Lactoferricin-induced apoptosis in estrogen-nonresponsive MDA-MB-435 breast cancer cells is enhanced by C6 ceramide or tamoxifen

Bovine lactoferricin (LfcinB) is a cationic peptide that selectively induces caspase-dependent apoptosis in human leukemia and carcinoma cell lines. Ceramide is a second messenger in apoptosis signaling that has been shown to increase the cytotoxicity of various anti-cancer drugs. In this study, we determined whether manipulation of intracellular ceramide levels enhanced LfcinB-induced apoptosis of estrogen-nonresponsive MDA-MB-435 breast carcinoma cells. LfcinB caused DNA fragmentation and morphological changes consistent with apoptosis in MDA-MB-435 breast cancer cell cultures, but did not affect the viability of untransformed mammary epithelial cells. MDA-MB-435 breast cancer cells also exhibited DNA fragmentation and morphological changes consistent with apoptosis following exposure to the cell-permeable ceramide analog C6. An additive increase in DNA fragmentation was observed when both LfcinB and C6 ceramide were added to MDA-MB-435 breast cancer cell cultures. A greater than additive increase in DNA fragmentation was seen when LfcinB was used in combination with tamoxifen, which prevents the metabolism of endogenous ceramide to glucosylceramide by glucosylceramide synthase, as well as blocking estrogen receptor signaling. However, a selective inhibitor of glucosylceramide synthase,1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, failed to further increase DNA fragmentation by LfcinB, suggesting that tamoxifen enhanced LfcinB-induced apoptosis in breast cancer cells via a mechanism that did not involve glucosylceramide synthase inhibition. We conclude that combination therapy with LfcinB and tamoxifen warrants further investigation for possible use in the treatment of breast cancer.     

Glucosylceramide synthase inhibition enhances vincristine-induced cytotoxicity

As a strategy to enhance tumor cell sensitivity to vincristine, we tested whether modulation of sphingolipid metabolism would alter vincristine cytotoxicity since this is linked to accumulation of the intermediate metabolite, ceramide. We blocked ceramide metabolism in a series of variably vincristine-resistant cell lines derived from CCRF-CEM leukemia cells using an inhibitor of glucosylceramide synthase, DL-threo-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP). PPPP alone (1.0 microM), while nearly completely blocking glucosylceramide synthesis, was not toxic and did not increase cellular ceramide levels. Vincristine alone was toxic, caused apoptosis or programmed cell death (PCD) and caused an elevation in ceramide levels. Strikingly, the combination of PPPP and vincristine resulted in a further increase, over that of vincristine alone, of (i) cellular ceramide concentration, (ii) cytotoxicity associated with PCD and (iii) G2/M cell-cycle arrest. PPPP had no effect on P-glycoprotein expression or function. We conclude that vincristine cytotoxicity occurs in part through a ceramide-dependent mechanism, resulting in both G2/M block as well as PCD, and that the blockade of glucosylceramide synthase, in itself not toxic, causes augmented accumulation of ceramide resulting from vincristine exposure, which in turn maximizes ceramide-dependent, vincristine-induced cytotoxicity. Inhibition of glucosylceramide synthesis may be a means of circumventing drug resistance by enhancing signaling through a cell-death pathway.     

Preferential killing of multidrug-resistant KB cells by inhibitors of glucosylceramide synthase.

This study has compared the preferential killing of three multidrug-resistant (MDR) KB cell lines, KB-C1, KB-A1 and KB-V1 by two inhibitors of glucosylceramide synthase, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) and 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP), to the killing produced by these compounds in the drug-sensitive cell line, KB-3-1. Both of the inhibitors caused much greater induction of apoptosis in each of the three MDR cell lines than in the drug-sensitive cell line, as judged by morphological assay and confirmed by poly-(ADP-ribose)-polymerase cleavage. The highest level of apoptosis was produced following 24-h exposure to 5 microM PPPP. This treatment produced 75.8 (+/- 7.1)%, 73.6 (+/- 9.8)% and 75.3 (+/- 6.4)% apoptotic cells in the three MDR cell lines respectively, compared to 19.0 (+/- 9.8)% in the drug-sensitive cell line. A reduction in glucosylceramide level following inhibitor treatment occurred in KB-3-1 cells as well as in the MDR cell lines, suggesting that the increased apoptotic response in the MDR cells reflected a different downstream response to changes in the levels of this lipid in these cells compared to that in the drug-sensitive cells. These results suggest that the manipulation of glucosylceramide levels may be a fruitful way of causing the preferential killing of MDR cells in vitro and possibly in vivo.     

Multidrug resistance modulators and doxorubicin synergize to elevate ceramide levels and elicit apoptosis in drug-resistant cancer cells.

BACKGROUND: To provide insight for the development of more effective clinical agents, the authors attempted to elucidate the mechanisms of action of multidrug resistance (MDR) modulators. Previously, the authors found that MDR modulators blocked the conversion of ceramide to glucosylceramide in MDR cells, thereby enhancing cytotoxicity. Because ceramide is a critical component of the apoptosis signaling cascade, the current study examined the impact of therapy using agents that elicit ceramide formation combined with agents that block ceramide glycosylation. METHODS: Doxorubicin-resistant human breast carcinoma cells (MCF-7-AdrR) were treated with either doxorubicin, tamoxifen, cyclosporine A, or the cyclosporine A analog SDZ PSC 833 (PSC 833) or with combinations thereof, and ceramide and glucosylceramide metabolisms were measured by cell radiolabeling. Cell viability was quantitated spectrophotometrically and apoptosis was evaluated analyzing DNA integrity by gel electrophoresis. RESULTS: Whereas cyclosporine A blocked the generation of glucosylceramide in MCF-7-AdrR cells, a chemical cousin, PSC 833, elicited a 3-fold increase in glucosylceramide and a 5-fold increase in ceramide levels at 24 hours. The PSC 833 response was time-dependent(as early as 30 minutes) and dose-dependent (as low as 0.1 microM). The appearance of ceramide foreran the generation of glucosylceramide. Sphingomyelin levels were not decreased in response to PSC 833; however, Fumonisin B1, a ceramide synthase inhibitor, blocked PSC 833-induced ceramide generation. Adding tamoxifen, which blocks ceramide glycosylation, to the PSC 833 regimen boosted ceramide levels 11-fold over controls and caused DNA fragmentation. A 3-component regimen comprised of tamoxifen, doxorubicin, and PSC 833 increased ceramide levels 26-fold and brought cell viability to zero. CONCLUSIONS: These results demonstrate that MDR modulators can be used separately, in combination, or in conjunction with chemotherapy at clinically relevant concentrations to manipulate cellular ceramide levels and restore sensitivity in the drug resistant setting. As such, this represents a new direction in the treatment of cancer.     

Bortezomib-induced apoptosis in cultured pancreatic cancer cells is associated with ceramide production

Purpose

The proteasome inhibitor bortezomib (PS-341) has displayed significant efficiency against pancreatic cancer cells. However, the underlying mechanisms are not fully understood. Here, we tested if ceramide production was involved in the bortezomib’s effect.

Methods

Two transformed pancreatic cancer cell lines (PANC-1 and Mia) and the primary pancreatic cancer cells were used. Cell death was analyzed by MTT viability assay and trypan blue staining. Cell apoptosis was analyzed by Histone DNA-ELISA assay and Annexin V FACS. Western blots were used to test signal protein changes. The cellular ceramide level after bortezomib treatment was also determined.

Results

In cultured pancreatic cancer cells, bortezomib increased cellular ceramide production to promote cell apoptosis. The ceramide de novo synthase inhibitor fumonisin B1 (F-B1) suppressed bortezomib-induced ceramide production and apoptosis, while exogenously added C6-ceramide facilitated bortezomib-induced pancreatic cancer cell death. Meanwhile, 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), the inhibitor of glucosylceramide synthetase as well as the sphingosine kinase 1 inhibitors (SKI-II and SKI-IV), facilitated bortezomib-induced ceramide production and subsequent cell apoptosis. Further, bortezomib-induced pro-apoptotic c-Jun N-terminal kinase (JNK) activation was also associated with ceramide production. JNK activation by bortezomib was suppressed by F-B1, but was enhanced by SKI-II and PDMP in pancreatic cancer cells. Finally, C6-ceramide, SKI-II, and PDMP dramatically enhanced bortezomib-induced cytotoxicity in primary cultured pancreatic cancer cells.

Conclusions

We found that bortezomib-induced apoptosis was associated with ceramide production in primary and transformed pancreatic cancer cells.     

Overcoming resistance to gamma-rays in squamous carcinoma cells by poly-drug elevation of ceramide levels

Recent strategies to sensitize radioresistant tumours are based on combining gamma-irradiation with inducers of apoptosis. We report that the combination of three inhibitors of sphingolipid metabolism, DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCl(DL-PDMP)+imipramine +/- D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-MAPP), with 10-Gy irradiation triggers both mitotic and apoptotic killing in radioresistant SQ20B squamous carcinoma cells. In these cells, apoptosis is defective due to a lack of ceramide generation upstream, which cannot be explained by sphingomyelinase (neutral and acidic) deficiency or rapid derivation to the sphingolipid pathway. We present evidence of a functional transduction death pathway when ceramide generation is restored, which involves the mitochondrial-mediated pathway coupled to alterations in redox status and to executive caspases activation. The poly-drug treatment restored apoptosis to levels similar to those observed in radiosensitive SCC61 squamous carcinoma cells. Simultaneous exposure to gamma-irradiation and poly-drug treatment acted synergistically in SQ20B cells to produce a marked increase in both mitochondrial dysfunction and caspase cleavage, which led to a 7.8-fold increase in apoptosis within 48 h, relative to irradiated cells. Moreover, the results suggest that the ceramide released by irradiation or poly-drug treatment converges upon common cellular targets. Modulation of endogenous ceramide levels by inhibitors of sphingolipid metabolism may represent a new cellular target for the sensitization of radioresistant tumours to gamma-ray therapy.     

Caspase-3-like activity is necessary but not sufficient for daunorubicin-induced apoptosis in Jurkat human lymphoblastic leukemia cells.

In the present study we have shown that the cancer therapeutic drug, daunorubicin, induces apoptosis in the human lymphoblastic leukemia cell line Jurkat E6.1. This effect was both dose-and time-dependent with nuclear fragmentation detectable by 8 h. Caspases have been implicated in pro-apoptotic events. By utilizing synthetic fluorochrome-linked substrates of the caspases, we observed that a caspase-3-like enzyme had dramatically increased activity (3340 130% with respect to basal levels) in response to daunorubicin treatment. Furthermore, by using an inhibitor to caspase-3, Ac-DEVD-CHO, we have shown that activation of a caspase-3-like enzyme appears to be necessary for nuclear fragmentation and apoptotic body formation, but is not required for chromatin condensation. In contrast, a general caspase inhibitor, Z-VAD-fmk, inhibited all apoptotic parameters measured. Ceramide has been implicated in daunorubicin-induced apoptosis in human myeloid leukemia cells. However, in Jurkat cells, caspase activation does not appear to be a consequence of ceramide generation since, although ceramide levels were elevated through the action of ceramide synthase in response to daunorubicin treatment, this occurred with slower kinetics than either nuclear fragmentation or caspase activation. In contrast, caspase inhibitors abrogated ceramide elevation induced by DNR treatment, suggesting that ceramide synthase may be a downstream target for caspase action. Therefore, daunorubicin-induced apoptosis does not appear to be mediated by ceramide in the lymphoblastic leukemia cell line, Jurkat E6.1. Instead, caspase 3 activity appears to be necessary, but not sufficient for this process.     

抗人DR5功能性抗体mDRA．6诱导人白血病Jurkat细胞凋亡的机制

背景与目的：肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factorrelated apoptosis-inducing ligand,TRAIL）及其死亡受体的功能性单克隆抗体具有杀伤肿瘤细胞的活性。我们研制了抗人死亡受体5（deathreceptor,DR5）功能性单克隆抗体（mDRA-6）,本研究对其诱导Jurkat细胞凋亡的Caspase分子机制进行初步探讨。方法：mDRA．6作用后,琼脂糖凝胶电泳检测Jurkat细胞的DNAladder：Mr丌法检测细胞存活情况;AnnexinV—FITC／PI双染流式细胞术定量分析细胞凋亡情况：观察Caspase-10、-9、-8、-3等的抑制剂对mDRA．6诱导细胞凋亡的抑制作用;免疫印迹技术检测凋亡信号蛋白Caspase-10、-9、-8、-3,多聚ADP核糖聚合酶（poly ADP-ribose polymerase．PARP）、BH3相关结构域死亡激动剂（BH3 interacting domain death agonist,Bid）、短缩的Bid（truncated Bid,tBid）、细胞色素C（cytochromec,CytoC）等活化裂解的情况。结果：琼脂糖凝胶电泳显示DNA呈现明显梯状带型：mDRA-6对Jurkat细胞具有明显的诱导凋亡作用且呈量-效关系。2．0μg／mLmDRA-6作用Jurkat细胞0．25h、0．5h、1h、2h．其凋亡率分别为16．2％、28．3％、69．2％、78．2％;Caspase-8抑制剂能明显抑制mDRA-6诱导的细胞凋亡,抑制率为77．9％,Caspase-3和Caspase-9抑制剂作用的抑制率分别为54．2％、8．7％,而Caspase-10的抑制剂无抑制作用;免疫印迹技术检测显示Caspase-8、-3、-9均呈现随时间延长酶原逐渐减少、活化片段增加的现象．PARP的降解片段亦增加,Bid激活降解为tbid,CytoC大量释放,而Caspase．10酶原无明显改变、无活化片段出现。结论：mDRA．6诱导Jurkat细胞凋亡主要是通过激活Caspase路径和线粒体路径来完成的。     

Acidic environment causes apoptosis by increasing caspase activity.

An exposure of HL-60 human promyelocytic leukaemia cells to acidic media with pH 6.2-6.6 caused an up-regulation of Bax protein expression within 2 h, which lasted for longer than 6 h. On the other hand, the apoptosis, as judged from PARP cleavage, DNA fragmentation and flow cytometric determination of cell population with sub-G1 DNA content, occurred after the cells were incubated in the acidic media for longer than 4 h. The PARP cleavage and DNA fragmentation in the cells exposed to an acidic environment could be effectively suppressed by inhibitors specific for ICE or CPP32, indicating that activation of these caspases is an essential step in acidic stress-induced apoptosis. It has been known that Bax is involved in the activation of caspases. Taken together, it appears that acidic stress first up-regulates Bax protein thereby activating caspases followed by PARP cleavage and DNA fragmentation. The observation that inhibition of either ICE or CPP32 could suppress acidic stress-induced apoptosis suggested that ICE activates pro-CPP32, which then cleaves PARP. Flow cytometric analysis indicated that acidic stress-induced apoptosis occurs mainly in G1 cells. The finding in the present study demonstrated that acidic intra-tumour environment may markedly perturb the tumour cell proliferation and tumour growth.     

Human mast cells induce caspase-independent DNA fragmentation in leukemic T cells

Mast cells are known to play an important role in inflammation and host defense. Recent evidence suggests that mast cells may also participate in immune surveillance against cancer cells. In this study, we demonstrate that human mast cells are able to trigger apoptosis in Jurkat T leukemia cells. Fragmentation of Jurkat cell DNA was detectable by JAM assay within 4 h of exposure to HMC-1 mast cells or cultured human cord blood-derived mast cells. HMC-1 mast cells that were fixed with paraformaldehyde retained the ability to induce apoptosis in Jurkat cells while cell-free conditioned supernatants from HMC-1 cell cultures failed to elicit DNA fragmentation, suggesting that mast cells mediate anti-tumor activity via a cell-surface molecule. Surprisingly, the apoptosis-inducing activity of HMC-1 mast cells was not mediated by known tumor necrosis factor (TNF) superfamily members (TNF-alpha, Fas ligand, TRAIL, TWEAK), nor did the fragmentation of Jurkat cell DNA by mast cells require the activity of caspase-3, -8, or -9. Collectively, these data indicate that mast cells trigger caspase-independent apoptosis in leukemic cells via a novel cell-surface molecule, and are consistent with a role for mast cells as anti-tumor effector cells.     

Regulation of G1/S transition and induction of apoptosis in HL-60 leukemia cells by fenretinide (4HPR)

We previously reported that all-trans retinoic acid (RA) and fenretinide (4HPR) suppress HL-60 leukemia cell growth and cause partial cell arrest in the G₁-to-S phase. Moreover, 4HPR but not RA induces apoptosis in HL-60 cells. To investigate further the observed biological effects, cyclin D1 and cdk4 expression and the level of phosphorylation of the retinoblastoma protein Rb were assessed. Cyclin D1 and cdk4 expression and Rb phosphorylation were significantly reduced, by 40–75%, after 24 hr of treatment with RA or 4HPR; these decreases were either transient, e.g., only at 24 hr for cdk4, or sustained for 72 hr. In general, more pronounced decreases were seen in the 4HPR-treated cells. Evidence for 4HPR-induced apoptosis comes from (1) cleavage of the enzyme poly(ADP-ribose) polymerase (PARP) to an 89-kDa truncated product, (2) appearance of DNA ladders on agarose gel electrophoresis, and (3) higher incorporation in situ of digoxigenin nucleotides into the free 3′-ends of DNA. Overnight pretreatment with 0.5–5.0 μM of the CPP32 inhibitor DEVD, but not the ICE inhibitor YVAD, significantly reduced the specific processing of PARP, suggesting that CPP32 is involved in the mechanism of action of 4HPR. Analysis of 2 lipid-derived second messengers, ceramide and diacylglycerol (DAG), as a function of time of treatment with RA or 4HPR, showed ceramide but not DAG to be significantly albeit transiently increased 2-fold at 3 hr, by 4HPR. To test further whether ceramide may be involved in the signaling cascade that culminates in the induction of apoptosis in 4HPR-treated HL-60 cells, the effects of fumonisin B₁, an inhibitor of ceramide synthase, were studied. Simultaneous treatment of cells with 4HPR and 25–100 μM fumonisin B₁ resulted in a dose-dependent reduction in the elevation in ceramide, the extent of PARP cleavage, and induction of apoptosis. Pretreatment with DEVD or YVAD, on the other hand, had no effect on the 4HPR-induced increase in ceramide. Int. J. Cancer 78:53–61, 1998.© 1998 Wiley-Liss, Inc.     

Therapeutic potential of targeting ceramide/glucosylceramide pathway in cancer

Sphingolipids including ceramides and its derivatives such as ceramide-1-phosphate, glucosylceramide (GlcCer), and sphingosine-1-phosphate are essential structural components of cell membranes. They now recognized as novel bioeffector molecules which control various aspects of cell growth, proliferation, apoptosis, and drug resistance. Ceramide, the central molecule of sphingolipid metabolism, generally mediates anti-proliferative responses such as inhibition of cell growth, induction of apoptosis, and/or modulation of senescence. There are two major classes of sphingolipids. One of them is glycosphingolipids which are synthesized from the hydrophobic molecule, ceramide. GlcCer, generated by glucosylceramide synthase (GCS) that transfers the glucose from UDP-glucose to ceramide, is an important glycosphingolipid metabolic intermediate. GCS regulates the balance between apoptotic ceramide and antiapoptotic GlcCer. Downregulation or inhibition of GCS results in increased apoptosis and decreased drug resistance. The mechanism underlying the drug resistance which develops with increased glucosylceramide expression is associated with P-glycoprotein. In various types of cancers, overexpression of GCS has been observed which renders GCS a good target for the treatment of cancer. This review summarizes our current knowledge on the structure and functions of glucosylceramide synthase and glucosylceramide and on the roles of glucosylceramide synthase in cancer therapy and drug resistance.     

The role of gangliosides in fenretinide-induced apoptosis of neuroblastoma

Fenretinide is thought to induce apoptosis via increases in ceramide levels but the mechanisms of ceramide generation and the link between ceramide and subsequent apoptosis in neuroblastoma cells is unclear. In SH-SY5Y neuroblastoma cells, evidence suggests that acid sphingomyelinase activity is essential for the induction of ceramide and apoptosis in response to fenretinide. Downstream of ceramide, apoptosis in response to fenretinide is mediated by increased glucosylceramide synthase activity resulting in increased levels of gangliosides GD3 and GD2 via GD3 synthase. GD3 is a key signalling intermediate leading to apoptosis via the activation of 12-Lipoxygenase, and the parallel induction of GD2 suggests that fenretinide might enhance the response of neuroblastoma to therapy with anti-GD2 antibodies.     

Selective susceptibility of transformed T lymphocytes to induction of apoptosis by PSC 833, an inhibitor of P-glycoprotein

P-glycoprotein is a cellular efflux pump. The P-glycoprotein inhibitor PSC 833 causes apoptosis of cancer cells and induces a rise in the intracellular levels of ceramide. Our aims were to determine whether a cause and effect relationship exists between these two actions of PSC 833, and to assess whether the PSC 833-induced apoptosis is restricted to transformed cells. Apoptosis was determined by flow cytometry and radioactive quantitation of DNA fragmentation. PSC 833 induced apoptosis in the human T leukemia cell lines: Molt-4 and Jurkat. Analysis of the apoptosis in Molt-4 and Jurkat cells revealed that PSC 833 induced a rise in the cellular ceramide levels (as measured by the DG kinase assay). PSC 833-induced apoptosis was significantly reduced by specific inhibitors of ceramide de novo synthesis (i.e., fumonisin B1 and L-cycloserine). On the other hand, PSC 833 did not induce apoptosis in normal peripheral blood T cells regardless of whether these cells were quiescent, activated, or proliferating. Our results suggest that PSC 833 induces apoptotic death in human transformed T lymphocytes through an increase in ceramide de novo synthesis. In addition, normal lymphocytes are not susceptible to induction of apoptosis by PSC 833. This difference between normal lymphocytes and leukemia cells presents a potential target for chemotherapy.     

Apoptotic activity of novel bile acid derivatives in human leukemic T cells through the activation of caspases

The therapeutic efficacies of bile acids, such as ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA), have been widely demonstrated in various liver diseases, suggesting that they might protect hepatocytes against common mechanisms of liver damage. Although they have been shown to prevent apoptotic cell death in certain cell lines, we have previously reported that a novel derivative (HS-1030) of UDCA significantly inhibited cell growth and induced apoptosis in cancer cells. To develop more effective agents, we synthesized several derivatives, named HS-1183, HS-1199 and HS-1200, based on the structure of UDCA and CDCA, and investigated them for anti-proliferative activity in Jurkat cells, a human leukemic T cell line. Whereas UDCA and CDCA had no significant effects on the growth of Jurkat cells in the concentration range tested, both HS-1199 and HS-1200 completely inhibited the cell proliferation, and HS-1183 showed only a weak inhibitory activity. Furthermore, chromatin condensation, DNA ladder formation and proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) were observed after treatment of novel bile acids, indicating the occurrence of apoptotic cell death, which was associated with down-regulation of caspase-3 and -8. The apoptotic manifestations such as PARP cleavage and DNA fragmentation were abolished in the presence of the tripeptide caspase inhibitor zVAD-fmk or the specific caspase-3 inhibitor DEVD-fmk. Our data thus demonstrate that novel bile acid derivatives-induced apoptosis of leukemic T cells is dependent on caspase activation.     

Anti-proliferative and apoptotic effects of celecoxib on human chronic myeloid leukemia in vitro

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, is the only non-steroidal anti-inflammatory drug so far which has been approved by the FDA for adjuvant treatment of patients with familial adenomatous polyposis. The molecular mechanism responsible for the anti-cancer effects of celecoxib is not fully understood. There is little data on the potential role of COX-2 in lymphoma pathogenesis. In view of the reported induction of apoptosis in cancer cells by cyclooxygenase-2 inhibitors, the present study is undertaken to test the effect of celecoxib on human chronic myeloid leukemia cell line, K562 and other hematopoietic cancer cell lines like Jurkat (human T lymphocytes), HL60 (human promyelocytic leukemia) and U937 (human macrophage). Treatment of these cells with celecoxib (10-100 microM) dose-dependently, reduced cell growth with arrest of the cell cycle at G0/G1 phase and induction of apoptosis. Further mechanism of apoptosis induction was elucidated in detail in K562 cell line. Apoptosis was mediated by release of cytochrome c into the cytoplasm and cleavage of poly (ADP-ribose) polymerase-1 (PARP-1). This was followed by DNA fragmentation. The level of anti-apoptotic protein Bcl-2 was decreased without any change in the pro-apoptotic Bax. Celecoxib also inhibited NF-kB activation. Celecoxib thus potentiates apoptosis as shown by MTT assay, cytochrome c leakage, PARP cleavage, DNA fragmentation, Bcl-2 downregulation and possibly by inhibiting NF-kB activation.     

Intracellular mediators of erucylphosphocholine-induced apoptosis

Induction of apoptosis contributes to the cytotoxic action of the intravenously applicable alkylphosphocholine erucylphosphocholine (ErPC). To define molecular requirements for ErPC-induced apoptosis, activation of caspases-8, -9 and -3 and cleavage of the caspase-3 substrates PARP and ICAD were tested in normal Jurkat T cells, Jurkat cells resistant to death receptor (CD95 or TNFalpha-related apoptosis inducing ligand (TRAIL)-induced apoptosis, Jurkat cells lacking caspase-8 or Fas-associated death domain (FADD) Jurkat cells expressing a dominant-negative caspase-9 or overexpressing Bcl-2 as well as BJAB B-lymphoma cells expressing a dominant-negative FADD (FADD-DN). ErPC induced a time- and dose-dependent apoptotic cell death in Jurkat and BJAB cells, which was characterized by breakdown of the phosphatidylserine asymmetry, depolarization of the mitochondrial membrane potential, release of cytochrome c, activation of caspases-9, -8 and -3, cleavage of PARP and ICAD, as well as chromatin condensation. ErPC-induced apoptosis was independent from CD95-receptor signaling and FADD since CD95- and TRAIL-resistant, caspase-8- and FADD-negative Jurkat cells, as well as BJAB cells expressing FADD-DN were sensitive to ErPC-induced apoptosis. In contrast, inhibition of caspase-9 and overexpression of Bcl-2 significantly reduced ErPC-induced caspase activation and apoptosis. Thus, ErPC triggers apoptosis via a Bcl-2-dependent mitochondrial but death receptor-independent pathway.