Characterization of an Mtbp Hypomorphic Allele in a Diethylnitrosamine-Induced Liver Carcinogenesis Model.

MTBP is implicated in cell cycle progression, DNA replication, and cancer metastasis. However, the function of MTBP remains enigmatic and is dependent on cellular contexts and its cellular localization. To understand the in vivo physiological role of MTBP, it is important to generate Mtbp knockout mice. However, complete deletion of the Mtbp gene in mice results in early embryonic lethality, while its heterozygous deletion shows modest biological phenotypes, including enhanced cancer metastasis. To overcome this and better characterize the in vivo physiological function of MTBP, we, for the first time, generated mice that carry an Mtbp hypomorphic allele (MtbpH) in which Mtbp protein is expressed at approximately 30% of that in the wild-type allele. We treated wild-type, Mtbp+/-, and MtbpH/- mice with a liver carcinogen, diethylnitrosamine (DEN), and found that the MtbpH/- mice showed worse overall survival when compared to the wild-type mice. Consistent with previous reports using human liver cancer cells, mouse embryonic fibroblasts (MEFs) from the MtbpH/- mice showed an increase in the nuclear localization of p-Erk1/2 and migratory potential. Thus, MtbpH/- mice and cells from MtbpH/- mice are valuable to understand the in vivo physiological role of Mtbp and validate the diverse functions of MTBP that have been observed in human cells.

Heat Transfer Characteristics of Pool Boiling with Scalable Plasma-Sprayed Aluminum Coatings.

To ensure adequate reliability in two-phase cooling systems involving boiling, it is essential to enhance the heat transfer coefficient and maximize the critical heat flux (CHF) limit. A key technique to avoid surface burnout and increase the CHF limit in pool boiling is the frequent coolant supply to the probable dry-out locations. In the present work, we have explored the plasma-spray coating as a surface modification technique for enhancing heat transfer coefficient and CHF value in pool boiling applications. Three plasma-coated aluminum surfaces (C-15, C-20, and C-25) are fabricated on a copper substrate at three different plasma powers of 15, 20, and 25 kW, respectively. Detailed surface morphologies of the plasma-sprayed coatings are presented, and their roles in pool boiling heat transfer mechanisms are analyzed. Plasma-coated surfaces exhibit wickability characteristics and enhanced wettability compared to the plain copper surface. Saturated pool boiling experiments are performed with DI (deionized) water at atmospheric pressure. Plasma spray-coated surfaces show favorable boiling incipience with less wall superheat and more active nucleation sites than the plain copper surface. Compared to the plain copper surface, enhancement values of nearly 68, 60.7, and 55.5% in the heat transfer coefficient are observed for C-15, C-20, and C-25 plasma-coated surfaces, respectively. Experiments could not be performed beyond the heat flux of 197 W/cm2 due to repeated failure of the cartridge heaters. Based on the experimental measurement of wickabilities, the CHF values of plasma-coated surfaces have been theoretically calculated. Compared to the plain copper surface, a maximum 2.39 times higher CHF value is observed for C-15 plasma-coated surface. Improved wettability and wickability are responsible for CHF enhancement in the case of plasma-coated surfaces. At higher heat flux, capillary wicking and frequent rewetting of the dryout locations delay the burnout phenomenon, enhancing CHF in plasma-coated surfaces. The plasma-spray coating is a robust and scalable process, which can be a potential candidate for high heat flux dissipation in various industrial applications.

DNAJA1- and conformational mutant p53-dependent inhibition of cancer cell migration by a novel compound identified through a virtual screen.

Cancers are frequently addicted to oncogenic missense mutant p53 (mutp53). DNAJA1, a member of heat shock protein 40 (HSP40), also known as J-domain proteins (JDPs), plays a crucial role in the stabilization and oncogenic activity of misfolded or conformational mutp53 by binding to and preventing mutp53 from proteasomal degradation. However, strategies to deplete mutp53 are not well-established, and no HSP40/JDPs inhibitors are clinically available. To identify compounds that bind to DNAJA1 and induce mutp53 degradation, we performed an in silico docking study of ~10 million of compounds from the ZINC database for the J-domain of DNAJA1. A compound 7-3 was identified, and its analogue A11 effectively reduced the levels of DNAJA1 and conformational mutp53 with minimal effects on the levels of wild-type p53 and DNA-contact mutp53. A11 suppressed migration and filopodia formation in a manner dependent on DNAJA1 and conformational mutp53. A mutant DNAJA1 with alanine mutations at predicted amino acids (tyrosine 7, lysine 44, and glutamine 47) failed to bind to A11. Cells expressing the mutant DNAJA1 became insensitive to A11-mediated depletion of DNAJA1 and mutp53 as well as A11-mediated inhibition of cell migration. Thus, A11 is the first HSP40/JDP inhibitor that has not been previously characterized for depleting DNAJA1 and subsequently conformational mutp53, leading to inhibition of cancer cell migration. A11 can be exploited for a novel treatment against cancers expressing conformational mutp53.

Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin.

Accumulation of missense mutant p53 (mutp53) in cancers promotes malignant progression. DNAJA1, a member of HSP40 (also known as J-domain proteins: JDPs), is shown to prevent misfolded or conformational mutp53 from proteasomal degradation. Given frequent addiction of cancers to oncogenic mutp53, depleting mutp53 by DNAJA1 inhibition is a promising approach for cancer therapy. However, there is no clinically available inhibitor for DNAJA1. Our in silico molecular docking study with a natural compound-derived small molecule library identified a plumbagin derivative, PLIHZ (plumbagin-isoniazid analog), as a potential compound binding to the J domain of DNAJA1. PLIHZ efficiently reduced the levels of DNAJA1 and several conformational mutp53 with minimal impact on DNA contact mutp53 and wild-type p53 (wtp53). An analog, called PLTFBH, which showed a similar activity to PLIHZ in reducing DNAJA1 and mutp53 levels, inhibited migration of cancer cells specifically carrying conformational mutp53, but not DNA contact mutp53, p53 null, and wtp53, which was attenuated by depletion of DNAJA1 or mutp53. Moreover, PLTFBH reduced levels of multiple other HSP40/JDPs with tyrosine 7 (Y7) and/or tyrosine 8 (Y8) but failed to deplete DNAJA1 mutants with alanine substitution of these amino acids. Our study suggests PLTFBH as a potential inhibitor for multiple HSP40/JDPs.

DNAJA1 promotes cancer metastasis through interaction with mutant p53.

Accumulation of mutant p53 (mutp53) is crucial for its oncogenic gain of function activity. DNAJA1, a member of J-domain containing proteins or heat shock protein 40, is shown to prevent unfolded mutp53 from proteasomal degradation. However, the biological function of DNAJA1 remains largely unknown. Here we show that DNAJA1 promotes tumor metastasis by accumulating unfolded mutp53. Levels of DNAJA1 in head and neck squamous cell carcinoma (HNSCC) tissues were higher than those in normal tissues. Knockdown of DNAJA1 in HNSCC cell lines carrying unfolded mutp53 significantly decreased the levels of mutp53, filopodia/lamellipodia formation, migratory potential, and active forms of CDC42/RAC1, which were not observed in HNSCC cells with DNA contact mutp53, wild-type p53, or p53 null. Such mutp53-dependent functions of DNAJA1 were supported by the observation that DNAJA1 selectively bound to unfolded mutp53. Moreover, DNAJA1 knockdown in HNSCC cells carrying unfolded mutp53 inhibited primary tumor growth and metastases to the lymph nodes and lungs. Our study suggests that DNAJA1 promotes HNSCC metastasis mainly in a manner dependent on mutp53 status, suggesting DNAJA1 as a potential therapeutic target for HNSCC harboring unfolded mutp53.

MTBP inhibits the Erk1/2-Elk-1 signaling in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and the prognosis of HCC patients, especially those with metastasis, remains extremely poor. This is partly due to unclear molecular mechanisms underlying HCC metastasis. Our previous study indicates that MDM2 Binding Protein (MTBP) suppresses migration and metastasis of HCC cells. However, signaling pathways regulated by MTBP remain unknown. To identify metastasis-associated signaling pathways governed by MTBP, we have performed unbiased luciferase reporter-based signal array analyses and found that MTBP suppresses the activity of the ETS-domain transcription factor Elk-1, a downstream target of Erk1/2 MAP kinases. MTBP also inhibits phosphorylation of Elk-1 and decreases mRNA expression of Elk-1 target genes. Reduced Elk-1 activity is caused by inhibited nuclear translocation of phosphorylated Erk1/2 (p-Erk) by MTBP and subsequent inhibition of Elk-1 phosphorylation. We also reveal that MTBP inhibits the interaction of p-Erk with importin-7/RanBP7 (IPO7), an importin family member which shuttles p-Erk into the nucleus, by binding to IPO7. Moreover, high levels of MTBP in human HCC tissues are correlated with cytoplasmic localization of p-Erk1/2. Our study suggests that MTBP suppresses metastasis, at least partially, by down-modulating the Erk1/2-Elk-1 signaling pathway, thus identifying a novel regulatory mechanism of HCC metastasis by regulating the subcellular localization of p-Erk.

Emerging Non-Canonical Functions and Regulation of p53.

The tumor suppressor p53 induces cell cycle arrest and/or apoptosis by transactivating numerous downstream target genes and also translocating to the mitochondrial outer membrane.

Akt1/NFκB signaling pathway activation by a small molecule DMA confers radioprotection to intestinal epithelium in xenograft model.

Normal tissue protection and recovery of radiation-induced damage are of paramount importance for development of radioprotector. Radioprotector which selectively protects normal tissues over cancerous tissues improves the therapeutic window of radiation therapy. In the present study, small bisbenzimidazole molecule, DMA (5-(4-methylpiperazin-1-yl)-2-[2'-(3,4-dimethoxy-phenyl)-5'-benzimidazolyl]-benzimidazole) was evaluated for in vivo radioprotective effects to selectively protect normal tissue over tumor with underlying molecular mechanism. Administration of single DMA dose prior to radiation has enhanced survival of Balb/c mice against sublethal and supralethal total body irradiation. DMA ameliorated radiation-induced damage of normal tissues such as hematopoietic (HP) and gastrointestinal tract (GI) system. Oxidative stress marker Malondialdehyde level was decreased by DMA whereas it maintained endogenous antioxidant status by increasing the level of reduced glutathione, glutathione reductase, glutathione-s-transferase, superoxide dismutase and total thiol content in hepatic tissue of irradiated mice. Mechanistic studies revealed that DMA treatment prior to radiation leads to Akt1/NFκB signaling which reduced radiation-induced genomic instability in normal cells. However, these pathways were not activated in tumor tissues when subjected to DMA treatment in similar conditions. Abrogation of Akt1 and NFκB genes resulted in no radioprotection by DMA and enhanced apoptosis against radiation. Plasma half-life of DMA was 3.5h and 2.65h at oral and intravenous dose respectively and 90% clearance was observed in 16h. In conclusion, these data suggests that DMA has potential to be developed as a safe radioprotective agent for radiation countermeasures and an adjuvant in cancer therapy.

Non-Canonical Cell Death Induced by p53.

Programmed cell death is a vital biological process for multicellular organisms to maintain cellular homeostasis, which is regulated in a complex manner. Over the past several years, apart from apoptosis, which is the principal mechanism of caspase-dependent cell death, research on non-apoptotic forms of programmed cell death has gained momentum. p53 is a well characterized tumor suppressor that controls cell proliferation and apoptosis and has also been linked to non-apoptotic, non-canonical cell death mechanisms. p53 impacts these non-canonical forms of cell death through transcriptional regulation of its downstream targets, as well as direct interactions with key players involved in these mechanisms, in a cell type- or tissue context-dependent manner. In this review article, we summarize and discuss the involvement of p53 in several non-canonical modes of cell death, including caspase-independent apoptosis (CIA), ferroptosis, necroptosis, autophagic cell death, mitotic catastrophe, paraptosis, and pyroptosis, as well as its role in efferocytosis which is the process of clearing dead or dying cells.

Genome-wide RNAi screening identifies TMIGD3 isoform1 as a suppressor of NF-κB and osteosarcoma progression.

The ability of cancer cells to survive and grow in anchorage- and serum-independent conditions is well correlated with their aggressiveness. Here, using a human whole-genome shRNA library, we identify TMIGD3 isoform1 (i1) as a factor that suppresses this ability in osteosarcoma (OS) cells, mainly by inhibiting NF-κB activity. Knockdown of TMIGD3 increases proliferation, tumour formation and metastasis of OS cells. Overexpression of TMIGD3 isoform1 (i1), but not isoform3 (i3) which shares a common C-terminal region, suppresses these malignant properties. Adenosine A3 receptor (A3AR) having an identical N-terminal region shows similar biological profiles to TMIGD3 i1. Protein expression of TMIGD3 and A3AR is lower in human OS tissues than normal tissues. Mechanistically, TMIGD3 i1 and A3AR commonly inhibit the PKA-Akt-NF-κB axis. However, TMIGD3 i1 only partially rescues phenotypes induced by A3AR knockdown, suggesting the presence of distinct pathways. Our findings reveal an unappreciated role for TMIGD3 i1 as a suppressor of NF-κB activity and OS progression.

DNAJA1 controls the fate of misfolded mutant p53 through the mevalonate pathway.

Stabilization of mutant p53 (mutp53) in tumours greatly contributes to malignant progression. However, little is known about the underlying mechanisms and therapeutic approaches to destabilize mutp53. Here, through high-throughput screening we identify statins, cholesterol-lowering drugs, as degradation inducers for conformational or misfolded p53 mutants with minimal effects on wild-type p53 (wtp53) and DNA contact mutants. Statins preferentially suppress mutp53-expressing cancer cell growth. Specific reduction of mevalonate-5-phosphate by statins or mevalonate kinase knockdown induces CHIP ubiquitin ligase-mediated nuclear export, ubiquitylation, and degradation of mutp53 by impairing interaction of mutp53 with DNAJA1, a Hsp40 family member. Knockdown of DNAJA1 also induces CHIP-mediated mutp53 degradation, while its overexpression antagonizes statin-induced mutp53 degradation. Our study reveals that DNAJA1 controls the fate of misfolded mutp53, provides insights into potential strategies to deplete mutp53 through the mevalonate pathway-DNAJA1 axis, and highlights the significance of p53 status in impacting statins' efficacy on cancer therapy.

Radioprotective Agents: Strategies and Translational Advances.

Radioprotectors are agents required to protect biological system exposed to radiation, either naturally or through radiation leakage, and they protect normal cells from radiation injury in cancer patients undergoing radiotherapy. It is imperative to study radioprotectors and their mechanism of action comprehensively, looking at their potential therapeutic applications. This review intimately chronicles the rich intellectual, pharmacological story of natural and synthetic radioprotectors. A continuous effort is going on by researchers to develop clinically promising radioprotective agents. In this article, for the first time we have discussed the impact of radioprotectors on different signaling pathways in cells, which will create a basis for scientific community working in this area to develop novel molecules with better therapeutic efficacy. The bright future of exceptionally noncytotoxic derivatives of bisbenzimidazoles is also described as radiomodulators. Amifostine, an effective radioprotectant, has been approved by the FDA for limited clinical use. However, due to its adverse side effects, it is not routinely used clinically. Recently, CBLB502 and several analog of a peptide are under clinical trial and showed high success against radiotherapy in cancer. This article reviews the different types of radioprotective agents with emphasis on the strategies for the development of novel radioprotectors for drug development. In addition, direction for future strategies relevant to the development of radioprotectors is also addressed.

Murine double minute 2, a potential p53-independent regulator of liver cancer metastasis.

Hepatocellular carcinoma (HCC) has emerged as one of the most commonly diagnosed forms of human cancer; yet, the mechanisms underlying HCC progression remain unclear. Unlike other cancers, systematic chemotherapy is not effective for HCC patients, while surgical resection and liver transplantation are the most viable treatment options. Thus, identifying factors or pathways that suppress HCC progression would be crucial for advancing treatment strategies for HCC. The murine double minute 2 (MDM2)-p53 pathway is impaired in most of the cancer types, including HCC, and MDM2 is overexpressed in approximately 30% of HCC. Overexpression of MDM2 is reported to be well correlated with metastasis, drug resistance, and poor prognosis of multiple cancer types, including HCC. Importantly, these correlations are observed even when p53 is mutated. Indeed, p53-independent functions of overexpressed MDM2 in cancer progression have been suitably demonstrated. In this review article, we summarize potential effectors of MDM2 that promote or suppress cancer metastasis and discuss the p53-independent roles of MDM2 in liver cancer metastasis from clinical as well as biological perspectives.

MTBP inhibits migration and metastasis of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with increasing incidence. Despite curative surgical resection and advanced chemotherapy, its survival rate remains low. The presence of microvascular invasion and occult metastasis is one of the major causes for this poor outcome. MDM2 Binding Protein (MTBP) has been implicated in the suppression of cell migration and cancer metastasis. However, clinical significance of MTBP, particularly in human cancer, is poorly understood. Specifically, clinical relevance of MTBP in human HCC has never been investigated. Here we demonstrated that expression of MTBP was significantly reduced in human HCC tissues compared to adjacent non-tumor tissues. MTBP expression was negatively correlated with capsular/vascular invasion and lymph node metastasis. Overexpression of MTBP resulted in the suppression of the migratory and metastatic potential of HCC cells, while its downregulation increased the migration. Consistent with the previous report, MTBP endogenously bound to alpha-actinin 4 (ACTN4) and suppressed ACTN4-mediated cell migration in multiple HCC cell lines. However, MTBP also inhibited migratory potential of PLC/PRF/5 HCC cells whose migration was not altered by manipulation of ACTN4 expression. These results suggest that mechanisms behind MTBP-mediated migration suppression may not be limited to the pathway involving ACTN4 in certain cellular contexts. Additionally, as a potential mechanism for reduced MTBP expression in tumors, we found that MTBP expression was increased following the treatment with histone deacetylase inhibitors (HDIs). Our study, for the first time, provides clinical relevance of MTBP in the suppression of HCC metastasis.

3,4-Dimethoxyphenyl bis-benzimidazole derivative, mitigates radiation-induced DNA damage.

Radiation-induced DNA damage initiates a series of overlapping responses that include DNA damage recognition and repair, induction of cell cycle checkpoints, senescence and/or apoptosis. This study assessed the DNA damage response and whole genome expression profile in two mammalian cell lines (HEK and U87) in response to (5-{4-methylpiperazin-1-yl}-2-[2'-(3,4-dimethoxyphenyl)-5'-benzimidazolyl] benzimidazole) DMA and ionizing radiation. DMA has been shown to act as a potent radiation protector, yielding significant levels of protection, i.e., 20.9% in HEK cells and 21.2% in U87 cells. Our findings revealed treatment with DMA significantly reduced γ-H2AX, 53BP1 and Rad51 foci formation after irradiation. MAP kinase, WNT signaling and p53 pathways were found to be activated in DMA-treated cells. In addition, the DNA damage response genes, HSP70, HSPD1, PRDX1, PRX, CALR, NPM, UBC, and SET showed differential regulation in DMA, DMA + radiation and radiation-treated cells. The data suggest that DMA-influenced repertoire of repair proteins, which are an indispensable part of the cell, interplay with each other to reduce DNA damage and maintain the genomic integrity of the cell.

Inhibition of 5'-UTR RNA conformational switching in HIV-1 using antisense PNAs.

BACKGROUND: The genome of retroviruses, including HIV-1, is packaged as two homologous (+) strand RNA molecules, noncovalently associated close to their 5'-end in a region called dimer linkage structure (DLS). Retroviral HIV-1 genomic RNAs dimerize through complex interactions between dimerization initiation sites (DIS) within the (5'-UTR). Dimer formation is prevented by so calledLong Distance Interaction (LDI) conformation, whereas Branched Multiple Hairpin (BMH) conformation leads to spontaneous dimerization. METHODS AND RESULTS: We evaluated the role of SL1 (DIS), PolyA Hairpin signal and a long distance U5-AUG interaction by in-vitro dimerization, conformer assay and coupled dimerization and template-switching assays using antisense PNAs. Our data suggests evidence that PNAs targeted against SL1 produced severe inhibitory effect on dimerization and template-switching processes while PNAs targeted against U5 region do not show significant effect on dimerization and template switching, while PNAs targeted against AUG region showed strong inhibition of dimerization and template switching processes. CONCLUSIONS: Our results demonstrate that PNA can be used successfully as an antisense to inhibit dimerization and template switching process in HIV -1 and both of the processes are closely linked to each other. Different PNA oligomers have ability of switching between two thermodynamically stable forms. PNA targeted against DIS and SL1 switch, LDI conformer to more dimerization friendly BMH form. PNAs targeted against PolyA haipin configuration did not show a significant change in dimerization and template switching process. The PNA oligomer directed against the AUG strand of U5-AUG duplex structure also showed a significant reduction in RNA dimerization as well as template- switching efficiency.The antisense PNA oligomers can be used to regulate the shift in the LDI/BMH equilibrium.

Inhibition of HIV-1 Integrase gene expression by 10-23 DNAzyme.

HIV Integrase (IN) is an enzyme that is responsible for the integration of the proviral genome into the human genome, and this integration step is the first step of the virus hijacking the human cell machinery for its propagation and replication. 10-23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. We have designed three novel DNAzymes, DIN54, DIN116, and DIN152, against HIV-1 Integrase gene using Mfold software and evaluated them for target site cleavage activity on the in vitro transcribed mRNA. All DNAzymes were tested for its inhibition of expression of HIV Integrase protein in the transiently transfected cell lines. DIN116 and DIN152 inhibited IN-EGFP expression by 80 percent and 70 percent respectively.

DMA, a bisbenzimidazole, offers radioprotection by promoting NFκB transactivation through NIK/IKK in human glioma cells.

BACKGROUND: Ionizing radiation (IR) exposure often occurs for human beings through occupational, medical, environmental, accidental and/or other sources. Thus, the role of radioprotector is essential to overcome the complex series of overlapping responses to radiation induced DNA damage. METHODS AND RESULTS: Treatment of human glioma U87 cells with DMA (5- {4-methylpiperazin-1-yl}-2-[2'-(3, 4-dimethoxyphenyl)-5'-benzimidazolyl] in the presence or absence of radiation uncovered differential regulation of an array of genes and proteins using microarray and 2D PAGE techniques. Pathway construction followed by relative quantitation of gene expression of the identified proteins and their interacting partners led to the identification of MAP3K14 (NFκB inducing kinase, NIK) as the candidate gene affected in response to DMA. Subsequently, over expression and knock down of NIK suggested that DMA affects NFκB inducing kinase mediated phosphorylation of IKKα and IKKß both alone and in the presence of ionizing radiation (IR). The TNF-α induced NFκB dependent luciferase reporter assay demonstrated 1.65, 2.26 and 3.62 fold increase in NFκB activation at 10, 25 and 50 µM DMA concentrations respectively, compared to control cells. This activation was further increased by 5.8 fold in drug + radiation (50 µM +8.5 Gy) treated cells in comparison to control. We observed 51% radioprotection in control siRNA transfected cells that attenuated to 15% in siRNA NIK treated U87 cells, irradiated in presence of DMA at 24 h. CONCLUSIONS: Our studies show that NIK/IKK mediated NFκB activation is more intensified in cells over expressing NIK and treated with DMA, alone or in combination with ionizing radiation, indicating that DMA promotes NIK mediated NFκB signaling. This subsequently leads to the radioprotective effect exhibited by DMA.