[Show abstract][Hide abstract] ABSTRACT: EHD3 [Eps15 homology (EH) domain-containing protein 3] is a protein that resides in tubular and vesicular membrane structures
and participates in endocytic recycling, although all its functions are unknown. Since Ehd3 is most abundantly expressed in brain tissues, we examined its role in brain cancer progression. Using immunohistochemistry,
we report loss of EHD3 expression in gliomas, including low-grade astrocytomas, suggesting that this is an early event in
gliomagenesis. EHD3 expression is also very low in most of glioma cell lines tested. In two cell lines, a bisulfite sequencing
method identifies promoter hypermethylation as a mechanism of Ehd3 silencing, and its expression was restored by the demethylating agent 5-Azacytidine. Doxycycline-inducible restoration of
EHD3 expression to glioma cells decreases their growth and invasiveness and induces cell cycle arrest and apoptosis. Furthermore,
shRNA-mediated Ehd3 silencing increases cell growth. Using a xenograft model, we demonstrate Ehd3 growth inhibitory functions in glioma cells in vivo. We suggest that Ehd3 functions as a tumor suppressor gene and loss of its expression is a very common event in gliomas. This is the first study
to highlight the importance of a member of the C-terminal EHD proteins in cancer and to link their functions to the cell cycle
[Show abstract][Hide abstract] ABSTRACT: The Eph and Ephrin proteins, which constitute the largest family of receptor tyrosine kinases, are involved in normal tissue development and cancer progression. Here, we examined the expression and role of the B-type Eph receptor EphB2 in breast cancers. By immunohistochemistry using a progression tissue microarray of human clinical samples, we found EphB2 to be expressed in benign tissues, but strongly increased in cancers particularly in invasive and metastatic carcinomas. Subsequently, we found evidence that EphB2, whose expression varies in established cell breast lines, possesses multiple functions. First, the use of a DOX-inducible system to restore EphB2 function to low expressers resulted in decreased tumor growth in vitro and in vivo, while its siRNA-mediated silencing in high expressers increased growth. This function involves the onset of apoptotic death paralleled by caspases 3 and 9 activation. Second, EphB2 was also found to induce autophagy, as assessed by immunofluorescence and/or immunoblotting examination of the LC3, ATG5 and ATG12 markers. Third, EphB2 also has a pro-invasive function in breast cancer cells that involves the regulation of MMP2 and MMP9 metalloproteases and can be blocked by treatment with respective neutralizing antibodies. Furthermore, EphB2-induced invasion is kinase-dependent and is impeded in cells expressing a kinase-dead mutant EphB2. In summary, we identified a mechanism involving a triple role for EphB2 in breast cancer progression, whereby it regulates apoptosis, autophagy, and invasion.
Full-text · Article · Nov 2013 · Experimental Cell Research
[Show abstract][Hide abstract] ABSTRACT: Nearly 30% of prostate cancer (PCa) patients treated with potentially curative doses relapse at the sites of irradiation. How some tumor cells acquire radioresistance is poorly understood. The platelet-type 12-lipoxygenases (12-LOX)-mediated arachidonic acid metabolism is important in PCa progression. Here we show that 12-LOX confers radioresistance upon PCa cells. Treatment with 12-LOX inhibitors baicalein or BMD122 sensitizes PCa cells to radiation, without radiosensitizing normal cells. 12-LOX inhibitors and radiation, when combined, have super additive or synergistic inhibitory effects on the colony formation of both androgen-dependent LNCaP and androgen-independent PC-3 PCa cells. In vivo, the combination therapy significantly reduced tumor growth.
[Show abstract][Hide abstract] ABSTRACT: Connexin43 (Cx43) expression is lost in cancer cells and many studies have reported that Cx43 is a tumor suppressor gene. Paradoxically, in a cellular NIH3T3 model, we have previously shown that Ha-Ras-mediated oncogenic transformation results in increased Cx43 expression. Although the examination of transcriptional regulation revealed essential regulatory elements, it could not solve this paradox. Here we studied post-transcriptional regulation of Cx43 expression in cancer using the same model in search of novel gene regulatory elements. Upon Ras transformation, both Cx43 mRNA stability and translation efficiency were increased. We investigated the role of Cx43 mRNA 3' and 5'Untranslated regions (UTRs) and found an opposing effect; a 5'UTR-driven positive regulation is observed in Ras-transformed cells (NIH-3T3(Ras)), while the 3'UTR is active only in normal NIH-3T3(Neo) cells and completely silenced in NIH-3T3(Ras) cells. Most importantly, we identified a previously unknown regulatory element within the 3'UTR, named S1516, which accounts for this 3'UTR-mediated regulation. We also examined the effect of other oncogenes and found that Ras- and Src-transformed cells show a different Cx43 UTRs post-transcriptional regulation than ErbB2-transformed cells, suggesting distinct regulatory pathways. Next, we detected different patterns of S1516 RNA-protein complexes in NIH-3T3(Neo) compared to NIH-3T3(Ras) cells. A proteomic approach identified most of the S1516-binding proteins as factors involved in post-transcriptional regulation. Building on our new findings, we propose a model to explain the discrepancy between the Cx43 expression in Ras-transformed NIH3T3 cells and the data in clinical specimens.
[Show abstract][Hide abstract] ABSTRACT: Despite ongoing attempts to improve the overall breast cancer (BC) survival rate, BC cells' (BCCs) predilection for metastasizing to the bone marrow has enabled BCCs to not only remain dormant, but also evade detection. BCCs are able to acquire quiescence by establishing gap junctional intercellular communication (GJIC) with the stroma through the assembly of connexins (Cxs). The chemoattractant CXCL12 also appears to play a role in GJIC based on its tendency to decrease when GJIC is formed between BCCs and bone marrow stroma. This study investigates the role CXCL12 has on Cx43 expression and PKC-mediated Cx43 phosphorylation. Cx43 gene reporter assays revealed that as the BCCs come in contact with each other and establish GJIC, there is an inverse relationship between CXCL12 level and Cx43 expression. Immunoblot analyses confirmed this relationship at the level of protein, showing decreased Cx43 and reduced Cx43 phosphorylation at higher CXCL12 concentrations. However, real-time PCR studies revealed little change in Cx43 mRNA levels, despite stimulation with different concentrations of CXCL12, indicating CXCL12's effect on Cx43 is post-translational, through phosphorylation. Immunoblot analyses and functional dye exchange studies showed activation of PKC by exogenous CXCL12 in the phosphorylation, which in turn, increased intercellular communication. These findings elucidate the importance of considering the microenvironment's role in micrometastasis in clinical studies pertaining to prospective breast cancer treatment.
[Show abstract][Hide abstract] ABSTRACT: Cancer cells rely on intercellular communication throughout the different stages of their transformation and progression into metastasis. They do so by co-opting different processes such as cell-cell junctions, growth factors, receptors, and vesicular release. Initially characterized in neuronal and vascular tissues, Ephs and Ephrins, the largest family of receptor tyrosine kinases, comprised of two classes (i.e., A and B types), is increasingly scrutinized by cancer researchers. These proteins possess the particular features of both the receptors and ligands being membrane-bound which, via mandatory direct cell-cell interactions, undergo a bidirectional signal transduction initiated from both the receptor and the ligand. Following cell-cell interactions, Ephs/Ephrins behave as guidance molecules which trigger both repulsive and attractive signals, so as to direct the movement of cells through their immediate microenvironment. They also direct processes which include sorting and positioning and cytoskeleton rearrangements, thus making them perfect candidates for the control of the metastatic process. In fact, the role of Ephs and Ephrins in cancer progression has been demonstrated for many of the family members and they, surprisingly, have both tumor promoter and suppressor functions in different cellular contexts. They are also able to coordinate between multiple processes including cell survival, proliferation, differentiation, adhesion, motility, and invasion. This review is an attempt to summarize the data available on these Ephs/Ephrins' biological functions which contribute to the onset of aggressive cancers. I will also provide an overview of the factors which could explain the functional differences demonstrated by Ephs and Ephrins at different stages of tumor progression and whose elucidation is warranted for any future therapeutic targeting of this signaling pathway in cancer metastasis.
No preview · Article · May 2012 · CANCER AND METASTASIS REVIEW
[Show abstract][Hide abstract] ABSTRACT: Connexins (Cxs) and gap junctional intercellular communications (GJICs) play roles in cancer development, growth and metastasis. Experimental studies suggest that targeting Cxs may be a novel technique, either to inhibit tumor cell growth directly or to sensitize to various therapies.
A brief introduction to the role of Cxs in cancer. The focus is mainly on data available in the literature regarding therapeutic aspects.
This article reviews the various strategies that take advantage of gap junctions and connexins to eliminate cancer cells, including use of the bystander effect (BE) in gene therapy, the effect of connexins on chemosensitization, the role of apoptotic processes and interactions with the microenvironment. Attempts to restore connexin expression at the transcriptional and post-transcriptional levels are described, as well as promising strategies recently explored. The potential and limitations of the approaches are discussed.
Connexins have multiple facets, singly, in hemichannel complexes, in gap junctions or interacting with different proteins. The regulation of their expression is not fully resolved and selective manipulation of Cxs expression is therefore a challenge. Although the therapeutic potential of connexins is undeniable, more effort is needed to study the regulation and functions of these proteins.
No preview · Article · Jul 2010 · Expert Opinion on Therapeutic Targets
[Show abstract][Hide abstract] ABSTRACT: EphB2 is a tyrosine kinase receptor that has been shown to be a tumor suppressor gene in various cancers. However the mechanisms of this function are unknown. We report that EphB2 induces a form of cell death that does not involve the formation of apoptotic bodies or nuclear fragmentation and is instead accompanied by extensive vacuolization. Transmission electron microscopy demonstrates cytoplasmic vacuoles in EphB2-overexpressing cells that resembled autophagosomes. Using an EYFP-LC3 fusion protein and immunoblotting, we detected LC3 aggregation and conversion from form I to form II, both hallmarks of autophagy, in EphB2-transfected cells. Silencing of the autophagy regulating genes ATG5 or ATG7 using shRNAs, strongly prevented EphB2-induced cell death, further confirming its autophagic nature. EphB2 expression results in mitochondrial depolarization and translocation of cytochrome c from the mitochondria to the cytosol. Mapping of signaling pathways revealed novel information about the mechanisms of action of EphB2. We demonstrated that the MAPK pathway is important in the pro-death action of EphB2, through ERK1/2 phosphorylation and inhibition of this pathway using PD98059 counters EphB2-driven cell death. In addition, we found that inhibition of class III PI3K pathway, using the autophagy inhibitor 3MA, but not class I PI3K inhibition using LY294002, also effectively blocks EphB2- induced cell death. Finally, EphB2 expression inactivates Akt, which is a known inhibitor of autophagy. In conclusion, the EphB2 receptor induces an autophagic cell death that is mediated through the ERK1/2 and PI3K/Akt pathways.
[Show abstract][Hide abstract] ABSTRACT: Prostate cancer is the first most common malignancy in men worldwide; this cancer is characterized by a marked propensity for invasion and spreading to local lymph nodes. On the other hand, Teucrium polium (TP) is a medicinal plant that has been used for more than two thousand years for treating many diseases such as abdominal pain, indigestion and diabetes in the Middle East. However, the effect of TP plant extract on human metastatic cancer cells especially prostate has not been investigated yet. In this study, we examined the effects of TP extract on selected parameters in PC3 and DU145 prostate cancer cell lines. Our results show that TP plant extract inhibits cell proliferation and provokes S cell cycle arrest and reduction of G0-G1 phase. In parallel, this extract induces differentiation to an epithelial phenotype "mesenchymal-epithelial transition" which is an important event in cell invasion and metastasis; thus TP plant extract causes a dramatic decrease in cell invasion and motility abilities of PC3 and DU145 cancer cells in comparison with untreated cells. These changes are accompanied by a re-localization of the expression patterns of E-cadherin and catenins. The molecular pathway analysis of the TP plant extract revealed that it inhibits the phosphorylation of beta-catenin, via Src dephosphorylation, and consequently converts its role from a transcriptional regulator to a cell-cell adhesion molecule. Our findings indicate that TP plant extract inhibits signaling pathways involved in regulating the E-cadherin/catenin complex and possibly other cell-cell adhesion genes via beta-catenin alteration, suggesting that this plant extract has therapeutic promise in the treatment of human metastatic prostate cancer.
Full-text · Article · Nov 2009 · Journal of ethnopharmacology
[Show abstract][Hide abstract] ABSTRACT: Nrf2 is a key transcription factor, which induces a cytoprotective gene array. Nrf2 is regulated at the posttranslational level through proteasomal degradation through an interaction with the adapter protein Keap1. High levels of Nrf2, resulting from a loss of function mutation in Keap1, were reported in chemoresistant non-small cell lung cancer. We observed very low levels of Nrf2 and of Nrf2-regulated detoxification proteins as a frequent phenotype in the more chemosensitive breast cancer, and when engineering increased Nrf2 levels, we found resistance to both doxorubicin and paclitaxel. We here show that basal Nrf2 levels in different cell lines correlate with their respective sensitivity to a common cytotoxic chemotherapy. Nrf2 and its regulated genes and proteins are the targets of a major strategy in cancer prevention. Molecules that interfere with the Nrf2-Keap1-Cul3 protein-protein interactions result in higher levels of Nrf2. Both naturally occurring and synthetic molecules with this effect have been suggested as clinical chemopreventive agents, including molecules derived from cruciferous vegetables such as the isothiocyanate sulforaphane and even green tea polyphenols. Here, we determine the impact of these putative chemopreventive agents on the sensitivity of established cancer cell lines to chemotherapy. We confirmed that these molecules do increase Nrf2 and detoxification enzyme levels in breast cancer cell lines with very low basal Nrf2 levels, and this is associated with significant chemoresistance to cytotoxic drugs. Both effects are less in another breast cancer cell line with intermediate Nrf2, and in lung cancer cells with high Nrf2, these same molecules have no effect on Nrf2 but do actually enhance chemoresistance. While the details of dose and schedule of these agents require further study in in vivo models, these data sound a cautionary note for the use of these agents in patients with established cancers who are undergoing chemotherapy treatment.
No preview · Article · Nov 2009 · Cancer Chemotherapy and Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Connexin43 (Cx43) is often deregulated in breast cancer tissue compared with normal adjacent tissue. Stable reexpression of Cx43 in cancer slows growth and renders the cells more sensitive to cytotoxic chemotherapeutics. Pseudogenes are often considered nonfunctional copies of DNA. The Cx43 pseudogene (PsiCx43) possesses all the features of an expressed gene and is exclusively transcribed in breast cancer cell lines and not in normal cells. PsiCx43 can be translated in vivo, and its protein exhibits growth-suppressive behavior similar to Cx43. We showed that PsiCx43 binds to the polyribosomes in breast cancer cells and that exogenous expression of PsiCx43 induces translational inhibition of Cx43. Furthermore, PsiCx43 is translated and binds more efficiently to the translational machinery than does Cx43 in an in vitro system. Following knockdown of PsiCx43 in breast cancer cells, we observed an increase in Cx43 RNA and protein. This results in increased cellular sensitivity to cytotoxic chemotherapy. Our results show that PsiCx43 acts as a posttranscriptional regulator of Cx43 in breast cancer cells, and that this represents an example of the regulation of genes by pseudogenes with potential therapeutic implications in cancer.
No preview · Article · May 2009 · Molecular Cancer Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Human papillomaviruses (HPVs) could be important risk factors for breast carcinogenesis and metastasis, as roughly 50% of breast cancers are positive for high-risk HPVs. To determine the role of high-risk HPVs in human breast carcinogenesis and metastasis, we examined the effect of E6/E7 of HPV type 16 in two non-invasive breast cancer cell lines, MCF7 and BT20. We report that E6/E7 of HPV type 16 induces cell invasive and metastatic abilities of MCF7 and BT20 in vitro and in vivo, respectively, in comparison with the wild type cells. This is accompanied by an upregulation of Id-1, a family member of helix-loop-helix (HLH) transcription factors, in MCF7 and BT20 cell lines which express E6/E7. Earlier studies have reported that Id-1 regulates cell invasion and metastasis of human breast cancer cells. To gauge the role of Id-1 in cell invasion and metastasis induced by E6/E7 of HPV type 16, we investigated the effect of E6/E7 in mouse normal embryonic fibroblast (NEF) and knockout Id-1 (Id-1(-/-)) cells. We establish that E6/E7 induces cell invasive ability in NEF but not Id-1(-/-) cells; moreover, we were able to inhibit the invasion ability of MCF7-E6/E7 and BT20-E6/E7 using Id-1 antisense retroviruses. Furthermore, we report that E6/E7 oncoproteins up-regulate Id-1 promoter activity in MCF7 and BT20 cells. We also found that HPV type 16 is present in all invasive and metastatic breast cancer and less frequently in in-situ breast cancer as opposed to normal mammary tissue. In parallel, we demonstrate that Id-1 overexpression is correlated with the presence of HPV type 16 in human invasive and metastatic breast cancer. These data suggest that high-risk HPV infections can induce cell invasion and metastasis in breast cancer through Id-1 regulation.
[Show abstract][Hide abstract] ABSTRACT: The connexin 43 gene (cx43, GJA1) mediates gap junctional intercellular communication (GJIC), which regulates tissue homeostasis. cx43 is frequently downregulated in prostate cancer. We investigated the role of a histone deacetylase (HDAC)-dependent mechanism in the transcriptional repression of cx43 in a panel of prostate cancer cells.
The impact of Trichostatin A (TSA), an inhibitor of HDAC, on exogenous and endogenous cx43 gene transcription was examined by the luciferase assay, Northern blot, nuclear run-on, Western blot, and chromatin immunoprecipitation assays.
Trichostatin A induces transcription of cx43 gene and GJIC. The co-activator p300/CBP synergizes with TSA for cx43 promoter activation. We identified a promoter region where cooperation between Ap1 and Sp1 elements was essential for TSA-induced cx43 transcription. TSA increased the level of hyperacetylated histones bound to cx43 promoter.
Our results highlight the potential utility of inhibitors of HDAC to restore cx43 gene expression in prostate cancer.
[Show abstract][Hide abstract] ABSTRACT: Pseudogenes are classically thought of as nonfunctional DNA sequences due to their inability to be translated, or to produce a functional protein. Gap junctions, a multiprotein complex made of proteins called connexins, are involved in intercellular communication and are deregulated in many cancers. Connexin43 (Cx43) is the only connexin for which a pseudogene has been reported so far. The Cx43 pseudogene (PsiCx43) has all of the features of an expressed gene. We identified the presence of a PsiCx43 mRNA transcript in several cancer cell lines and in none of the normal mammary epithelial cells studied. Using an in vitro translation assay, we found that the PsiCx43 coding plasmid could be translated into a 43 kDa protein. This was further confirmed by expressing a PsiCx43-green fluorescence protein fusion protein in breast cancer MCF-7 cells. We then examined the functional significance of the PsiCx43. In both MTT growth and colony formation assays, significant growth inhibition was observed, a feature common to cells overexpressing the Cx43 gene. However, using a scrape-loading assay, we could not detect any effect on gap junctional intercellular communication. Based on our findings, PsiCx43 joins and enlarges the thus far restricted group of functionally transcribed and translated pseudogenes.
[Show abstract][Hide abstract] ABSTRACT: Many naturally occurred or synthetic compounds can modulate the body's drug-metabolizing enzymes to enhance carcinogen detoxification, and some have demonstrated remarkable cancer prevention effects. Understanding the molecular mechanism behind each candidate agent is critically important in designing rational cancer chemoprevention strategies. In this work, we have employed a set of molecular mechanism-based assays and characterized eight classes of known drug-metabolizing enzyme (DME) modulators in a cellular system. Examination of mRNA and protein levels of representative phase I and phase II enzymes validated the results obtained in our cell-based system. Our data confirmed that the antioxidant ethoxyquin (EQ) and the isothiolcyanate sulfurophane (SFP) exclusively activate the antioxidant response element (ARE), and thus represent monofunctional inducers. We were also able to reclassify some compounds, and to use the system to identify structure-activity relationships among structurally related but different compounds. Finally, this cell-based system permitted us to identify a potential novel mechanism for cross-talk between the ARE and the xenobiotic response element (XRE)-mediated pathways.
No preview · Article · Jun 2004 · Biochemical Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Oltipraz, a promising cancer chemopreventive agent, has been recognized as a monofunctional inducer selectively activating phase II carcinogen-detoxifying enzymes via the antioxidant responsive element (ARE). However, we report here that oltipraz also induces rat glutathione S-transferase A5 (GSTA5), a potent phase II detoxifying enzyme, by means of the xenobiotic responsive element (XRE). Although an ARE sequence exists in the 5' upstream of the rGSTA5 gene, this cis-acting regulatory element loses its responsiveness to oltipraz treatment because of extensive mutations in its distal-half site. Our data indicate that a XRE sequence, located downstream of the transcription initiation site of the gene, is another oltipraz-responsive element. Electrophoretic mobility shift assay showed that oltipraz steadily induces XRE-aryl hydrocarbon receptor (AhR) binding, which can be blocked specifically by excess XRE oligonucleotides or by AhR antibody. By cloning different XREs into the pGL3-promoter vector, we found that oltipraz can activate XRE enhancers from several phase II drug metabolism enzymes, including rGSTA5, rGSTA2, NAD(P)H:quinone reductase, and it also activates XRE from the phase I metabolism enzyme CYP1A1. Oltipraz's effect on XRE is AhR-dependent and is independent of the presence of active CYP1A1. Reverse transcriptase-polymerase chain reaction experiments revealed that oltipraz induces gene expression of both phase I and II drug-metabolizing enzymes in rat hepatoma cells. Thus, we conclude that, like ARE, the XRE pathway constitutes an important part of the molecular mechanism contributing to oltipraz-induced expression of the phase II metabolism enzymes. Oltipraz is a bifunctional inducer, modulating both phase I and II drug-metabolizing enzymes to enhance carcinogen detoxification.
No preview · Article · Sep 2003 · Molecular Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Platelet-type arachidonate 12-lipoxygenase (12-LOX) is highly expressed in many types of cancers and plays an important role in cancer pathophysiology. Arachidonic acid metabolism by 12-LOX results in the stable end product 12(S)-hydroxy eicosatetraenoic acid (12(S)-HETE), which is a signaling molecule with effects on cell proliferation, motility, invasiveness, angiogenesis, and inhibition of apoptosis. The myriad biological activities manifested by 12(S)-HETE appear to be mediated, at least in part, by the activation of NF-kappaB. Overexpression of the 12-LOX in PC-3 prostate cancer cells resulted in the constitutive activation of the transcription factor. The enzymatic product of arachidonic acid metabolism, 12(S)-HETE, mediates the activation of NF-kappaB by the 12-LOX. 12(S)-HETE treatment of PC-3 cells induced the degradation of IkappaB by the S6 proteasomal pathway and the activated NF-kappaB translocated to the nucleus causing kappaB-induced transcription. Specificity of the NF-kappaB activation by 12(S)-HETE was established by the use of a 12-LOX-specific inhibitor and 13(S)-HODE, a known 12(S)-HETE antagonist. Considering the known involvement of MAP kinase pathway in NF-kappaB activation and that of 12(S)-HETE in MAP kinase pathway, 12-LOX present in prostate cancer tissues may contribute to the constitutive activation of NF-kappaB in prostate cancer cells.
No preview · Article · Aug 2003 · Prostaglandins & other lipid mediators
[Show abstract][Hide abstract] ABSTRACT: Connexin 43 (Cx43) is essential for survival and is tightly regulated at the transcriptional and post-transcriptional levels. A number of previous studies have demonstrated altered expression in malignant tissues, and in the presence of carcinogenic factors. We examined the effect of protooncogenes of Cx43 expression, and found no effect on Cx43 promoter activity in cells transformed with Src or erbB2. On the other hand, we identified and characterized a novel sequence that mediates Cx43 promoter regulation in cell lines engineered to overexpress H-Ras. Compared with wild-type NIH3T3 cells, both Cx43 mRNA and protein levels are increased in NIH3T3-Ras cells. The H-Ras+ cells also have enhanced Cx43 promoter activation, which is inhibited by the MEK1 inhibitor 2'-amino-3'-methoxyflavone (PD98059), suggesting that Ras-mediated Cx43 overexpression is via the mitogen activated protein kinase kinase/extracellular signal-regulated pathway. Deletion analysis of the Cx43 promoter revealed a 200-bp region downstream of the Cx43 transcription start site as the minimal sequence essential for the Ras-mediated Cx43 up-regulation. Using this 200-base pair fragment in electrophoretic mobility shift assays, we identified one main protein complex that binds efficiently and is more abundant in nuclear extracts from NIH3T3-Ras and MCF7-Ras cells compared with their matched controls. This complex selectively recognizes a consensus sequence, AGTTCAATCA, located at positions +149 to +158 of the Cx43 promoter. Supershift assays identified the 90-kDa heat shock protein (HSP90) and c-Myc as constituents of this DNA-binding complex. Treatment of cells with the HSP90 inhibitor geldanamycin resulted in repression of the Cx43 promoter activity, and inhibits binding of the complex to the Cx43 promoter. Coimmunoprecipitation studies confirmed the interaction between endogenous HSP90 and c-Myc. This study provides evidence that the transcriptional up-regulation of Cx43 by Ras-Raf-MAPK is mediated via the interaction of a novel Cx43 promoter element with a protein complex that contains both HSP90 and c-Myc.
No preview · Article · May 2003 · Molecular Pharmacology
[Show abstract][Hide abstract] ABSTRACT: Extensive studies have implicated the role of dietary fatty acids in prostatecancer progression. Platelet-type 12-Lipoxygenase (12-LOX) has beenshown to regulate growth, metastasis, and angiogenesis of prostate cancer. The effect of two 12-LOX inhibitors, Baicalein and N-benzyl-N-hydroxy-5-phenylpentamide (BHPP), on the mechanisms controlling cell cycle progression and apoptosis were examined in two prostate cancer cell lines, PC3 and DU-145. Treatment with Baicalein or BHPP resulted in a dose-dependent decrease in cell proliferation, as measured by BrdUrd incorporation. This growth arrest was shown to be because of cell cycle inhibition at G0/G1, and was associated with suppression of cyclin D1 and D3 protein levels. PC3 cells also showed a strong decrease in phosphorylated retinoblastoma (pRB) protein, whereas the other retinoblastoma-associated proteins, p107 and p130, were inhibited in DU-145 cells. Treatment with 12-hydroxyeicosatetraenoic acid in the presence of Baicalein blocked loss of pRB, whereas 12(S)-HETE alone induced pRB expression. Treatment with either Baicalein or BHPP resulted in significant apoptosis in both cell lines as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. DU-145 cells underwent apoptosis more rapidly than PC-3 cells. The mechanisms involved were decreased phosphorylation of Akt, loss of survivin and subsequent activation of caspase-3 and caspase-7 in each cell line, decreased Bcl-2 and Bcl-X(L) expression in DU-145, and a shift in Bcl-2/Bax levels favoring apoptosis in PC-3 cells. Addition of 12(S)-HETE protected both cell lines from Baicalein-induced apoptosis, whereas other LOX metabolites, 5(S)-HETE, or 15(S)-HETE did not. These results show that the 12-LOX pathway is a critical regulator of prostate cancer progression and apoptosis, by affecting various proteins regulating these processes. Therefore, inhibition of 12-LOX is a potential therapeutic agent in the treatment of prostate cancer.