Biomedical subjects

Bobby Bhatia

Publications and source records attributed to Bobby Bhatia.

8 recordsLinked to original sources

PubMed2007

Prostate cancer stem/progenitor cells: identification, characterization, and implications.

Several solid tumors have now been shown to contain stem cell-like cells called cancer stem cells (CSC). These cells, although generally rare, appear to be highly tumorigenic and may be the cells that drive tumor formation, maintain tumor homeostasis, and mediate tumor metastasis. In this Perspective, we first provide our insight on how a CSC should be defined. We then summarize our current knowledge of stem/progenitor cells in the normal human prostate (NHP), an organ highly susceptible to hyperproliferative diseases such as benign prostate hyperplasia (BPH) and prostate cancer (PCa). We further review the evidence that cultured PCa cells, xenograft prostate tumors, and patient tumors may contain stem/progenitor cells. Along with our discussion, we present several methodologies that can be potentially used to identify putative tumor-reinitiating CSC. Finally, we present a hypothetical model for the hierarchical organization of human PCa cells and discuss the implications of this model in helping understand prostate carcinogenesis and design novel diagnostic, prognostic, and therapeutic approaches.

Cell Differentiation↗

PubMed2006

15-lipoxygenase 2 (15-LOX2) is a functional tumor suppressor that regulates human prostate epithelial cell differentiation, senescence, and growth (size).

15-Lipoxygenase 2 (15-LOX2) is the major mammalian lipoxygenase expressed in normal human adult prostate and its expression is decreased or lost in high-grade prostate intraepithelial neoplasia (HGPIN) and prostate cancer (PCa). Our recent work has demonstrated that (1) 15-LOX2 has multiple alternatively spliced isoforms and is a negative cell-cycle regulator in normal human prostate (NHP) epithelial cells; (2) 15-LOX2 in NHP cells is positively regulated by Sp1 and negatively regulated by Sp3; (3) 15-LOX2 in NHP cells may be partially involved in cell differentiation; (4) 15-LOX2 is cell-autonomously upregulated in cultured NHP cells and its induction is associated with NHP cell senescence; and (5) 15-LOX2 is a functional prostate tumor suppressor. Here we summarize these new findings to provide a concise view of the potential biological functions of 15-LOX2 in NHP cells and of its deregulation in PCa development.

Adult↗

PubMed2005

Cell-autonomous induction of functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) contributes to replicative senescence of human prostate progenitor cells.

Normal human prostatic (NHP) epithelial cells undergo senescence in vitro and in vivo, but little is known about the tissue-specific molecular mechanisms. Here we first characterize young primary NHP cells as CK5(+)/CK18(+) intermediate basal cells that also express several other putative stem/progenitor cell markers including p63, CD44, alpha2beta1, and hTERT. When cultured in serum- and androgen-free medium, NHP cells gradually lose the expression of these markers, slow down in proliferation, and enter senescence. Several pieces of evidence implicate 15-lipoxygenase 2 (15-LOX2), a molecule with a restricted tissue expression and most abundantly expressed in adult human prostate, in the replicative senescence of NHP cells. First, the 15-LOX2 promoter activity and the mRNA and protein levels of 15-LOX2 and its multiple splice variants are upregulated in serially passaged NHP cells, which precede replicative senescence and occur in a cell-autonomous manner. Second, all immortalized prostate epithelial cells and prostate cancer cells do not express 15-LOX2. Third, PCa cells stably transfected with 15-LOX2 or 15-LOX2sv-b, a splice variant that does not possess arachidonate-metabolizing activity, show a passage-related senescence-like phenotype. Fourth, infection of early-passage NHP cells with retroviral vectors encoding 15-LOX2 or 15-LOX2sv-b induces partial cell-cycle arrest and big and flat senescence-like phenotype. Finally, 15-LOX2 protein expression in human prostate correlates with age. Together, these data suggest that 15-LOX2 may represent an endogenous prostate senescence gene and its tumor-suppressing functions might be associated with its ability to induce cell senescence.

Adolescent↗

PubMed2004

Evidence that Sp1 positively and Sp3 negatively regulate and androgen does not directly regulate functional tumor suppressor 15-lipoxygenase 2 (15-LOX2) gene expression in normal human prostate epithelial cells.

In this project, we studied the gene regulation of 15-lipoxygenase 2 (15-LOX2), the most abundant arachidonate-metabolizing LOX in adult human prostate and a negative cell-cycle regulator in normal human prostate (NHP) epithelial cells. Through detailed in silico promoter examination and promoter deletion and activity analysis, we found that several Sp1 sites (i.e., three GC boxes and one CACCC box) in the proximal promoter region play a critical role in regulating 15-LOX2 expression in NHP cells. Several pieces of evidence further suggest that the Sp1 and Sp3 proteins play a physiologically important role in positively and negatively regulating the 15-LOX2 gene expression, respectively. First, mutations in the GC boxes affected the 15-LOX2 promoter activity. Second, both Sp1 and Sp3 proteins were detected in the protein complexes that bound the GC boxes revealed by electrophoretic mobility shift assay. Third, importantly, inhibition of Sp1 activity or overexpression of Sp3 both inhibited the endogenous 15-LOX2 mRNA expression. Since 15-LOX2 is normally expressed in the prostate luminal epithelial cells, we subsequently explored whether androgen/androgen receptor may directly regulate its gene expression. The results indicate that androgen does not directly regulate 15-LOX2 gene expression. Together, these observations provide insight on how 15-LOX2 gene expression may be regulated in NHP cells.

Androgens↗

PubMed2004

Association of active caspase 8 with the mitochondrial membrane during apoptosis: potential roles in cleaving BAP31 and caspase 3 and mediating mitochondrion-endoplasmic reticulum cross talk in etoposide-induced cell death.

It was recently demonstrated that during apoptosis, active caspase 9 and caspase 3 rapidly accumulate in the mitochondrion-enriched membrane fraction (D. Chandra and D. G. Tang, J. Biol. Chem.278:17408-17420, 2003). We now show that active caspase 8 also becomes associated with the membranes in apoptosis caused by multiple stimuli. In MDA-MB231 breast cancer cells treated with etoposide (VP16), active caspase 8 is detected only in the membrane fraction, which contains both mitochondria and endoplasmic reticulum (ER), as revealed by fractionation studies. Immunofluorescence microscopy, however, shows that procaspase 8 and active caspase 8 predominantly colocalize with the mitochondria. Biochemical analysis demonstrates that both procaspase 8 and active caspase 8 are localized mainly on the outer mitochondrial membrane (OMM) as integral proteins. Functional analyses with dominant-negative mutants, small interfering RNAs, peptide inhibitors, and Fas-associated death domain (FADD)- and caspase 8-deficient Jurkat T cells establish that the mitochondrion-localized active caspase 8 results mainly from the FADD-dependent and tumor necrosis factor receptor-associated death domain-dependent mechanisms and that caspase 8 activation plays a causal role in VP16-induced caspase 3 activation and cell death. Finally, we present evidence that the OMM-localized active caspase 8 can activate cytosolic caspase 3 and ER-localized BAP31. Cleavage of BAP31 leads to the generation of ER- localized, proapoptotic BAP20, which may mediate mitochondrion-ER cross talk through a Ca(2+)-dependent mechanism.

Antineoplastic Agents, Phytogenic↗

PubMed2003

Subcellular localization and tumor-suppressive functions of 15-lipoxygenase 2 (15-LOX2) and its splice variants.

15-Lipoxygenase 2 (15-LOX2), the most abundant arachidonate (AA)-metabolizing enzyme expressed in adult human prostate, is a negative cell-cycle regulator in normal human prostate epithelial cells. Here we study the subcellular distribution of 15-LOX2 and report its tumor-suppressive functions. Immunocytochemistry and biochemical fractionation reveal that 15-LOX2 is expressed at multiple subcellular locations, including cytoplasm, cytoskeleton, cell-cell border, and nucleus. Surprisingly, the three splice variants of 15-LOX2 we previously cloned, i.e. 15-LOX2sv-a/b/c, are mostly excluded from the nucleus. A potential bi-partite nuclear localization signal (NLS),203RKGLWRSLNEMKRIFNFRR221, is identified in the N terminus of 15-LOX2, which is retained in all splice variants. Site-directed mutagenesis reveals that this putative NLS is only partially involved in the nuclear import of 15-LOX2. To elucidate the relationship between nuclear localization, enzymatic activity, and tumor suppressive functions, we established PCa cell clones stably expressing 15-LOX2 or 15-LOX2sv-b. The 15-LOX2 clones express 15-LOX2 in the nuclei and possess robust enzymatic activity, whereas 15-LOX2sv-b clones show neither nuclear protein localization nor AA-metabolizing activity. To our surprise, both 15-LOX2- and 15-LOX2sv-b-stable clones proliferate much slower in vitro when compared with control clones. More importantly, when orthotopically implanted in nude mouse prostate, both 15-LOX2 and 15-LOX2sv-b suppress PC3 tumor growth in vivo. Together, these results suggest that both 15-LOX2 and 15-LOX2sv-b suppress prostate tumor development, and the tumor-suppressive functions apparently do not necessarily depend on AA-metabolizing activity and nuclear localization.

Arachidonate 15-Lipoxygenase↗

PubMed2003

Annexin II expression is reduced or lost in prostate cancer cells and its re-expression inhibits prostate cancer cell migration.

While studying Bim, a BH3-only proapoptotic protein, we identified an approximately 36 kDa protein, which was abundantly expressed in all five strains of primary normal human prostate (NHP) epithelial cells but significantly reduced or lost in seven prostate cancer cell lines. The approximately 36 kDa protein was subsequently identified as annexin II by proteomic approach and confirmed by Western blotting using an annexin II-specific antibody. Conventional and 2D SDS-PAGE, together with Western blotting, also revealed reduced or lost expression of annexin I in prostate cancer cells. Subcellular localization studies revealed that in NHP cells, annexin II was distributed both in the cytosol and underneath the plasma membrane, but not on the cell surface. Prostate cancer cells showed reduced levels as well as altered expression patterns of annexin II. Since annexins play important roles in maintaining Ca(2+) homeostasis and regulating the cytoskeleton and cell motility, we hypothesized that the reduced or lost expression of annexin I/II might promote certain aggressive phenotypes of prostate cancer cells. In subsequent experiments, we indeed observed that restoration of annexin II expression inhibited the migration of the transfected prostate cancer cells without affecting cell proliferation or apoptosis. Hence, our results suggest that annexin II, and, likely, annexin I, may be endogenous suppressors of prostate cancer cell migration and their reduced or lost expression may contribute to prostate cancer development and progression.

Amino Acid Sequence↗

PubMed2002

Evidence that arachidonate 15-lipoxygenase 2 is a negative cell cycle regulator in normal prostate epithelial cells.

15-Lipoxygenase 2 (15-LOX2) is a recently cloned human lipoxygenase that shows tissue-restricted expression in prostate, lung, skin, and cornea. The protein level and enzymatic activity of 15-LOX2 have been shown to be down-regulated in prostate cancers compared with normal and benign prostate tissues. The biological function of 15-LOX2 and the role of loss of 15-LOX2 expression in prostate tumorigenesis, however, remain unknown. We report the cloning and functional characterization of 15-LOX2 and its three splice variants (termed 15-LOX2sv-a, 15-LOX2sv-b, and 15-LOX2sv-c) from primary prostate epithelial cells. Western blotting with multiple primary prostate cell strains and prostate cancer cell lines reveals that the expression of 15-LOX2 is lost in all prostate cancer cell lines, accompanied by decreased enzymatic activity revealed by liquid chromatography/tandem mass spectrometry analyses. Further experiments show that the loss of 15-LOX2 expression results from transcriptional repression caused by mechanism(s) other than promoter hypermethylation or histone deacetylation. Subsequent functional studies indicate the following: 1) the 15-LOX2 product, 15(S)-hydroxyeicosatetraenoic acid, inhibits prostate cancer cell cycle progression; 2) 15-LOX2 expression in primary prostate epithelial cells is inversely correlated with cell cycle; and 3) restoration of 15-LOX2 expression in prostate cancer cells partially inhibits cell cycle progression. Taken together, these results suggest that 15-LOX2 could be a suppressor of prostate cancer development, which functions by restricting cell cycle progression.

Alternative Splicing↗