a novel vicenin-2 and oral docetaxel combinatorial regimen in androgen-independent prostate cancer.

e13534 Background: A major challenge in the management of androgen-independent prostate cancer (AIPC) is the emergence of dose-limiting toxicities like febrile neutropenia while on i.v. docetaxel (DTL) treatment which has lead to investigation of alternate routes of DTL administration and drug combinations. Hence, we tested the potency of a novel flavonoid vicenin-2 (VCN-2) alone and in combination with oral DTL in AIPC. METHODS Cell survival was tested at various drug concentrations to determine in vivo doses by MTT and colony forming assays in PC-3 cells which have been established from the bone metastases of a 62 yr old male Caucasian with grade IV metastatic AIPC. Protein levels were analyzed by Western blot. For in vivo model studies, nu/nu nude mice (n=20) were implanted with 2 x 106 PC-3 cells subcutaneously into one flank and divided in to 4 groups. From tenth day after implantation, each group (n=5) were treated with corn oil (control), VCN-2 (3 mg/m2), DTL (0.03 mg/m2) and VCN-2 plus DTL in corn oil orally on alternate day. Tumor growth and body weight were monitored everyday. On day 60, tumors were excised for histopathological examination. RESULTS VCN-2 alone inhibited the survival of AIPC (p<0.01). The combination of VCN-2 and DTL induced synergistic effect in AIPC both in vitro and in vivo (~90% inhibition, CI <1 [Chou-Talalay test], p<0.0001). Tumor lysate analysis revealed that VCN-2 and DTL inhibited critical signaling proteins like pIGF1R, pRb, pAkt, PCNA and cyclin D1 to a greater extent in combination than either drugs alone. Histology of tumor sections revealed decreased levels of PSMA, ki67, CD31 and increase in the pro-differentiation marker E-cadherin. No overt toxicity was observed due to co-administration of VCN-2 and DTL. CONCLUSIONS Oral administration of VCN-2 and DTL is effective and tolerable in vivo model of advanced AIPC. The salient feature of this study was the potent synergistic effect of VCN-2 and a low dose of DTL (0.03 mg/m2 of DTL and 3 mg/m2 of VCN-2 orally on alternate days compared to clinical dose in AIPC: 75 mg/m2 of DTL i.v. once in 3 wks. plus 5 mg prednisone twice daily) which strongly supports further development of VCN-2 and DTL combinatorial regimens for clinical use in AIPC.