Tumour necrosis factor-alpha induces translocation of protein kinase C in tumour necrosis factor-sensitive cell lines.

In this study we investigated whether the anti-proliferative effect of tumour necrosis factor-alpha (TNF-alpha) was associated with the activation of protein kinase C (PKC), using PANC-1 cells (TNF-alpha sensitive) and LoVo cells (TNF-alpha resistant). In combination with 12-0-tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, TNF-alpha caused marked inhibition of the growth of LoVo cells. Inhibition of PANC-1 cell growth by TNF-alpha was blocked by pretreatment with TPA for 24 hr, along with down-regulation of PKC activity. Intracellular translocation of PKC from cytosol to membrane was induced by TNF-alpha treatment in PANC-1 cells but not in LoVo cells.     

Involvement of annexin V in the antiproliferative effect of GnRH agonist on cultured human uterine leiomyoma cells

The objective of this study was to elucidate the role of annexin V, an endogenous inhibitor of protein kinase C (PKC), with regard to the antiproliferative effect of gonadotrophin-releasing hormone (GnRH) agonist (buserelin) on cultured human uterine leiomyoma cells. Uterine leiomyoma tissue was collected from the surgical specimens of patients and cells from 37 specimens (15 cases) were cultured. For up to 96 h after the addition of buserelin to the cultured cells, a time-dependent antiproliferative effect was noted in the group to which 10(-5) mol/l buserelin was added. Both the intracellular concentration of annexin V and the expression of annexin V mRNA increased time-dependently with the addition of buserelin. The intracellular concentration of annexin V increased with the addition of PKC activator (12-O:-tetradecanoylphorbor-13-acetate; TPA) much as it did with the addition of buserelin, and the rise in the concentration caused by the addition of buserelin was completely attenuated by pretreatment with PKC inhibitor (calphostin C). Our findings suggest that buserelin inhibits cell proliferation in cultured human uterine leiomyoma cells accompanied with an increase in the intracellular concentration of annexin V, mediated, at least in part, by the activation of PKC.     

Comparison of interleukin 2 and 12-O-tetradecanoyl-phorbol 13-acetate as signals for protein kinase C activation in purified human T lymphocytes

Interleukin 2 (IL2) and 12-O-tetradecanoylphorbol 13-acetate (TPA) have been compared for their ability to induce translocation of protein kinase C (PKC) in T lymphocytes prestimulated with anti-CD3 monoclonal antibody (mAb), either in the presence or absence of monocytes. TPA alone did not promote purified T cell growth, but it was able to induce a transient, within 30 min, translocation of PKC activity. The profiles of PKC association with the membrane of the T cells under TPA stimulation were quite similar when either the anti-CD3 mAb or the fixed monocytes, or both, were added to the T cells. The decrease of cytosolic PKC under TPA stimulation was less pronounced for the purified T cells stimulated with anti-CD3 mAb, fixed monocytes alone or both than for unstimulated purified T cells. Even in the absence of monocytes, the addition of exogenous IL2 to the anti-CD3 mAb-treated T cells resulted in PKC translocation, with a transient increase in the PKC activity found in both the particulate and cytosolic fractions. When exogenous IL2 was added to the proliferating T cells, the association of PKC with the membrane was prolonged and the activity did not reach a plateau during the first 2 h after the IL2 stimulation. In parallel, the level of PKC associated with the membrane was higher in proliferating cells than in resting cells even 4 days after stimulation. These results suggest that activation of PKC by IL2 might be different from the direct activation of PKC by TPA and that a specific activation pathway, at least kinetically distinct from the classical phosphatidyl inositol diphosphate degradation by phospholipase C, might be involved during IL2 stimulation of T lymphocytes through high-affinity IL2 receptors.     

12-O-tetradecanoyl phorbol 13-acetate, protein kinase C (PKC) activator, protects human leukemia HL-60 cells from taxol-induced apoptosis: possible role for extracellular signal-regulated kinase

The purpose of this study was to evaluate whether the mitogen-activated protein kinase / extracellular signal-regulated kinase (MEK) signaling pathway contributes to 12-O-tertadecanoyl phorbol 13-acetate (TPA)mediated protection from taxol-induced apoptosis of human leukemia HL-60 cells. Treatment of cells with taxol for 12 h resulted in apoptosis of HL-60 cells. TPA was protective against taxol-induced apoptosis and this anti-apoptotic effect was reversible when TPA was used in conjunction with staurosporine and H-7, PKC inhibitors, suggesting that TPA may protect HL-60 cells against taxol-induced apoptosis via the PKC-dependent pathway. Since TPA stimulates MEK signal transduction pathway in HL-60 cells, we postulated that MEK pathway may be playing a role in the ability of TPA to inhibit taxol-induced apoptosis. PD098059, a specific MEK kinase inhibitor, abolished the ability of TPA to inhibit taxol-induced apoptosis. These results suggest that activation of PKC in HL-60 cells confers protection against taxol-induced apoptosis and that MEK mediates anti-apoptotic signaling of PKC.     

Isolation and characterization of a T lymphocyte mutant defective in the protein kinase C signal transduction pathway

The phorbol ester TPA is a potent protein kinase C (PKC) activator and a cofactor in the activation of the human Jurkat leukemic T cell line. We have studied the implication of the PKC signaling pathway in the process of T cell activation by generating TPA resistant mutants of Jurkat. These mutants were obtained by recovery of cells that survived a growth arrest induced by TPA. Several cellular phenomena dependent on TPA were dramatically altered in the mutated cells. The mutants were unable to form homoaggregates upon TPA stimulation. Moreover, they did not produce interleukin-2 after activation through engagement of the T cell receptor, in the presence of TPA. These results suggest that the PKC signaling pathway activated by TPA is defective in these cells. In an attempt to define and locate the defect present in the mutants, we have analysed the biochemical properties of PKC, the cellular receptor of TPA. The increase in kinase activity and the translocation of the enzyme to the plasma membrane after stimulation by TPA appeared to be normal in the mutants. We hypothesize that a metabolic step, critical for the completion of T cell activation, distinct from protein kinase C, is impaired in the mutant cells.     

12-O-Tetradecanoyl Phorbol 13-Acetate,Protein Kinase C (PKC) Activator,Protects Human Leukemia HL-60 Cells from Taxol-Induced Apoptosis: Possible Role for Extracellular Signal-Regulated Kinase

Abstract

The purpose of this study was to evaluate whether the mitogen-activated protein kinase / extracellular signal-regulated kinase (MEK) signaling pathway contributes to 12-O-tertadecanoyl phorbol 13-acetate (TPA)mediated protection from taxol-induced apoptosis of human leukemia HL-60 cells. Treatment of cells with taxol for 12 h resulted in apoptosis of HL-60 cells. TPA was protective against taxol-induced apoptosis and this anti-apoptotic effect was reversible when TPA was used in conjunction with staurosporine and H-7, PKC inhibitors, suggesting that TPA may protect HL-60 cells against taxol-induced apoptosis via the PKC-dependent pathway. Since TPA stimulates MEK signal transduction pathway in HL-60 cells, we postulated that MEK pathway may be playing a role in the ability of TPA to inhibit taxol-induced apoptosis. PD098059, a specific MEK kinase inhibitor, abolished the ability of TPA to inhibit taxol-induced apoptosis. These results suggest that activation of PKC in HL-60 cells confers protection against taxol-induced apoptosis and that MEK mediates anti-apoptotic signaling of PKC.     

Role of the nitric oxide synthase pathway in inhibition of growth of interferon-sensitive and interferon-resistant Rickettsia prowazekii strains in L929 cells treated with tumor necrosis factor alpha and gamma interferon.

The ability of tumor necrosis factor alpha (TNF-alpha) alone and in combination with gamma interferon (IFN-gamma) to inhibit the growth of interferon-sensitive and -resistant Rickettsia prowazekii strains in mouse L929 cells was examined, and the possible role of the nitric oxide synthase pathway in the suppression of rickettsial growth induced by TNF-alpha, IFN-gamma, or both cytokines was evaluated. TNF-alpha inhibited the growth of strains Madrid E (IFN-gamma sensitive and alpha/beta interferon [IFN-alpha/beta] sensitive) and Breinl (IFN-gamma sensitive and IFN-alpha/beta resistant), but not that of strain 83-2P (IFN-gamma resistant and IFN-alpha/beta resistant), in L929 cells. Inhibition of the growth of the Madrid E strain in L929 cells treated with TNF-alpha and IFN-gamma in combination was greater than that observed with either TNF-alpha or IFN-gamma alone. Similarly, inhibition of the growth of the Breinl strain in L929 cells treated with both cytokines was greater than that observed with TNF-alpha alone; however, it did not differ significantly from the inhibition observed with IFN-gamma alone. Although strain 83-2P was resistant to TNF-alpha or IFN-gamma alone, its growth was inhibited in L929 cells treated with TNF-alpha and IFN-gamma in combination. Nitrite production was measured in mock-infected and infected L929 cell cultures, and the nitric oxide synthase inhibitors NG-methyl-L-arginine (NGMA) and aminoguanidine were used to evaluate the role of the nitric oxide synthase pathway in cytokine-induced inhibition of rickettsial growth. Nitrite production was induced in mock-infected or R. prowazekii-infected L929 cell cultures treated with IFN-gamma plus TNF-alpha, but not in mock-infected cultures that were untreated or treated with IFN-gamma or TNF-alpha alone. Nitrite production was also not induced in untreated, R. prowazekii-infected cultures; however, in some instances, it was induced in infected cultures treated with IFN-gamma or TNF-alpha alone. Nitrite production was blocked by NGMA or aminoguanidine, and these compounds markedly relieved the synergistic inhibitory effect of IFN-gamma plus TNF-alpha on the growth of strain 83-2P in L929 cells. In contrast, NGMA did not alleviate the inhibition of the growth of the Madrid E strain in L929 cells treated with IFN-gamma or TNF-alpha alone; however, it slightly and variably relieved the inhibition of the growth of the Madrid E strain in L929 cells treated with IFN-gamma and TNF-alpha in combination.(ABSTRACT TRUNCATED AT 400 WORDS)     

Affinity alteration of insulin receptor induced by a phorbol ester

The effect of a phorbol ester tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA) on 125I-insulin binding to human cells was examined. TPA markedly inhibits insulin binding to cultured human lymphocytes and macrophages but has a minimal effect on human fibroblasts. This inhibition is temperature, time, and concentration dependent. The inhibition of insulin binding to the cells at 37 degrees C occurs within minutes and diminishes by 6 h of incubation. Insulin binding is decreased by TPA whether the phorbol ester is added before, after, or simultaneously with 125I-insulin to the cell suspension. Scatchard analysis of binding to IM-9 lymphocytes indicates that TPA affects the affinity rather than the number of insulin receptors. The phorbol ester has only a small effect on 125I-human growth hormone binding in cultured human lymphocytes. TPA perturbs the insulin receptor of cultured human lymphocytes in a fashion similar to its effect on the epidermal growth factor receptor of several other cell types. The specific mechanism of TPA action that affects the receptor of these two potent growth factors (i.e., insulin and epidermal growth factor), however, is unknown.     

Regulation of the 12-o-tetradecanoyl-phorbol-13-acetate-induced inhibition of intercellular communication

Effects of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and protein kinase C (PKC) inhibitors on cell-cell communication were studied in a normal rat liver cell line, clone 9. Communication was observed and quantitated with microspectrofluorometric and image analysis techniques following scrape-loading of the cells with lucifer yellow. Lucifer yellow migrated as far as ten cells away from the scraped edge in control populations. Two minute TPA (25–50 μg/ml) treatment inhibited dye movement such that the dye remained mainly in the cells at the cut edge. The TPA-induced inhibition of cell-cell communication could be partially blocked by 15 min treatment of the cell populations with the PKC inhibitors trifluoperazine (30 μg/ml), staurosporine (2 × 10^?8 or 2 × 10^?6 M), sangivamycin (15 or 200 μM), or a PKC inhibitor peptide (20 μg/ml) scraped in at the same time as lucifer yellow. Normal communication was observed in cultures treated only with PKC inhibitors. Lower concentrations of TPA (50 ng/ml-1 μg/ml) used for 2 min did not inhibit dye communication. Our results demonstrate the phorbol ester-induced interruption of cell-cell communication. The inhibition of PKC by inhibitors eliminates the effect of TPA on communication. Our data are consistent with a role of PKC in the control of junctional communication. © 1993 Wiley-Liss, Inc.     

浅蓝菌素诱发人结肠癌细胞凋亡的实验研究

目的　探讨脂肪酸合酶抑制剂———浅蓝菌素能否阻遏人结肠癌细胞增殖与诱发凋亡。方法　采用人结肠癌细胞系LoVo ,应用细胞形态学观察 ,噻唑蓝法 (MTT法 ) ,片断DNA琼脂糖凝胶电泳及流式细胞仪等方法进行检测和观察。结果　LoVo细胞在浅蓝菌素作用下 ,细胞增殖被阻遏 ,并呈现剂量效应关系 ,浅蓝菌素浓度 10 -9～ 10 -5mol/L增殖抑制率由 ( 2 1.0± 15 .9) %到 ( 96 .3± 2 7) %(P <0 .0 5或 0 .0 1)。同时诱发细胞发生凋亡。凋亡细胞表现为细胞固缩 ,核染色质凝聚、边集或断裂。细胞DNA裂解片段呈典型的“阶梯状”排列的条带 ,流式细胞仪显示“凋亡”峰 ,细胞周期分析浅蓝菌素能阻滞肿瘤细胞从S期进入G2 M期 ,并诱导其细胞凋亡。浅蓝菌素对人成纤维细胞增殖无明显影响。结论　脂肪酸合酶抑制剂可能通过抑制LoVo细胞内源性脂肪酸合成 ,诱发细胞凋亡来阻遏LoVo细胞的增殖     

尼妥珠单抗联合化疗药物对人结肠癌细胞株LoVo的实验研究

目的探讨表皮生长因子受体（EGFR）的单克隆抗体尼妥珠单（nimotuzumab，h-R3）与化疗药物联合对结肠癌细胞株kvo的作用，初步探讨其可能的作用机制。方法采用MTT法测定不同药物单药、两药联合分别对结肠癌细胞株LoVo的IC50值；计算不同药物联合的q值，以确定药物联合的作用；流式细胞仪检测不同药物单药或者联合对细胞周期分布及凋亡比例的影响。结果联合h-R3能显著提高伊立替康对LoVo细胞的细胞抑制率，诱导更多肿瘤细胞发生凋亡以及产生G0／G1期、G2／M期阻滞，与5-氟尿嘧啶联合具有类似的作用，但较伊立替康不明显，而与奥沙利铂联合无明显增效作用。结论h-R3通过协同或相加作用提高伊立替康、氟尿嘧啶的细胞杀伤作用．其机制与促进细胞凋亡和影响细胞周期分布有关，奥沙利铂联合对LoVo细胞无明显增效作用。为进一步的体内及体外实验提供了一定的指导作用。     

Protein kinase C-activating phorbol esters augment expression of T cell receptor genes

Tumor-promoting phorbol esters (PE) can modulate cellular functions and cell surface determinant expression in a variety of cell types, including T lymphocytes, presumably by activating the enzyme, protein kinase C (PKC). To examine whether PKC might be involved in regulating the expression of genes encoding the antigen-specific T cell receptor (TCR), we cultured the murine thymoma line, EL4, in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA) and analyzed the expression of TCR alpha or beta-chain genes by Northern blots. TPA stimulation of an interleukin 2 (IL 2)-producing variant, EL4+, induced a 3-4-fold increase in TCR beta, but not alpha, chain mRNA. Maximal increase was obtained with 3 ng/ml TPA and 12 h of stimulation. This effect appeared related to PKC activation because other tumor-promoting PE known to be PKC activators, but not inactive PE, induced the same increase. TPA stimulation of EL4+ cells also induced de novo expression of the IL 2 gene and subsequent secretion of this lymphokine. However, the increased expression of the TCR beta-chain gene and the induction of the IL 2 gene were not linked since expression of TCR beta-chain mRNA was increased to a similar degree in EL4+ and IL 2-nonproducing EL4- sublines, and cyclosporin A selectively blocked TPA-induced IL 2-gene expression in EL4+ cells without affecting the increase in TCR beta-chain mRNA. These findings suggest that PKC activation, an event that supposedly occurs after antigen-mediated triggering of the TCR, can regulate the expression of at least some of the genes encoding this receptor.     

Regulation of the cAMP signal transduction pathway by protein kinase C in rat submandibular cells

Treatment of rat submandibular acinar cell extracts with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused the dose-dependent activation of protein kinase C (PKC), assessed by the phosphorylation of a novel and highly specific substrate. This effect was duplicated by a diacylglycerol, but not by the 4-phorbol ester 4-phorbol 12,13-didecanoate. The TPA elevation of PKC was blocked by the PKC inhibitors H-7 and sangivamycin. In intact cells, TPA caused the translocation of PKC from cytosol to membrane, consistent with its known mode of activation. The -adrenergic agonist, isoproterenol, stimulated cAMP levels which were significantly reduced by preactivation of PKC. This inhibitory PKC effect was reversed by H-7. When cAMP was stimulated at the post-receptor level, however, by forskolin, NaF or GTP[S], PKC did not inhibit, but rather enhanced the cyclic nucleotide response. Since PKC phosphorylated an endogenous protein of 55 kDa, the size of the ₁ receptor, these findings indicate that, as in other cell types, PKC can desensitize adenylate cyclase by direct phosphorylation of the receptor, but potentiate the cAMP response by a post-receptor mechanism. In mucin secretion studies in the model, TPA alone caused the cAMP-independent release of up to 44% total mucin, which was much less than additive with the isoproterenol response. When the cAMP-mucosecretory response was stimulated at the adenylate cyclase level by forskolin, however, the TPA + forskolin effects were additive. These findings on the modulation of cAMP by PKC indicate cross-talk regulation in the phosphoinositide-cAMP signal transduction pathways in submandibular acinar cells.     

Cell genetic evidence of correlation of intracellular translocation of protein kinase C (PKC) and PKC-mediated phosphorylation of 80-kDa protein with mitogenic action of tumor promoters

Recently, we isolated a series of 3T3-L1 cell variants that are unable to respond to mitogenic stimulation by the tumor promoter, 12-O-tetradecanoylphorbol acetate (TPA). Since protein kinase C (PKC) is the major receptor for TPA and appears to play a key role in cellular proliferation, we have examined the distribution of PKC in the parental 3T3-L1 cells and the variant VT-1 cells. PKC was located predominantly in the cytosol of growth-arrested confluent 3T3-L1 cells, and upon TPA treatment it was rapidly translocated into the plasma membrane. In contrast, PKC was located predominantly in the plasma membrane of confluent VT-1 variant cells and was no longer activated by TPA. Two-dimensional gel analysis showed that a Mr 80,000 acidic protein (80-kDa protein) was rapidly phosphorylated in 3T3-L1 cells upon TPA treatment, whereas phosphorylation of this protein was barely detected in VT-1 cells. In growing cultures, the majority of PKC was found in the plasma membrane of both cell lines, and no change occurred upon TPA treatment. Hydroxyapatite column chromatography revealed the presence of -type PKC as the major component in both cell lines. These results suggest that the intracellular translocation of -type PKC and the PKC-mediated phosphorylation of the 80-kDa protein may be involved in the mechanism of mitogenic signal transfer.     

Helicobacter pylori -induced apoptosis in gastric epithelial cells is blocked by protein kinase C activation

Apoptosis plays a major role in gastrointestinal epithelial cell turnover. We have examined induction of apoptosis by Helicobacter pylori in gastric AGS cells and the role of protein kinase C (PKC) which has been shown to modulate programmed cell death. Incubation of AGS cells with H. pylori resulted in an activation of caspases 3 and 9 and induced programmed cell death. The PKC activator 12- O -tetradecanoylphorbol-13-acetate (TPA) caused translocation of PKC gamma, delta and var epsilon, prevented H. pylori -induced caspase activation and programmed cell death. Cocultivation of AGS cells with H. pylori resulted in a translocation of the atypical PKC isoform PKC lambda. We suggest that inhibition of H. pylori induced apoptosis by PKC activation can play a role in the process of neoplastic transformation.     

12-O-tetradecanoylphorbol-13-acetata (TPA) directly inhibits spontaneous immunoglobulin secretion byin vivo antigen-induced human lymphoblastoid B cells

Tetanus toxoid (Tet) booster immunization induces the transient appearance in the circulation of lymphoblastoid (LB) B cells which spontaneously produce anti-tetanus toxoid IgG antibody (IgG-Tet) during a 3-dayin vitro culture. In this study we have examined the effects of TPA on the ability of LB cells to secrete antibody and have found that as little as 10 ng/ml of TPA provoked a marked inhibition of the induced LB cells' IgG-Tet production. This inhibitory effect was observed only when TPA was added early in the culture and could be achieved by pretreating the B cells with TPA for as little as 1 hr. Only marginal inhibition of IgG-Tet production was observed if the addition of TPA was delayed 14–24 hr. The TPA inhibition was not mediated by contaminant T cells as the addition of increasing numbers of T cells to LB cell cultures proportionally reversed the TPA inhibitory effect. Likewise, the inhibition of antibody synthesis was not due to a monocyte-dependent mechanism since (1) substantial depletion of adherent cells did not reverse the inhibition of antibody synthesis, (2) the addition of a monocyte-enriched population to monocyte-depleted B cells did not enhance, but in fact partially reversed, the inhibition caused by TPA, and (3) the addition of monocyte populations pretreated with TPA to monocyte-depleted B-cell fractions did not inhibit subsequent IgG-Tet production by the LB cells.     

丙泊酚对结直肠癌细胞生物学行为的影响

目的:研究丙泊酚对结直肠癌细胞活力、侵袭和凋亡的影响,并探讨其作用机制。方法:10、25、50和100μmol/L的丙泊酚处理Lo Vo细胞72 h,或以100μmol/L的丙泊酚处理细胞12、24、48和72 h,CCK-8实验检测细胞活力;Transwell小室检测经100μmol/L丙泊酚处理72 h的细胞的侵袭能力;流式细胞术检测细胞周期及细胞凋亡率;Western blot检测基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)、活化的胱天蛋白酶3(cleaved caspase-3)、Notch1和发状分裂相关增强子1(Hes1)的蛋白表达。结果:丙泊酚可抑制结直肠癌细胞活力;丙泊酚组细胞侵袭能力、S期细胞及MMP-2、MMP-9、Notch1和Hes1蛋白表达均显著低于对照组,而细胞凋亡率、G0/G1期细胞及cleaved caspase-3的蛋白水平均显著高于对照组(P0.01)。结论:丙泊酚可抑制结直肠癌Lo Vo细胞生长及侵袭能力,阻滞细胞周期,并诱导细胞凋亡,其机制与下调Notch1信号通路有关。     

Differential Effect of TPA on Cell Growth and Epstein-Barr Virus Reactivation in Epithelial Cell Lines Derived from Gastric Tissues and B Cell Line Raji

We characterized the cell growth and Epstein-Barr virus (EBV) reactivation for EBV infected epithelial cell lines, GT38, GT39, and GTC-4 using 12-O-tetradecanoylphorbol-13-acetate (TPA). These cell lines grew similarly in liquid medium, and formed colonies in soft agar. The cell growth was inhibited with TPA, dose-dependently in liquid medium. The colony formation was enhanced with low concentrations of TPA, but was inhibited with high concentrations. The latent EBV was reactivated with high concentrations of TPA as shown by the expression of EBV BZLF1 gene product ZEBRA. The effects of TPA on GTC-4 were compared with a Burkitt's lymphoma cell line Raji. The mode of actions of TPA in GTC-4 was different from Raji in terms of cell growth and EBV reactivation. The effective concentrations of TPA for cell growth inhibition and EBV reactivation were higher in Raji than GTC-4. Cell cycle analysis showed that TPA (20 ng/ml) induced cell cycle arrest to Raji but not to GTC-4; however, the rate of trypan blue stained cells increased in the TPA treated GTC-4 but not Raji. These results demonstrated that TPA affects differentially for the stimulation and inhibition of cell growth, and also EBV reactivation depends on TPA concentrations and cell types.     

PKCα inhibited apoptosis by decreasing the activity of JNK in MCF-7/ADR cells

The development of multidrug resistance (MDR) in breast cancer patients is a serious therapeutic problem. The role of signal transduction in the development of MDR has drawn intensive attention recently. In this study, the role of c-Jun N-terminal kinase (JNK) pathway in MDR, specifically regulated by PKCα, was investigated in MCF-7/ADR cells. MTT, DNA ladder and flow cytometry were used to detect cell growth inhibition or apoptosis while Western blot was used to detect the activation of proteins. Compared with MCF-7 cells, the cell growth inhibition and apoptosis induced by tamoxifen (TAM) could not be detected in MCF-7/ADR cells, but the expression of PKCα in MCF-7/ADR cells was higher. And, Western blot results showed that JNK was activated by TAM in MCF-7 cells while not in MCF-7/ADR cells, even at very high doses. In addition, sp600125, the inhibitor of JNK, decreased the percentage of apoptosis induced by TAM in MCF-7 cells. These data showed that PKCα and JNK were key regulators in the apoptosis of MCF-7/ADR cells. Furthermore, PKCα being the upstream of JNK in inhibiting apoptosis was suggested by using Go6976, the specific PKCα inhibitor, in the presence or absence of sp600125. This study highlighted an important signaling pathway involved in MDR regulated by PKCα in MCF-7/ADR breast cancer cells and implied that JNK might be an important downstream target of PKCα in this cellular context.     

Lipoxygenase inhibitors prevent urological cancer cell growth

Recent studies have demonstrated that lipoxygenase (LOX) inhibitor induces growth arrest of cancer cells through apoptosis. In this study, we examined the effects of LOX inhibitors on cell proliferation in renal cell carcinoma (RCC), bladder tumor (BT), and prostate cancer (PC) cell lines. We investigated the inhibitory effect of LOX inhibitors, 5-, 12- and non-specific LOX inhibitor on RCC, BT and PC-derived cell lines using MTT assay and Hoechst staining. All LOX inhibitors induced the reduction of cell viability with the half-maximal concentration of growth inhibition of RCC, BT, and PC cell lines. Furthermore, counting cells at days 1, 2 and 3, clearly showed marked inhibition of cell proliferation by treatment with non-specific and 5-LOX inhibitor. All LOX inhibitors stopped the growth of all RCC, BT and PC cells. The effect of non-specific LOX inhibitor was strongest. The effect of 5-LOX inhibitor was stronger than 12-LOX inhibitor. All LOX inhibitors caused marked inhibition of urological cancer cells through apoptosis. LOX inhibitor may mediate potent antiproliferative effects against RCC, BT and PC cells through differentiation. Thus, LOX inhibitor, especially 5-LOX inhibitor may become a new target in treatment of urological cancers.