Approaches for Enhancing Therapeutic Efficacy of a Novel IL-10 Gene Family Member: MDA-7/IL-24

Melanoma differentiation associated gene-7 (mda-7) was discovered in the Fisher laboratory by subtraction hybridization of temporally spaced subtracted cDNA libraries prepared from terminally differentiated human melanoma cells treated with human fibroblast interferon (IFN-β) and the protein kinase C activator mezerein (MEZ), an approach called ‘differentiation induction subtraction hybridization’ (DISH). mda-7 is located in human chromosome 1q32–33 and based on sequence homology, chromosomal localization, and its functional properties, the mda-7 gene is now classified as a member of the IL-10 family of cytokines and named IL-24. The mda-7/IL-24 cDNA encodes a protein of 206-amino acids with a predicted size of ~24-kDa, which contains an interleukin (IL)-10 signature motif at amino acids 101–121 (SDAESCYLVHTLLEFYLKTVF) shared by other members of the IL-10 family of cytokines. Sequence analysis revealed the presence of a 49-amino acid signal peptide suggesting that the molecule could be cleaved and secreted. Expression of MDA-7/IL-24 protein was detected in cells of the immune system (mainly by expression in tissues associated with the immune system, such as spleen, thymus and PBMC) and normal human melanocytes. Of interest, a progressive loss of MDA-7/IL-24 expression during melanoma progression suggests an inverse relationship between MDA-7/IL-24 expression and the evolution of melanocytes to various stages of melanoma. mda-7/IL-24 induces growth suppression in human melanoma and other cancer cells, without affecting normal cells. Subsequent studies provided consistent evidence that ectopic expression of mda-7/IL-24 employing a replication incompetent adenovirus (Ad.mda-7) resulted in apoptosis induction and cell death in a wide variety of solid tumors including melanoma, malignant glioma, carcinomas of the breast, kidney, cervix, colorectum , liver, lung, ovary and prostate sparing normal cellular counterparts, i.e., such as normal melanocytes, astrocytes, fibroblasts, and mesothelial and epithelial cells. The in vitro antitumor activity of mda-7/IL-24 readily translated into the in vivo situation in animal models containing human breast, prostate, lung and colorectal carcinomas and in malignant glioma xenografts. Moreover, the ability of mda-7/IL-24 to induce a potent “bystander cancer-specific killing effect” provides an unprecedented opportunity to use this molecule to target for destruction not only primary tumors, but also metastases. Based on its profound cancer-selective tropism, substantiated by in vivo human xenograft studies in nude mice, mda-7/IL-24 (administered as Ad.mda-7) was evaluated in a Phase I clinical trial in patients with melanomas and solid cancers. These studies document that mda-7/IL-24 is well tolerated and demonstrates evidence of significant (44%) clinical activity. This review focuses on the recent enhancements in our understanding of the mode of action of mda-7/IL-24 and its potential applications as a unique and promising effective cytokine-based gene therapy for human cancers. The first chapter explored the efficacy of a tropism-modified Ad-based cancer gene therapy approach for eradicating low CAR colorectal cancer cells. We show that in low CAR human colorectal cancer cells (RKO), a recombinant Ad.5/3 virus delivering mda-7/IL-24 (Ad.5/3-mda-7) is more efficient than Ad.5 delivering mda-7 (Ad.5-mda-7) in expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis and inhibiting in vivo tumor growth in a nude mouse xenograft model. Additionally, our in vitro and in vivo data confirms that BI-97C1 (Sabutoclax) profoundly sensitizes mda-7/IL-24 mediated toxicity in colorectal cancer. Thus, Ad.5/3-mda-7, alone and/or in combination with BI-97C1 (Sabutoclax), might represent an improved and more effective therapeutic approach for colorectal and other cancers. In view of the essential roles of anti-apoptotic Bcl-2 family proteins in tumorigenesis and chemoresistance, efforts are focused on developing small molecule inhibitors of Bcl-2 family proteins as potential therapeutics for cancer. Unfortunately, due to the unique structure of Mcl-1 as compared with Bcl-2 and Bcl-xL, currently employed inhibitors, such as ABT-737 or its clinical counterpart, ABT-263, display limited affinity for Mcl-1. Using nuclear magnetic resonance (NMR) binding assays and computational docking studies, we have recently identified a series of new Apogossypol derivatives, compound 3 (BI-79D10) and compound 11 (BI-97C1), with pan-Bcl-2- inhibitory potency. BI-79D10 binds to Bcl- xL, Bcl-2, and Mcl-1 with IC50 values of 190, 360, and 520 nmol/L, respectively. BI-97C1 (Sabutoclax) is an optically pure individual Apogossypol derivative that retains all the properties of BI-79D10 along with superior in vitro and in vivo efficacy. Because Mcl-1 is over-expressed in the majority of PCs, we hypothesized that suppressing Mcl-1 by treating human PC cells with BI-97C1 (Sabutoclax) would sensitize them to mda-7/IL-24-mediated cytotoxicity. The second chapter study highlights the noteworthy potential of a combinatorial approach involving mda-7/IL-24, a broad-acting anticancer gene, and BI-97C1 (Sabutoclax), which targets Mcl-1, to sensitize PC to mda-7/IL-24-mediated cytotoxicity, thereby enhancing therapeutic efficacy. Our data suggests that treatment with the combination regimen of mda-7/IL-24 and BI-97C1 (Sabutoclax) induces autophagy that facilitates apoptosis in association with up regulation of NOXA, accumulation of Bim, and activation of Bax and Bak. Treatment with mda-7/IL-24 and BI-97C1 (Sabutoclax) inhibited the growth of PC xenografts and suppressed PC development in an immunocompetent transgenic mouse model of PC. The third chapter study explored the efficacy of a tropism-modified CRCA cancer gene therapy approach for eradicating low CAR prostate cancer cells. We showed that in low CAR PC3 cells Ad.5/3-CTV is more efficient than Ad.5-CTV in delivering transgene (mda-7/IL-24), infecting tumor cells, expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis, inhibiting in vivo tumor growth and exerting an antitumor ‘bystander’ effect in a nude mouse human prostate cancer xenograft and suppressed PC development in an immunocompetent transgenic mouse model of PC model

Autophagy switches to apoptosis in prostate cancer cells infected with melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24)

MDA-7/IL-24 has noteworthy potential as an anticancer therapeutic because of its diversity of antitumor properties, its lack of toxicity toward normal cells and tissues, and its safety and efficacy as evidenced in a phase I clinical trial. In a recent study, we document that Ad.mda-7-induced ER stress and ceramide production leads to early autophagy that subsequently switches to apoptosis in human prostate cancer cells. During the apoptotic phase, the MDA-7/IL-24 protein physically interacts with Beclin 1 and this interaction might inhibit Beclin 1 function culminating in apoptosis. Conversely, Ad.mda-7 infection leads to calpain-mediated cleavage of the Atg5 protein that might also facilitate a biochemical switch from autophagy to apoptosis. Our recent paper reveals novel aspects of the interplay between autophagy and apoptosis that underlie the cytotoxic action of MDA-7/IL-24 in prostate cancer cells. These new insights into MDA-7/IL-24 action provide intriguing leads for developing innovative combinatorial approaches for prostate cancer therapy

Effects of Dual Peroxisome Proliferator-Activated Receptors α and γ Activation in Two Rat Models of Neuropathic Pain

Neuropathic pain is a growing healthcare problem causing a global burden. Currently used analgesics such as opioids are associated with adverse effects; urging the need for safer alternatives. Here we aimed to investigate the potential analgesic effects of tesaglitazar; dual peroxisome proliferator-activated receptors α and γ (PPARα and γ) agonist in rat models of neuropathic pain. This study also aimed to investigate the modulation of the transient receptor potential vanilloid 1 (TRPV1) receptor activity by tesaglitazar which could provide a potential mechanism that underlie tesaglitazar antinociceptive effects. Von Frey filaments were used to determine the paw withdrawal threshold (PWT) in adult male Sprague Dawley rats (180-250g) following i.p. injection of streptozotocin (STZ) or cisplatin, which were used as models of neuropathic pain. Antinociceptive effects of tesaglitazar were determined 6 hours after drug administration. Cobalt influx assays in cultured dorsal root ganglia (DRG) neurons were used to study the effects of tesaglitazar preincubation on capsaicin-evoked cobalt influx. Both cisplatin and STZ produced a significant decrease in PWT. The higher dose of tesaglitazar (20μg/kg) significantly restored PWT in both neuropathic pain models (P<0.05). 10μM capsaicin produced a robust cobalt response in DRG neurons. Preincubation of DRG neurones with tesaglitazar 6 hours prior to stimulation with capsaicin significantly reduce capsaicin-evoked cobalt responses in a PPARα and PPARγ dependent fashion (P<0.05). In conclusion, tesaglitazar produced significant analgesic effects in STZ and cisplatin-induced neuropathy, possibly by modulating TRPV1 receptor activity. This may be of potential benefit in clinical practice dealing with peripheral neuropathy

Novel frameshift variant in the IDUA gene underlies Mucopolysaccharidoses type I in a consanguineous Yemeni pedigree

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive storage disorder that result as a consequence of a deficiency in the lysosomal hydrolase, a-L-iduronidase enzyme encoded by IDUA gene. Over a hundred causative variants in IDUA have been identified, which result in a progressive multi-systemic disease with a broad range of severity and disease progression reported across affected individuals. The aim of this study was the detection and interpretation of IDUA mutation in a family with two children affected with lethal MPS I. The IDUA gene was sequenced in the parents of two deceased children who had a clinical diagnosis of MPS I, to assess their carrier status and to help inform on risk in future children. The sequencing analysis was performed by PCR and bidirectional Sanger sequencing of the coding region and exon-intron splice junctions at Labor MVZ Westmecklenburg molecular diagnostics laboratory. A heterozygous c.657delA variant in exon 6 was identified in each parent, which is the most likely explanation for disease in their children. This report represents the first Yemeni family to have a molecular diagnosis for MPS I

Genotoxicity of cisplatin and carboplatin in cultured human lymphocytes: a comparative study

Cisplatin and carboplatin are integral parts of many antineoplastic management regimens. Both platinum analogues are potent DNA alkylating agents that robustly induce genomic instability and promote apoptosis in tumor cells. Although the mechanism of action of both drugs is similar, cisplatin appears to be more cytotoxic. In this study, the genotoxic potential of cisplatin and carboplatin was compared using chromosomal aberrations (CAs) and sister-chromatid exchange (SCE) assays in cultured human lymphocytes. Results showed that cisplatin and carboplatin induced a significant increase in CAs and SCEs compared to the control group (p0.05). In conclusion, cisplatin was found to be more genotoxic than carboplatin in the SCE assay in cultured human lymphocytes, and that might explain the higher cytotoxicity of cisplatin

The desensitization of the transient receptor potential vanilloid 1 by nonpungent agonists and its resensitization by bradykinin

Transient receptor potential vanilloid type-1 (TRPV1) channels have crucial roles in inflammatory hyperalgesia. Different inflammatory mediators can modulate TRPV1 sensitization. Bradykinin is an algogenic substance released at the site of inflammation. The aim of the present study is to investigate the desensitization of TRPV1 receptor by nonpungent agonists and to determine how bradykinin and prostaglandin E2 receptors (EP3 and EP4) modulate the resensitization of TRPV1 receptor after being desensitized by nonpungent agonists. Tail flick test was used to investigate capsaicin-induced thermal hyperalgesia and the desensitization of TRPV1 by the nonpungent agonists (olvanil and arvanil) in male BALB/c mice weighed (22-25 g). Resensitization of TRPV1 by bradykinin and the role of prostaglandin receptors in mediating sensitization of TRPV1 were also investigated. Intraplantar injection of capsaicin (0.3 µg) produced a robust thermal hyperalgesia in mice, while olvanil (0.3 µg) or arvanil (0.3 µg) produced no hyperalgesia, emphasizing their lack of pungency. Olvanil and arvanil significantly attenuated capsaicin-induced thermal hyperalgesia in mice. Bradykinin significantly reversed the desensitizing effects of arvanil, but not olvanil. EP4 but not EP3 receptors mediate the sensitization of TRPV1 By bradykinin in vivo. The present study provides evidence for a novel signaling pathway through which bradykinin can regulate the TRPV1 ion channel function via EP4 receptor.Scopu

Identification of APTX disease-causing mutation in two unrelated Jordanian families with cerebellar ataxia and sensitivity to DNA damaging agents.

BackgroundAtaxia with oculomotor apraxia type 1 (AOA1) is a rare autosomal recessive cerebellar ataxia, caused by mutations in the APTX gene. The disease is characterized by early-onset cerebellar ataxia, oculomotor apraxia and severe axonal polyneuropathy. The aim of this study was to detect the disease-causing variants in two unrelated consanguineous Jordanian families with cerebellar ataxia using whole exome sequencing (WES), and to correlate the identified mutation(s) with the clinical and cellular phenotypes.MethodsWES was performed in three affected individuals and segregation analysis of p.W279* APTX candidate variant was performed. Expression levels of APTX were measured in patients' skin fibroblasts and peripheral blood mononuclear cells, followed by western blot analysis in skin fibroblasts. Genotoxicity assay was performed to detect the sensitivity of APTX mutated cells to H2O2, MMC, MMS and etoposide.ResultsA recurrent homozygous nonsense variant in APTX gene (c.837G>A, p.W279*) was revealed in all affected individuals. qRT-PCR showed normal APTX levels in peripheral blood and lower levels in fibroblast cells. However, western blot showed the absence of APTX protein in patients' skin fibroblasts. Significant hypersensitivity to H2O2, MMC and etoposide and lack of sensitivity to MMS were noted.ConclusionsThis is the first study to report the identification of a nonsense variant in the APTX gene (c.837G>A; p.W279*) in AOA1 patients within the Jordanian population. This study confirmed the need of WES to assist in the diagnosis of cerebellar ataxia and it emphasizes the importance of studying the pathophysiology of the APTX gene