That 12-O-tetradecanoylphorbol-13-acetate Research Articles

Cell Morphology-Based Screening Identified Vitetrifolin D from <i>Vitex Rotundifolia</i> as an Inhibitor of Phorbol Ester–Induced Downregulation of E-Cadherin in HHUA Endometrial Cells

The epithelial–mesenchymal transition (EMT) of endometrial cells contributes to the development of endometrial cancer, endometriosis, and adenomyosis. We have recently reported that 12-O-Tetradecanoylphorbol 13-acetate (TPA), a protein kinase C activator, induces EMT in HHUA endometrial cells cultured on collagen type I gels. HHUA cells showed an obvious morphological change from spheroids to scattered spindle cells during the TPA-induced EMT. In this study, we searched for inhibitors of the TPA-induced morphological change, and isolated vitetrifolin D from Vitex rotundifolia leaves. Vitetrifolin D suppressed the TPA-induced downregulation of E-cadherin, a major component of the epithelial adherens junction. Since vitetrifolin D showed a modest cytotoxicity at the concentration required to inhibit E-cadherin downregulation, it would not be suitable as a drug to treat endometrial disorders. However, our cell morphology-based screening could contribute to the discovery of new compounds that inhibit the loss of epithelial cell junctions.

12-O-Tetradecanoylphorbol-13-Acetate Induces Up-Regulated Transcription of Variant 1 but Not Variant 2 of VIL2 in Esophageal Squamous Cell Carcinoma Cells via ERK1/2/AP-1/Sp1 Signaling.

The membrane-cytoskeleton link organizer ezrin may be the most “dramatic” tumor marker, being strongly over-expressed in nearly one-third of human malignancies. However, the molecular mechanisms of aberrant ezrin expression still need to be clarified. Ezrin, encoded by the VIL2 gene, has two transcript variants that differ in the transcriptional start site (TSS): V1 and V2. Both V1 and V2 encode the same protein. Here, we found that 12-O-tetradecanoylphorbol-13-acetate (TPA) induced over-expression of human VIL2 in esophageal squamous cell carcinoma (ESCC) cells. Furthermore, VIL2 V1 but not V2 was up-regulated after TPA stimulation in a time-dependent manner. AP-1 and Sp1 binding sites within the promoter region of VIL2 V1 acted not only as basal transcriptional elements but also as a composite TPA-responsive element (TRE) for the transcription of VIL2 V1. TPA stimulation enhanced c-Jun and Sp1 binding to the TRE via activation of the ERK1/2 pathway and increased protein levels of c-Jun, c-Fos, and Sp1, resulting in over-expression of VIL2 V1, whereas the MEK1/2 inhibitor U0126 blocked these events. Finally, we showed that TPA promoted the migration of ESCC cells whereas MEK1/2 inhibitor or ezrin silencing could partially inverse this alteration. Taken together, these results suggest that TPA is able to induce VIL2 V1 over-expression in ESCC cells by activating MEK/ERK1/2 signaling and increasing binding of Sp1 and c-Jun to the TRE of the VIL2 V1 promoter, and that VIL2 is an important TPA-induced effector.

TPA‐induced invasion of HT29 cells are mediated through ROS production and AMPK deactivation (1055.5)

AMP‐activated protein kinase (AMPK) is a member of serine/threonine protein kinase which serves as an energy sensor in all eukaryotic cells. In the present study, the role of AMPK in cancer cell metastasis was investigated. We found that 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA), a tumor promoter, induced invasive behavior of HT‐29 colorectal cancer cells, which corresponded to increased ROS production and MMP‐7 expression, whereas TPA suppressed phosphorylation of AMPK in the cells. The TPA‐induced reactive oxygen species (ROS) production and invasion of HT29 cells were inhibited by NADPH oxidase inhibitors (apocynin and diphenyleneiodinium), ROS scavengers (Vitamin C and Vitamin E), and AMPK activator (AICAR). Taken together, TPA induces transition of epithelial HT29 colon carcinoma cells to invasive mesenchymal phenotype through NADPH oxidase activation and AMPK inactivation.Grant Funding Source: This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea

12-O-Tetradecanoylphorbol-13-acetate Promotes Breast Cancer Cell Motility by Increasing S100A14 Level in a Kruppel-like Transcription Factor 4 (KLF4)-dependent Manner

The S100 protein family represents the largest subgroup of calcium binding EF-hand type proteins. These proteins have been reported to be involved in a wide range of biological functions that are related to normal cell development and tumorigenesis. S100A14 is a recently identified member of the S100 protein family and differentially expressed in a number of different human malignancies. However, the transcriptional regulation of S100A14 and its role in breast cancer needs to be further investigated. Here, we determined that 12-O-tetradecanoylphorbol-13-acetate (TPA) up-regulated the expression of KLF4 and facilitated its binding directly to two conserved GC-rich DNA segments within the S100A14 promoter, which is essential for the transactivation of KLF4 induced S100A14 expression. Furthermore, stable silencing of KLF4 significantly suppressed breast cancer cell migration induced by TPA. Collectively, these results offer insights into the fact that TPA provokes cell motility through regulating the expression and function of S100A14 in a KLF4-dependent manner.

Abstract P5-07-04: Aurora kinase regulates PKC-mediated MMP-9 expression and invasion in MCF-7 breast cancer cells

Abstract Aurora kinase is a novel family of serine/threonine kinases. Elevated expression of aurora kinase A and B is observed in many tumor cells, and dysregulation of aurora kinase has been linked to tumorigenesis. Therefore, a number of studies focused in their oncogene activities as anti-tumor targets. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix (ECM) and it is important process for breast cancer cell invasion. MMP-9 can be stimulated by activation of various cellular signaling pathways including protein kinase C (PKC), and mitogen-activated protein kinase (MAPKs), activator protein-1 (AP-1) and nuclear factor-kappaB (NF-κB) signaling pathways. Here, we show that 12-O-tetradecanoylphorbol-13-acetate (TPA), a directly PKC activator stimulation resulted in an up-regulation and phosphorlyation of aurora kinases in MCF-7 breast cancer cells. Also, Results showed that inhibition of the aurora kinases suppressed TPA-induced MMP-9 secretion/expression and cell invasion through suppression of NF-κB, AP-1, and MAPKs in MCF-7 breast cancer cell. In conclusion, this study provides new insight into the novel role of aurora kinase for expression of MMP-9 by TPA and regulation of aurora kinase by TPA through MAPKs signaling pathway. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-07-04.

Abstract 5082: c-Abl function in TPA- and radiation-induced apoptosis in human prostate cancer cells

Abstract We previously reported that 12-O-tetradecanoylphorbol 13-acetate (TPA) decreased transcription and protein levels of Ataxia-Telangiectasia Mutated (ATM) in human prostate cancer cell lines LNCaP and CWR22-Rv1, de-repressing the enzyme ceramide synthase (CerS) to promote apoptosis. Here we analyzed the expression and subcellular distribution of the six mammalian CerS isoforms and their specific ceramide species generated within mitochondrial-associated membranes (MAM) and within the mitochondria in these cells before and after TPA±radiation treatments. This analysis revealed that TPA±radiation induces de novo synthesis of ceramide by specifically activating CerS isoforms 5 and/or 6 generating pro-apoptotic C16-ceramide species in mitochondrial membranes. In addition we demonstrate that LNCaP cells transfected with a c-Abl kinase dead (KD) mutant showed a significant decrease in both TPA and TPA + radiation-induced apoptosis compared with c-Abl (WT) transfected LNCaP cells. Thus, this result indicates that the kinase activity of c-Abl is necessary for TPA±radiation-induced apoptosis in LNCaP cells. In LNCaP cells transfected with WT-c-Abl, ceramide elevation was observed in response to TPA coupled to a synergistic increase of ceramide after TPA+radiation treatment. However, ceramide elevation was abolished in LNCaP transfected with the KD-c-Abl, for both TPA and TPA+radiation treatments. These findings identify kinase active c-Abl as a pro-apoptotic factor in these cells, which utilizes the activation of the CerS pathway to transduce this apoptotic response. Whether c-Abl directly or indirectly activates CerS5 and/or 6 activities, responsible for the generation of the pro-apoptotic ceramide species in response to TPA±radiation, remains to be elucidated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5082. doi:10.1158/1538-7445.AM2011-5082

Phosphorylation of Histone H3 by Protein Kinase C Signaling Plays a Critical Role in the Regulation of the Developmentally Important TBX2 Gene

The mechanism(s) regulating the expression of the TBX2 gene, a key regulator of development, is poorly understood and thus limits an understanding of its function(s). Here we demonstrate that 12-O-tetradecanoylphorbol-13-acetate (TPA) induces TBX2 expression in normal human fibroblasts in a protein kinase C (PKC)-dependent and MAPK-independent manner. Our data further reveal that TPA activates transcription of TBX2 through activating MSK1, which leads to an increase in phosphorylated histone H3 and the recruitment of Sp1 to the TBX2 gene. In addition, TPA was shown to activate MSK1 in a PKC-dependent and MAPK-independent manner. This study is the first to provide evidence that phosphorylation of histone H3 leads to the transcriptional activation of the TBX2 gene and to link MSK1 to PKC.

Protein kinase C activation stabilizes LDL receptor mRNA via the JNK pathway in HepG2 cells

LDL is the most abundant cholesterol transport vehicle in plasma and a major prognostic indicator of atherosclerosis. Hepatic LDL receptors limit circulating LDL levels, since cholesterol internalized by the liver can be excreted. As such, mechanisms regulating LDL receptor expression in liver cells are appealing targets for cholesterol-lowering therapeutic strategies. Activation of HepG2 cells with phorbol esters enhances LDL receptor mRNA levels through transcriptional and posttranscriptional mechanisms. Here, we show that 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced stabilization of receptor mRNA requires the activity of protein kinase C and is accompanied by activation of the major mitogen activated protein kinase pathways. Inhibitor studies demonstrated that receptor mRNA stabilization is independent of the extracellular signal-regulated kinase or p38(MAPK), but requires activation of the c-Jun N-terminal kinase (JNK). An essential role for JNK in stabilizing receptor mRNA was further confirmed through small interfering RNA (siRNA) experiments and by activating JNK through two protein kinase C-independent mechanisms. Finally, prolonged JNK activation increased steady-state levels of receptor mRNA and protein, and significantly enhanced cellular LDL-binding activity. These data suggest that JNK may play an important role in posttranscriptional control of LDL receptor expression, thus constituting a novel mechanism to enhance plasma LDL clearance by liver cells.

Identification and analysis of the promoter region of the human PLC-δ4 gene

The delta4 isoform of phospholipase C (PLC-delta4) is thought to be associated with various cellular functions and disease status. However, little is known about how its function is controlled in cells, particularly in terms of the regulation of its expression. To understand the regulation mechanisms of the PLC-delta4 gene transcription, the 5'-flanking region (-2046 approximately +5) (the nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank/DDBJ data bank under accession numbers DQ302751) of the human PLC-delta4 gene was isolated from human genomic DNA. It was a TATA-less promoter with very GC-rich sequences near the transcription start site. The activity of the PLC-delta4 promoter was shown in various human and mouse cell lines by luciferase reporter assay. Serial deletion analysis identified the core promoter region as being between -402 and -67, in which an E-box and an AP-1 binding site played important roles in the promoter activity. In addition, we also showed that 12-O-tetradecanoylphorbol-1,3-acetate (TPA), a PKC activator and tumor promoter, induced the activity of the PLC-delta4 promoter via the AP-1 binding site. In summary, this study identified a core promoter region of the hPLC-delta4 gene and the factor binding sites responsible for the promoter activity. These results will provide important new information to further understand the regulatory mechanism of the PLC-delta4 function.

Establishment of a Kit‐negative cell line of melanocyte precursors from mouse neural crest cells

We previously established a mouse neural crest cell line named NCCmelb4, which is positive for Kit and negative for tyrosinase. NCCmelb4 cells were useful to study the effects of extrinsic factors such as retinoic acids and vitamin D(3) on melanocyte differentiation, but in order to study the development of melanocytes from multipotent neural crest cells, cell lines of melanocyte progenitors in earlier developmental stages are needed. In the present study, we established an immortal cell line named NCCmelb4M5 that was derived from NCCmelb4 cells. NCCmelb4M5 cells do not express Kit and are immortal and stable in the absence of Kit ligand. They are positive for melanocyte markers such as tyrosinase-related protein 1 and DOPAchrome tautomerase and they contain stage I melanosomes. Interestingly, glial fibrillary acidic protein, which is a marker for glial cells, is also positive in NCCmelb4M5 cells, while NCCmelb4 cells are negative for this protein. Immunostaining and a cell ELISA assay revealed that 12-O-tetradecanoylphorbol 13-acetate (TPA) and cholera toxin (CT) induce Kit expression in NCCmelb4M5 cells. Real-time polymerase chain reaction analysis also demonstrated the induction of Kit mRNA by TPA and CT. Microphthalmia-associated transcription factor mRNA is simultaneously enhanced by the same treatment. Kit induced by TPA/CT in NCCmelb4M5 cells disappeared after the cells were subcultured and incubated without TPA/CT. These findings show that NCCmelb4M5 cells have the potential to differentiate into Kit-positive melanocyte precursors and may be useful to study mechanisms of development and differentiation of melanocytes in mouse neural crest cells.

INK4 Family —A promising target for ‘gene-regulating chemoprevention’ and ‘molecular-targeting prevention’ of cancer

Inactivation of the p16(INK4a) gene is one of the most frequent defects that contribute to oncogenesis in human cancer, since it is a tumor-suppressor gene. Therefore, functional restoration of p16(INK4a) is one of the most effective methods for cancer prevention. We proposed the concept of 'gene-regulating chemoprevention' and 'molecular-targeting prevention' of cancer, which assumes that transcriptional regulation by drugs on tumor-suppressor genes or functionally similar genes to the tumor-suppressor genes contributes to the prevention of human malignancies. The p16(INK4a) homologs p15(INK4b), p18(INK4c) and p19(INK4d) have been recently identified, and these four members constitute the INK4 family of proteins. All directly bind to cyclin D-cyclin dependent kinase (CDK) 4/6 and are therefore specific inhibitors of these complexes. We recently showed that histone deacetylase (HDAC) inhibitors, promising chemopreventive and chemotherapeutical agents, induce p15(INK4b) and p19(INK4d) gene expression and cause growth arrest, suggesting that both genes are important molecular targets for HDAC inhibitors. Furthermore, we found that 12-O-tetradecanoylphorbol-13-acetate (TPA), which is widely used as a tumor promoter and protein kinase C activator, promotes human cancer cell growth through the down-regulation of p18(INK4c) gene expression. This suggests that a mouse two-stage carcinogenesis model using TPA might partially represent the most common human carcinogenesis pathway related to RB. Our results suggest that the INK4 family consists of attractive and promising molecular targets for the 'gene-regulating chemoprevention' and 'molecular-targeting prevention' of cancer.

Synergistic stimulatory effect of 12-O-tetradecanoylphorbol-13-acetate and capsaicin on macrophage differentiation in HL-60 and HL-525 human myeloid leukemia cells

Our previous studies demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) had pharmacological activity for the treatment of myeloid leukemia patients. In the present study, we investigated the effects of TPA alone or in combination with capsaicin (8-methyl-N-vanillyl-6-nonenamide) on growth and differentiation in myeloid leukemia HL-60 cells and in a TPA-resistant HL-60 variant cell line termed HL-525. Treatment of HL-60 cells with TPA (0.16-1.6 nM) for 48 h resulted in concentration-dependent growth inhibition and cell differentiation (via the macrophage pathway). Capsaicin (5-50 microM) inhibited the growth of HL-60 cells in a concentration-dependent manner. Treatment of HL-60 cells with capsaicin alone only resulted in a small increase in the number of differentiated cells but treatment of the cells with TPA in combination with capsaicin synergistically increased differentiation. Moreover, inhibitors of protein kinase C (PKC), 7-hydroxystaurosporin (UCN-01; 100 nM) and chelerythrine (0.5 microM), significantly decreased HL-60 cell differentiation induced by the combination of TPA and capsaicin. These results suggest that PKC may be involved in HL-60 cell differentiation induced by TPA in combination with capsaicin. Capsaicin alone caused a very small increase in differentiation in the TPA-resistant HL-525 cells. However, treatment of HL-525 cells with combinations of TPA (0.16 nM) and capsaicin (10-50 microM) caused a strong synergistic increase in differentiation. Results from the present study suggest that a combination of TPA and capsaicin may improve the therapeutic efficacy of TPA and overcome resistance to TPA in some myeloid leukemia patients.

Prostaglandin D 2 and J 2 induce apoptosis in human leukemia cells via activation of the caspase 3 cascade and production of reactive oxygen species

The presence of prostaglandins (PGs) has been demonstrated in the processes of carcinogenesis and inflammation. In the present study, we found that 12- o-tetradecanoylphorbol 13-acetate (TPA) induced cyclooxygenase 2 (COX-2), but not COX-1, protein expression in HL-60 cells, and the addition of arachidonic acid (AA) in the presence or absence of TPA significantly reduced the viability of HL-60 cells, an effect that was blocked by adding the COX inhibitors, NS398 and aspirin. The AA metabolites, PGD 2 and PGJ 2, but not PGE 2 or PGF 2α, reduced the viability of the human HL60 and Jurkat leukemia cells according to the MTT assay and LDH release assay. Apoptotic characteristics including DNA fragmentation, apoptotic bodies, and hypodiploid cells were observed in PGD 2- and PGJ 2-treated leukemia cells. A dose- and time-dependent induction of caspase 3 protein procession, and PARP and D4-GDI protein cleavage with activation of caspase 3, but not caspase 1, enzyme activity was detected in HL-60 cells treated with PGD 2 or PGJ 2. Additionally, DNA ladders induced by PGD 2 and PGJ 2 were significantly inhibited by the caspase 3 peptidyl inhibitor, Ac-DEVD-FMK, but not by the caspase 1 peptidyl inhibitor, Ac-YVAD-FMK, in accordance with the blocking of caspase 3, PARP, and D4-GDI protein procession. An increase in intracellular peroxide levels by PGD 2 and PGJ 2 was identified by the DCHF-DA assay, and anti-oxidant N-acetyl cysteine (NAC), mannitol (MAN), and tiron significantly inhibited cell death induced by PGD 2 and PGJ 2 by reducing reactive oxygen species (ROS) production. The PGJ 2 metabolites, 15-deoxy-Δ 12,14-PGJ 2 and Δ 12-PGJ 2, exhibited effective apoptosis-inducing activity in HL-60 cells through ROS production via activation of the caspase 3 cascade. The proliferator-activated receptor-γ (PPAR-γ) agonists, rosiglitazone (RO), troglitazone (TR), and ciglitazone (CI), induced apoptosis in cells which was blocked by the addition of the PPAR-γ antagonists, GW9662 and BADGE, via blocking of caspase 3 and PARP cleavage. However, neither GW9662 nor BADGE showed any protective effect on PGD 2- and PGJ 2-induced apoptosis. A differential apoptotic effect of PGs through ROS production, followed by activation of the caspase 3 cascade, was demonstrated.

Biokinetics and subchronic toxic effects of oral arsenite, arsenate, monomethylarsonic acid, and dimethylarsinic acid in v-Ha-ras transgenic (Tg.AC) mice.

Previous research demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment increased the number of skin papillomas in v-Ha-ras transgenic (Tg.AC) mice that had received sodium arsenite [(As(III)] in drinking water, indicating that this model is useful for studying the toxic effects of arsenic in vivo. Because the liver is a known target of arsenic, we examined the pathophysiologic and molecular effects of inorganic and organic arsenical exposure on Tg.AC mouse liver in this study. Tg.AC mice were provided drinking water containing As(III), sodium arsenate [As(V)], monomethylarsonic acid [(MMA(V)], and 1,000 ppm dimethylarsinic acid [DMA(V)] at dosages of 150, 200, 1,500, or 1,000 ppm as arsenic, respectively, for 17 weeks. Control mice received unaltered water. Four weeks after initiation of arsenic treatment, TPA at a dose of 1.25 μg/200 μL acetone was applied twice a week for 2 weeks to the shaved dorsal skin of all mice, including the controls not receiving arsenic. In some cases arsenic exposure reduced body weight gain and caused mortality (including moribundity). Arsenical exposure resulted in a dose-dependent accumulation of arsenic in the liver that was unexpectedly independent of chemical species and produced hepatic global DNA hypomethylation. cDNA microarray and reverse transcriptase–polymerase chain reaction analysis revealed that all arsenicals altered the expression of numerous genes associated with toxicity and cancer. However, organic arsenicals [MMA(V) and DMA(V)] induced a pattern of gene expression dissimilar to that of inorganic arsenicals. In summary, subchronic exposure of Tg.AC mice to inorganic or organic arsenicals resulted in toxic manifestations, hepatic arsenic accumulation, global DNA hypomethylation, and numerous gene expression changes. These effects may play a role in arsenic-induced hepatotoxicity and carcinogenesis and may be of particular toxicologic relevance.

Activation of protein kinase C promotes human cancer cell growth through downregulation of p18(INK4c).

p18(INK4c), a member of INK4 family of cyclin-dependent kinase inhibitors, negatively regulates the cyclin D-cyclin-dependent kinase 4/6 complexes which promote G1/S transition by phosphorylating the retinoblastoma tumor-suppressor gene product. Several recent studies using p18(INK4c)-null mice revealed that the p18(INK4c) plays an important role in cell proliferation and tumor development. We report here that 12-O-tetradecanoylphorbol-13-acetate (TPA), widely used as a protein kinase C (PKC) activator, suppresses the expression of p18(INK4c) through its promoter, accompanied by the induction of human cancer cell growth. Reduction of p18(INK4c) using small interfering RNA (siRNA) also enhanced cell growth, suggesting that p18(INK4c) is a critical target of TPA. Ro 31-8425, a potent and highly specific PKC inhibitor abrogated the suppressive effect of TPA on p18(INK4c) gene expression. However, the expression of dominant-negative c-Jun (TAM-67) did not inhibit the action of TPA on p18(INK4c). These findings suggest that activation of PKC promotes human cancer cell growth through downregulation of p18(INK4c) in an AP-1 activation-independent manner. These results suggest that the accelerated cellular proliferation of some human tumors caused by enhanced PKC activity at least partially involves the suppression of p18(INK4c), which is a ubiquitously expressed cyclin-dependent kinase inhibitor.

TR3 Orphan Nuclear Receptor Mediates Apoptosis through Up-regulating E2F1 in Human Prostate Cancer LNCaP Cells

Early studies suggested both TR3 orphan receptor (TR3) and apoptosis mediator E2F1 might play an important role in mediating prostate cancer cell apoptosis. Their linkage and relationship, however, remain unclear. Here we found that 12-O-tetradecanoylphorbol-13-acetate (TPA) could induce cell apoptosis via induction of TR3 and E2F1 expression in LNCaP prostate cancer cells. Addition of antisense E2F1 could partially rescue the TR3-mediated cell apoptosis, and transfection of the TR3 dominant-negative plasmid could block the TR3-induced E2F1 expression. These data suggest that TPA is able to induce LNCaP cell apoptosis via induction of TR3 resulting in the induction of E2F1. Promoter reporter assays show that TR3 can induce E2F1 expression via binding to the TR3 response element (TR3RE) in the E2F1 promoter -316 to -324 bp region. TR3 can bind specifically to this TR3RE with a Kd of 6.29 nm, and mutations of this E2F1-TR3RE can partially block the TR3-mediated E2F1 expression. Taken together, these data suggest that TPA is able to induce cell apoptosis via a TPA --> TR3 --> E2F1 --> apoptosis pathway in LNCaP cells. Further studies of how to modulate this pathway may allow us to better understand how to control the prostate cancer growth.

Induction of plasminogen activator inhibitor-2 is associated with suppression of invasive activity in TPA-mediated differentiation of human prostate cancer cells

We previously reported that 12- O-tetra-decanoylphorbol-13-acetate (TPA) induces microglia-like differentiation and decreases malignancy in human prostate cancer TSU-Pr1 cells. To investigate the mechanism underlying differentiation and decrease of malignancy in TSU-Pr1 cells treated with TPA, we attempted to identify genes expressed differentially during the differentiation using differential display. We successfully detected plasminogen activator inhibitor type-2 (PAI-2) as one gene up-regulated by TPA treatment. The change in expression of PAI-2 by TPA was blocked by treatment with protein kinase C or mitogen-activated protein kinase inhibitors. We also found that secretion of PAI-2 protein was increased by TPA treatment. Moreover, we demonstrated that suppression of invasive activity of TSU-Pr1 cells by TPA treatment was blocked by co-treatment with anti-PAI-2 antibody. These results suggest that induction of PAI-2 is associated with suppression of invasive activity in TSU-Pr1 cells treated with TPA.

Differential expression of genes during TPA-induced differentiation of human prostatic cancer TSU-Pr1 cells

We are interested in the possibility of a new prostate cancer therapy that would control tumor malignancy via induction of terminal cell differentiation. We previously reported that 12-O-tetra-decanoylphorbol-13-acetate (TPA) induces differentiation into cells with characteristics of microglia and decreases the malignant characteristics of human prostatic cancer TSU-Pr1 cells. To investigate the mechanism underlying differentiation of TSU-Pr1 cells, we attempted to identify genes expressed during differentiation using differential display. We identified four genes expressed differentially after TPA treatment. Levels of expression of two genes, human flavoprotein subunit of complex II and JKTBP, were downregulated by TPA, and expression of two genes, human golgin p245 and bcl-xL, was upregulated. Moreover, we found that the changes in expression of flavo-protein, JKTBP and bcl-xL induced by TPA were blocked by treatment with protein kinase C (PKC) or mitogen-activated protein (MAP) kinase inhibitors that prevent TPA-induced differentiation of TSU-Pr1 cells. These results suggest that the differential expression of these genes is associated with TPA-induced differentiation of TSU-Pr1 cells. We expect that understanding the roles of these genes during differentiation will provide for new approaches and therapeutic targets for treatment of prostate cancer.

Enhancement of anchorage-independent growth of human pancreatic carcinoma MIA PaCa-2 cells by signaling from protein kinase C to mitogen-activated protein kinase.

We found that 12-O-tetradecanoylphorbol-13-acetate (TPA) promoted anchorage-independent growth but did not affect anchorage-dependent growth of MIA PaCa-2 human pancreatic carcinoma cells. TPA markedly activated mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase in an anchorage-independent manner. Two protein kinase C (PKC) isoforms, conventional PKC (cPKC) and novel PKC (nPKC), but not apical PKC, translocated from the cytosolic to the particulate fraction upon TPA treatment. To identify the PKC isoforms involved in the regulation of anchorage-independent growth, four PKC isoforms (alpha, delta, epsilon, and zeta) were forced to be expressed in MIA PaCa-2 cells with an adenovirus vector. Overexpression of nPKCdelta or nPKC epsilon activated MAPK and promoted anchorage-independent growth. Overexpression of cPKCalpha alone did not influence anchorage-independent growth but lowered the concentration of TPA that was required to enhance such growth. Expression of constitutively active MAPK kinase-1 (MEK1) also promoted anchorage-independent growth. Furthermore, PKC inhibitors or an MEK inhibitor completely suppressed both TPA-induced activation of MAPK and promotion of anchorage-independent growth, but a cPKC-selective inhibitor partially suppressed TPA-induced promotion of the growth. Based on these results, we suggest that MAPK activation, mediated by certain isoforms of PKC, plays a part in oncogenic growth of MIA PaCa-2 cells. In summary, our data indicated that specific inhibitors of the cPKC and nPKC signaling pathway might be selective anti-oncogenic growth agents for some types of human pancreatic cancer.

Blockade of AP1 Transactivation Abrogates the Abnormal Expression of Breast Cancer-specific Gene 1 in Breast Cancer Cells

Breast cancer-specific gene 1 (BCSG1) is not expressed in normal breast tissue but is highly expressed in the vast majority of invasive and metastatic breast carcinomas. When over-expressed, BCSG1 significantly stimulates the proliferation and invasion of breast cancer cells. The accumulated evidence suggests that the aberrant expression of BCSG1 in breast carcinomas is caused by transcriptional activation of the BCSG1 gene. However, the transcription factors that activate BCSG1 transcription have not been identified. In this study, we extensively investigated the role of AP1 in BCSG1 expression in breast cancer cells. We demonstrate that there are two closely located AP1 binding sites residing in the first intron of the BCSG1 gene. Mutation of either AP1 motif on the BCSG1 promoter constructs markedly reduces the promoter activity. We further show that 12-O-tetradecanoylphorbol-13-acetate (TPA) increases BCSG1 mRNA expression and up-regulates BCSG1 promoter activity through the intronic AP1 sites. The effect of TPA on BCSG1 transcription is also demonstrated under in vivo conditions in intact cells by using chromatin immunoprecipitation assays that show the TPA-induced binding of c-Jun to the chromatin region encompassing the intronic AP1 sites. Finally, to examine the direct effect of AP1 transactivation on BCSG1 expression, we established stable cell lines of T47D that express the dominant negative mutant of c-Jun, TAM67. RT-PCR and Western blot analyses demonstrated that levels of BCSG1 mRNA and protein in TAM67 transfectants were drastically reduced as compared with mock-transfected cells. Furthermore, inhibition of BCSG1 expression by blocking AP1 transactivation produced a similar repressive effect on cell growth as that by expressing BCSG1 antisense mRNA. We show that the anchorage-independent growth of T47D cells expressing either TAM67 or BCSG1 antisense mRNA is significantly inhibited. Taken together, we provide strong evidence that AP1 plays an overriding role in the transcription of the BCSG1 gene and that blockade of AP1 transactivation down-regulates BCSG1 expression and suppresses tumor phenotype.