CopA3 peptide inhibits MDM2-p53 complex stability in colorectal cancers and activates p53 mediated cell death machinery.

The diverse expression patterns of the tumor suppressor p53 in cancer cells reflect the regulatory efficiency of multiple cellular pathways. By contrast, many human tumors are reported to develop in the presence of wild-type p53. Recently, several oncogene inhibitors have been used clinically to suppress tumor development by functionally reactivating other oncoproteins. On the other hand, p53 reactivation therapies have not been well established, as few of the p53-MDM2 complex inhibitors such as Nutlin-3 induces mutation in p53 gene upon prolonged usage. Therefore, in this study CopA3, a 9-mer dimeric D-type peptide with anticancer activity against the human colorectal cancer cells, was used to explore the efficacy of p53 reactivation in-vitro and in-vivo. The anticancer activity of CopA3 was more selective towards the wild-type p53 expressing cells than the p53 deficient or mutant colorectal cancer cells. In response to this, this study investigated the signaling pathway in vitro and validated its anti-tumor activity in-vivo. The protein-peptide interaction and molecular docking efficiently provided insight into the specific binding affinity of CopA3 to the p53-binding pocket of the MDM2 protein, which efficiently blocked the p53 and MDM2 interaction. CopA3 plays a crucial role in the binding with MDM2 and enhanced the nuclear translocation of the p53 protein, which sequentially activated the downstream targets to trigger the autophagic mediated cell death machinery through the JNK/Beclin-1 mediated pathway. Collectively, CopA3 affected the MDM2-p53 interaction, which suppressed tumor development. This study may provide a novel inhibitor candidate for the MDM2-p53 complex, which could ultimately suppress the growth of colorectal cancer cells without being cytotoxic to the healthy neighboring cells present around the tumor microenvironment.

Silencing ESRP1 expression promotes caspase-independent cell death via nuclear translocation of AIF in colon cancer cells.

Epithelial splicing regulatory protein 1 (ESRP1) is overexpressed in the majority of cancer types, while downregulated in a few cancers, thus it has emerged as a tumorigenic or a tumor suppressor depending on disease context and cell type. Moreover, the underlying molecular mechanism of ESRP1 is poorly understood in cancer progression. Here, we initially analyzed Clinical Proteomic Tumor Analysis Consortium (CPTAC), colon tissue microarray, and colon cancer cells to evaluate the ESRP1 expression levels in colorectal cancer subtypes. The association between the expression of ESRP1 and cell death signaling pathways was evaluated in colon cancer cells. Furthermore, silencing ESRP1 was performed to detect the relation between ESRP1 and apoptosis-inducing factor (AIF). Subsequently, translocation of AIF and apoptosis were analyzed by immunofluorescence assay and FACS, respectively. ESRP1 is found to be expressed at high levels in the early stage, and gradually decreases with the increasing colorectal cancer stage, wherein epithelial cell to mesenchymal cell transition (EMT) occurs during cancer progression. Moreover, ESRP1 silencing in HCT116 colorectal cancer cells reveals the translocation of the caspase-independent cell death marker AIF to the nucleus, thereby enhancing the DNA damage response, which inevitably induces cancer cell death. Our results demonstrate that silencing ESRP1 in colorectal cancer cells promotes HCT116 cell death by inducing caspase-independent cell death via regulation of CD44 alternative splicing. Collectively, our findings provide an insight into ESRP1 as a therapeutic target in colon cancer.

Synergistic therapy with tangeretin and 5-fluorouracil accelerates the ROS/JNK mediated apoptotic pathway in human colorectal cancer cell.

Synergistic therapy is emerging as a promising strategy for improving the chemotherapeutic efficacy of anticancer drugs. Addition of adjuvants with standard anticancer drugs has shown successful reduction of adverse side effects. The synthetic drug 5-Fluorouracil (5-FU) shows several side effects upon prolonged chemotherapy, thereby restricting its long-term clinical application. Several studies have reported anticancer potential and anti-inflammatory activity of tangeretin (TAN) towards mammalian cells. Therefore, we investigate whether the combination of TAN with 5-FU increases their anticancer potential against colorectal cancer. In this study, we examined the synergistic activity of TAN and 5-FU on the viability of several human cancer and normal cells. Several possible mechanistic pathways were screened, and found that co-exposure of TAN and 5-FU accelerates oxidative-stress and increases endogenous-ROS generation, which sequentially triggers the DNA damage response and activates the apoptotic pathway, by down-regulating autophagy and DNA repair system in HCT-116 cells. TAN and 5-FU co-treatment also remarkably reduces the mitochondrial membrane potential, and sequentially decreases ATPase activity. Collectively, results indicate that combination of TAN and 5-FU significantly accelerates apoptosis via JNK mediated pathway. To our knowledge gained from literature, this study is the first to describe synergistic activity of TAN and 5-FU against colorectal cancer cells.

Emerging Multi-cancer Regulatory Role of ESRP1: Orchestration of Alternative Splicing to Control EMT.

RNA binding proteins (RBPs) associate with nascent and mature RNAs to perform biological functions such as alternative splicing and RNA stability. Having unique RNA recognition binding motifs, RBPs form complexes with RNA in a sequence- and structure-based manner. Aberrant expressions of several RBPs have been identified in tumorigenesis and cancer progression. These uncontrolled RBPs affect several mechanisms, including cell proliferation, tumor growth, invasion, metastasis and chemoresistance. Epithelial splicing regulatory protein 1 (ESRP1) is a member of the hnRNP family of proteins that play a crucial role in regulating numerous cellular processes, including alternative splicing and translation of multiple genes during organogenesis. Abnormal expression of ESRP1 alters the cell morphology, and leads to cell proliferation and tumor growth during cancer progression. ESRP1 mediated alternative splicing of target genes, including CD44, FGFR, PTBP1, LYN, ENAH, SPAG1 and ZMYND8, results in cancer progression. In addition, ESRP1 also regulates circularization and biogenesis of circular RNAs such as circUHRF1, circNOL10 and circANKS1B, whose expressions have been identified as key factors in various cancers. This multi-functional protein is also involved in imposing stability of target mRNAs such as cyclin A2, and thereby cell cycle regulation. The scope of this review is to examine recent scientific data, outcomes of the up- and down-regulated proteins, and the role of ESRP1 in various cancers. We conclude by summarizing ESRP1 dysregulation and its consequences on target genes in various human cancers. Collectively, the consequences of ESRP1 mediated splicing in cancer cells suggest the role of ESRP1 in cell proliferation and chemoresistance via apoptosis and autophagy modulation, which could, therefore, be potential targets for cancer therapeutics.

PTBP2 exon 10 inclusion is associated with the progression of CML and it is BCR-ABL1 dependent.

Altered or aberrant expression of several splicing factors leads to the progression of different cancers. Though there are several ongoing studies underscoring the role of the splicing regulator polypyrimidine tract binding protein 2 (PTBP2) in neuronal cells, we unveil the role of PTBP2 in chronic myeloid leukemia (CML). Different RNA binding proteins (RBP's) earlier reported in chronic myeloid leukemia blast crisis (CML-BC) cases (n = 28) from Radich Oncomine leukemia dataset, were compared. We observed increased expression of MSI2 followed by PTBP2 in BC cases and increased PTBP2 expression in relapsed cases (n = 10) from the same dataset compared to other RBPs. We also observed increased PTBP2 exon 10 inclusion in KCL22, a granulocytic lineage CML cell line when compared to other CML cell lines of different lineages. As PTBP2 protein expression is associated with PTBP2 exon 10 inclusion, we observed in cell lines and in a set of progressed cases (n = 4) that increased BCR-ABL1 expression potentiates PTBP2 exon 10 inclusion and thus confers the existence of a functional protein. Inhibition of BCR-ABL1 with imatinib not only blocks the inclusion of exon 10 but also deregulates PTBP2 expression in CML cells. Knockdown of PTBP2 in KCL22 cells leads to reduced cell proliferation, increased G2/M cell cycle arrest and increased apoptosis. Taken together our study portrays PTBP2 as a new possible target for CML and progressive inclusion/exclusion of PTBP2 exon 10 might play an important role in CML progression.

Molecular modeling, dynamics, and an insight into the structural inhibition of cofactor independent phosphoglycerate mutase isoform 1 from Wuchereria bancrofti using cheminformatics and mutational studies.

Phosphoglycerate mutase catalyzes the interconversion between 2-phosphoglycerate and 3-phosphoglycerate in the glycolytic and gluconeogenic pathways. They exist in two unrelated forms, that is either cofactor (2,3-diphosphoglycerate) dependent or cofactor-independent. These two enzymes have no similarity in amino acid sequence, tertiary structure, and in catalytic mechanism. Wuchereria bancrofti (WB) contains the cofactor-independent form, whereas other organisms can possess the dependent form or both. Since, independent phosphoglycerate mutase (iPGM) is an essential gene for the survival of nematodes, and it has no sequence or structural similarity to the cofactor-dependent phosphoglycerate mutase found in mammals, it represents an attractive drug target for the filarial nematodes. In this current study, a putative cofactor-iPGM gene was identified in the protein sequence of the WB. In the absence of crystal structure, a three-dimensional structure was determined using the homology modeling approximation, and the most stable protein conformation was identified through the molecular dynamics simulation studies, using GROMACS 4.5. Further, the functional or characteristic residues were identified through the sequence analysis, potential inhibitors were short-listed and validated, and potential inhibitors were ranked using the cheminformatics and molecular dynamics simulations studies, Prime MM-GBSA approach, respectively.