Statins decrease the expression of c-Myc protein in cancer cell lines.

Statins are potent inhibitors of the mevalonate/cholesterol biosynthetic pathway and are widely prescribed for the prevention of cardiovascular diseases. Here, we carried out a comprehensive analysis of the effects of three statins, simvastatin, atorvastatin, and lovastatin, on six different cancer cell lines that include a P-glycoprotein-expressing, multidrug resistant variant of an ovarian cancer cell line. Incubation of all cancer cell lines with statins resulted in suppression of cell proliferation without inducing apoptotic cell death. The cell proliferation arrest could be reversed upon transfer of cells to statin-free growth media as well as by the supplementation of the growth media with mevalonate. Further analysis suggested that statins induced cell cycle arrest at G0/G1 phase in four cancer cell lines and the loss of c-Myc protein in three cancer cell lines. The c-Myc expression and the progression of cell division cycle were restored upon the addition of mevalonate to the culture media containing statins. Finally, cells incubated with statins contained an increased level of phosphorylated histone H2AX, an observation previously correlated to cellular senescence. Together, these data demonstrate that statins inhibit the mevalonate pathway which is tightly coupled to oxidative branch of the pentose phosphate pathway, c-Myc expression, cell division cycle progression, and cellular senescence. Implications of these observations in the application of statins as cancer therapeutics are discussed.

A 6-week laboratory research rotation in pharmacogenomics: a model for preparing pharmacy students to practice precision medicine.

Comparison of human genome sequences from different individuals has unraveled that genes involved in the drug efficacy and metabolism are polymorphic, harboring mutations, splicing variations and other alterations. These data provide a reasonable explanation for the inter-individual variations observed in drug therapy. Thus, a detailed molecular analysis and an in-depth knowledge of these genes is a prerequisite to practice pharmacogenomics-based medicine. We have introduced a 6-week laboratory research rotation to train students in the expression analysis of different pharmacogenes combined with bioinformatics tools. Students were first introduced to the bioinformatics tools to identify appropriate DNA primers to amplify specific pharmacogenes from the laboratory cancer cell lines. The amplified DNA fragments were sequenced. Finally, students were trained in bioinformatics tools to establish the identity of these DNA sequences. The possible implications of this laboratory training in developing problem-solving skills needed in the implementation of pharmacogenomics knowledge in the clinic, are discussed.

Surface-bound galectin-4 regulates gene transcription and secretion of chemokines in human colorectal cancer cell lines.

One long-term complication of chronic intestinal inflammation is the development of colorectal cancer. However, the mechanisms linking inflammation to the colorectal tumorigenesis are poorly defined. Previously, we have demonstrated that galectin-4 is predominantly expressed in the luminal epithelia of the gastrointestinal tract, and its loss of expression plays a key role in the colorectal tumorigenesis. However, the mechanism by which galectin-4 regulates inflammation-induced tumorigenesis is unclear. Here, we show that galectin-4 secreted by the colorectal cancer cell lines was bound to the cell surface. Neutralization of surface-bound galectin-4 with anti-galectin-4 antibody resulted in increased cell proliferation with concomitant secretion of several chemokines into the extracellular medium. Neutralization of the surface-bound galectin-4 also resulted in the up-regulation of transcription of 29 genes, several of which are components of multiple inflammation signaling pathways. In an alternate experiment, binding of recombinant galectin-4 protein to cell surface of the galectin-4-negative colorectal cancer cells resulted in increased p27, and decreased cyclin D1 and c-Myc levels, leading to cell cycle arrest and apoptosis. Together, these data demonstrated that surface-bound galectin-4 is a dual function protein-down-regulating cell proliferation and chemokine secretion in galectin-4-expressing colorectal cancer cells on one hand and inducing apoptosis in galectin-4-negative colorectal cancer cells on the other hand.

Heterogeneity in the expression of receptors in the human breast cancer metastasized to the brain.

Assessment of the human epidermal growth factor receptor-2 (Her2/ErbB2) and estrogen receptor (ER) and progesterone receptor (PR) expression in breast cancer has been an accepted standard to predict clinical outcome. Expression of these receptors in primary breast cancer has also been an important predictor of visceral organ metastasis. Many studies of breast cancer have reported risk factors for brain metastasis that include Her2/ErbB2 positivity, ER negativity, and negativity for all the above three receptors. However, it is not clear whether expression of these receptors would persist subsequent to brain metastasis. To address this possibility, we analyzed different breast cancer brain metastases (BCBM) for the expression of Her2/ErbB2, ER, and PR by immunohistochemistry procedure. The results showed that BCBM are heterogeneous in the receptor expression: Five BCBMs were Her2/ErbB2-positive and one negative; four BCBMs were ER-positive, and two were negative; five BCBMs were PR-positive and one negative. However, expression of these receptors in their combination is also heterogeneous: Four BCBMs were positive for all of the Her2/ErbB2, ER, and PR; one BCBM was positive for Her2/ErbB2 and PR but negative for ER; one BCBM was positive for PR but negative for Her2/ErbB2/ER. Similar heterogeneity in the expression of these receptors was also observed in primary tumors. Importantly, BCBM tumors that were assigned as ER- and PR-positive contained tumor cells that lacked expression of these receptors in other regions of the biopsies. Taken together, our findings indicate that the BCBM exhibit heterogeneity in the expression amounts of Her2/ErbB2, ER, and PR, which could be a result of the influence of tumor microenvironment in the brain or different tumor cells populating the metastatic region.

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

BACKGROUND AND AIM: 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. METHODS: The human biopsies of adenocarcinomas in colon and CRC liver and lung metastases were analyzed by immunohistochemistry for the expression of CEA. Expression of E-cadherin and ß-catenin was also analyzed to localize the CRC neoplastic glands in metastatic tissues. RESULTS: The CRC neoplastic glands in colon and liver expressed significantly higher amount of CEA compared with crypts in normal colon. In contrast, CRC neoplastic glands formed in lung expressed low CEA level. However, CEA expression was high in areas of tumor necrosis in lung. E-cadherin and ß-catenin were cell membrane-bound in normal crypts and CRC neoplastic glands in colon and liver. Although these two proteins were also cell membrane-bound in a majority of CRC neoplastic glands in lungs, a significant proportion of these expressed ß-catenin in the nucleus, which lacked either E-cadherin or ß-catenin at the cell membrane. CONCLUSION: Our findings indicate that lung microenvironment is unique in that it suppresses the expression of CEA by CRC cells forming neoplastic glands. In addition, lung microenvironment promotes nuclear localization of ß-catenin, suggesting that the Wnt signaling pathway is relatively active highly in CRC metastasized to lung, when compared with liver or colon.

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/Mito(R) 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-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.

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 (1)Met coupled to N-acetylation of (2)Ser, and phosphorylation of (41)Ser. Interestingly, (41)Ser 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.

Bap29varP, a variant of Bap29, influences the cell surface expression of the human P-glycoprotein.

P-glycoprotein (Pgp), a plasma membrane (PM) glycoprotein, is responsible for the development of multidrug resistance. The mechanism by which Pgp is targeted to the PM is not defined. To identify proteins that influence Pgp trafficking, we utilized the yeast two-hybrid analysis procedure, which identified a new isoform of endoplasmic reticulum (ER)-bound Bap29, termed Bap29varP, as an interacting protein with the N-terminus of Pgp. The drug-resistant human breast cancer MCF-7 (MCF-7/Adr(R)) cells express both Bap29varP and approximately 170 kDa Pgp, which are however absent in the drug-sensitive MCF-7 cells. When Bap29varP was overexpressed in MCF-7/Adr(R) cells, Pgp was predominantly localized in the ER and intracellular vesicles, suggesting Bap29varP influences Pgp trafficking. When Pgp was expressed in MCF-7 cells, it was exclusively found in the ER with a molecular mass of approximately 160 kDa slightly smaller than that of the molecular mass of Pgp expressed in MCF-7/Adr(R) cells. On the other hand, when Pgp was expressed in Bap29varP-containing human colon adenocarcinoma HT-29 cells, it was localized at the PM. These findings together suggest that Bap29varP acts as an essential chaperone, influencing the processing and trafficking of Pgp to the cell surface.

Varied expression and localization of multiple galectins in different cancer cell lines.

Galectins play a key role in oncogenic processes. Although several galectins are known, their relative expression at the mRNA and protein levels, the subcellular localization, and their relationship to the oncogenic manifestation remains unclear. Herein we report a comprehensive characterization of the expression of major galectins in human breast cancer (drug-sensitive MCF-7 and drug-resistant MCF-7/Adr(R)), colon cancer (HCT-116 and HT-29), and glioma (T98G) cell lines, as these cells are common model systems for studying oncogenic processes. The expected approximately 14.5 kDa galectin-1, predominantly cytosolic, was detected in the cancer and normal cell lines. Notably, two different molecular forms of galectin-1 with molecular masses of approximately 13.5 and 15 kDa were detected in T98G cells, the latter being in the extracellular medium, perhaps a result of post-translational processing. Immunocytochemistry indicated that the extracellular galectin-1 bound to the cell surface was punctated in appearance, suggesting that it was bound to specific receptors. Immunohistological studies indicated that metastasizing carcinomas express high levels of galectin-1. On the other hand, galectin-3 was readily detectable in all cancer cell lines but undetectable in normal cell lines, indicating that galectin-3 is a cancer-specific biomarker protein. Galectin-3 was a cytosolic protein but was not detected in the extracellular medium, indicating that cancer cells do not secrete this galectin. Finally, despite the RT-PCR analysis suggesting the presence of two transcripts of galectin-8 in all cancer cell lines, the corresponding approximately 36 kDa protein was only detectable in the nuclear and cytosolic fractions upon cell fractionation. Notably, a different molecular form of galectin-8 of approximately 18 kDa was immunoprecipitated from the extracellular media, suggesting that the secreted galectin-8 undergoes post-translational processing. These results highlight the expression of galectins in different molecular forms in cancers, warranting caution in interpreting the results of functional studies of individual galectins, particularly because these proteins function redundantly in cancer pathways.

E-cadherin phosphorylation by protein kinase D1/protein kinase C{mu} is associated with altered cellular aggregation and motility in prostate cancer.

The cadherin family of transmembrane glycoproteins plays a critical role in cell-to-cell adhesion and cadherin dysregulation is strongly associated with cancer metastasis and progression. In this study, we report a novel interaction between protein kinase D1 [PKD1; formerly known as protein kinase C mu (PKCmu)] and E-cadherin. PKD1 is a serine/threonine-specific kinase known to play a role in multiple cellular processes including apoptosis, cytoskeleton remodeling, and invasion. Our study shows that PKD1 colocalizes with E-cadherin at cell junctions in LNCaP prostate cancer cells and coimmunoprecipitates with E-cadherin from lysates of LNCaP cells. In vitro kinase assays have shown that PKD1 phosphorylates E-cadherin. Inhibition of PKD1 activity by the selective inhibitor Gö6976 in LNCaP cells resulted in decreased cellular aggregation and overexpression of PKD1 in C4-2 prostate cancer cells increased cellular aggregation and decreased cellular motility. We also validated the PKD1 and E-cadherin colocalization in human prostate cancer tissue by confocal laser scanning microscopy. Our study has identified E-cadherin as a novel substrate of PKD1, and phosphorylation of E-cadherin by PKD1 is associated with increased cellular aggregation and decreased cellular motility in prostate cancer. Because both E-cadherin and PKD1 are known to be dysregulated in prostate cancer, our study identified an important protein-protein interaction influencing the signal transduction system associated with cell adhesion in prostate cancer.

RNF2 interacts with the linker region of the human P-glycoprotein.

The human P-glycoprotein (Pgp) is a drug-efflux pump responsible for innate or acquired multidrug resistance in many cancers. Pgp contains a unique approximately 75 amino acid long linker region in its middle, which is critically important for its drug transport and ATPase functions. To identify cellular proteins that bind to this linker region and modulate Pgp function, a yeast two-hybrid analysis was carried out. This procedure identified RNF2 (RING finger protein 2), an E3 ubiquitin ligase, as a prominent Pgp-interacting protein. Co-expression of RNF2 with Pgp in Sf9 insect cells resulted in decreased ATPase activity and proteolytic protection of the transporter protein. Immunoprecipitation experiments confirmed the physical interaction between these two proteins. Confocal microscopy showed the presence of RNF2 in the cytoplasm of the Pgp-negative, drug-sensitive MCF-7 breast cancer cells. However, it was undetectable in the Pgp-positive and drug-resistant MCF-7 cells. We suggest that RNF2 regulates the cellular abundance of Pgp, and plays a key role in the development of cancer drug resistance through its own down-regulation.

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 NH2 11 antibody. We compared the properties of NH2 11 antibody with that of the well-characterized Pgp-specific antibody, C219, by Western blotting, immunoprecipitation, immunocytochemistry, and immunohistochemistry. RESULTS: Immunoblotting analysis suggested that NH2 11 antibody efficiently interacts with both recombinant and constitutively expressed Pgp in cancerous and noncancerous human cells. Immunoprecipitation reactions indicated that the NH2 11 antibody selectively immunoprecipitates Pgp. Immunocytochemical analyses indicated that the NH2 11 antibody detects Pgp in drug-resistant breast cancer cells as well as in human prostate and breast adenocarcinoma tissue sections. CONCLUSION: As the NH2 11 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.

Tissue transglutaminase interacts with protein kinase A anchor protein 13 in prostate cancer.

We have previously described that tissue transglutaminase (tTG) is a high level phenotypic biomarker in prostate cancer, which is down regulated in prostate cancer and surrounding premalignant field compared to benign prostate glands. To understand the function of tTG in prostate cancer, we sought to identify proteins that interact with the transglutaminase moiety of tTG using a human prostate cancer complementary deoxyribonucleic acid library in a Yeast 2-Hybrid system. The Yeast 2-Hybrid experiments identified a strong and novel interaction between the transglutaminase moiety and protein kinase A anchor protein 13 (AKAP13), which was quantified by beta-galactosidase assay, confirmed in vitro by immunoprecipitation experiments using PC3 prostate cancer cell lysates, and in vivo colocalization was confirmed by immunofluorescence studies in PC3 cells. Because AKAP plays a major role in protein kinase A and Rho protein mediated signaling, functional studies are underway to elucidate the significance of tTG-AKAP13 interaction in prostate cancer.

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.

Microarray analysis of differential gene expression in androgen independent prostate cancer using a metastatic human prostate cancer cell line model.

Progression to androgen independence (AI) leading to uncontrolled cell growth is the main cause of death in prostate cancer. While almost all patients with metastatic prostate cancer will initially respond to anti-androgen treatments, the majority will fail hormonal treatments in less than 2 yrs. Both genetic and epigenetic alterations in gene expression contribute significantly to the development of AI. To investigate this we have used an in vitro cell line model of AI prostate cancer from which we have identified a number of differentially expressed genes associated with progression to AI in prostate cancer. We used an in vitro cell line model of AI prostate cancer, to study differential gene expression using cDNA microarray analysis and corroborated the microarray results with Ribonuclease Protection Assay (RPA). Approximately 4480 out of 7075 (63.3%) cDNA cloned genes were differentially expressed, of which, 6 genes were differentially expressed by at least fivefold. RPA was used to corroborate the microarray results for the five most highly differentially expressed genes. Using an in vitro cell line model and microarray analysis we have identified a number of candidate genes for further investigation in AI prostate cancer.

Protein kinase C mu is down-regulated in androgen-independent prostate cancer.

Progression to androgen independence (AI) is the main cause of death in prostate cancer. Our prior differential gene expression studies by microarray analysis in progressive prostate cancer cell line model identified dysregulation of protein kinase C mu (PKCmu) expression in prostate cancer. In this study, quantitative ribonuclease protection assay and immunoblot analysis demonstrate down regulation of PKCmu at transcription and translational level, respectively, in AI C4-2 cells compared to its parental androgen dependent (AD) LNCaP prostate cancer cells. Significantly lower PKCmu kinase activity was confirmed in C4-2 cells by in vitro kinase assay. Immunohistochemical studies of prostate cancer tissue from patient progressing to AI prostate cancer demonstrated that PKCmu expression is decreased in 100% of AI human prostate cancers. The consistent down regulation of PKCmu in cell line models and human prostate cancer tissues suggests a possible functionally significant role for PKCmu in progression to AI in prostate cancer.

Metallothionein 2A interacts with the kinase domain of PKCmu in prostate cancer.

Prostate cancer (PC) patients die from progression to androgen independence (AI) and chemoresistance (CR). Protein kinase Cmu (PKCmu) a novel member of the PKC family of signal transduction proteins is downregulated in AI PC. Studying PKCmu interactors in the yeast two-hybrid system identified metallothionein 2A (MT 2A) as an interactor of PKCmu kinase domain (KD) in PC, which was quantified by beta-gal assay, confirmed in PC cells by immunoprecipitation, and PKCmu-MT 2A co-localization in vivo by immunofluorescence studies. PKCmu domain interaction studies revealed that MT 2A interacted strongly with KD, relatively weakly with C1, and failed to interact with C2, PH or kinase mutant domains. Peptide library and in silico analysis strongly suggest that MT 2A is a novel substrate of PKCmu and our data indicate that the PKCmu-MT 2A interaction depends on PKCmu kinase activity. Because metallothioneins are associated with cell proliferation and CR, the PKCmu-MT 2A interaction may contribute to CR and/or AI in PC.