Identification and Functional Characterization of Nuclear Mortalin in Human Carcinogenesis

The Hsp70 family protein mortalin is an essential chaperone that is frequently enriched in cancer cells and exists in various subcellular sites, including the mitochondrion, plasma membrane, endoplasmic reticulum, and cytosol. Although the molecular mechanisms underlying its multiple subcellular localizations are not yet clear, their functional significance has been revealed by several studies. In this study, we examined the nuclear fractions of human cells and found that the malignantly transformed cells have more mortalin than the normal cells. We then generated a mortalin mutant that lacked a mitochondrial targeting signal peptide. It was largely localized in the nucleus, and, hence, is called nuclear mortalin (mot-N). Functional characterization of mot-N revealed that it efficiently protects cancer cells against endogenous and exogenous oxidative stress. Furthermore, compared with the full-length mortalin overexpressing cancer cells, mot-N derivatives showed increased malignant properties, including higher proliferation rate, colony forming efficacy, motility, and tumor forming capacity both in in vitro and in vivo assays. We demonstrate that mot-N promotes carcinogenesis and cancer cell metastasis by inactivation of tumor suppressor protein p53 functions and by interaction and functional activation of telomerase and heterogeneous ribonucleoprotein K (hnRNP-K) proteins.     

Identifications small molecules inhibitor of p53-mortalin complex for cancer drug using virtual screening

Mortalin was over expressed in tumor cells and bind to p53 protein. This interaction was suggested to promote sequestration of p53 in the cytoplasm, thereby inhibiting its nuclear activity. The p53 is a tumor suppressor that is essential for the prevention of cancer development and loss of p53 function is one of the early events in immortalization of human cells. Therefore, abrogation p53-mortalin interaction using small molecule is guaranteed stop cancer cell grow. However study interaction of p53-mortalin, and its inhibition using small molecule is still challenging because specific site of mortalin that bind to p53, vice versa, is still debatable. This study has aims to analyze the p53-binding site of mortalin using molecular docking and to screen drug-like compounds that have potential as inhibitors of p53-mortalin interaction using virtual screening. The result showed that the lowest energy binding of p53-mortalin complex is -31.89 kcal/mol, and p53 protein bind to substrate binding domain of mortalin (THR433; VAL435; LEU436; LEU437; PRO442; ILE558; LYS555). Furthermore, the p53-binding domain of mortalin was used as receptor to screen 9000 drug-like compounds from ZINC database using molecular docking program Auto Dock Vina in PyRx 0.8 (Virtual Screening Tools). Here, we have identified three drug-like compounds that are ZINC01019934, ZINC00624418 and ZINC00664532 adequate to interrupt stability of p53-mortalin complex that warrant for anticancer agent.     

Patterns of p53, p73 and mortalin gene expression associated with haemocyte polyploidy in the soft-shell clam, Mya arenaria

The molecular mechanisms by which haemocytes of clams are transformed in the course of haemic neoplasia remain by far unknown. The aim of this study was to quantify the expression of p53/p73 and mortalin genes, in relation with the ploidy status of clam haemocytes and to correlate the p53 expression with mortalin expression. For this purpose, soft-shell clams, Mya arenaria, were collected from an endemic zone for neoplasia. The ploidy of haemocytes was assessed for each individual clam by flow cytometry using a propidium iodide protocol, while p53/p73 and mortalin gene expressions were quantified by real-time RT-PCR. Results show that haemocytes of some clams with a moderate percentage (15-50%) of tetraploid cells have a significantly high level of p53 and p73 in comparison with clams belonging to categories with low (<15%) or high levels (>50%) of tetraploid cells, where low levels of expression of these genes were observed. Furthermore, mortalin gene expression is strongly correlated (r² = 0.68, p < 0.01) with p53 gene expression level. This reinforces the hypothesis of a cytoplasmic p53 sequestration mechanism in clam haemic neoplasia. Further studies are needed to confirm these preliminary results and further unravel the molecular pathways involved in this process. Our results are believed to provide phenotypic foundation for such studies to be undertaken.     

Anti-tumor activity of the X-linked inhibitor of apoptosis (XIAP) inhibitor embelin in gastric cancer cells

This study investigated the anticancer effects of embelin in human gastric cancer cells and the underlying molecular mechanisms. Gastric cancer cells were treated with embelin and 5-FU for methyl-thiazolyl-tetrazolium bromide cell viability assay and flow cytometric detection of cell viability and apoptosis. Protein pathway array (PPA) and Western blot were used to investigate differentially expressed proteins in embelin-treated gastric cancer cells. Embelin reduced gastric cancer cell viability, induced apoptosis, and enhanced 5-FU antitumor activity in gastric cancer cells. Mechanistically, embelin induced cell cycle arrest at the S and G2/M phases. Molecularly, embelin downregulated expression of X-linked inhibitor of apoptosis and cell cycle-regulatory proteins, such as CDK1, CDC25B, CDC25C, cyclinB1, and CDK2. PPA analysis showed that embelin modulated several pathways that are associated with cell growth and apoptosis, such as PI3K/AKT, JAK/STAT, p38 MAPK, and p53. The data from the current study implied that reduction of gastric cancer cell viability after treatment with embelin was through cell cycle arrest at the S and G2/M phases and apoptosis.     

XIAP inhibitor and antiestrogen embelin abrogates metastasis and augments apoptosis in estrogen receptor positive human breast adenocarcinoma cell line MCF-7

Tamoxifen therapy for the treatment of hormone responsive breast cancer has limitations due to acquired resistance in the case of recurrences. Embelin, a known inhibitor of X-linked inhibitor of apoptosis protein (XIAP) was also reported to exhibit strong antiestrogenic effects in animal models. Dual role of embelin as a proapoptotic and antiestrogenic agent may have potential benefits in the therapy of breast cancer. In this study, the effects of embelin treatment on estrogen receptor positive Human breast adenocarcinoma (MCF-7) cells was investigated to primarily understand if embelin being an antiestrogen and XIAP inhibitor could be a potential alternative to tamoxifen therapy. Results revealed that, embelin at a concentration of 65 μg/ml attenuated proliferation, inhibited metastatic migration, modulated the expression of Bcl2, Caspases and induced apoptosis in MCF-7 cells which was found to be p53 mediated. Hence, chemotherapy with embelin could be a promising strategy to be experimented in hormone responsive breast cancers.     

Embelin Suppresses Growth of Human Pancreatic Cancer Xenografts,and Pancreatic Cancer Cells Isolated from KrasG12D Mice by Inhibiting Akt and Sonic Hedgehog Pathways

Pancreatic cancer is a deadly disease, and therefore effective treatment and/or prevention strategies are urgently needed. The objectives of this study were to examine the molecular mechanisms by which embelin inhibited human pancreatic cancer cell growth in vitro, and xenografts in Balb C nude mice, and pancreatic cancer cell growth isolated from Kras^G12D transgenic mice. XTT assays were performed to measure cell viability. AsPC-1 cells were injected subcutaneously into Balb c nude mice and treated with embelin. Cell proliferation and apoptosis were measured by Ki67 and TUNEL staining, respectively. The expression of Akt, and Sonic Hedgehog (Shh) and their target gene products were measured by the immunohistochemistry, and Western blot analysis. The effects of embelin on pancreatic cancer cells isolated from 10-months old Kras^G12D mice were also examined. Embelin inhibited cell viability in pancreatic cancer AsPC-1, PANC-1, MIA PaCa-2 and Hs 766T cell lines, and these inhibitory effects were blocked either by constitutively active Akt or Shh protein. Embelin-treated mice showed significant inhibition in tumor growth which was associated with reduced expression of markers of cell proliferation (Ki67, PCNA and Bcl-2) and cell cycle (cyclin D1, CDK2, and CDK6), and induction of apoptosis (activation of caspase-3 and cleavage of PARP, and increased expression of Bax). In addition, embelin inhibited the expression of markers of angiogenesis (COX-2, VEGF, VEGFR, and IL-8), and metastasis (MMP-2 and MMP-9) in tumor tissues. Antitumor activity of embelin was associated with inhibition of Akt and Shh pathways in xenografts, and pancreatic cancer cells isolated from Kras^G12D mice. Furthermore, embelin also inhibited epithelial-to-mesenchymal transition (EMT) by up-regulating E-cadherin and inhibiting the expression of Snail, Slug, and ZEB1. These data suggest that embelin can inhibit pancreatic cancer growth, angiogenesis and metastasis by suppressing Akt and Shh pathways, and can be developed for the treatment and/or prevention of pancreatic cancer.     

Nutlin-3a, an MDM2 antagonist and p53 activator, helps to preserve the replicative potential of cancer cells treated with a genotoxic dose of resveratrol

Resveratrol is a natural compound that has been intensely studied due to its role in cancer prevention and potential as an anti-cancer therapy. Its effects include induction of apoptosis and senescence-like growth inhibition. Here, we report that two cancer cell lines (U-2 OS and A549) differ significantly in their molecular responses to resveratrol. Specifically, in U-2 OS cells, the activation of the p53 pathway is attenuated when compared to the activation in A549 cells. This attenuation is accompanied by a point mutation (458: CGA→TGA) in the PPM1D gene and overexpression of the encoded protein, which is a negative regulator of p53. Experimentally induced knockdown of PPM1D in U-2 OS cells resulted in slightly increased activation of the p53 pathway, most clearly visible as stronger phosphorylation of p53 Ser³⁷. When treated with nutlin-3a, a non-genotoxic activator of p53, U-2 OS and A549 cells both responded with substantial activation of the p53 pathway. Nutlin-3a improved the clonogenic survival of both cell lines treated with resveratrol. This improvement was associated with lower activation of DNA-damage signaling (phosphorylation of ATM, CHK2, and histone H2AX) and higher accumulation of cells in the G1 phase of the cell cycle. Thus, the hyperactivation of p53 by nutlin-3a helps to preserve the replicative potential of cells exposed to resveratrol.     

p130Cas Is Required for Mammary Tumor Growth and Transforming Growth Factor-��-mediated Metastasis through Regulation of Smad2/3 Activity

During breast cancer progression, transforming growth factor-β (TGF-β) switches from a tumor suppressor to a pro-metastatic molecule. Several recent studies suggest that this conversion in TGF-β function depends upon fundamental changes in the TGF-β signaling system. We show here that these changes in TGF-β signaling are concomitant with aberrant expression of the focal adhesion protein, p130Cas. Indeed, elevating expression of either the full-length (FL) or just the carboxyl terminus (CT) of p130Cas in mammary epithelial cells (MECs) diminished the ability of TGF-β1 to activate Smad2/3, but increased its coupling to p38 MAPK. This shift in TGF-β signaling evoked (i) resistance to TGF-β-induced growth arrest, and (ii) acinar filling upon three-dimensional organotypic cultures of p130Cas-FL or -CT expressing MECs. Furthermore, rendering metastatic MECs deficient in p130Cas enhanced TGF-β-stimulated Smad2/3 activity, which restored TGF-β-induced growth inhibition both in vitro and in mammary tumors produced in mice. Additionally, whereas elevating TβR-II expression in metastatic MECs had no affect on their phosphorylation of Smad2/3, this event markedly enhanced their activation of p38 MAPK, leading to increased MEC invasion and metastasis. Importantly, depleting p130Cas expression in TβR-II-expressing metastatic MECs significantly increased their activation of Smad2/3, which (i) reestablished the physiologic balance between canonical and noncanonical TGF-β signaling, and (ii) reversed cellular invasion and early mammary tumor cell dissemination stimulated by TGF-β. Collectively, our findings identify p130Cas as a molecular rheostat that regulates the delicate balance between canonical and noncanonical TGF-β signaling, a balance that is critical to maintaining the tumor suppressor function of TGF-β during breast cancer progression.