The Effects Of 12-O-tetradecanoylphorbol-13-acetate Research Articles

Praeruptorin A Inhibits Human Cervical Cancer Cell Growth and Invasion by Suppressing MMP-2 Expression and ERK1/2 Signaling.

Praeruptorin A (PA) is a pyranocumarin present in the dried root of Peucedanumpraeruptorum Dunn that has anticancer effects against several types of cells. However, the effect of PA on human cervical cancer cells is unknown. Our results indicate that PA significantly inhibited cell proliferation, colony formation, migration, invasion, and wound closure of HeLa and SiHa cells, induced cell cycle arrest at G0/G1 phase, upregulated Rb, p16, p21 and p27 proteins and downregulated cyclin D1 and S-phase kinase-associated protein 2 (Skp2) proteins. PA also significantly reduced expression of matrix metalloproteinase-2 (MMP-2) and increased expression of tissue inhibitor of metalloproteinase-2 (TIMP-2). In addition, PA suppressed ERK1/2 activation and increased the effect of PD98059 (a specific MEK1/2 inhibitor) in downregulation of MMP-2 and upregulation of TIMP-2. PA treatment inhibited the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on upregulation of ERK1/2 activation, MMP-2 expression, cellular migration, and invasion of HeLa cells. Our findings are the first to demonstrate the activity of PA against cervical cancer cells, and suggest this agent has promise as a therapeutic agent in treatment of human cervical cancer.

Combination of 12-O-tetradecanoylphorbol-13-acetate with diethyldithiocarbamate markedly inhibits pancreatic cancer cell growth in 3D culture and in immunodeficient mice.

The aim of the present study was to determine the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and diethyldithiocarbamate (DDTC) alone or in combination on human pancreatic cancer cells cultured in vitro and grown as xenograft tumors in nude mice. Pancreatic cancer cells were treated with either DDTC or TPA alone, or in combination and the number of viable cells was then determined by trypan blue ecxlusion assay and the number of apoptotic cells was determined by morphological assessment by staining the cells with propidium idiode and examining them under a fluorescence microscope. Treatment with DDTC or TPA alone inhibited the growth and promoted the apoptosis of pancreatic cancer cells in a concentration-dependent manner. These effects were more prominent following treatment with TPA in combination with DDTC than following treatment with either agent alone in PANC-1 cells in monolayer cultures and in 3 dimensional (3D) cultures. The potent effects of the combination treatment on PANC-1 cells were associated with the inhibition of nuclear factor-κB (NF-κB) activation and the decreased expression of Bcl-2 induced by DDTC, as shown by NF-κB-dependent reporter gene expression assay and western blot analysis. Furthermore, treatment of nude mice with DDTC + TPA strongly inhibited the growth of PANC-1 xenograft tumors. The results of the present study indicate that the administration of TPA and DDTC in combination may be an effective strategy for inhibiting the growth of pancreatic cancer.

Protein kinase C-α downregulates estrogen receptor-α by suppressing c-Jun phosphorylation in estrogen receptor-positive breast cancer cells

Protein kinase C (PKC) activity is elevated in malignant compared with that in normal human breast tissue. In the present study, we investigated the regulatory mechanism and the co-relationship between PKC-α and estrogen receptor-α (ER-α) in ER-α-positive and tamoxifen-resistant (TAMR) breast cancer cells. Our results showed that the level of ER-α expression was significantly decreased in TAMR when compared with that in tamoxifen-sensitive (TAMS) breast cancer cells. However, PKC-α phosphorylation was increased in TAMR breast cancer cells when compared to that in TAMS breast cancer cells. Additionally, ER-α expression was significantly decreased due to the overexpression of constitutively active PKC-α (CA-PKC-α). Next, we investigated the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a reversible activator of PKC, on ER-α expression in ER-α-positive breast cancer cells. TPA decreased the levels of ER-α expression in a time- and dose-dependent manner. In contrast, the TPA-induced downregulation of ER-α was prevented by Go6983, a specific PKC inhibitor. Notably, we found that CA-PKC-α suppressed c-JUN phosphorylation, which is a major activating protein-1 factor, and TPA-induced downregulation of ER-α was prevented by SR11302, a specific activator protein-1 inhibitor. Taken together, we demonstrated that PKC-α activity suppressed the level of ER-α expression by inhibiting c-JUN phosphorylation in ER-α-positive breast cancer cells. Therefore, we suggest that PKC-α may be a potential therapeutic target for treating ER-positive and TAMR breast cancer.

Effects of 12-O-tetradecanoylphorbol-13-acetate in combination with gemcitabine on Panc-1 pancreatic cancer cells cultured in vitro or Panc-1 tumors grown in immunodeficient mice

In the present study, the effects of 12-O-tetra-decanoylphorbol-13-acetate (TPA) alone or in combination with gemcitabine on the growth of Panc-1 pancreatic cancer cells cultured invitro or grown in NCr immunodeficient nude mice were investigated. Combinations of TPA and gemcitabine synergi-stically inhibited the growth and induced apoptosis in Panc-1 cells. The combination of TPA (0.16nM) and gemcitabine (0.5µM) induced a marked increase in phosphorylated c-Jun NH2-terminal kinase (JNK) in the Panc-1 cells. In animal experiments, NCr nude mice with established Panc-1 tumors received daily intraperitoneal (i.p.) injections of TPA (50ng/g body weight/day) or gemcitabine (0.5µg/g body weight/day) alone or in combination for 26 days. Treatment with daily i.p. injections of low doses of TPA or gemcitabine alone had a modest inhibitory effect on the growth of the tumors. However, the combination of low doses of TPA and gemcitabine more potently inhibited the growth of Panc-1 tumors than either agent used individually. Treatment with TPA or gemcitabine alone or in combination did not affect the body weight of the animals. Clinical trials with TPA alone or in combination with gemcitabine on patients with pancreatic cancer are warranted in order to confirm our results.

Inhibition of metabolic cooperation in Chinese hamster cells by various chemical including tumor promoters.

The effects of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and other chemicals on the contact-feeding phenomenon were investigated. The death of 6-TGr cells was prevented by TPA even upon culture with 3 times as many wild-type cells as in TPA-free medium. Seven out of 18 chemicals tested were shown to inhibit the phenomenon of metabolic cooperation. They were concanavalin A, lithocholic acid, chaetoglobosin A, 3-hydroxyanthranilic acid, urea, anthralin and saccharin. Among known tumor promoters, sodium deoxycholate, estradiol and phenobarbital did not inhibit the phenomenon of metabolic cooperation.

Proteasome-dependent inactivation of Akt is essential for 12-O-tetradecanoylphorbol 13-acetate-induced apoptosis in vascular smooth muscle cells

Apoptosis of vascular smooth muscle cells (SMCs) is a prominent feature of blood vessel remodeling. Here we investigated the effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on SMC apoptosis. We found that TPA treatment induced SMC apoptosis through the rapid downregulation of Akt phosphorylation. The inhibition of Akt activation by TPA was markedly reduced by inhibitors of protein phosphatase 2A and proteasome. Moreover, TPA promoted the ubiquitination of p-Akt, whereas inhibition of TPA-induced PKC activation suppressed the downregulation and ubiquitination of p-Akt. Taken together, these results demonstrate that TPA triggers inactivation of Akt, at least in part, through PKC and Ubiquitin-proteasome degradation, thereby contributing to SMC apoptosis.

Inhibition of NF-κB by (E)3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile (BAY11-7082; BAY) is associated with enhanced 12-O-tetradecanoylphorbol-13-acetate-induced growth suppression and apoptosis in human prostate cancer PC-3 cells

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) alone or in combination with an NF-kappaB inhibitor, (E)3-[(4-methylphenyl)-sulfonyl]-2-propenenitrile (BAY 11-7082; BAY), on the growth and apoptosis of human prostate cancer PC-3 cells cultured in vitro or grown in immunodeficient mice were studied. Treatment of cultured PC-3 cells with TPA (0.2-10 ng/ml) for 96 h resulted in growth inhibition and apoptosis in a concentration-dependent manner. BAY inhibited NF-kappaB activity in PC-3 cells as determined by a luciferase reporter assay and enhanced TPA-induced growth inhibition and apoptosis in cultured PC-3 cells. In animal studies, NCr immunodeficient mice were injected subcutaneously with PC-3 cells in Matrigel. Mice with well-established tumors received daily i.p. injections with TPA (100 ng/g body weight/day), BAY (4 microg/g/day), or a combination of TPA (100 ng/g/day) and BAY (4 microg/g/day) for 36 days. Tumor growth occurred in all of the vehicle-treated control mice. The percent of animals with some tumor regression after 36 days of treatment was 0% for the control group, 40% for the TPA group, 50% for the BAY group and 100% for the TPA + BAY group. Mechanistic studies indicated that treatment of the mice with TPA or TPA + BAY decreased proliferation and increased apoptosis in the tumors. Results from our studies indicate that inhibition of NF-kappaB activity is associated with enhanced TPA-induced growth inhibition and apoptosis in PC-3 cells. Inhibition of NF-kappaB activity by suitable pharmacological inhibitors may be an effective strategy for improving the therapeutic efficacy of TPA in prostate cancer.

Phorbol ester potentiates the growth inhibitory effects of troglitazone via up-regulation of PPARγ in A549 cells

The activation of peroxisome proliferator-activated receptor gamma (PPARγ) has been shown to induce growth arrest and differentiation of various cancer cells. In the current study, we investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of PPARγ and proliferation of A549 cells. TPA elicited a dose- and time-dependent increase in PPARγ mRNA and protein levels. PPARγ expression in response to TPA was attenuated by pretreatment with bisindolylmaleimide I, N-acetyl-L-cysteine (NAC) and PD98059. TPA-induced protein kinase C (PKC) activation was linked to the generation of reactive oxygen species (ROS), both of which were indispensable for PPARγ expression in A549 cells. Pretreatment with bisindolylmaleimide I or NAC blocked TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK), suggesting that ERK-mediated signaling is also involved in the induction of PPARγ. Furthermore, the growth inhibitory effect of troglitazone was significantly potentiated by prolonged incubation with TPA and was attenuated in the presence of GW9662, a specific inhibitor of PPARγ. These effects were associated with an induction of cell cycle arrest at G0/G1 phase, which was accompanied by the induction of p21Waf1/Cip1 expression and decreased cyclin D1 expression. Taken together, these observations indicate that TPA synergizes with PPARγ ligand to inhibit cell growth through up-regulation of PPARγ expression.

Protein kinase C inhibits human hair follicle growth and hair fibre production in organ culture.

In this study we have used a human hair follicle whole-organ culture system to examine the effects of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent activator of protein kinase C (PKC), on hair follicle growth and hair fibre production. Anagen hair follicles were isolated from human facial skin by microdissection and placed in suspension culture in supplemented Williams E medium. Hair follicle and hair fibre lengths were measured daily using an inverted microscope and cumulative growth values were calculated. Treatment with TPA resulted in a potent, dose-dependent inhibition of total cumulative hair follicle growth (IC50 = 1 nM). Hair follicles grew at a comparable rate for 4 days in the presence or absence of 10 nM TPA, after which growth of TPA-treated follicles ceased while control follicles grew by a further 0.8 mm over the subsequent 6 days. In contrast, 10 nM TPA treatment did not affect hair fibre elongation for a period of 8 days, after which TPA-treated fibre production ceased while control fibres grew by a further 0.79 mm over the subsequent 7 days. Incubation of hair follicles with TPA resulted in a 41% inhibition of hair fibre protein synthesis, as measured biochemically from the incorporation of 3H-leucine using a differential akali extraction method. The inhibitory effect of TPA on follicle growth was partially prevented by preincubation with the selective PKC inhibitor H-7, and almost completely prevented by preincubation with the more potent PKC inhibitor Ro 31-7549. Neither agent alone significantly affected follicle growth at concentrations that reversed the TPA response. These findings indicate that PKC is a negative regulator of hair follicle growth, and suggest that PKC may play a part in the transduction of follicular growth-inhibitory signals.

Radiofrequency radiation does not induce stress response in human T-lymphocytes and rat primary astrocytes

Heat shock proteins (HSPs) are rapidly induced by a variety of stressors, including heat shock, ethanol, heavy metals, UV, and gamma-radiation. Mitogen-activated protein kinases (MAPKs) are also involved in the stress transduction pathways in all eukaryotes. In this study, we attempted to determine whether radiofrequency (RF) radiation is able to induce a non-thermal stress response. Human T-lymphocyte Jurkat cells and rat primary astrocytes were exposed to 1763 MHz of RF radiation at an average specific absorption rate (SAR) of either 2 W/kg or 20 W/kg, for 30 min or 1 h. Temperature was completely controlled at 37 +/- 0.2 degrees C throughout the exposure period. The sham exposures were performed under exactly identical experimental conditions without exposure to RF radiation. We assessed alterations in the expression of HSPs and the activation of MAPKs in the RF-exposed cells. No detectable difference was observed in the expression levels of HSP90, HSP70, and HSP27. The phosphorylation status of MAPKs, extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal protein kinases (JNK1/2), or p38, did not change significantly. In order to determine whether RF radiation can promote the effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on stress response, cells were exposed to RF radiation coupled with TPA treatment. When TPA alone was applied, the MAPKs were found to be phosphorylated in a dose-dependent manner. However, RF radiation did not result in any enhancement of TPA-induced MAPK phosphorylation. Neither TPA nor RF radiation exerted any detectable effect on the induction of HSPs. These results indicate that 1763 MHz RF radiation alone did not elicit any stress response, nor did it have any effect on TPA-induced MAPK phosphorylation, under our experimental conditions.

Analysis of Epstein-Barr virus gene expression upon phorbol ester and hydroxyurea treatment by real-time quantitative PCR

Epstein-Barr virus (EBV) has the potential to undergo latent and lytic pathways during infection. However, expression of many of the viral genes during the lytic-latent transition remains unclear. In this study, we investigated the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) and hydroxyurea (HU), two commonly used modulators of EBV life cycle, on the expression profiles of the entire genome of EBV persistent infected in B95-8 cells. After treatment with TPA for 48 h, the copy number of EBV genome in the cells increased about 2.5 fold, whereas HU treatment resulted in a reduction to approximately two-thirds of the original level. Except a small set of genes, the amounts of EBV mRNA are generally less abundant than that of beta-actin. The expression of a large fraction of the 80 EBV genes was found to be activated after TPA treatment with a noticeable increase of 19 and 21 fold, respectively in BSLF1 and BBLF4. In contrast, treatment of the B95-8 cells with HU, a nucleotide synthesis inhibitor, dramatically suppressed the expression of EBV lytic genes. In summary, we have demonstrated that real-time quantitative PCR is a reliable method to monitor the influence of drug-treatment in EBV genes regulation. Our results also provide a basis for further investigation on how the virus coordinates its own gene expression during latent-lytic pathway transition.

Nitric oxide induces expression of cyclooxygenase-2 in mouse skin through activation of NF-kappaB.

Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are frequently overexpressed in tumor tissues or transformed cells. In the present work, we assessed the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on expression of iNOS and COX-2 in mouse skin. Topical application to the dorsal skin of female ICR mice of 10 nmol TPA led to maximal induction of iNOS and COX-2 protein expression at approximately 2 and 4 h, respectively. When applied topically onto shaven backs of mice 30 min prior to TPA, the NOS inhibitor aminoguanidine (AG) inhibited the expression of COX-2 protein at the pharmacologically effective dose. Pretreatment with a more specific iNOS inhibitor, N(G)-nitro-l-arginine-methyl ester, also suppressed TPA-induced COX-2 expression. Immunohistochemical analysis of TPA-treated mouse skin using an anti-nitrotyrosine antibody reveals enhanced levels of nitrotyrosine protein localized in epidermal and dermal layers. Topical application of NO donors, such as sodium nitroprusside (SNP) and S-nitroso-N-acetyl-d,l-penicillamine, induced expression of COX-2 in mouse skin, which was attenuated by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide. SNP treatment stimulated NF-kappaB activation in mouse skin, which was associated with the degradation of IkappaBalpha. Topical application of inhibitors of NF-kappaB, such as pyrrolidine dithiocarbamate or N-alpha-p-tosyl-l-lysine chloromethylketone, inhibited the SNP-induced COX-2 expression. SNP induced a weak but concentration-related increase in COX-2 expression in cultured mouse keratinocytes, which was abolished by treatment with SN50, a specific inhibitor of nuclear translocation of NF-kappaB. Mouse keratinocytes treated with SNP exhibited an elevated NF-kappaB-driven COX-2 promoter activity. Topical application of AG (10 micro mol) prior to each TPA treatment after initiation reduced the multiplicity of papillomas by 44% at 22 weeks. In conclusion, up-regulation of COX-2 by NO may be mediated by activation of NF-kappaB in mouse skin, which provides a molecular mechanism by which COX-2 is induced during tumor promotion.

Notch Inhibits AP-1

The activation of Notch requires its proteolytic cleavage, which results in the translocation of the Notch intracellular domain (NICD) to the nucleus. NICD binds to the repressor protein CSL [C promoter binding factor1/RBP-Jκ, Su(H), and Lag-1] and acts to derepress the expression of several specific genes. Chu et al . have found that, independent of its role in gene activation, NICD can inhibit the expression of activator protein-1 (AP-1)-dependent genes. Overexpression of NICD in cells transfected with AP-1 component genes greatly decreased AP-1-mediated gene expression, but had no effect on nuclear factor kappa B (NF-κB)-dependent gene expression. This suggests that NICD specifically inhibited AP-1-dependent transactivation rather than affecting global gene expression. The effect of 12- O -tetradecanoylphorbol-13-acetate (TPA) in enhancing the endogenous expression of the AP-1-regulated genes interleukin-8 and matrix metalloproteinase-1 in cells treated with TPA was also inhibited by NICD. Localization of NICD to the nucleus correlated with the repression of AP-1. Additionally, NICD did not affect the activation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1 (ERK1) or ERK2, p38, or c-Jun NH 2 -terminal kinase (JNK), indicating that NICD most likely exerts its inhibitory effects in the nucleus. Thus, these data suggest that the inhibitory effects of NICD on AP-1-mediated transcription may be important for the proper regulation of cellular responses to Notch. J. Chu, S. Jeffries, J. E. Norton, A. J. Capobianco, E. H. Bresnick, Repression of activator protein-1-mediated transcriptional activation by the Notch-1 intracellular domain. J. Biol. Chem. 277 , 7587-7597 (2002). [Abstract] [Full Text]

Notch Inhibits AP-1

The activation of Notch requires its proteolytic cleavage, which results in the translocation of the Notch intracellular domain (NICD) to the nucleus. NICD binds to the repressor protein CSL [C promoter binding factor1/RBP-Jκ, Su(H), and Lag-1] and acts to derepress the expression of several specific genes. Chu et al. have found that, independent of its role in gene activation, NICD can inhibit the expression of activator protein-1 (AP-1)-dependent genes. Overexpression of NICD in cells transfected with AP-1 component genes greatly decreased AP-1-mediated gene expression, but had no effect on nuclear factor kappa B (NF-κB)-dependent gene expression. This suggests that NICD specifically inhibited AP-1-dependent transactivation rather than affecting global gene expression. The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) in enhancing the endogenous expression of the AP-1-regulated genes interleukin-8 and matrix metalloproteinase-1 in cells treated with TPA was also inhibited by NICD. Localization of NICD to the nucleus correlated with the repression of AP-1. Additionally, NICD did not affect the activation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1 (ERK1) or ERK2, p38, or c-Jun NH2-terminal kinase (JNK), indicating that NICD most likely exerts its inhibitory effects in the nucleus. Thus, these data suggest that the inhibitory effects of NICD on AP-1-mediated transcription may be important for the proper regulation of cellular responses to Notch.J. Chu, S. Jeffries, J. E. Norton, A. J. Capobianco, E. H. Bresnick, Repression of activator protein-1-mediated transcriptional activation by the Notch-1 intracellular domain. J. Biol. Chem. 277, 7587-7597 (2002).[Abstract][Full Text]

Survival mechanisms induced by 12-O-tetradecanoylphorbol-13-acetate in normal human melanocytes include inhibition of apoptosis and increased Bcl-2 expression.

Phorbol esters, which activate protein kinase C, stimulate the growth of normal human melanocytes yet inhibit the growth of most melanoma cells. We investigated whether apoptosis mediates the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on melanocyte and melanoma cell growth. Few apoptotic cells were present when melanocytes were cultured with TPA. Upon removal of TPA, the number of apoptotic cells increased over 10 days. Addition of TPA did not induce apoptosis in a metastatic melanoma cell line, Demel, although it strongly inhibited its growth. Protection of normal melanocytes from apoptosis was associated with high levels of Bcl-2. Following withdrawal of TPA from melanocytes, the expression of Bcl-2 decreased steadily. Bax and Bcl-X(L) levels did not differ between melanoma cells or melanocytes and were unaffected by the addition of TPA. These results suggest that TPA plays an important role in stimulating the growth of melanocytes by promoting anti-apoptotic mechanisms associated with high levels of Bcl-2.

Phorbol ester-induced production of cytostatic factors by normal and oncogenic Ha- ras -transformed human breast cell lines

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell cycle progression were examined in the human breast cell line MCF10A-Neo and a derivative line which expresses a Ha-ras oncogene (MCF10A-NeoT cells). Exposure of MCF10A-Neo cultures to TPA induced a G(1) arrest that lasted approximately 16-24 h (IC(50) approximately 0.5 nM). TPA-treated cultures produced a cytostatic conditioned medium. Cytostatic activity was detectable within 1 h of TPA treatment, peaked 3-7 h later and disappeared between 16 and 24 h post-treatment. However, cytostatic conditioned medium could be quickly regenerated by re-feeding previously treated cultures with new medium. Removal of latent transforming growth factor beta (TGF beta) from the culture medium, supplementing the culture medium with anti-TGFbeta or soluble TGF beta(II) receptor, or pre-absorption of conditioned medium with anti-TGF beta all reduced the cytostatic effects of TPA or conditioned medium on MCF10A-Neo proliferation by approximately 50%. Co-treatment with the serine protease inhibitors aprotinin or plasminogen activator inhibitor-1 also suppressed the cytostatic activity of TPA approximately 50%. Conditioned medium isolated from TPA-treated MCF10A-Neo cultures was transiently cytostatic to MCF10A-NeoT cells. The proliferation of MCF10A-NeoT cultures, in contrast to MCF10A-Neo cells, was suppressed at least 72 h following TPA exposure. Conditioned medium isolated from TPA-treated MCF10A-NeoT cultures also suppressed MCF10A-NeoT proliferation for approximately 72 h, but suppressed MCF10A-Neo proliferation for <24 h. These studies suggest that TPA quickly activates proteolytic processes in MCF10A-Neo cells leading to the activation of latent TGF beta supplied by the serum in the culture medium. TPA also stimulates the production of an additional cytostatic factor(s) which signals via a mechanism not involving the TGF beta(II) receptor. Lastly, expression of an activated Ha-ras oncogene alters both the types of cytostatic factors produced following TPA treatment and responsiveness to these factors.

Phorbol ester-induced production of cytostatic factors by normal and oncogenic Ha-ras-transformed human breast cell lines

The effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on cell cycle progression were examined in the human breast cell line MCF10A-Neo and a derivative line which expresses a Ha-ras oncogene (MCF10A-NeoT cells). Exposure of MCF10A-Neo cultures to TPA induced a G1 arrest that lasted ~16–24 h (IC50 ~0.5 nM). TPA-treated cultures produced a cytostatic conditioned medium. Cytostatic activity was detectable within 1 h of TPA treatment, peaked 3–7 h later and disappeared between 16 and 24 h post-treatment. However, cytostatic conditioned medium could be quickly regenerated by re-feeding previously treated cultures with new medium. Removal of latent transforming growth factor β (TGFβ) from the culture medium, supplementing the culture medium with anti-TGFβ or soluble TGFβII receptor, or pre-absorption of conditioned medium with anti-TGFβ all reduced the cytostatic effects of TPA or conditioned medium on MCF10A-Neo proliferation by ~50%. Co-treatment with the serine protease inhibitors aprotinin or plasminogen activator inhibitor-1 also suppressed the cytostatic activity of TPA ~50%. Conditioned medium isolated from TPA-treated MCF10A-Neo cultures was transiently cytostatic to MCF10A-NeoT cells. The proliferation of MCF10A-NeoT cultures, in contrast to MCF10A-Neo cells, was suppressed at least 72 h following TPA exposure. Conditioned medium isolated from TPA-treated MCF10A-NeoT cultures also suppressed MCF10A-NeoT proliferation for ~72 h, but suppressed MCF10A-Neo proliferation for <24 h. These studies suggest that TPA quickly activates proteolytic processes in MCF10A-Neo cells leading to the activation of latent TGFβ supplied by the serum in the culture medium. TPA also stimulates the production of an additional cytostatic factor(s) which signals via a mechanism not involving the TGFβII receptor. Lastly, expression of an activated Ha-ras oncogene alters both the types of cytostatic factors produced following TPA treatment and responsiveness to these factors.

Overexpression of novel protein kinase C δ in BL6 murine melanoma cells inhibits the proliferative capacity in vitro but enhances the metastatic potential in vivo

In this study we analysed the effect of overexpressing novel protein kinase C delta isoform (n-PKC delta) on melanin synthesis and metastatic potential in the highly metastatic BL6 murine melanoma cells. The proliferative capacity in vitro and into matrigel in vivo were also examined. Although murine melanocytes express the n-PKC delta isoform, BL6 cells do not express this isoform at levels detectable by Western blot analysis. In untransfected and transfected cells we also studied the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a modulator of specific isoforms of PKC, and of bryostatin 1, a potent immunomodulator and antineoplastic drug and a partial agonist of PKC. Our results demonstrate a pivotal role for this isoform in melanin synthesis and the close relationship between n-PKC delta expression and its association with the particulate fraction, melanogenesis and metastatic potential. In fact, heterogeneous BL6 cells overexpressing n-PKC delta and all the clones isolated showed increased intracellular melanin and metastatic capacity. TPA and bryostatin 1 decreased n-PKC delta expression, the intracellular melanin level and metastatic capacity in both cell lines. Therefore both treatments were able to abolish the effects of overexpressing n-PKC delta.

Phorbol ester-induced production of prostaglandin E2 from phosphatidylcholine through the activation of phospholipase D in UMR-106 cells

To determine the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA) on phospholipase D (PLD) activity in osteoblast-like UMR-106 cells, we used cells prelabeled with [3H] myristic acid or [3H] arachidonic acid, which were preferentially incorporated to phosphatidylcholine. The treatment of [3H] myristate-labeled cells with TPA in the presence of 1% ethanol caused a dose-dependent formation of [3H] phosphatidylethanol (PEt), a product specific to PLD, suggesting an activation of this enzyme. Pretreatment of the cells with protein kinase C (PKC) inhibitors (GF109203X, staurosporine or H-7) abolished the TPA-dependent formation of PEt. The PEt formation in response to TPA treatment was not observed after the pretreatment of the cells with TPA to downregulate PKC. These results suggest the involvement of PKC in the TPA-induced activation of PLD. With [3H] arachidonate-labeled cells, TPA treatment in the absence of ethanol resulted in the liberation of [3H] arachidonic acid, which was gradually converted to prostaglandin E2 (PGE2), but the accumulations of [3H] phosphatidic acid (PA) and [3H] diacylglycerol (DAG) were very small and temporary. In contrast, PA was linearly accumulated following TPA treatment, when the cells were pretreated with an inhibitor of phosphatidate phosphohydrolase (PAP), propranolol, with no accumulation of either DAG or arachidonic acid. The TPA treatment of the cells pretreated with a DAG lipase inhibitor, RHC-80267, caused the generation of DAG after a lag period of approximately 5 min, with a very small and temporary accumulation of PA. The TPA treatment of cells pretreated with a cyclooxygenase (COX) inhibitor, indomethacin, blocked the PGE2 production. The TPA-induced PGE2 production was not affected by the pretreatment of cells with a phospholipase A2 inhibitor, p-bromophenacylbromide, or with a phospholipase C inhibitor, D-609. TPA also stimulated PGE2 production in osteoblastic cells that were enzymatically isolated from adult rat calvaria, and the experiments with lipid metabolizing enzyme inhibitors gave the same profile of inhibition of TPA-induced PGE2 production as was observed in UMR-106 cells. These results suggest that PA formed as a consequence of the activation of PLD by TPA is rapidly converted to arachidonic acid via a PAP/DAG lipase pathway, followed by a gradual conversion of arachidonic acid to PGE2 by COX in both UMR-106 cells and isolated adult osteoblastic cells, and that neither phospholipase A2 nor phospholipase C is involved in the TPA-induced PGE2 production. To the best of our knowledge, this is the first report that shows that the activation of PKC in osteoblastic cells leads to the production of PGE2 via a PLD/PAP/DAG lipase/COX pathway.

12-O-tetradecanoylphorbol-13-acetate upregulates the Ah receptor and differentially alters CYP1B1 and CYP1A1 expression in MCF-7 breast cancer cells

Elevated expression of cytochrome P450 1B1 (CYP1B1) and estradiol 4-hydroxylation have been reported to be biomarkers of tumorigenesis in humans. The aromatic hydrocarbon receptor (AhR) regulates expression of human cytochrome P450 1A1 (CYP1A1) and CYP1B1, 17beta-estradiol (E2) 2- and 4-hydroxylases, respectively. There is also evidence that expression of estrogen receptor alpha (ERalpha) potentiates CYP1A1 inducibility in breast cancer cells. To characterize these relationships further, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), which downregulates ERalpha, and the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the expression of AhR, ERalpha, CYP1A1, and CYP1B1 in MCF-7 human breast cancer cells. Treatment with TPA, which suppressed ERalpha mRNA levels, caused a greater than fourfold elevation of AhR mRNA and protein levels, whereas treatment with TCDD caused a decrease in AhR protein but no change in ERalpha or AhR mRNA levels. In MCF-7 cells treated with TPA prior to treatment with TCDD, the AhR mRNA level was elevated, the ERalpha mRNA level remained suppressed, and the ratio of CYP1B1 to CYP1A1 mRNA was increased compared with treatment with TCDD alone. A corresponding increase in the ratio of the rates of 4- to 2-hydroxylation pathways of E2 metabolism was also observed in response to pretreatment with TPA prior to the addition of TCDD. These results demonstrate differential regulation of the human CYP1A1 and CYP1B1 genes and provide a cellular model to investigate further the mechanisms that may be involved in the elevated expression of CYP1B1 in tumorigenesis.