Biomedical subjects

Dimpy Koul

Publications and source records attributed to Dimpy Koul.

13 recordsLinked to original sources

PubMed2006

PTEN enhances TNF-induced apoptosis through modulation of nuclear factor-kappaB signaling pathway in human glioma cells.

The PTEN tumor suppressor gene modulates cell growth and survival known to be regulated by the activation of the transcription factor NFkappaB, suggesting PTEN might affect the NFkappaB activation pathway. We found that PTEN inhibited NFkappaB activation induced by TNF. The suppression of NFkappaB activation correlated with sequential inhibition of the tumor necrosis factor-induced expression of NFkappaB-regulated anti-apoptotic (IAP1, IAP2, Bcl-2, Bcl-xL, cFLIP, Bfl-1/A1, and survivin) gene products. Downregulation of the antiapoptotic genes by PTEN increased TNF-induced apoptosis, as indicated by caspase activation, TUNEL, annexin staining, and esterase assay. We conclude that the ectopic expression of PTEN enhances TNF-induced apoptosis and downregulates the proliferation of glioma cells through the suppression of various molecules including NFkappaB, and various mediators of cellular survival and proliferation, and that this targets might be essential for its central role in the growth and survival of glioma cancer cells.

Apoptosis↗

PubMed2006

Inhibition of Akt survival pathway by a small-molecule inhibitor in human glioblastoma.

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and Akt are important regulators of the phosphatidylinositol 3-kinase (PI3K) pathway and thus are important to the regulation of a wide spectrum of tumor-related biological processes. Akt regulates several critical cellular functions, including cell cycle progression; cell migration, invasion, and survival; and angiogenesis. Decreased expression of PTEN and overexpression of the Akt proto-oncogene, which is located downstream of PI3K, have been shown in a variety of cancers, including glioblastoma. Novel small-molecule inhibitors of receptors and signaling pathways, including inhibitors of the PI3K pathway, have shown antitumor activity, but inhibitors of Akt have not been examined. In this study, we tested our hypothesis that the pharmacologic inhibition of Akt has an antiproliferative effect on gliomas. We showed that two newly developed Akt inhibitors, KP-372-1 and KP-372-2 (herein called KP-1 and KP-2), effectively inhibited the PI3K/Akt signaling cascade. KP-1 and KP-2 blocked both the basal and epidermal growth factor-induced phosphorylation of Akt Ser473 at 125 and 250 nmol/L, which, in turn, reduced the activation of intracellular downstream targets of Akt, including GSK-3beta and p70s6k. Furthermore, the treatment of U87 and U251 glioma cells with 125 to 250 nmol/L KP-1 and KP2 for 48 hours inhibited cell growth by approximately 50%. This decrease in cell growth stemmed from the induction of apoptosis. Collectively, these results provide a strong rationale for the pharmacologic targeting of Akt for the treatment of gliomas.

Apoptosis Regulatory Proteins↗

PubMed2005

Se-methylselenocysteine inhibits phosphatidylinositol 3-kinase activity of mouse mammary epithelial tumor cells in vitro.

INTRODUCTION: Se-methylselenocysteine (MSC), a naturally occurring selenium compound, is a promising chemopreventive agent against in vivo and in vitro models of carcinogen-induced mouse and rat mammary tumorigenesis. We have demonstrated previously that MSC induces apoptosis after a cell growth arrest in S phase in a mouse mammary epithelial tumor cell model (TM6 cells) in vitro. The present study was designed to examine the involvement of the phosphatidylinositol 3-kinase (PI3-K) pathway in TM6 tumor model in vitro after treatment with MSC. METHODS: Synchronized TM6 cells treated with MSC and collected at different time points were examined for PI3-K activity and Akt phosphorylation along with phosphorylations of Raf, MAP kinase/ERK kinase (MEK), extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). The growth inhibition was determined with a [3H]thymidine incorporation assay. Immunoblotting and a kinase assay were used to examine the molecules of the survival pathway. RESULTS: PI3-K activity was inhibited by MSC followed by dephosphorylation of Akt. The phosphorylation of p38 MAPK was also downregulated after these cells were treated with MSC. In parallel experiments MSC inhibited the Raf-MEK-ERK signaling pathway. CONCLUSION: These studies suggest that MSC blocks multiple signaling pathways in mouse mammary tumor cells. MSC inhibits cell growth by inhibiting the activity of PI3-K and its downstream effector molecules in mouse mammary tumor cells in vitro.

Animals↗

PubMed2005

Integrated array-comparative genomic hybridization and expression array profiles identify clinically relevant molecular subtypes of glioblastoma.

Glioblastoma, the most aggressive primary brain tumor in humans, exhibits a large degree of molecular heterogeneity. Understanding the molecular pathology of a tumor and its linkage to behavior is an important foundation for developing and evaluating approaches to clinical management. Here we integrate array-comparative genomic hybridization and array-based gene expression profiles to identify relationships between DNA copy number aberrations, gene expression alterations, and survival in 34 patients with glioblastoma. Unsupervised clustering on either profile resulted in similar groups of patients, and groups defined by either method were associated with survival. The high concordance between these separate molecular classifications suggested a strong association between alterations on the DNA and RNA levels. We therefore investigated relationships between DNA copy number and gene expression changes. Loss of chromosome 10, a predominant genetic change, was associated not only with changes in the expression of genes located on chromosome 10 but also with genome-wide differences in gene expression. We found that CHI3L1/YKL-40 was significantly associated with both chromosome 10 copy number loss and poorer survival. Immortalized human astrocytes stably transfected with CHI3L1/YKL-40 exhibited changes in gene expression similar to patterns observed in human tumors and conferred radioresistance and increased invasion in vitro. Taken together, the results indicate that integrating DNA and mRNA-based tumor profiles offers the potential for a clinically relevant classification more robust than either method alone and provides a basis for identifying genes important in glioma pathogenesis.

Adipokines↗

PubMed2005

Targeting integrin-linked kinase inhibits Akt signaling pathways and decreases tumor progression of human glioblastoma.

The phosphatidylinositol 3-kinase pathway is an important regulator of a wide spectrum of tumor-related biological processes, including cell proliferation, survival, and motility, as well as neovascularization. Protein kinase B/Akt is activated in a complex manner through the phosphorylation of protein kinase B/Akt on Thr308 and Ser473. Although protein-dependent kinase-1 has been shown to phosphorylate Akt at Thr308, it is not clear whether there is a distinct kinase that exclusively phosphorylates Akt at Ser473. A possible candidate is integrin-linked kinase (ILK), which has been shown to phosphorylate Akt at Ser473 in vitro. ILK is a multidomain focal adhesion protein that is believed to be involved in signal transmission from integrin and growth factor receptors. Further, ILK is implicated in the regulation of anchorage-dependent cell growth/survival, cell cycle progression, invasion and migration, and tumor angiogenesis. In this study, we tested the hypothesis that ILK inhibition would inhibit these processes in gliomas in which it is constitutively expressed. We found that a newly developed small-molecule compound (QLT0267) effectively inhibited signaling through the ILK/Akt cascade in glioma cells by blocking the phosphorylation of Akt and downstream targets, including mammalian target of rapamycin and glycogen synthase kinase-3beta. Treatment of glioma cells with 12.5 micromol/L QLT0267 inhibited cell growth by 50% at 48 hours. An anchorage-dependent cell growth assay confirmed the cell growth-inhibitory effect of QLT0267. Further, the decrease in cell growth was associated with a dramatic accumulation of cells in the G2-M phase of the cell cycle. Although the cell growth-inhibitory effects of the ILK inhibitor were achieved only at a high concentration, the QLT0267 was able to reduce cellular invasion and angiogenesis at much lower concentrations as shown by in vitro invasion assays and vascular endothelial growth factor secretion. Thus, blocking the ILK/Akt pathway is a potential strategy for molecular targeted therapy for gliomas.

Blotting, Western↗

PubMed2004

Prognostic significance of tissue transglutaminase in drug resistant and metastatic breast cancer.

PURPOSE: Drug resistance and metastasis pose major impediments in the successful treatment of cancer. We previously reported that multidrug-resistant breast cancer cells exhibit high levels of tissue transglutaminase (TG2; EC 2.3.2.13). Because the drug-resistant and metastatic phenotypes are thought to share some common pathways, we sought to determine whether metastatic breast cancer cells express high levels of TG2. EXPERIMENTAL DESIGN: The metastatic breast cancer cell line MDA-MB-231 and the sublines derived from it were tested for TG2 expression. Similarly, several sublines derived from an immortal but normal breast epithelial cell line, MCF10A, representing various stages in breast cancer progression were studied for TG2 expression. The primary and nodal tumor samples from 30 patients with breast cancer were also studied for TG2 expression. RESULTS: The MDA-MB-231 cells expressed high basal levels of TG2. Two clones derived from this cell line, MDA231/cl.9 and MDA231/cl.16, showed a 10- to 15-fold difference in TG2 level. TG2-deficient MDA231/cl.9 cells exhibited higher sensitivity to doxorubicin and were less invasive than were the TG2-sufficient MDA231/cl.16 cells. The MCF10A-derived sublines had increased TG2 expression as they advanced from noninvasive to an invasive phenotype. Importantly, the metastatic lymph node tumors from patients with breast cancer showed significant higher levels of TG2 expression compared with the primary tumors from the same patients. CONCLUSIONS: TG2 expression is up-regulated in drug-resistant and metastatic breast cancer cells, and it can serve as a valuable prognostic marker for these phenotypes.

Animals↗

PubMed2004

Up-regulation of Flotillin-2 is associated with melanoma progression and modulates expression of the thrombin receptor protease activated receptor 1.

Flotillin 2 (flot-2) is a highly conserved protein isolated from caveolae/lipid raft domains that tether growth factor receptors linked to signal transduction pathways. Flot-2 protein and mRNA were increased in tumorigenic and metastatic melanoma cell lines in vitro, and the immunostaining intensity increased substantially across a tissue array of melanocytic lesions. Flot-2 transfection transformed SB2 melanoma cells from nontumorigenic, nonmetastatic to highly tumorigenic and metastatic in a nude mouse xenograft model. SB2 cells stably transfected with the flot-2 cDNA (SB2-flot)-2 cells proliferated faster in the absence of serum, and their migration through Matrigel was additionally enhanced by thrombin. When SB2-flot-2 cells were compared with SB2-vector-control cells on a cancer gene pathway array, SB2-flot-2 cells had increased expression of protease activated receptor 1 (PAR-1) mRNA, a transmembrane, G-protein-coupled receptor involved in melanoma progression. PAR-1 and flot-2 were coimmunoprecipitated from SB2-flot-2 cells. Up-regulation of PAR-1 was additionally confirmed in SB2-flot-2 cells and melanoma cell lines. SB2-flot-2 cells transfected with flot-2-specific small-interfering RNAs made substantially less flot-2 and PAR-1 mRNA. In conclusion, flot-2 overexpression is associated with melanoma progression, with increased PAR-1 expression, and with transformation of SB2 melanoma cells to a highly metastatic line. Flot-2 binds to PAR-1, a known upstream mediator of major signal transduction pathways implicated in cell growth and metastasis, and may thereby influence tumor progression.

Animals↗

PubMed2004

Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma: correlation with suppression of COX-2 synthesis.

The cyclooxygenase 2 (COX-2) inhibitor celecoxib (also called celebrex), approved for the treatment of colon carcinogenesis, rheumatoid arthritis, and other inflammatory diseases, has been shown to induce apoptosis and inhibit angiogenesis. Because NF-kappa B plays a major role in regulation of apoptosis, angiogenesis, carcinogenesis, and inflammation, we postulated that celecoxib modulates NF-kappa B. In the present study, we investigated the effect of this drug on the activation of NF-kappa B by a wide variety of agents. We found that celecoxib suppressed NF-kappa B activation induced by various carcinogens, including TNF, phorbol ester, okadaic acid, LPS, and IL-1 beta. Celecoxib inhibited TNF-induced I kappa B alpha kinase activation, leading to suppression of I kappa B alpha phosphorylation and degradation. Celecoxib suppressed both inducible and constitutive NF-kappa B without cell type specificity. Celecoxib also suppressed p65 phosphorylation and nuclear translocation. Akt activation, which is required for TNF-induced NF-kappa B activation, was also suppressed by this drug. Celecoxib also inhibited the TNF-induced interaction of Akt with I kappa B alpha kinase (IKK). Celecoxib abrogated the NF-kappa B-dependent reporter gene expression activated by TNF, TNF receptor, TNF receptor-associated death domain, TNF receptor-associated factor 2, NF-kappa B-inducing kinase, and IKK, but not that activated by p65. The COX-2 promoter, which is regulated by NF-kappa B, was also inhibited by celecoxib, and this inhibition correlated with suppression of TNF-induced COX-2 expression. Besides NF-kappa B, celecoxib also suppressed TNF-induced JNK, p38 MAPK, and ERK activation. Thus, overall, our results indicate that celecoxib inhibits NF-kappa B activation through inhibition of IKK and Akt activation, leading to down-regulation of synthesis of COX-2 and other genes needed for inflammation, proliferation, and carcinogenesis.

Carcinogens↗

PubMed2004

Differential activation of the Fas/CD95 pathway by Ad-p53 in human gliomas.

Adenoviral p53 gene transfer (Ad-p53) induces apoptosis in glioma cells expressing mutant p53, but fails in cells with wild-type p53. Endogenously, gliomas express varied levels of Fas/CD95, yet constitutively high levels of Fas/CD95 ligand. Because the mechanism behind the differential apoptotic response to Ad-p53 infection remains elusive, we examined how the Fas/CD95 pathway is involved in U87MG (wt-p53), D54 (wt-p53), U251MG (mutant-p53), and U373MG (mutant-p53) glioma cell lines. Ad-p53 infection did not alter the levels of Fas/CD95 ligand in either wild-type or mutant p53-expressing cell lines. In contrast, Ad-p53 infection led to an approximately 3-fold increase in Fas/CD95 mRNA expression in mutant p53-bearing cell lines but not in their wild-type (wt) counterparts, as assessed in an RNase protection assay. Fas/CD95 mRNA induction appeared to be regulated at the transcriptional level because Ad-p53 infection resulted in up to a 4-fold increase in Fas/CD95 promoter reporter activity. Subsequently, flow cytometric analysis revealed a 2- to 4-fold increase in surface Fas/CD95 expression following Ad-p53 infection in mutant-p53-containing cell lines. Use of the protein transport inhibitor Brefeldin A significantly inhibited Ad-p53-induced surface Fas/CD95 expression, but only partially inhibited apoptosis in mutant-p53 cell lines. These results suggest that p53 regulates Fas/CD95 expression at the transcriptional level and through protein trafficking in mutant-p53 cell lines. Fluorogenic activity assays demonstrated that induction of caspase-8 activity following Ad-p53 infection correlated with increases in Fas/CD95 expression. Incubating cells with a caspase-8-specific inhibitor Ac-IETD-CHO prior to Ad-p53 infection inhibited caspase-8 activity and apoptosis. Together, our results suggest that regulation of the Fas/CD95 pathway is partly responsible for Ad-p53-induced apoptosis in glioma cells, which depends on the p53 status of the involved cells. Additionally, the inability of Ad-p53 to activate the Fas/CD95 pathway in wt-p53 glioma cells coincides with their apoptotic-resistant phenotype. Further elucidation of the nature of this resistance could ultimately augment the efficacy of Ad-p53 gene therapy.

Adenoviridae↗

PubMed2003

Src family protein-tyrosine kinases alter the function of PTEN to regulate phosphatidylinositol 3-kinase/AKT cascades.

Src family protein-tyrosine kinases, which play an important role in signal integration, have been implicated in tumorigenesis in multiple lineages, including breast cancer. We demonstrate, herein, that Src kinases regulate the phosphatidylinositol 3-kinase (PI3K) signaling cascade via altering the function of the PTEN tumor suppressor. Overexpression of activated Src protein-tyrosine kinases in PTEN-deficient breast cancer cells does not alter AKT phosphorylation, an indicator of signal transduction through the PI3K pathway. However, in the presence of functional PTEN, Src reverses the activity of PTEN, resulting in an increase in AKT phosphorylation. Activated Src reduces the ability of PTEN to dephosphorylate phosphatidylinositols in micelles and promotes AKT translocation to cellular plasma membranes but does not alter PTEN activity toward water-soluble phosphatidylinositols. Thus, Src may alter the capacity of the PTEN C2 domain to bind cellular membranes rather than directly interfering with PTEN enzymatic activity. Tyrosine phosphorylation of PTEN is increased in breast cancer cells treated with pervanadate, suggesting that PTEN contains sites for tyrosine phosphorylation. Src kinase inhibitors markedly decreased pervanadate-mediated tyrosine phosphorylation of PTEN. Further, expression of activated Src results in marked tyrosine phosphorylation of PTEN. SHP-1, a SH2 domain-containing protein-tyrosine phosphatase, selectively binds and dephosphorylates PTEN in Src transfected cells. Both Src inhibitors and SHP-1 overexpression reverse Src-induced loss of PTEN function. Coexpression of PTEN with activated Src reduces the stability of PTEN. Taken together, the data indicate that activated Src inhibits PTEN function leading to alterations in signaling through the PI3K/AKT pathway.

Animals↗

PubMed2002

Motif analysis of the tumor suppressor gene MMAC/PTEN identifies tyrosines critical for tumor suppression and lipid phosphatase activity.

The tumor suppressor gene, MMAC/PTEN, has phosphatase, C2, and PDZ-binding domains as well as potential sites of regulation by phosphorylation, including tyrosine phosphorylation, which may contribute to its ability to modulate cell growth and viability. Several obvious and significant motifs were found in MMAC/PTEN, including most notably, a catalytic domain of tyrosine phosphatase (IHCxxGxxRS/T) and several potential tyrosine phosphorylation sites. To examine the functional significance of tyrosine phosphorylation of MMAC/PTEN, retroviral constructs were generated with mutations at two putative tyrosine phosphorylation sites (Y240A/Y240F and Y315A/Y315F). Stable expression of wild-type MMAC/PTEN in U251 human glioma cells (which do not normally produce a functional MMAC/PTEN gene product) resulted in a significant reduction of tumor growth in nude mice, decreased growth rate, saturation density, and colony formation in vitro, as well as dephosphorylation of D3-phosphorylated phosphatidylinositols (PtdIns) in vitro. Mutation of Y240 or Y315 to either alanine or phenylalanine abrogated the ability of MMAC/PTEN to alter growth rate, saturation density, and colony formation in vitro. The ability of MMAC/PTEN to limit tumor growth in nude mice was markedly decreased but not abrogated by mutation of Y240 or Y315 to alanine. Thus, Y240 and Y315 are required for MMAC/PTEN to decrease tumor growth in vitro and in vivo. In contrast to wild-type MMAC/PTEN, mutant MMAC/PTEN containing Y240A or Y315A was unable to dephosphorylate D3-phosphorylated PtdIns in vitro. Thus, Y240A and Y315A are involved in the ability of MMAC/PTEN to dephosphorylate PtdIns and regulate tumor cell growth in vitro and in vivo.

Amino Acid Motifs↗

PubMed2002

Promoter analysis of tumor suppressor gene PTEN: identification of minimum promoter region.

Mutations of PTEN, a tumor suppressor gene located on chromosome 10, which encodes a protein-tyrosine and lipid-phosphatase, are prevalent in various human cancers, including glioblastoma. Despite extensive characterization of PTEN mutations in human cancers and a relatively good understanding of the molecular roles of PTEN in the control of cellular processes, little is known about modes of PTEN regulation. To understand the regulation of expression of the tumor suppressor gene PTEN, we isolated a 2212 bp fragment from the human BAC clone 46B12 DNA. The 3' end of this fragment starts at the Not I site of -745 relative to the first translation codon ATG (+1) and ends at the Sal I site of -2957 at the 5' end. Using classical 5'RACE and primer extension techniques, nine start sites were observed between -817 and -984 upstream of the ATG start site. We located a 137 bp fragment (-958/-821) as the minimum promoter region using promoter deletion and luciferase assays. A 704 bp fragment (-33/-737) downstream of the 2212 bp fragment was also cloned. As indicated by luciferase assays, the data show that this region possesses no promoter function. Interestingly, a p53 binding sequence is located within the 599 bp fragment (-1344/-745), although p53 expression had a minimal effect on PTEN, demonstrating its insignificant role in PTEN gene expression.

5' Flanking Region↗

PubMed2002

MMAC/PTEN tumor suppressor gene regulates vascular endothelial growth factor-mediated angiogenesis in prostate cancer.

Prostate cancer presents with a broad spectrum of biologic behavior, ranging from being an indolent, incidental finding to an aggressively invasive and metastatic disease. An improved understanding of the events involved in prostate cancer progression is critically important to its diagnosis and staging, as well as to the development of new therapies. Tumor progression, particularly in aggressive and malignant tumors, is associated with the induction of an angiogenic, gene-driven switch. In prostate cancer, one of the most powerful stimulators of angiogenesis is the vascular endothelial growth factor (VEGF). VEGF transcription can be induced by hypoxia through activation of the PI3 kinase pathway and hypoxia-inducible factor alpha. MMAC/PTEN (henceforth referred to as PTEN) is a recently identified tumor suppressor gene residing on chromosome 10q23, which is frequently inactivated in a wide range of human tumors, including advanced prostate cancer. The goal of this study was to determine whether PTEN inhibits angiogenesis by modulating VEGF activity. Our results showed that reintroduction of the PTEN gene into human prostate PC-3 and LNCaP cells decreased VEGF secretion, which was accompanied by various biologic activities, including inhibited endothelial cell growth and migration. PTEN expression also down-regulated VEGF mRNA levels, as detected by RT-PCR analysis. Concomitant with lessened VEGF expression was the reduction of VEGF promoter activity in PTEN-expressing cells. Our findings suggest that PTEN modulates angiogenesis by regulating VEGF expression.

Adenocarcinoma↗