Ramakrishna Vadde

Anthocyanin-containing purple-fleshed potatoes suppress colon tumorigenesis via elimination of colon cancer stem cells

Cancer stem cells (CSCs) are shown to be responsible for initiation and progression of tumors in a variety of cancers. We previously showed that anthocyanin-containing baked purple-fleshed potato (PP) extracts (PA) suppressed early and advanced human colon cancer cell proliferation and induced apoptosis, but their effect on colon CSCs is not known. Considering the evidence of bioactive compounds, such as anthocyanins, against cancers, there is a critical need to study anticancer activity of PP, a global food crop, against colon CSCs. Thus, isolated colon CSCs (positive for CD44, CD133 and ALDH1b1 markers) with functioning p53 and shRNA-attenuated p53 were treated with PA at 5.0 μg/ml. Effects of baked PP (20% wt/wt) against colon CSCs were also tested in vivo in mice with azoxymethane-induced colon tumorigenesis. Effects of PA/PP were compared to positive control sulindac. In vitro, PA suppressed proliferation and elevated apoptosis in a p53-independent manner in colon CSCs. PA, but not sulindac, suppressed levels of Wnt pathway effector β-catenin (a critical regulator of CSC proliferation) and its downstream proteins (c-Myc and cyclin D1) and elevated Bax and cytochrome c, proteins-mediating mitochondrial apoptosis. In vivo, PP reduced the number of crypts containing cells with nuclear β-catenin (an indicator of colon CSCs) via induction of apoptosis and suppressed tumor incidence similar to that of sulindac. Combined, our data suggest that PP may contribute to reduced colon CSCs number and tumor incidence in vivo via suppression of Wnt/β-catenin signaling and elevation of mitochondria-mediated apoptosis.

Synthesis, pharmacological assessment, molecular modeling and in silico studies of fused tricyclic coumarin derivatives as a new family of multifunctional anti-Alzheimer agents.

A series of fused tricyclic coumarin derivatives bearing iminopyran ring connected to various amido moieties were developed as potential multifunctional anti-Alzheimer agents for their cholinesterase inhibitory and radical scavenging activities. In vitro studies revealed that most of these compounds exhibited high inhibitory activity on acetylcholinesterase (AChE), with IC50 values ranging from 0.003 to 0.357 μM which is 2-220 folds more potent than the positive control, galantamine. Their inhibition selectivity against AChE over butyrylcholinesterase (BuChE) has increased about 194 fold compared with galantamine. The developed compounds also showed potent ABTS radical scavenging activity (IC50 7.98-15.99 μM). Specifically, the most potent AChE inhibitor 6n (IC50 0.003 ± 0.0007 μM) has an excellent antioxidant profile as determined by the ABTS method (IC50 7.98 ± 0.77 μM). Moreover, cell viability studies in SK N SH cells showed that the compounds 6m-q have significant neuroprotective effects against H2O2-induced cell death, and are not neurotoxic at all concentrations except 6n and 6q. The kinetic analysis of compound 6n proved that it is a mixed-type inhibitor for EeAChE (Ki1 0.0103 μM and Ki2 0.0193 μM). Accordingly, the molecular modeling study demonstrated that 6m-q with substituted benzyl amido moiety possessed an optimal docking pose with interactions at catalytic active site (CAS) and peripheral anionic site (PAS) of AChE simultaneously and thereby they might prevent aggregation of Aβ induced by AChE. Furthermore, in silico ADMET prediction studies indicated that these compounds satisfied all the characteristics of CNS acting drugs. Most active inhibitor 6n is permeable to BBB as determined in the in vivo brain AChE activity. To sum up, the multipotent therapuetic profile of these novel tricyclic coumarins makes them promising leads for developing anti-Alzheimer agents.

Eugenia jambolana (Java Plum) Fruit Extract Exhibits Anti-Cancer Activity against Early Stage Human HCT-116 Colon Cancer Cells and Colon Cancer Stem Cells

The World Health Organization predicts over a 70% increase in cancer incidents in developing nations over the next decade. Although these nations have limited access to novel therapeutics, they do have access to foods that contain chemopreventive bioactive compounds such as anthocyanins, and as such, consumption of these foods can be encouraged to combat cancer. We and others have previously characterized the anti-colon cancer properties of dietary anthocyanins from different sources. Eugenia jambolana (Java plum) is a tropical medicinal fruit rich in anthocyanins, however, its anti-colon cancer properties are not well characterized. Furthermore, recent evidence suggests that colon cancer stem cells (colon CSCs) promote resistance to chemotherapy, relapse of tumors and contribute to poor prognosis. The objectives of this study were to 1) characterize the anthocyanin profile of Java plum using HPLC-MS; and 2) determine the anti-proliferative (cell counting and MTT) and pro-apoptotic (TUNEL and caspase 3/7 glo assay) properties of Java plum fruit extract (JPE) using HCT-116 colon cancer cell line and colon CSCs (positive for CD 44, CD 133 and ALDH1b1 markers). HPLC-MS analysis showed that JPE contains a variety of anthocyanins including glucosides of delphinidin, cyanidin, petunidin, peonidin and malvidin. JPE anthocyanins suppressed (p < 0.05) proliferation in HCT-116 cells and elevated (p < 0.05) apoptosis in both HCT-116 cells and colon CSCs. JPE also suppressed the stemness in colon CSCs as evaluated using colony formation assay. These results warrant further assessment of the anti-cancer activity of JPE, and its molecular mechanisms using pre-clinical models of colon cancer.

Triphala Extract Suppresses Proliferation and Induces Apoptosis in Human Colon Cancer Stem Cells via Suppressing c-Myc/Cyclin D1 and Elevation of Bax/Bcl-2 Ratio

Colon cancer is the second leading cause of cancer related deaths in the USA. Cancer stem cells (CSCs) have the ability to drive continued expansion of the population of malignant cells. Therefore, strategies that target CSCs could be effective against colon cancer and in reducing the risk of relapse and metastasis. In this study, we evaluated the antiproliferative and proapoptotic effects of triphala, a widely used formulation in Indian traditional medicine, on HCT116 colon cancer cells and human colon cancer stem cells (HCCSCs). The total phenolic content, antioxidant activity, and phytochemical composition (LC-MS-MS) of methanol extract of triphala (MET) were also measured. We observed that MET contains a variety of phenolics including naringin, quercetin, homoorientin, and isorhamnetin. MET suppressed proliferation independent of p53 status in HCT116 and in HCCSCs. MET also induced p53-independent apoptosis in HCCSCs as indicated by elevated levels of cleaved PARP. Western blotting data suggested that MET suppressed protein levels of c-Myc and cyclin D1, key proteins involved in proliferation, and induced apoptosis through elevation of Bax/Bcl-2 ratio. Furthermore, MET inhibited HCCSCs colony formation, a measure of CSCs self-renewal ability. Anticancer effects of triphala observed in our study warrant future studies to determine its efficacy in vivo.

Grape compounds suppress colon cancer stem cells in vitro and in a rodent model of colon carcinogenesis

BackgroundWe have previously shown that the grape bioactive compound resveratrol (RSV) potentiates grape seed extract (GSE)-induced colon cancer cell apoptosis at physiologically relevant concentrations. However, RSV-GSE combination efficacy against colon cancer stem cells (CSCs), which play a key role in chemotherapy and radiation resistance, is not known.MethodsWe tested the anti-cancer efficacy of the RSV-GSE against colon CSCs using isolated human colon CSCs in vitro and an azoxymethane-induced mouse model of colon carcinogenesis in vivo.ResultsRSV-GSE suppressed tumor incidence similar to sulindac, without any gastrointestinal toxicity. Additionally, RSV-GSE treatment reduced the number of crypts containing cells with nuclear β-catenin (an indicator of colon CSCs) via induction of apoptosis. In vitro, RSV-GSE suppressed - proliferation, sphere formation, nuclear translocation of β-catenin (a critical regulator of CSC proliferation) similar to sulindac in isolated human colon CSCs. RSV-GSE, but not sulindac, suppressed downstream protein levels of Wnt/β-catenin pathway, c-Myc and cyclin D1. RSV-GSE also induced mitochondrial-mediated apoptosis in colon CSCs characterized by elevated p53, Bax/Bcl-2 ratio and cleaved PARP. Furthermore, shRNA-mediated knockdown of p53, a tumor suppressor gene, in colon CSCs did not alter efficacy of RSV-GSE.ConclusionThe suppression of Wnt/β-catenin signaling and elevated mitochondrial-mediated apoptosis in colon CSCs support potential clinical testing/application of grape bioactives for colon cancer prevention and/or therapy.

Role of hypoxia-inducible factors (HIF) in the maintenance of stemness and malignancy of colorectal cancer

Hypoxia is a condition of insufficient tissue oxygenation, which is observed during normal development as well as tumorigenesis and its response at the cellular level is primarily mediated through hypoxia inducible factors (HIFs). HIFs have a significant role in the maintenance of stemness in both stem cells as well as in cancer stem cells (CSC) by acting as transcription factors. The CSCs are proposed to be the driving force of colon tumorigenesis and malignancy. These HIFs play a significant role in a wide range of diseases including colon cancer. HIFs signaling functions with stemness, and maintaining Wnt/β-catenin signaling pathways. Due to HIFs functional significance in stemness maintenance in malignancy, targeting HIFs might provide a new approach for development of new therapy for colon cancer. In this review, we will be briefing on the colon and its stem cells, various molecular signaling pathways involved in stemness preservation, and the role hypoxia and its HIFs in the maintenance of stemness in colon stem cells and colon cancer stem cells.

Synthesis, Biological Evaluation, and Molecular Docking of 8-imino-2-oxo-2H,8H-pyrano[2,3-f]chromene Analogs: New Dual AChE Inhibitors as Potential Drugs for the Treatment of Alzheimer's Disease

Alzheimers disease onset and progression are associated with the dysregulation of multiple and complex physiological processes, and a successful therapeutic approach should therefore address more than one target. In line with this modern paradigm, a series of 8‐imino‐2‐oxo‐2H,8H‐pyrano[2,3‐f]chromene analogs (4a–q) were synthesized and evaluated for their multitarget‐directed activity on acetylcholinesterase, butyrylcholinesterase (BuChE), 2,2’‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid) (ABTS) radical, and amyloid‐β peptide (Aβ) specific targets for Alzheimers disease therapy. Most of the synthesized compounds showed remarkable acetylcholinesterase inhibitory activities in low nm concentrations and good ABTS radical scavenging activity, however, no evidence of BuChE inhibitory activity. Among them, 3‐bromobenzylamide derivative 4m exhibited the best acetylcholinesterase inhibitory activity with IC50 value of 13 ± 1.4 nm which is 51‐fold superior to galantamine, a reference drug. Kinetic and molecular docking studies indicated 4m as mixed‐type inhibitor, binding simultaneously to catalytic active and peripheral anionic sites of acetylcholinesterase. Five compounds 4e, 4f, 4g, 4j, and 4k have shown 1.4‐ to 2.5‐fold of higher antioxidant activities than trolox. Interestingly, the most active compound 4m demonstrated dosage‐dependent acceleration of Aβ1−42 aggregation, which may reduce toxicity of oligomers. Overall, these results lead to discovery of fused tricyclic coumarins as promising dual binding site inhibitors of acetylcholinesterase and afford multifunctional compounds with potential impact for further pharmacological development in Alzheimers therapy.

Phytochemical profiling and in vitro screening for anticholinesterase, antioxidant, antiglucosidase and neuroprotective effect of three traditional medicinal plants for Alzheimer’s Disease and Diabetes Mellitus dual therapy

BackgroundExtensive epidemiological and clinical studies revealed that Alzheimer’s Disease (AD) and Type 2 Diabetes Mellitus (T2D) are most likely to appear simultaneously in aged people as T2D is a major risk factor for AD. Therefore, development of potential multifunctional agents for dual therapy of AD and T2D has received much attention. Buchanania axillaris, Hemidesmus indicus and Rhus mysorensis have been used extensively in popular medicine. The present study was aimed at phytochemical profiling and evaluating multifunctional ability of titled plants in the AD and T2D dual therapy.MethodsMethanolic extracts and their derived fractions were evaluated for their inhibitory capacities against acetylcholinesterase (AChE) & butyrylcholinesterase (BuChE), and α– & β–glucosidase besides kinetic analysis of inhibition using methods of Elmann and Shibano, respectively. Antioxidant potency of active fractions was assessed by their DPPH and ABTS radical scavenging activities. Active fractions were tested by the MTT assay to verify cytotoxicity and neuroprotective ability in human nueroblastoma cell lines. Phytochemical screening was done with the aid of spectrophotometric methods.ResultsAll the methanolic extracts of test plants (BAM, HIM, RMM) showed concentration dependent inhibitory activities against AChE, BuChE, α– and β–glucosidase enzymes. Subsequent fractionation and evaluation revealed that chloroform fractions BAC, HIC and RMC with IC50 values of 12.29±2.14, 9.94±2.14, 16.65±1.99 and 27.38±1.24; 28.14±0.9, 5.16±0.22, 11.03±0.5 and 87.64±15.41; 41.35±1.6, 15.86±7.3, 26.04±0.37 and 25.33±0.3 were most prominent with regard to inhibition potential against AChE, BuChE, α– and β–glucosidase, respectively. Kinetic analysis of these active fractions proved that they disclosed mixed-type inhibition against AChE, BuChE, α– and β–glucosidase enzymes. In the MTT assay, active fractions BAC, HIC, RMC showed significant cell viability at high concentrations (400 μg). Moreover, in MTT assay, the active fractions displayed excellent neuroprotective effects against oxidative stress induced cell death and significant cell viability in SK N SH cells at all concentrations.ConclusionThe strong anticholinesterase, antiglucosidase, antioxidant and neuroprotective activities of methanolic extracts and their derived chloroform fractions indicate the potential of Buchanania axillaris, Hemidesmus indicus and Rhus mysorensis as multifunctional therapeutic remedies for the dual therapy of T2D and AD.

Ligand-Based Pharmacophore Modeling and Virtual Screening of RAD9 Inhibitors

Human RAD9 is a key cell-cycle checkpoint protein that participates in DNA repair, activation of multiple cell cycle phase checkpoints, and apoptosis. Aberrant RAD9 expression has been linked to breast, lung, thyroid, skin, and prostate tumorigenesis. Overexpression of RAD9 interacts with BCL-2 proteins and blocks the binding sites of BCL-2 family proteins to interact with chemotherapeutic drugs and leads to drug resistance. Focusing on this interaction, the present study was designed to identify the interaction sites of RAD9 to bind BCL-2 protein and also to inhibit RAD9-BCL-2 interactions by designing novel small molecule inhibitors using pharmacophore modeling and to restore BCL-2 for interacting with anticancer drugs. The bioactive molecules of natural origin act as excellent leads for new drug development. Thus, in the present study, we used the compounds of natural origin like camptothecin, ascididemin, and Dolastatin and also compared them with synthetic molecule NSC15520. The results revealed that camptothecin can act as an effective inhibitor among all the ligands taken and can be used as an RAD9 inhibitor. The amino acids ARG45 and ALA134 of RAD9 protein are interacting commonly with the drugs and BCL-2 protein.

In silico analysis of differential gene expressions in biliary stricture and hepatic carcinoma.

In-silico attempt was made to identify the key hub genes which get differentially expressed in biliary stricture and hepatic carcinoma. Gene expression data, GSE34166, was downloaded from the GEO database, which contains 10 biliary stricture samples (4 benign control and 6 malignant carcinoma), for screening of key hub genes associated with the disease. R packages scripts were identified 85 differentially expressed genes. Further these genes were uploaded in WebGestalt database and identified nine key genes. Using STRING database and Gephi software, the protein-protein interaction networks were constructed and also studied gene ontology through WebGestalt. Finally, we identified four key genes (CXCR4, ADH1C, ABCB1 and ADH1A) are associated with liver carcinoma and further cross-validated with Liverome, Protein Atlas database and bibliography. In addition, transcription factors and their binding sites also studied. These identified hub genes and their transcription factors are the probable potential targets for possible future drug design.