U. Subrahmanyeswara Rao

Galectin‐4 functions as a tumor suppressor of human colorectal cancer

Development of colorectal cancer (CRC) involves a series of genetic alterations with altered expression of proteins and cell signaling pathways. Here, we identified that galectin‐4 (gal‐4), a marker of differentiation, was down‐regulated in CRC. The goal of this work was to determine the function of gal‐4 in CRC. Toward this goal, the human colon biopsies and tissue microarrays containing a gradient of pathology were analyzed for gal‐4 expression by immunohistochemistry. Cell proliferation, migration, motility, forced expression, knockdown, cell cycle and apoptosis assays were used to characterize gal‐4 function. Immunohistochemistry identified that gal‐4 expression was significantly down‐regulated in adenomas and was essentially absent in invasive carcinomas. Forced expression of gal‐4 in gal‐4 −ve cells induced cell cycle arrest and retarded cell migration and motility. Further, gal‐4 sensitized the cells to camptothecin‐induced apoptosis. Gal‐4 knockdown resulted in increased cell proliferation, migration and motility. Gal‐4 was found to be associated with Wnt signaling proteins. Finally, gal‐4 expression led to down‐regulation of Wnt signaling target genes. This study demonstrates that loss of gal‐4 is a common and specific event in CRC. This study also shows that gal‐4 exhibits tumor suppressive effects in CRC cells in vitro. Through its ability to interact with and down‐regulate the functions of Wnt signaling pathway, gal‐4 reveals a new dimension in the control of the Wnt signaling pathway. Thus, gal‐4 may prove to be an important molecule in understanding the biology of CRC.

Luteolin induces apoptosis in multidrug resistant cancer cells without affecting the drug transporter function: involvement of cell line-specific apoptotic mechanisms.

Bioflavonoids are of considerable interest to human health as these serve as antioxidant and anticancer agents. Although epidemiological and experimental studies suggest that luteolin, a natural bioflavonoid, exhibits chemopreventive properties, its effectiveness as an antiproliferative agent against multidrug resistant (MDR) cancers is unclear. Thus, we assessed the antiproliferative effects of luteolin and associated molecular mechanisms using two MDR cancer cell lines that express high levels of P‐glycoprotein and ABCG2. In this article, we demonstrate that luteolin induces apoptosis in P‐glycoprotein‐ and ABCG2‐expressing MDR cancer cells without affecting the transport functions of these drug transporters. Analysis of various proliferative signaling pathways indicated that luteolin‐induced apoptosis involves reactive oxygen species generation, DNA damage, activation of ATR → Chk2 → p53 signaling pathway, inhibition of NF‐kB signaling pathway, activation of p38 pathway and depletion of antiapoptotic proteins. Importantly, use of luteolin in these analyses also identified specific molecular characteristics of NCI‐ADR/RES and MCF‐7/MitoR cells that highlight their different tissue origins. These results suggest that luteolin possesses therapeutic potential to control the proliferation of MDR cancers without affecting the physiological function of drug transporters in the body tissues.

Galectin-1 is silenced by promoter hypermethylation and its re-expression induces apoptosis in human colorectal cancer cells

Galectin-1 (gal-1) is an important molecule secreted by many tumors, which induces apoptosis in activated T-cells and promotes tumor angiogenesis, both of which phenomena facilitate successful establishment of tumor in the body. However, little is known about the function of intracellular gal-1 or its transcriptional regulation in colorectal cancer (CRC). Here, we demonstrate that gal-1 expression is epigenetically regulated in CRC through promoter hypermethylation. Intracellular gal-1 induces cell cycle arrest and apoptosis in CRC cells with concomitant down-regulation of Wnt and NF-κB signaling pathways. Together, these data suggested that gal-1 silencing imparts CRC with the ability to proliferate and escape apoptosis.

RNF2 is the target for phosphorylation by the p38 MAPK and ERK signaling pathways

RNF2, a member of polycomb group (PcG) proteins, is involved in chromatin remodeling. However, mechanisms that regulate RNF2 function are unknown. To identify such mechanisms, RNF2 was expressed in HEK‐293 cells and analyzed by 2‐D electrophoresis. RNF2 was resolved into at least seven protein spots, migrating toward the lower pI from its expected pI of 6.38, suggesting that RNF2 undergoes post‐translational modifications. Western blotting indicated that majority of these RNF2 spots contained phosphoserine(s), which were completely dephosphorylated upon treatment with a phosphatase. SB203580, a specific inhibitor of p38 MAPK, inhibited RNF2 phosphorylation at one site. On the other hand, PD98059, an inhibitor of MEK1/2, inhibited majority of the phosphorylation events in RNF2. Mass spectrometry analysis identified that RNF2 expressed in Sf9 insect cells undergoes co‐translational excision of 1Met coupled to N‐acetylation of 2Ser, and phosphorylation of 41Ser. Interestingly, 41Ser is a predicted p38/MAPK phosphorylation site, consistent with the loss of phosphorylation induced by SB203580. Further analysis indicated that RNF2 phosphorylation differentially modulates the expression of transcription factors and histone 2B acetylation. These results provide first evidence for phosphorylation of RNF2, and suggest that the mitogen activated protein kinases including p38 MAPK and ERK1/2 regulate growth, stress response, differentiation and other cellular processes, through phosphorylation of RNF2.

Strategies for implementation of an effective pharmacogenomics program in pharmacy education.

Sequencing of the human genome and the evidence correlating specific genetic variations to diseases have opened up the potential of genomics to more effective and less harmful interventions of human diseases. A wealth of pharmacogenomics knowledge is in place for the practice of precision medicine. However, this knowledge is not fully realized in clinical practice. One reason for this impasse is the lack of in-depth understanding of the potential of pharmacogenomics among the healthcare professionals. Pharmacists are the point-of-care providers and are expected to advise clinicians on matters relating to the implementation of pharmacogenomics in patient care. However, current pharmacogenomics instruction in pharmacy schools fails to produce pharmacists with the required knowledge or practical training in this discipline. In this perspective, we provide several strategies to overcome limitations faced by pharmacy schools. Once implemented, pharmacy schools will produce precision medicine-ready pharmacists.

Characterization of a New Antibody Raised against the NH2 Terminus of P-Glycoprotein

Purpose: Cancers exposed to chemotherapy develop multidrug resistance, a major cause for chemotherapy failure. One mechanism of multidrug resistance development is due to overexpression of P-glycoprotein (Pgp) in these cancer cells. Thus, a prechemotherapy evaluation of Pgp in cancer cells aids in the design of alternative regimens that can circumvent such failure. As few Pgp-specific antibodies are available in detecting low levels of Pgp, there is a need for preparing an antibody that allows the detection of Pgp by various immunologic methods. Experimental Design: We selected the amino acid stretch 11 to 34 in the cytoplasmically located NH2 terminus of Pgp as antigen, which was chemically synthesized and used to raise an antibody in a rabbit, termed NH211 antibody. We compared the properties of NH211 antibody with that of the well-characterized Pgp-specific antibody, C219, by Western blotting, immunoprecipitation, immunocytochemistry, and immunohistochemistry. Results: Immunoblotting analysis suggested that NH211 antibody efficiently interacts with both recombinant and constitutively expressed Pgp in cancerous and noncancerous human cells. Immunoprecipitation reactions indicated that the NH211 antibody selectively immunoprecipitates Pgp. Immunocytochemical analyses indicated that the NH211 antibody detects Pgp in drug-resistant breast cancer cells as well as in human prostate and breast adenocarcinoma tissue sections. Conclusion: As the NH211 antibody detects Pgp present in cells and tissues, we conclude that the amino acid sequence to which this antibody was raised is highly antigenic and the antibody is useful in the detection of Pgp by a variety of immunologic methods.

The influence of metastatic site on the expression of CEA and cellular localization of β-catenin in colorectal cancer

The usefulness of carcinoembryonic antigen (CEA) in the diagnosis and prognosis of colorectal cancer (CRC) is unclear. The aim was to analyze changes in the expression of CEA during CRC progression and metastasis, so as to determine the influence of tumor metastatic organ on the CEA expression by CRC cells.

Abstract 2729: Galectin-4 in colorectal cancer

Introduction: Colorectal cancer (CRC) is one of the leading diseases in the western world and is curable if diagnosed at an early stage. The normal-adenoma-carcinoma sequence concept suggests that the normal epithelia in the colorectal region transform into adenomas, which progress to invasive carcinomas. The role of galectin-4 (gal-4) a protein abundantly expressed in normal colonic epithelia, hereto considered a differentiation marker, on the onset or progression of colorectal cancer (CRC) is unknown. The main aim of this study is to evaluate gal-4 as a potential biomarker in detecting the onset of CRC and to determine the function of gal-4 in CRC development. Methods: We analyzed the paraffin-embedded normal, adenomatous polyps and invasive carcinomas of the human colon biopsies and tissue microarrays for gal-4 expression by immunohistochemistry. Gal-4 expression in CRC cell lines was characterized by RT-PCR, western blotting, immunofluorescence microscopy and FACS analysis. The functional role of gal-4 was examined by cell proliferation, migration, motility, transfection and RNAi experiments. Results: Biopsies immunostained for gal-4 were quantified using ImageJ software. The normal tissue as well as normal tissue adjacent to cancer contained highest amount of gal-4. Gal-4 intensity decreased in all the disease-states, which was found to be statistically significant (P Conclusions: We conclude that galectin-4 plays a key role the maintenance of epithelial cells in a differentiated state, and its loss allows the cells to de-differentiate, leading to increased cell proliferation, migration and motility, which are hallmark features of cancer. Galectin-4 brings about these changes through cell cycle regulation by modulating the cyclin D1, p15 and p21 levels. Finally, our findings that extracellular galectin-4 retards the migratory potential raise a possibility for it to serve as a therapeutic agent in controlling CRC progression. Thus beyond shedding light on the role of tumor-suppressor gal-4 in CRC, this study underscores the potential clinical value of gal-4 in prognosis and diagnosis of CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2729.