COX-2 inhibitor, NS398, enhances Fas-mediated apoptosis via modulation of the PTEN-Akt pathway in human gastric carcinoma cell lines

A variety of human cancer cells are resistant to Fas ligand and anti-Fas antibody induced apoptosis. Previously, we reported that human gastric carcinoma cell lines were resistant to the anti-Fas antibody, CH-11, without interferon-gamma pretreatment in vitro. Cyclooxygenase (COX)-2 is known to be expressed in many human malignancies, and is correlated with tumor progression and resistance to apoptosis. This study examined whether NS398, a COX-2 inhibitor, inhibited cell proliferation and increased Fas-mediated apoptosis in human gastric carcinoma cell lines. Treatment of NS398 inhibited cell proliferation in MKN-45, which expressed the highest level of COX-2 among seven human gastric carcinoma cell lines, in a dose- and time-dependent manner, in contrast to less prominent effects in KATO-III, which expresses no COX-2. Although the treatment of CH-11 induced apoptosis in both cells, the simultaneous treatment of NS398 and CH-11 remarkably induced apoptosis, as confirmed by Hoechst 33258 staining and the terminal deoxynucleotidyl transferase- mediated dUTP-digoxigenin nick-end labeling (TUNEL) method in MKN-45. Flow cytometric analysis also revealed the increased pre-G1 fraction by the simultaneous treatment. The treatment of NS398 induced upregulation of Bad and PTEN, and downregulation of phosphorylated Akt (Thr308). These findings suggest that COX-2 might inhibit Fas-mediated apoptosis in human gastric carcinoma cell lines, especially MKN-45, by modulating PTEN and Akt.     

Prostaglandin E2 receptor EP1 transactivates EGFR/MET receptor tyrosine kinases and enhances invasiveness in human hepatocellular carcinoma cells

Recent evidence indicates that cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) are involved in hepatocarcinogenesis. This study was designed to evaluate the possible interaction between the COX-2 and EGFR signaling pathways in human hepatocellular carcinoma (HCC) cells. Immunohistochemical analysis using serial sections of human HCC tissues revealed positive correlation between COX-2 and EGFR in HCC cells (P < 0.01). Overexpression of COX-2 in cultured HCC cells (Hep3B) or treatment with PGE(2) or the selective EP(1) receptor agonist, ONO-DI-004, increased EGFR phosphorylation and tumor cell invasion. The PGE(2)-induced EGFR phosphorylation and cell invasiveness were blocked by the EP(1) receptor siRNA or antagonist ONO-8711 and by two EGFR tyrosine kinase inhibitors, AG1478 and PD153035. The EP(1)-induced EGFR transactivation and cell invasion involves c-Src, in light of the presence of native binding complex of EP(1)/Src/EGFR and the inhibition of PGE(2)-induced EGFR phosphorylation and cell invasion by the Src siRNA and the Src inhibitor, PP2. Further, overexpression of COX-2 or treatment with PGE(2) also induced phosphorylation of c-Met, another receptor tyrosine kinase critical for HCC cell invasion. Moreover, activation of EGFR by EGF increased COX-2 promoter activity and protein expression in Hep3B and Huh-7 cells, whereas blocking PGE(2) synthesis or EP(1) attenuated EGFR phosphorylation induced by EGF, suggesting that the COX-2/PGE(2)/EP(1) pathway also modulate the activation of EGFR by its cognate ligand. These findings disclose a cross-talk between the COX-2/PGE(2)/EP(1) and EGFR/c-Met signaling pathways that coordinately regulate human HCC cell invasion.     

Bcl-2 and Bcl-xL are important for the induction of paclitaxel resistance in human hepatocellular carcinoma cells

In this study we have investigated the mechanism underlying resistance to the chemotherapeutic drug paclitaxel in tumors of hepatocellular carcinoma (HCC) patients. Treatment with paclitaxel led to potent inhibition of growth of Hep3B hepatoma cells, but did not affect the growth properties of SNU-368 and SNU-398 cell lines that were established from primary HCC tumors. The growth inhibitory effect induced by paclitaxel correlated with levels of intracellular p21 and resulted in cell cycle arrest at the G2/M phase. However, paclitaxel treatment did not alter intracellular p53 levels. Instead, SNU-398 cells express high levels of the anti-apoptotic Bcl-2 and Bcl-x(L) proteins and the level of Bcl-x(L) could be further induced upon paclitaxel treatment. In contrast, Hep3B cells express pro-apoptotic members of the Bcl family and fail to induce Bcl-x(L) upon paclitaxel treatment. Therefore, these results strongly suggest that Bcl-2 and Bcl-x(L) play an important role in mediating resistance to paclitaxel.     

Resveratrol causes COX-2- and p53-dependent apoptosis in head and neck squamous cell cancer cells

Cyclooxygenase-2 (COX-2) content is increased in many types of tumor cells. We have investigated the mechanism by which resveratrol, a stilbene that is pro-apoptotic in many tumor cell lines, causes apoptosis in human head and neck squamous cell carcinoma UMSCC-22B cells by a mechanism involving cellular COX-2. UMSCC-22B cells treated with resveratrol for 24 h, with or without selected inhibitors, were examined: (1) for the presence of nuclear activated ERK1/2, p53 and COX-2, (2) for evidence of apoptosis, and (3) by chromatin immunoprecipitation to demonstrate p53 binding to the p21 promoter. Stilbene-induced apoptosis was concentration-dependent, and associated with ERK1/2 activation, serine-15 p53 phosphorylation and nuclear accumulation of these proteins. These effects were blocked by inhibition of either ERK1/2 or p53 activation. Resveratrol also caused p53 binding to the p21 promoter and increased abundance of COX-2 protein in UMSCC-22B cell nuclei. Resveratrol-induced nuclear COX-2 accumulation was dependent upon ERK1/2 activation, but not p53 activation. Activation of p53 and p53-dependent apoptosis were blocked by the COX-2 inhibitor, NS398, and by transfection of cells with COX-2-siRNA. In UMSCC-22B cells, resveratrol-induced apoptosis and induction of nuclear COX-2 accumulation share dependence on the ERK1/2 signal transduction pathway. Resveratrol-inducible nuclear accumulation of COX-2 is essential for p53 activation and p53-dependent apoptosis in these cancer cells.     

Proapoptotic activity of NAG-1 is cell type specific and not related to COX-2 expression

Non-steroidal anti-inflammatory drugs (NSAIDs) activated gene (NAG-1) is a newly identified member of the transforming growth factor-β (TGF-β) superfamily. Members of the TGF-β family are multifunctional growth factors, and the nature of their effects depends on the cellular context and cell type. NAG-1 has antitumorigenic and proapoptotic activities in colon and gastric cancer cells lacking endogenous cyclooxgenase-2 (COX-2) expression. In contrast, COX-2 overexpression is related to antiapoptotic activity. The purpose of this study is to evaluate the proapoptotic activity of NAG-1 according to COX-2 expression and cell type. NAG-1 cDNA was transfected in SNU668 cells with endogenous COX-2 expression, SNU601 cells with forced COX-2 expression and Hep3B hepatocellular carcinoma cells. SNU668 cells with ectopic expression of NAG-1 showed markedly elevated subG1 population, induced death receptor-4 (DR-4) and DR-5, and revealed smaller active fragments of caspase-3. Forced COX-2 expression in SNU601 cells did not inhibit apoptosis caused by NAG-1 expression. Sulindac sulfide caused apoptosis, and induced expression of DR-5 and NAG-1 in Hep3B cells. However, Hep3B cells ectopically expressing NAG-1 did not cause apoptosis, and smaller active fragments of caspase-3 and an 85 kDa band of poly ADP-ribose polymerase (PARP) did not appear in the transfected cells, either. This study suggests that proapoptotic activity of NAG-1 is cell type specific and not related to COX-2 expression.     

Inhibition of CTRP6 prevented survival and migration in hepatocellular carcinoma through inactivating the AKT signaling pathway

C1qTNF-related proteins (CTRPs) are a member of the adiponectin paralogs family, which are implicated in regulation of various biological processes. Recently, CTRP6 was found upregulated in human hepatocellular carcinomas (HCC). However, the specific roles and molecular mechanisms of CTRP6 in HCC remain unclear. Here, we investigated the effects of CTRP6 on the vitality, apoptosis, migration, and invasion of HCC cells. Firstly, we measured the levels of CTRP6 in HCC tissues and cell lines. Our results showed that CTRP6 was markedly upregulated in HCC tissues and Hep3B cells. Then, the CTRP6 siRNA was transfected into Hep3B cells. Cell counting kit-8 (CCK-8) assay and flow cytometry analysis revealed that silencing CTRP6-induced cell viability inhibition, and apoptosis. The wound-healing and transwell assay showed that CTRP6 deficiency suppressed the migration and invasion of Hep3B cells. Meanwhile, the AKT phosphorylation level was reduced by CTRP6 silencing. Next, Hep3B cells were pretreated with insulin-like growth factor-1 (IGF-1) (an activator of AKT), and then transfected with CTRP6 siRNA, and the cell vitality, apoptosis, migration, and invasion were measured again. We found that all these alterations caused by CTRP6 inhibition could be reversed by IGF-1 treatment. Taken together, CTRP6 suppression blocked cell survival, migration, and invasion and promoted cell apoptosis through inactivating the AKT signaling pathway. Our findings present a novel potential molecular target for HCC therapy.     

选择性环氧合酶-2抑制剂联合奥曲肽诱导人胃癌细胞的凋亡

目的探讨选择性环氧合酶-2抑制剂NS-398与奥曲肽联合应用对人胃癌细胞株BGC-823生长、凋亡的影响。方法体外培养BGC-823细胞,分别用NS-398(100μmol/L)与奥曲肽(1μmol/L)单独及联合处理不同时间后,倒置显微镜观察细胞形态学变化;观察生长曲线的变化;流式细胞仪检测细胞凋亡率;实时定量(Real-time)PCR检测COX-2mRNA的表达;Western blot法检测Caspase-3蛋白表达。结果倒置显微镜下,对照组BGC-823细胞生长良好,药物处理后,细胞变小、变圆,悬浮,联合组细胞形态学改变显著强于单纯用药组;药物作用后,细胞生长受抑制,出现负增长,联合组作用明显强于单纯用药组;流式细胞仪检测表明联合用药组诱导BGC-823细胞的凋亡率明显高于单一用药组和对照组(P0.01);各处理组均使BGC-823细胞COX-2mRNA表达下调(P0.05);药物处理后细胞Caspase-3蛋白表达明显增加。结论 NS-398、奥曲肽联合可协同抑制BGC-823细胞生长、增殖,其机制可能与下调COX-2mRNA表达、诱导肿瘤细胞凋亡相关。     

Breast Cancer Metastasis Suppressor 1 Regulates Hepatocellular Carcinoma Cell Apoptosis via Suppressing Osteopontin Expression

Breast cancer metastasis suppressor 1 (BRMS1) was originally identified as an active metastasis suppressor in human breast cancer. Loss of BRMS1 expression correlates with tumor progression, and BRMS1 suppresses several steps required for tumor metastasis. However, the role of BRMS1 in hepatocellular carcinoma (HCC) remains elusive. In this study, we found that the expression level of BRMS1 was significantly down-regulated in HCC tissues. Expression of BRMS1 in SK-Hep1 cells did not affect cell growth under normal culture conditions, but sensitized cells to apoptosis induced by serum deprivation or anoikis. Consistently, knockdown of endogenous BRMS1 expression in Hep3B cells suppressed cell apoptosis. We identified that BRMS1 suppresses osteopontin (OPN) expression in HCC cells and that there is a negative correlation between BRMS1 and OPN mRNA expression in HCC tissues. Moreover, knockdown of endogenous OPN expression reversed the anti-apoptosis effect achieved by knockdown of BRMS1. Taken together, our results show that BRMS1 sensitizes HCC cells to apoptosis through suppressing OPN expression, suggesting a potential role of BRMS1 in regulating HCC apoptosis and metastasis.     

The cyclooxygenase-2 inhibitor celecoxib induces apoptosis by blocking Akt activation in human prostate cancer cells independently of Bcl-2

This study investigates the apoptotic activity of the cyclooxygenase-2 (COX-2) inhibitor celecoxib in prostate carcinoma cells. COX-2 is constitutively expressed in androgen-responsive LNCaP and androgen-nonresponsive PC-3 cells. Exposure of these cells to celecoxib induces characteristic features of apoptosis, including morphological changes, DNA laddering, and caspase-3 activation, whereas piroxicam, a COX-1-specific inhibitor, displays no appreciable effect on either cancer cell line even after prolonged exposure. Moreover, the potency of celecoxib in apoptosis induction is significantly higher than that of other COX-2 inhibitors examined despite the observation that these inhibitors exhibit similar IC(50) in COX-2 inhibition. It is noteworthy that normal human prostate epithelial cells, expressing a marginally detectable level of COX-2, are insensitive to the induction of apoptosis by celecoxib. These data suggest a correlation between COX-2 expression and sensitivity to the apoptotic effect of the COX-2 inhibitor. In an effort to delineate the underlying mechanism, we examined the effect of celecoxib on the expression of Bcl-2 as well as the activation of the key anti-apoptotic kinase Akt. In contrast to an earlier report that attributed the apoptotic activity of NS398 in LNCaP cells to Bcl-2 down-regulation, we provide evidence that the induction of apoptosis by celecoxib in LNCaP and PC-3 cells is independent of Bcl-2. First, treatment with celecoxib does not alter the cellular Bcl-2 level in both cell lines. Second, enforced Bcl-2 expression in PC-3 cells does not confer protection against the induction of apoptosis by celecoxib. Our data show that celecoxib treatment blocks the phosphorylation of Akt. This correlation is supported by studies showing that overexpression of constitutively active Akt protects PC-3 cells from celecoxib-induced apoptosis. Nevertheless, how celecoxib down-regulates Akt is not clear because the drug does not adversely affect phosphoinositide 3-kinase activity in vivo and okadaic acid, a protein phosphatase 2A inhibitor, cannot rescue the inhibition. In summary, our data demonstrate that inhibition of Akt activation may play a crucial role in the induction of apoptosis by celecoxib.     

The nonsteroidal anti-inflammatory drug NS398 reactivates SPARC expression via promoter demethylation to attenuate invasiveness of lung cancer cells

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to exhibit potent anticancer effects in vitro and in vivo. One of the mechanisms by which NSAIDs suppress tumorigenesis is inhibition of angiogenesis and metastasis. In this study, we used a microarray system to study the change of expression profile of metastasis-related genes regulated by NS398, a NSAID and a cyclooxygenase-2 (COX-2) inhibitor. We found that several negative regulators of cell invasion, including secreted protein acidic and rich in cysteine (SPARC), thrombospondin 1 (TSP-1), thrombospondin 3 (TSP-3), and tissue inhibitors of matrix metalloproteinase-2 (TIMP-2) are upregulated by NS398. In addition, we demonstrated that upregulation of SPARC expression by NS398 in human lung cancer cells is mediated by promoter demethylation and associated with a decrease in DNA methyltransferase (DNMT) expression. This is the first report to show that NS398 can inhibit the expression of DNMT1 and 3b. Functional assay indicated that SPARC is a critical mediator for NS398 to inhibit cell invasion. Our results provide new insights for the understanding of the anticancer actions of NSAIDs.     

Susceptibility of Hep3B cells in different phases of cell cycle to tBid

tBid is a pro-apoptotic molecule. Apoptosis inducers usually act in a cell cycle-specific fashion. The aim of this study was to elucidate whether effect of tBid on hepatocellular carcinoma (HCC) Hep3B cells was cell cycle phase specific. We synchronized Hep3B cells at G0/G1, S or G2/M phases by chemicals or flow sorting and tested the susceptibility of the cells to recombinant tBid. Cell viability was measured by MTT assay and apoptosis by TUNEL. The results revealed that tBid primarily targeted the cells at G0/G1 phase of cell cycle, and it also increased the cells at the G2/M phase. 5-Fluorouracil (5-FU), on the other hand, arrested Hep3B cells at the G0/G1 phase, but significantly reduced cells at G2/M phase. The levels of cell cycle-related proteins and caspases were altered in line with the change in the cell cycle. The combination of tBid with 5-FU caused more cells to be apoptotic than either agent alone. Therefore, the complementary effect of tBid and 5-FU on different phases of the cell cycle may explain their synergistric effect on Hep3B cells. The elucidation of the phase-specific effect of tBid points to a possible therapeutic option that combines different phase specific agents to overcome resistance of HCC.     

MAPK (ERK2) kinase--a key target for NSAIDs-induced inhibition of gastric cancer cell proliferation and growth.

Limited clinical and experimental studies indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) may inhibit gastric cancer growth. However, the mechanisms involved are not completely understood and cannot be explained by COX-2 inhibition alone. MAPK signaling pathway is essential for cell proliferation, but the effect of NSAIDs on MAPK activity and phosphorylation in gastric cancer has never been studied. Since increased and unregulated cell proliferation and reduced cell apoptosis are important features of cancer growth, we studied whether NS-398, a selective COX-2 inhibitor and/ or indomethacin (IND), a non-selective NSAID: 1) inhibit gastric cancer cell proliferation, 2) whether this inhibition is mediated via MAPK (ERK2), and 3) whether NSAIDs enhance apoptosis in gastric cancer cells. Human gastric epithelial cells (MKN28) derived from gastric tubular adenocarcinoma were cultured and treated with either vehicle, IND (0.25-0.5mM) or NS-398 (50-100 microM) for 6, 16, 24 and 48h. Studies: 1) Cellular proliferation was determined by 3H-thymidine uptake. 2) MAPK activity was measured by incorporation of radiolabeled phosphate into myelin basic protein. 3) Apoptosis was evaluated using TUNEL assay. IND and NS-398 significantly inhibited the proliferation of MKN28 cells at 24h by 3.5 - 5 fold (p<0.002) and at 48h by 2.5 - 10 fold (p<0.02). Both NSAIDs also significantly inhibited ERK2 activity: IND >53% inhibition, NS-398, 100 microM >72% inhibition; all p<0.05. Both IND and NS-398 significantly increased apoptotic index. In conclusion, IND and NS-398 significantly inhibit proliferation and growth of human gastric cancer cell line MKN28. This effect is mediated by NSAID-induced inhibition of MAPK (ERK2) kinase signaling pathway, essential for cell proliferation. NSAIDs also increase apoptosis in MKN28 cells. In addition to inhibiting cyclooxygenase, NSAIDs inhibit phosphorylating enzymes--kinases essential for signaling cell proliferation.     

COX-2 inhibition affects growth rate of Chlamydia muridarum within epithelial cells

Chlamydiae alter apoptosis of host target cells, which regulates their growth. Cyclooxygenase-2 (COX-2), the rate-limiting enzyme for prostaglandin E2 (PGE2) production, modulates epithelial cell survival. We addressed whether endogenous PGE2 alters chlamydial growth or apoptosis of epithelial cells infected with Chlamydia muridarum. PGE2 is secreted by infected host cells in the genital tract (GT). Using immunohistochemical techniques, we found that COX-2 enzyme was localized to epithelial cells in the GT in vivo. Pellets of the COX-2 enzyme inhibitor, NS-398, and placebo were implanted in mice subcutaneously and released a constant amount of these chemicals throughout the infection. NS-398-treated mice were found to exhibit 10-fold lower bacterial load than the placebo group on day 3 post infection, suggesting disruption of the chlamydial developmental cycle. To prove this, the human lung adenocarcinoma cell line A549 was then infected with different MOIs of C. muridarum in the presence of multiple concentrations of NS-398 in vitro. There was no difference in inclusion forming units (IFUs) between NS-389-treated and untreated cells. We also found no alterations in C. muridarum IFUs in A549 cells transfected with a 2.0 kb cDNA fragment of human COX-2 cloned in the sense (S) or anti-sense (AS) orientation. However, the inclusion size was reduced and the number of EB was significantly diminished during reinfection in AS-transfected cells. In addition, the absence of COX-2 did not significantly modify apoptosis in infected cells. In total, COX-2 deficiency reduces the infectious burden in vivo and may modulate transmission of the organism.     

shRNA靶向干扰COX-2抑制人肝癌裸鼠皮下移植瘤及其血管生成

目的:探讨重组干扰质粒pshRNA-COX-2对人肝癌细胞Hep3B裸鼠皮下移植瘤生长和肿瘤血管生成的抑制作用。方法:重组干扰质粒pshRNA-COX-2转染Hep3B细胞并筛选后,RT-PCR和Western blot检测COX-2mRNA和蛋白表达,RT-PCR检测VEGFmRNA表达。将被成功转染的Hep3B细胞种植于裸鼠皮下,测量肿瘤大小,4周后处死裸鼠,免疫组织化学法检测肿瘤组织中COX-2蛋白表达和肿瘤微血管密度(MVD)。结果:与未转染细胞相比,干扰组COX-2mRNA和蛋白表达抑制率分别为65.3%和52.8%(P<0.05),干扰组VEGFmRNA表达抑制率为56.5%(P<0.05)。干扰组瘤体大小明显小于阴性组和空白组(P<0.01)。干扰组COX-2得分和MVD均明显低于阴性组和空白组(P<0.01)。结论:pshRNA-COX-2通过抑制COX-2表达明显抑制人肝癌细胞Hep3B裸鼠皮下移植瘤生长和肿瘤血管生成。     

Camptothecin-induced apoptosis in non-small cell lung cancer is independent of cyclooxygenase expression

Recent observations show a positive correlation between the expression of cyclooxygenase (COX), especially COX-2), and cancer development. Here we tested the hypothesis that expression of COX-2 could influence apoptosis in lung cancer cell lines. To address this question, we determined the effects of camptothecin-induced apoptosis on three lung cancer cell lines which over express COX-1 (CORL23), COX-2 (MOR-P) and neither isoform (H-460), and determine if these effects were prostaglandin mediated. We also compared the effects of non-selective and isoenzyme selective COX-2 inhibitors on camptothecin-induced apoptosis in these three cell lines. Camptothecin induced apoptosis in all three cell lines independently of COX-1 or COX-2 expression. Indomethacin, a non-selective COX inhibitor and NS398, a selective COX-2 inhibitor had no effect on camptothecin-induced apoptosis at concentrations that abolished prostaglandin production. In conclusion, these finding suggest that the COX pathway is not involved in camptothecin-induced apoptosis of non-small cell lung cancer cell lines.     

Salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis through DR5 and survivin-dependent mechanisms

Ras activation is a frequent event in human hepatocarcinoma that may contribute to resistance towards apoptosis. Salirasib is a ras and mTOR inhibitor that induces a pro-apoptotic phenotype in human hepatocarcinoma cell lines. In this work, we evaluate whether salirasib sensitizes those cells to TRAIL-induced apoptosis. Cell viability, cell death and apoptosis were evaluated in vitro in HepG2, Hep3B and Huh7 cells treated with DMSO, salirasib and YM155 (a survivin inhibitor), alone or in combination with recombinant TRAIL. Our results show that pretreatment with salirasib sensitized human hepatocarcinoma cell lines, but not normal human hepatocytes, to TRAIL-induced apoptosis. Indeed, FACS analysis showed that 25 (Huh7) to 50 (HepG2 and Hep3B) percent of the cells treated with both drugs were apoptotic. This occurred through activation of the extrinsic and the intrinsic pathways, as evidenced by a marked increase in caspase 3/7 (five to ninefold), caspase 8 (four to sevenfold) and caspase 9 (eight to 12-fold) activities in cells treated with salirasib and TRAIL compared with control. Survivin inhibition had an important role in this process and was sufficient to sensitize hepatocarcinoma cells to apoptosis. Furthermore, TRAIL-induced apoptosis in HCC cells pretreated with salirasib was dependent on activation of death receptor (DR) 5. In conclusion, salirasib sensitizes hepatocarcinoma cells to TRAIL-induced apoptosis by a mechanism involving the DR5 receptor and survivin inhibition. These results in human hepatocarcinoma cell lines and primary hepatocytes provide a rationale for testing the combination of salirasib and TRAIL agonists in human hepatocarcinoma.