Upregulation of miR-22-3p contributes to plumbagin-mediated inhibition of Wnt signaling in human colorectal cancer cells.

The emergence of resistance and side effects for currently available drugs warrant the need for an alternative treatment regime for colorectal cancer (CRC). Several natural compounds, including plumbagin, have exhibited promising anti-cancer effects in different cancer models, although the molecular mechanisms underlying their effects remain at large. We previously reported plumbagin-mediated inhibition of Wnt signaling in CRC cells and to unravel the actual mechanism, we hypothesized the involvement of miRNAs as they have emerged as critical regulators of gene expression. We performed miRNA-microarray to check if plumbagin-mediated effects are through alteration of miRNA expression and found 32 DE-miRNAs (11 upregulated and 21 downregulated), and KEGG enrichment analysis indicated that their target mRNAs are associated with several pathways relevant to CRC and Wnt signaling. miRNA-mRNA network analysis by miRNET revealed a highly upregulated miRNA upon plumbagin treatment, miR-22-3p, having multiple Wnt-associated mRNAs interacting partners. 3'UTR luciferase assays revealed that miR-22-3p directly targeted the 3'UTR of BCL9L (a coactivator of CTNNB1). miR-22-3p inhibited Wnt signaling by downregulating the levels of BCL9L and that of CTNNB1 and several Wnt-associated proteins (TCF4, c-Myc, CCND1, Snail, Slug, and vimentin) in CRC cells. We also demonstrated that both miR-22-3p and plumbagin reduced colony formation and caused apoptotic cell death (detected by Annexin V/PI dual staining), possibly through increased ROS (detection by Dihydroethidium staining) and decreased MMP (detection by MitoTracker™ Orange staining) in CRC cells. These effects of plumbagin were partially rescued by antimiR-22-3p, suggesting the involvement of miR-22-3p in plumbagin-mediated effects. The present study revealed that alteration in miR-22-3p levels by plumbagin contributes to the induction of apoptosis and its inhibitory effects on Wnt signaling and colony formation, thus providing a mechanistic basis behind its anti-cancer potential.

An integrated approach to understand fluid shear stress-driven and reactive oxygen species-mediated metastasis of colon adenocarcinoma through mRNA-miRNA-lncRNA-circRNA networks.

Development of colon adenocarcinoma (COAD) metastasis involves several mediators including fluid shear stress (FSS), intracellular ROS levels, and non-coding RNAs. In our present study, we identified and investigated the role of regulatory non-coding RNA molecules specifically involved in COAD metastasis and their association with FSS and ROS. Interactions between the mRNAs associated with FSS and ROS, the corresponding microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) in COAD metastasis were used to generate the mRNA-miRNA-lncRNA-circRNA network. Experimental validation of the identified RNA hubs using quantitative real-time PCR demonstrated a direct effect of the FSS on their expression levels in cancer cells. FSS resulted in the downregulation of HMGA1 and RAN, as well as the upregulation of HSP90AA1, PMAIP1 and BIRC5. Application of shear stress also led to downregulation of hsa-miR-26b-5p and hsa-miR-34a-5p levels in HCT116 cells. Further, functional enrichment and survival analysis of the significant miRNAs, as well as the OncoPrint and the survival analyses of the selected mRNAs were performed. Subsequently, their functional role was also corroborated with existing literature. Ten significant miRNA hubs were identified, out of which hsa-miR-17-5p and hsa-miR-20a-5p were found to interact with lncRNA (CCAT2) while hsa-miR-335 was found to interact with four circRNAs. Fifteen significant miRNAs were identified in 10 different modules suggesting their importance in FSS and ROS-mediated COAD metastasis. Finally, 10 miRNAs and 3 mRNAs associated with FSS and/or ROS were identified as significant overall survival markers; 33 mRNAs were also identified as metastasis-free survival markers whereas 15 mRNAs showed > 10% gene alterations in TCGA-COAD data and may serve as promising therapeutic biomarkers in the COAD metastasis.

HMGB3 inhibition by miR-142-3p/sh-RNA modulates autophagy and induces apoptosis via ROS accumulation and mitochondrial dysfunction and reduces the tumorigenic potential of human breast cancer cells.

AIMS: High mobility group box (HMGB) family proteins, HMGB1, HMGB2, HMGB3, and HMGB4 are oncogenic. The oncogenic nature of HMGB1 is characterized by its association with autophagy, ROS, and MMP. Since HMGB3 is its paralog, we hypothesized that it might also modulate autophagy, ROS, and MMP. Hence, we targeted HMGB3 using its shRNA or miR-142-3p and assessed the changes in autophagy, ROS, MMP, and tumorigenic properties of human breast cancer cells. MAIN METHODS: Cell viability was assessed by resazurin staining and annexin-V/PI dual staining was used for confirming apoptosis. Colony formation, transwell migration, invasion and luciferase reporter (for miRNA-target validation) assays were also performed. ROS and MMP were detected using DHE and MitoTracker dyes, respectively. A zebrafish xenograft model was used to assess the role of miR-142-3p on in vivo metastatic potential of breast cancer cells. KEY FINDINGS: Breast cancer tissues from Indian patients and TCGA samples exhibit overexpression of HMGB3. miR-142-3p binds to 3' UTR of HMGB3, leading to its downregulation that subsequently inhibits colony formation and induces apoptosis involving increased ROS accumulation and decreased MMP, phospho-mTOR and STAT3. Our findings show that HMGB3 is directly involved in the miR-142-3p-mediated disruption of autophagy and induction of apoptotic cell death via modulation of LC3, cleaved PARP and Bcl-xL. In addition, miR-142-3p inhibited migration, invasion and metastatic potential of breast cancer cells. SIGNIFICANCE: Our findings highlighted the role of HMGB3, for the first time, in the modulation of autophagy and apoptosis in human breast cancer cells, and these results have therapeutic implications.

Design, synthesis and evaluation of 4,7-disubstituted 8-methoxyquinazoline derivatives as potential cytotoxic agents targeting ß-catenin/TCF4 signaling pathway.

Overactivation of Wnt/ß-catenin signaling by accumulated ß-catenin in the nucleus has been shown to play a crucial role in the etiology of cancer. Interaction of ß-catenin with Transcription factor 4 (TCF4) is a key step for the activation of Wnt genes in response to upstream signals of the Wnt/ß-catenin pathway. Hence, down regulation of Wnt/ß-catenin signaling or targeting downstream events by selective ß-catenin/TCF4 protein-protein interaction inhibitors could be a potential therapeutic strategy against such cancers. In this study structure-based drug design approach was followed to design novel 4,7-disubstituted 8-methoxyquinazoline-based derivatives which could act as potential cytotoxic agents inhibiting the ß-catenin/TCF4 protein-protein interactions. Fifteen compounds possessing 4,7-disubstituted 8-methoxyquinazoline scaffold were synthesized. Cytotoxic potential of the synthesised derivatives were determined against constitutively activated ß-catenin/TCF4 signaling pathway cancer cells (HCT116 and HepG2) using the sulforhodamine B assay. The most potent compound (18B) was selected for detailed biological evaluation. Cell morphology, Hoechst 33342 and Annexin V/PI staining were used to detect apoptosis, while inhibition of cell migration was assessed by in vitro wound healing assay against HCT116 and HepG2 cells. Effect on ß-catenin/TCF mediated transcriptional activity was assessed by TOPFlash/FOPFlash assay, TCF4 and ß-catenin protein expression by immunocytofluorescence, and Wnt target genes (like c-MYC and Cyclin D1) mRNA levels by RT-PCR against HCT116 cells. Cytotoxic potency of the most potential compound (18B) against primary human gallbladder cancer cells was also evaluated. The derivatives showed interactions with active site residues of ß-catenin and were capable of hindering the TCF4 binding, thereby disrupting ß-catenin/TCF4 interactions. Cytotoxic potencies (IC50) of these derivatives ranged from 5.64 ± 0.68 to 23.18 ± 0.45 µM against HCT116 and HepG2 cells respectively. Compound (18B), the most potent compound among the series, induced apoptosis and inhibited cell migration against HCT116 and HepG2 cells. Mechanistic studies indicated that compound (18B) downregulated ß-catenin/TCF4 signaling pathway, ß-catenin and TCF4 protein expression, and mRNA levels of c-MYC andCyclin D1 in HCT116 cells and showed cytotoxicity against primary human gallbladder cancer cells with IC50 value of 8.50 ± 1.44 µM. Thus, novel 4,7-disubstituted 8-methoxyquinazoline derivatives were identified as potential cytotoxic agents with potencies comparable to that of imatinib mesylate. Compound (18B) represents a promising lead molecule as anticancer agent against colon, hepatocellular and gallbladder cancers targeting ß-catenin/TCF4 signaling pathway.

Non-coding RNAs as emerging regulators and biomarkers in colorectal cancer.

CRC is the third most common cancer occurring worldwide and the second leading cause of cancer deaths. In the year 2020, 1,931,590 new cases of CRC and 935,173 deaths were reported. The last two decades have witnessed an intensive study of noncoding RNAs and their implications in various pathological conditions including cancer. Noncoding RNAs such as miRNAs, tsRNAs, piRNAs, lncRNAs, pseudogenes, and circRNAs have emerged as promising prognostic and diagnostic biomarkers in preclinical studies of cancer. Some of these noncoding RNAs have also been shown as promising therapeutic targets for cancer treatment. In this review, we have discussed the emerging roles of various types of noncoding RNAs in CRC and their future implications in colorectal cancer management and research.

RETRA induces necroptosis in cervical cancer cells through RIPK1, RIPK3, MLKL and increased ROS production.

Cervical cancer is the fourth most prevalent cancer in women worldwide, predominantly infected with human papillomavirus (HPV). The current chemo and radiotherapies are mostly futile due to acquired resistance to apoptosis and warrant new therapeutic approaches targeting potent non-apoptotic cell death pathways to eliminate cervical cancer cells. Induction of necroptosis by pharmaceutical interventions is emerging as a promising tool in multiple apoptotic resistant cancer cells. RETRA (REactivation of Transcriptional Reporter Activity) is a small molecule known to induce expression of p53 regulated genes in mutant (mt) p53 cells but, detailed mechanisms of its anticancer effects are poorly known. The present study investigated the potentials of RETRA as an anticancer agent and found that it induces necroptosis selectively in cervical cancer cells irrespective of p53 status through the phosphorylation of receptor-interacting protein kinase 1,3 (RIPK1, RIPK3) and mixed lineage kinase domain-like protein (MLKL) with no cytotoxic effects in normal human peripheral blood mononuclear cells (PBMCs). RETRA-treated cells also displayed necroptotic morphology of disintegrated plasma membranes with intact nuclei and also showed cell cycle arrest at the S phase with the upregulation of p21 and downregulation of cyclin-D3. Intriguingly, the combinatorial approach of using RETRA with Necrostain-1, a known inhibitor of necroptosis, reversed the effect of RETRA and rescued cell death. Moreover, induction of necroptosis by RETRA is associated with mitochondrial hyperpolarization and elevated ROS production. Collectively, these findings suggest that RETRA induces cell death via necroptosis with increased production of ROS, accentuating the therapeutic implication of RETRA in cervical cancer cells.

Metabolic modeling of host-microbe interactions for therapeutics in colorectal cancer.

The onset of colorectal cancer (CRC) is often attributed to gut bacterial dysbiosis, and thus gut microbiota are highly relevant in devising treatment strategies. Certain gut microbes, like Enterococcus spp., exhibit remarkable anti-neoplastic and probiotic properties, which can aid in silver nanoparticle (AgNPs) induced reactive oxygen species (ROS)-based CRC treatment. However, the effects of AgNPs on gut microbial metabolism have not been reported thus far. In this study, a detailed systems-level understanding of ROS metabolism in Enterococcus durans (E. durans), a representative gut microbe, was gained using constraint-based modeling, wherein, the critical association between ROS and folate metabolism was established. Experimental studies involving low AgNP concentration treatment of E. durans cultures confirmed these modeling predictions (an increased extracellular folate concentration by 52%, at the 9th h of microbial growth, was observed). Besides, the computational studies established various metabolic pathways involving amino acids, energy metabolites, nucleotides, and SCFAs as the key players in elevating folate levels on ROS exposure. The anti-cancer potential of E. durans was also studied through MTT analysis of HCT 116 cells treated with microbial culture (AgNP treated) supernatant. A decrease in cell viability by 19% implicated the role of microbial metabolites (primarily folate) in causing cell death. The genome-scale modeling approach was then extended to extensively model CRC metabolism, as well as CRC-E. durans interactions in the context of CRC treatment, using tissue-specific metabolic models of CRC and healthy colon. These findings on further validation can facilitate the development of robust and effective cancer therapy.

miR-142-3p simultaneously targets HMGA1, HMGA2, HMGB1, and HMGB3 and inhibits tumorigenic properties and in-vivo metastatic potential of human cervical cancer cells.

AIMS: High-mobility group (HMG) proteins are oncogenic in different cancers, including cervical cancer; silencing their individual expression using sh-RNAs, siRNAs, and miRNAs has had anti-tumorigenic effects, but the consequences of their collective downregulation are not known. Since multiple gene targeting is generally very effective in cancer therapy, the present study highlighted the consequences of silencing the expression of HMGA1, A2, B1, and B3 using sh-RNAs or miR-142-3p (that can potentially target HMGA1, A2, B1, and B3) in cervical cancer cell lines. MAIN METHODS: 3' UTR luciferase reporter assays were performed to validate HMGA1, A2, B1, and B3 as targets of miR-142-3p in human cervical cancer cells. Annexin V/PI dual staining and flow cytometry analyses were used to detect apoptotic cells. miR-142-3p-mediated regulation of cell death, colony formation, migration, and invasion was investigated in human cervical cancer cells together with in vivo metastasis in zebrafish. KEY FINDINGS: Concurrent knockdown of HMGA1, A2, B1, and B3 through their corresponding sh-RNAs inhibited cell viability and colony formation but induced apoptosis, and these effects were relatively reduced upon their individual knockdown. miR-142-3p targeted HMGA1, A2, B1, and B3 by binding to their 3'UTRs and induced apoptosis but inhibited proliferation, migration, and invasion of human cervical cancer cells. In addition, miR-142-3p expression decreased phospho-p65 and EMT-related proteins in cervical cancer cells and their in vivo metastatic potential upon implantation in zebrafish. SIGNIFICANCE: These findings suggest that miR-142-3p acts as a tumor-suppressive miRNA by targeting HMGA1, A2, B1, and B3 and may serve as a potential therapeutic agent in human cervical cancer.

Synergistic effect of metformin and vemurufenib (PLX4032) as a molecular targeted therapy in anaplastic thyroid cancer: an in vitro study.

BACKGROUND: Survival rate of patients affected with anaplastic thyroid carcinoma (ATC) is less than 5% with current treatment. In ATC, BRAFV600E mutation is the major mutation that results in the transformation of normal cells in to an undifferentiated cancer cells via aberrant molecular signaling mechanisms. Although vemurufenib is a selective oral drug for the BRAFV600E mutant kinase with a response rate of nearly 50% in metastatic melanoma, our study has showed resistance to this drug in ATC. Hence the rationale of the study is to explore combinational therapeutic effect to improve the efficacy of vemurafenib along with metformin. Metformin, a diabetic drug is an AMPK activator and has recently proved to be involved in preventing or treating several types of cancer. METHODS AND RESULTS: Using iGEMDock software, a protein-ligand interaction was successful between Metformin and TSHR (receptor present in the thyroid follicular cells). Our study demonstrates that combination of vemurufenib with metformin has synergistic anti-cancer effects which was evaluated through MTT assay (cytotoxicity), colony formation assay (antiproliferation evaluation) and suppressed the progression of ATC cells growth by inducing significant apoptosis, proven by Annexin V-FITC assay (Early Apoptosis Detection). Downregulation of ERK signaling, upregulation of AMPK pathway and precision in epithelial-mesenchymal transition (EMT) pathway which were assessed by RT-PCR and Western blot provide the evidence that the combination of drugs involved in the precision of altered molecular signaling Further our results suggest that Metformin act as a demethylating agent in anaplastic thyroid cancer cells by inducing the expression of NIS and TSHR. Our study for the first time explored cAMP signaling in ATC wherein cAMP signaling is downregulated due to decrease in intracellular cAMP level upon metformin treatment. CONCLUSION: To conclude, our findings demonstrate novel therapeutic targets and treatment strategies for undifferentiated ATC.

SLC7A11/ xCT is a target of miR-5096 and its restoration partially rescues miR-5096-mediated ferroptosis and anti-tumor effects in human breast cancer cells.

Breast cancer cells evade cell death by overexpressing SLC7A11, which functions by transporting cystine into cells in exchange for intracellular glutamate facilitating glutathione synthesis and reducing reactive oxygen species (ROS)-mediated stress. Using an in silico approach, we predicted an miRNA (miR-5096) that can target and downregulate SLC7A11. We demonstrated SLC7A11 as a target of miR-5096 by 3'UTR luciferase assay and further validated it by identifying reduced mRNA and protein levels of SLC7A11 upon miR-5096 overexpression. miR-5096-induced ferroptotic cell death in human breast cancer cells was confirmed by concurrently increased ROS, OH-, lipid ROS, and iron accumulation levels and decreased GSH and mitochondrial membrane potential (MitoTracker™ Orange) with mitochondrial shrinkage and partial cristae loss (observed by TEM). miR-5096 inhibited colony formation, transwell migration, and breast cancer cell invasion, whereas antimiR-5096 promoted these tumorigenic properties. Ectopic expression of SLC7A11 partly reversed miR-5096-mediated effects on cell survival, ROS, lipid peroxides, iron accumulation, GSH, hydroxyl radicals, mitochondrial membrane potential, and colony formation. miR-5096 modulated the expression of epithelial-mesenchymal transition markers in vitro and inhibited the metastatic potential of MDA-MB-231 cells in a tumor xenograft model of zebrafish larvae. Our results demonstrate that miR-5096 is a tumor-suppressive miRNA in breast cancer cells, and this paper discusses its therapeutic implications.

MicroRNA Interactome Multiomics Characterization for Cancer Research and Personalized Medicine: An Expert Review.

MicroRNAs (miRNAs) that are mutually modulated by their interacting partners (interactome) are being increasingly noted for their significant role in pathogenesis and treatment of various human cancers. Recently, miRNA interactome dissected with multiomics approaches has been the subject of focus since individual tools or methods failed to provide the necessary comprehensive clues on the complete interactome. Even though single-omics technologies such as proteomics can uncover part of the interactome, the biological and clinical understanding still remain incomplete. In this study, we present an expert review of studies involving multiomics approaches to identification of miRNA interactome and its application in mechanistic characterization, classification, and therapeutic target identification in a variety of cancers, and with a focus on proteomics. We also discuss individual or multiple miRNA-based interactome identification in various pathological conditions of relevance to clinical medicine. Various new single-omics methods that can be integrated into multiomics cancer research and the computational approaches to analyze and predict miRNA interactome are also highlighted in this review. In all, we contextulize the power of multiomics approaches and the importance of the miRNA interactome to achieve the vision and practice of predictive, preventive, and personalized medicine in cancer research and clinical oncology.

Clinicopathologic implications of epithelial cell adhesion molecule expression across molecular subtypes of breast carcinoma.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM), a type I transmembrane protein of the epithelial tissues and known cell adhesion molecule, has been demonstrated to have critical role in carcinogenesis. In breast cancer, EpCAM expression has been associated with poor prognosis. The expression pattern of EpCAM across molecular subtypes of breast carcinoma has been studied in patients reporting to a South Indian multispecialty tertiary care hospital. The prognostic significance of EpCAM expression pattern and probable response to therapy has also been addressed. MATERIALS AND METHODS: EpCAM expression was assessed by immunohistochemical studies on 200 breast carcinoma tissue samples of different molecular subtypes, including luminal A, luminal B, Her2Neu, and triple-negative breast cancer (TNBC). The expression was scored using the standard scoring system. A correlation was drawn with detailed clinicopathologic annotation and available outcomes data to analyze the influence of EpCAM on prognosis. RESULTS: EpCAM expression varied significantly in the different intrinsic subtypes of breast carcinoma. Differential expression was also established with different grades of breast carcinoma with varying levels of differentiation. We observed strong EpCAM expression in TNBC among other subtypes. CONCLUSION: The differential expression of EpCAM among intrinsic subtypes of breast cancer and the correlation of EpCAM expression with high-grade breast carcinoma shown in the study have important implications in understanding the role of EpCAM and might form the basis for developing targeted therapies in breast cancer in the future.

Identification of mRNA and non-coding RNA hubs using network analysis in organ tropism regulated triple negative breast cancer metastasis.

Triple negative breast cancer (TNBC) is aggressive in nature, resistant to conventional therapy and often ends in organ specific metastasis. In this study, publicly available datasets were used to identify miRNA, mRNA and lncRNA hubs. Using validated mRNA-miRNA, mRNA-mRNA and lncRNA-miRNA interaction information obtained from various databases, RNA interaction networks for TNBC and its subtype specific as well as organ tropism regulated metastasis were generated. Further, miRNA-mRNA-lncRNA triad classification was performed using social network analysis from subnetworks and visualized using Cytoscape. Survival analysis of the RNA hubs, oncoprint analysis for mRNAs and pathway analysis of the lncRNAs were also performed. Results indicated that two lncRNAs (NEAT1 and CASC7) and four miRNAs (hsa-miR-106b-5p, hsa-miR-148a-3p, hsa-miR-25-3p and hsa-let-7i-5p) were common between hubs identified in TNBC and TNBC associated metastasis. The exclusive hubs for TNBC associated metastasis were hsa-miR-200b-3p, SP1, HSPA4 and RAB1B. HMGA1 was the top ranked hub in mesenchymal subtype associated lung metastasis, while hsa-miR-27a-3p was identified as the top ranked hub mRNA in luminal androgen receptor subtype associated bone metastasis. When lncRNA associated pathway analysis was performed, Hs Cytoplasmic Ribosomal Protein pathway was found to be the most significant and among the selected hubs, CTNND1, SON and hsa-miR-29c emerged as TNBC survival markers. TP53, FOXA1, MTDH and HDGF were found as the top ranked mRNAs in oncoprint analysis. The pipeline proposed for the first time in this study with validated RNA interaction data integration and graph-based learning for miRNA-mRNA-lncRNA triad classification from RNA hubs may aid experimental cost reduction and its successful execution will allow it to be extended to other diseases too.

An integrated approach for mining precise RNA-based cervical cancer staging biomarkers.

Since international federation of gynecology and obstetrics (FIGO) staging is mainly based on clinical assessment, an integrated approach for mining RNA based biomarkers for understanding the molecular deregulation of signaling pathways and RNAs in cervical cancer was proposed in this study. Publicly available data were mined for identifying significant RNAs after patient staging. Significant miRNA families were identified from mRNA-miRNA and lncRNA-miRNA interaction network analyses followed by stage specific mRNA-miRNA-lncRNA association network generation. Integrated bioinformatic analyses of selected mRNAs and lncRNAs were performed. Results suggest that HBA1, HBA2, HBB, SLC2A1, CXCL10 (stage I), PKIA (stage III) and S100A7 (stage IV) were important. miRNA family enrichment of interacting miRNA partners of selected RNAs indicated the enrichment of let-7 family. Assembly of collagen fibrils and other multimeric structures_Homosapiens_R-HSA-2022090 in pathway analysis and progesterone_CTD_00006624 in DSigDB analysis were the most significant and SLC2A1, hsa-miR-188-3p, hsa-miR-378a-3p and hsa-miR-150-5p were selected as survival markers.

Vitamin K3 (menadione) suppresses epithelial-mesenchymal-transition and Wnt signaling pathway in human colorectal cancer cells.

Tumor recurrence and metastasis decrease the survival rate of colorectal cancer (CRC) patients. Menadione reduces the numbers and incidences of 1,2-dimethylhydrazine induced colon tumors in mouse but the mechanism of anticancer activity of menadione in colorectal cancer is not very clear. Since Wnt signaling is constitutively active in CRC and it aggravates the epithelial mesenchymal transition (EMT), the regulation of EMT and Wnt signaling by menadione (vitamin K3) was investigated in CRC cells. Menadione showed cytotoxicity against human CRC cells (SW480 and SW620) and human primary colon cancer cells but was relatively ineffective against the cells from human normal colon (CRL-1790) and human primary colon epithelial cells. Menadione suppressed invasion, migration and epithelial-mesenchymal transition in human CRC cells by upregulating the expression of E-cadherin (CDH1), ZO-1 and downregulating that of N-cadherin (CDH2), Vimentin (VIM), ZEB1, MMP2 and MMP9. Menadione decreased TOPFlash/FOPFlash luciferase activity and expression of several downstream targets of Wnt signaling and coactivators such as ß-catenin (CTNNB1), TCF7L2, Bcl9l, p300 (EP300) and cyclin D1 (CCND1) was suppressed. Menadione induced differentiation and increased apoptotic cell population in SubG0 phase of cell cycle in SW480 and SW620 cells. The ability of menadione to suppress EMT, migration, invasion, Wnt signaling, cell proliferation and induce Sub G0 arrest, highlights its potential to be considered for intensive preclinical and clinical investigation in CRC.

A novel reporter system for cyclic AMP mediated gene expression in mammalian cells based on synthetic transgene expression system.

Cyclic AMP (cAMP) is an important second messenger that mediates various biological functions in both prokaryotes and eukaryotes. Due to the ever increasing significance in studying the function and modulation of cAMP-based signaling, it is important to develop a protein-based biosensor that reports the cAMP mediated gene expression. Based on a synthetic transgene approach, an artificial mammalian transactivator was developed by fusing a transcriptional regulatory element cAMP receptor protein (CRP) of Escherichia coli to the VP16 transactivation domain of Herpes simplex virus in a mammalian expression vector (pLA1) that activates CRP specific operator site present in a chimeric promoter (OCRP- PhCMVmin- Luciferase) in a concentration dependent manner in mammalian cells. Our results reveal that the engineered transactivator report the gene expression mediated by cAMP directly in mammalian cells and this cAMP reporter system works irrespective of Protein kinase A (PKA) - cyclic AMP response element binding protein (CREB) - cyclic AMP response element (CRE) signaling since the luciferase activity mediated by synthetic gene construct is seen even in the presence of PKA inhibitor H-89 (derived from H-8 (N-[2-(methylamino) ethyl]-5-isoquinoline-sulfonamide). Furthermore this synthetic transcription factor plays a significant role in reporting and mediating cAMP signaling in tumorigenic cells which possess an aberrant cAMP signaling due to PKA and CREB mutations.

Entrainment of superoxide rhythm by menadione in HCT116 colon cancer cells.

Reactive oxygen species (ROS) are primary effectors of cytotoxicity induced by many anti-cancer drugs. Rhythms in the pseudo-steady-state (PSS) levels of particular intracellular ROS in cancer cells and their relevance to drug effectiveness are unknown thus far. We report that the PSS levels of intracellular superoxide (SOX), an important ROS, exhibit an inherent rhythm in HCT116 colon cancer cells, which is entrained (reset) by the SOX inducer, menadione (MD). This reset was dependent on the expression of p53, and it doubled the sensitivity of the cells to MD. The period of oscillation was found to have a linear correlation with MD concentration, given by the equation, T, in h = 23.52 - 1.05 [MD concentration in µM]. Further, we developed a mathematical model to better understand the molecular mechanisms involved in rhythm reset. Biologically meaningful parameters were obtained through parameter estimation techniques; the model can predict experimental profiles of SOX, establish qualitative relations between interacting species in the system and serves as an important tool to understand the profiles of various species. The model was also able to successfully predict the rhythm reset in MD treated hepatoma cell line, HepG2.

A Feedback Loop between MicroRNA 155 (miR-155), Programmed Cell Death 4, and Activation Protein 1 Modulates the Expression of miR-155 and Tumorigenesis in Tongue Cancer.

MicroRNA 155 (miR-155) is an oncomir, generated as a noncoding RNA from the BIC gene whose promoter activity is mainly controlled via activation protein 1 (AP-1) and NF-κB transcription factors. We found that the expression levels of miR-155 and programmed cell death 4 (Pdcd4) exhibit inverse relationships in tongue cancer cells (SAS and AWL) and tumor tissues compared to their relationships in normal FBM cells and normal tongue tissues, respectively. In silico and in vitro studies with the 3' untranslated region (UTR) of Pdcd4 via luciferase reporter assays, quantitative PCR (qPCR), and Western blotting showed that miR-155 directly targets Pdcd4 mRNA and blocks its expression. Ectopic expression of Pdcd4 or knockdown of miR-155 in tongue cancer cells predominantly reduces AP-1-dependent transcriptional activity of the BIC promoter and decreases miR-155 expression. In this study, we demonstrate that miR-155 expression is modulated by a feedback loop between Pdcd4, AP-1, and miR-155 which results in enhanced expression of miR-155 with a consequent progression of tongue tumorigenesis. Further, miR-155 knockdown increases apoptosis, arrests the cell cycle, regresses tumor size in xenograft nude mice, and reduces cell viability and colony formation in soft-agar and clonogenic assays. Thus, the restoration of Pdcd4 levels by the use of molecular manipulation such as using a miR-155 sponge has an essential role in the therapeutic intervention of cancers, including tongue cancer.

microRNA-145 downregulates SIP1-expression but differentially regulates proliferation, migration, invasion and Wnt signaling in SW480 and SW620 cells.

microRNA-145 (miR-145) has been shown to act as a tumor suppressor in colorectal cancer but its role in the regulation of epithelial-mesenchymal transition (EMT) is unclear. Ectopic expression of miR-145 suppressed the proliferation, migration and invasion in SW480 but surprisingly enhanced these traits in its metastatic counterpart, SW620 cells, while, antimiR-145 reversed the effects of miR-145 in both of these human colorectal cancer cells. In SW480 and SW620 cells, SMAD-interacting protein 1 (SIP1), was identified as a target of miR-145, and its expression was suppressed both at mRNA and protein levels, and siRNA-SIP1 mimicked the effects of miR-145. Further, re-introduction of SIP1 alone or its co-expression with miR-145, rescued SW480 and SW620 cells from the effects of miR-145, indicating that the distinct functions of miR-145 might be mediated, in part, through SIP1. Since Wnt signaling plays an essential role in EMT in CRC progression, the effects of miR-145 on the expression of Wnt signaling intermediates and EMT markers were studied. Re-expression of miR-145 was found to downregulate the expression of CTNNB1, TCF4, CCND1, VIM, and SNAI, but, upregulated CDH1 expression in SW480 cells. On the other hand, miR-145 exhibited an oncogenic potential in SW620 cells by actuating Wnt signaling and the expression of EMT-relevant markers. These results strongly hint that the paradoxical functions of miR-145 in the regulation of proliferation, migration and invasion might be mediated through downregulation of SIP1, and differential tuning of Wnt signaling and EMT-mediators.