NS398 inhibits the growth of Hep3B human hepatocellular carcinoma cells via caspase-independent apoptosis

Overexpression of cyclooxygenase 2 (COX-2) is associated with the development of a number of human cancers including hepatocellular carcinoma (HCC). In addition, NS398, a selective COX-2 inhibitor, has been found to inhibit the growth of COX-2 expressing HCC cell lines. However, the mechanism of this effect remains unclear. Here, we report that NS398 inhibits the growth of the Hep 3B human HCC cell line and that inhibition results from the induction of apoptosis with no evidence of cell cycle arrest. We also show that the extent of apoptosis is greatly influenced by culture conditions. The NS398-induced apoptosis in Hep 3B cells is caspase-independent. Our data point to the feasibility of preventing HCC by means of COX-2 inhibitors, and show that the environment influences the cytotoxic effect of NS398 on cancer cells.     

Expression of genes that regulate Fas signalling and Fas-mediated apoptosis in colon carcinoma cells

The expression of genes that regulate Fas-induced apoptosis has been examined in 10 human cultured colon carcinoma cell lines with defined and varied sensitivity to the cytolytic anti-Fas MoAb CH-11. Four lines demonstrated sensitivity to CH-11 (HT29, GC3/c1, TS-, Thy4), and six were resistant to the induction of apoptosis vis Fas. In nine lines expressing Fas, PCR-sequencing indicated that the death domain contained wt sequences. Downstream of Fas, expression of FADD/MORT1 and FLICE, essential components of the DISC, and negative regulators of Fas signalling including sFas, FAP-1 and Bcl-2, showed no correlation between levels of expression and sensitivity to Fas-mediated cytotoxicity. However, levels of the Fas antigen varied by >1000-fold, and correlated with CH-11 sensitivity. Following fourfold elevation in Fas expression in HT29 cells treated with interferon-gamma, a synergistic effect on Fas-mediated apoptosis was obtained when CH-11 and interferon-gamma were combined.     

Effect of epiberberine from Coptis chinensis Franch on inhibition of tumor growth in MKN-45 xenograft mice

Background and purposeGastric cancer is one of the major malignancies worldwide. Epiberberine (EPI) is a major alkaloid from Coptis chinensis Franch and the antitumor property of EPI remains poorly understood.MethodThe inhibition on gastric cancer cells was observed by MTT assays and colony formation experiments. The apoptosis, cell cycle, and reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) in gastric cancer cells were analyzed by Flow cytometry. The anti-tumor effect of EPI was evaluated with the MKN-45-beraring nude mice, and the potential mechanisms were explored by RNA-seq, qPCR, siRNA silencing and western blotting.ResultsEPI inhibited the proliferation of human gastric cancer cell lines MKN-45 (harboring wild-type p53) and HGC-27 (harboring mutant p53) in a dose dependent manner. EPI induced the apoptosis and cell cycle arrest in these two cell lines, of which MKN-45 cells are more sensitive to EPI than HGC-27 cells. Further experiments indicated that EPI induced the accumulation of ROS and decreased of ΔΨm in MKN-45 cells. The significant differentially expressed genes obtained by RNA-seq were distinctly enriched in the p53 signaling pathway. The apoptosis induced by EPI in MKN-45 cells would be effectively inhibited with the treatment of p53 siRNA and p53 inhibitor PFT-α. Western blotting demonstrated that EPI diminished the expression of Bcl-2 and XIAP, and increased those of p53, Bax, p21, p27, Cytochrome C and Cleaved-caspase 3. Animal experiments confirmed that EPI significantly alleviated tumor growth in MKN-45 xenograft mice via p53/Bax pathway.ConclusionsThese data indicated that EPI could be a novel anti-tumor candidate against MKN-45-related gastric cancer via targeting p53-dependent mitochondria-associated pathway.     

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.     

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.     

Activation of caspase-8 is critical for sensitivity to cytotoxic anti-Fas antibody-induced apoptosis in human ovarian cancer cells

Two ovarian cancer cell lines named NOS4 and SKOV-3 have been shown to have different sensitivities to a cytotoxic anti-Fas antibody, CH-11. Although both cell lines express Fas molecules on the cell surfaces at the same intensities, apoptosis is induced by CH-11 in NOS4 cells but not in SKOV-3 cells. In this study, the different apoptosis-sensitivities of these cells were assessed. Both cell lines express almost the same levels of FADD, RIP, c-FLIP, FAP-1, Bax, Bcl-2 and Bcl-XL. Evidence of caspase-8, caspase-9 and caspase-3 activation and of cleavage of PARP and Bid was obtained in NOS4 cells but not in SKOV-3 cells. When triggered by FasL protein, DNA fragmentation and caspase-8 activation were observed in SKOV-3 cells, though they were not as clear as in NOS4 cells. All the anti-Fas antibody-mediated signals for apoptosis induction in NOS4 cells were completely blocked by a caspase-8-specific inhibitor, Z-IETD-FMK. These results indicate that the different sensitivities to the anti-Fas antibody are solely dependent on the activation of caspase-8, which could be influenced by yet unknown qualitative or quantitative abnormalities in molecules involved in DISC formation.     

Transfer of Fas (CD95) protein from the cell surface to the surface of polystyrene beads coated with anti-Fas antibody clone CH-11

Mouse monoclonal anti-Fas (CD95) antibody clone CH-11 has been widely used in research on apoptosis. CH-11 has the ability to bind to Fas protein on cell surface and induce apoptosis. Here, we used polystyrene beads coated with CH-11 to investigate the role of lipid rafts in Fas-mediated apoptosis in SKW6.4 cells. Unexpectedly, by treatment of the cells with CH-11-coated beads Fas protein was detached from cell surface and transferred to the surface of CH-11-coated beads. Western blot analysis showed that Fas protein containing both extracellular and intracellular domains was attached to the beads. Fas protein was not transferred from the cells to the surface of the beads coated with other anti-Fas antibodies or Fas ligand. Similar phenomenon was observed in Jurkat T cells. Furthermore, CH-11-induced apoptosis was suppressed by pretreatment with CH-11-coated beads in Jurkat cells. These results suggest that CH-11 might possess distinct properties on Fas protein compared with other anti-Fas antibodies or Fas ligand, and also suggest that caution should be needed to use polystyrene beads coated with antibodies such as CH-11.Key words: Fas, CD95, CH-11, apoptosis, Fas ligand, polystyrene beads.     

Cyclooxygenase-2 positively regulates Akt signalling and enhances survival of erythroleukemia cells exposed to anticancer agents

Cyclooxygenase-2 (COX-2) has been found to be highly expressed in many types of cancers and to contribute to tumorigenesis via the inhibition of apoptosis, increased angiogenesis and invasiveness. In hematological malignancies, COX-2 expression was found to correlate with poor patient prognosis. However, the exact role of COX-2 expression in these malignancies, and particularly in erythroleukemias, remains unclear. The aim of this work was to describe and understand the relationships between COX-2 expression and apoptosis rate in erythroleukemia cells after apoptosis induction by several anticancer agents. We used three different erythroleukemia cell lines in which COX-2 expression was modulated by transfection with either COX-2 siRNA or COX-2 cDNA. These cellular models were then treated with apoptosis inducers and apoptosis onset and intensity was followed. Cell signalling was evaluated in unstimulated transfected cells or after apoptosis induction. We found that COX-2 inhibition rendered erythroleukemia cells more sensitive to apoptosis induction and that in cells overexpressing COX-2 apoptosis induction was reduced. We demonstrated that COX-2 inhibition decreased the pro-survival Akt signalling and activated the negative regulator of Akt signalling, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Conversely, in COX-2 overexpressing cells, Akt signalling was activated and PTEN was inhibited. In these last cells, inhibition of casein kinase 2 or Akt signalling restored sensitivity to apoptotic agents. Our findings highlighted that COX-2 can positively regulate Akt signalling mostly through PTEN inhibition, partly via casein kinase 2 activation, and enhances survival of erythroleukemia cells exposed to anticancer agents.     

Ribotoxic stress sensitizes glioblastoma cells to death receptor induced apoptosis: requirements for c-Jun NH2-terminal kinase and Bim

A prominent feature of glioblastoma is its resistance to death receptor-mediated apoptosis. In this study, we explored the possibility of modulating death receptor-induced cell death with the c-Jun-NH2-terminal kinase (JNK) activator anisomycin. Anisomycin activates JNK by inactivating the ribosome and inducing "ribotoxic stress." We found that anisomycin and death receptor ligand anti-Fas antibody CH-11 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) synergistically induce apoptosis in multiple human glioblastoma cell lines. For example, in U87 cells, anisomycin reduced the IC50 of CH-11 by more than 20-fold (from 500 to 25 ng/mL). Cell viability in response to anisomycin, CH-11, and their combination was 79%, 91%, and 28% (P<0.001), respectively. Anisomycin and TRAIL were found to be similarly synergistic in glioblastoma cells maintained as tumor xenografts. The potentiation of death receptor-dependent cell death by anisomycin was specific because emetine, another ribosome inhibitor that does not induce ribotoxic stress or activate JNK, did not have a similar effect. Synergistic cell death was predominantly apoptotic involving both extrinsic and intrinsic pathways. Expression of Fas, FasL, FLIP, and Fas-associated death domain (FADD) was not changed following treatment with anisomycin+CH-11. JNK was activated 10- to 22-fold by anisomycin+CH-11 in U87 cells. Inhibiting JNK activation with pharmacologic inhibitors of JNKK and JNK or with dominant negative mitogen-activated protein kinase (MAPK) kinase kinase 2 (MEKK2) significantly prevented cell death induced by the combination of anisomycin+CH-11. We further found that anisomycin+CH-11 up-regulated the proapoptotic protein Bim by approximately 14-fold. Simultaneously inhibiting Bim expression and JNK activation additively desensitized U87 cells to anisomycin+CH-11. These findings show that anisomycin-induced ribotoxic stress sensitizes glioblastoma cells to death receptor-induced apoptosis via a specific mechanism requiring both JNK activation and Bim induction.     

Celecoxib increases retinoid sensitivity in human colon cancer cell lines

Retinoid resistance has limited the clinical application of retinoids as differentiation-inducing and apoptosis-inducing drugs. This study was designed to investigate whether celecoxib, a selective COX-2 inhibitor, has effects on retinoid sensitivity in human colon cancer cell lines, and to determine the possible mechanism of said effects. Cell viability was measured using the MTT assay. Apoptosis was detected via Annexin-V/PI staining and the flow cytometry assay. PGE₂ production was measured with the ELISA assay. The expression of RARβ was assayed via western blotting. The results showed that celecoxib enhanced the inhibitory effect of ATRA in both COX-2 high-expressing HT-29 and COX-2 low-expressing SW480 cell lines. Further study showed the ATRA and celecoxib combination induced greater apoptosis, but that the addition of PGE₂ did not affect the enhanced growth-inhibitory and apoptosis-inducing effects of the combination. Moreover, NS398 (another selective COX-2 inhibitor) did not affect the inhibitory effects of ATRA in the two cell lines. Western blotting showed that the expression of RARβ in HT-29 cell lines was increased by celecoxib, but not by NS398, and that the addition of PGE₂ did not affect the celecoxib-induced expression of the retinoic acid receptor beta. In conclusion, celecoxib increased the expression of RARβ and the level of cellular ATRA sensitivity through COX-2-independent mechanisms. This finding may provide a potential strategy for combination therapy.     

The drug resistance proteins, multidrug resistance-associated protein and P-glycoprotein, do not confer resistance to Fas-induced cell death

BACKGROUND: Multidrug resistance (MDR) is mediated by the drug resistance proteins, the multidrug resistance-associated protein (MRP) and P-glycoprotein, both of which confer resistance by the active efflux of chemotherapeutic drugs from the cell. Reduced Fas (CD95/APO-1) expression and resistance to Fas-mediated apoptosis have also been correlated with P-glycoprotein-mediated MDR. METHODS: We investigated cell surface Fas expression (using anti-Fas monoclonal antibody DX2.1) in a series of MRP-expressing drug-resistant leukemia sublines, and P-glycoprotein-expressing leukemia sublines, and their susceptibility to apoptosis induced by anti-Fas treatment (CH-11 monoclonal antibody). Caspase-3 activation was detected by Western blot and apoptosis was determined by flow cytometry with 7-aminoactinomycin D (7-AAD) staining of cells. RESULTS: Fas expression was not reduced in either the MRP- or P-glycoprotein-expressing drug-resistant cell lines, although expression was reduced by 15% in one low-level drug-resistant subline. Expression of MRP or P-glycoprotein did not confer resistance to caspase-3 activation or to anti-Fas-induced cell death. CONCLUSIONS: MDR mediated by the drug transport proteins MRP and P-glycoprotein does not correlate with resistance to Fas-mediated cell death or resistance to caspase-3 activation.     

5-Fluorouracil (5-FU) induced apoptosis in gastric cancer cell lines: role of the p53 gene

We examined chemosensitivity to 5-fluorouracil (5-FU) in four human gastric cancer cell lines, by analyzing the expression of p53 and its related genes. Treatment with 1mM 5-FU induced variable degrees of apoptosis in the cultured cells. The apoptotic indices 72 h after treatment were approximately 14% in MKN-74 (wild-type p53 gene), 12% in MKN-45 (wild-type), 3% in MKN-28 (mutated) and 0.5% in KATO-III cells (deleted), respectively. On the other hand, 50 M 5-FU had little effect on the induction of apoptosis in MKN-74 cells, the value being approximately 2% after 72 h. Induction of P53 expression was noted 3 h after initiating the treatment, followed by the induction of P21/Waf1 after 6 h in both MKN-74 and MKN-45 cells. The same expression mode was noted in MKN-74 treated with 50 M 5-FU. Conversely, the level of P53 expression was constant in MKN-28 cells and absent in KATO-III cells, in which P21/Waf1 had never been induced. The Bax/Bcl-2 expression ratio was gradually elevated for up to 72 h in MKN-74 and MKN-45 cells treated with 1mM 5-FU; in contrast, it was unchanged in MKN-28 and KATO-III cells, and MKN-74 treated with 50 M 5-FU. These results might indicate that (1) 1mM 5-FU induces apoptosis in cultured gastric cancer cells carrying the wild-type p53 gene, but not those carrying the mutated type or a gene deletion, and (2) the elevated Bax/Bcl-2 expression ratio plays a more crucial role than the higher expression of P21/Waf1 in the induction of p53- gene dependent apoptosis.     

Cell death induced by Pteris semipinnata L. is associated with p53 and oxidant stress in gastric cancer cells

In this study, we demonstrated that Ent-11alpha-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) had stronger cytotoxicity against MKN-45, a gastric cancer cell line bearing wild-type p53 than MKN-28, another gastric cancer cell line containing missense mutation in p53. The rapid increase of ROS level was involved in the mechanism of cytotoxicity. Classical features of apoptosis induced by 5F were observed in MKN-45 cells only or more significant in MKN-45 cells than MKN-28 cells. Translocation of Bax from cytosol to mitochondria, reduction of delta psi m and DNA fragmentation were induced by 5F in the p53-dependent manner. We conclude that the expression of Bax and its downstream molecules requires the presentation of a wild-type p53 in the cells treated by 5F.     

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.     

The role of COX-2/PGE2 in gossypol-induced apoptosis of colorectal carcinoma cells

Our previous study showed that gossypol (GOS) exhibits potent cytotoxic effects via apoptosis induction against human colorectal carcinoma cells; however, the role of cyclooxygenase (COX)-2/prostaglandin (PG)E(2) on GOS-induced apoptosis is still unknown. In the present study, 12-O-tetradecanoylphorbol-13-acetate (TPA) addition significantly inhibited GOS-induced apoptosis in human colorectal carcinoma HT-29 cells in accordance with inducing COX-2 protein/PGE(2) production. TPA inhibition of GOS-induced apoptosis was blocked by adding protein kinase (PK)C inhibitors including staurosporine (ST), GF109203X (GF), and H7, characterized by the occurrence of cleaved caspase 3 proteins and a decrease in COX-2 protein/PGE(2) production in HT-29 cells. The addition of COX activity inhibitors, including NS398 (NS), aspirin (AS), diclofenac (DI), and indomethacin (IN), suppressed TPA protection of GOS-induced apoptosis with decreased PGE(2) production in HT-29 cells. Application of PGE(2), but not it analogs PGD(2), PGJ(2), or PGF(2α), protected HT-29 cells from GOS-induced DNA ladders, and the E-prostanoid (EP(1)) receptor agonist, 17PT-PGE(2), mimicked the protection induced by PGE(2), whereas the selective EP(2) receptor agonist, butaprostol (BUT), the EP(3) receptor agonist, sulprostol (SUL), and the EP(4) receptor agonist, PGE(1) alcohol (PGE(1)), showed no significant effects on GOS-induced apoptosis in HT-29 cells. PGE(2) 's protection against GOS-induced apoptosis was reversed by adding the selective EP(1) receptor antagonist, SC-19220. Furthermore, GOS had an effective apoptotic effect on COLO205 colorectal carcinoma cells which expressed undetectable level of endogenous COX-2 protein than HT-29 cells, and the decreased COX-2 protein level via COX-2 siRNA or addition of COX-2 activity inhibitor NS significantly elevated GOS-induced cell death in HT-29 cells. COLO205-T cells were established through sustained TPA incubation of COLO205 cells, and COLO205-T cells showed a lower sensitivity to GOS-induced cell death with increased COX-2 (not Bcl-2 and Mcl-1) protein than parental COLO-205 cells. A decrease in COX-2 protein expression in COLO205-T cells by COX-2 siRNA transfection or enhanced GOS-induced cell death according to MTT assay and DNA integrity assay. The notion of COX-2/PGE(2) activation against GOS-induced apoptosis in colon carcinoma cells was demonstrated, and the combination of GOS and COX-2 inhibitors to treat colon carcinoma possesses clinical potential worthy of further investigation.     

Rapid induction of apoptosis in human gastric cancer cell lines by sorbitol

Most solid tumors, including gastric cancers, respond poorly to non-surgical treatments which are expected to induce an apoptosis-dependent involution. We hypothesize that the apoptotic machinery in solid tumors is either defective or in a suppressed condition. Overcoming the ineffective induction of apoptosis may improve the responsiveness of solid tumors to non-surgical treatments. Recently, sorbitol, a kind of hexose, has been found to be an effective inducer of apoptosis in HEp-2 cells. Therefore, it is of particular interest to examine the effect of sorbitol-treatment on gastric cancer cells. In the present study, we selected 4 gastric cancer cell lines which have been reported to exhibit different abilities in regard to apoptosis induction, and examined the effect of sorbitol-treatment on apoptosis induction. Within 3 hr after sorbitol-treatment, apoptosis was induced comparably in all cell lines examined. Cell death in MKN-1, MKN-28 or MKN-74 proceeded in a biphasic manner, while cell death in KATO-III was monophasic. The cell death partially depended on caspase activity. Treatments with sorbitol in combination with 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly suppressed the apoptotic cell death, suggesting a role of protein kinase-C-dependent process. To our knowledge, this is the most rapid induction of apoptosis in human gastric cancer cells reported to date.