Potentiation of acetaminophen hepatotoxicity by doxapram in mouse primary cultured hepatocytes

The augmentation by doxapram (DOP) of the reduction in viability and of the apoptosis of cells induced by acetaminophen (AA) was examined in mouse primary cultured hepatocytes. Loss of viability on exposure to AA and/or DOP in cultured hepatocytes was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and the apoptosis of cultured hepatocytes was detected by nuclear morphologic observation and from a ladder-like DNA fragmentation pattern. The combination of AA (5 mM) and DOP (10, 20, 50 or 100 microM) potentiated the reduction in cell viability and increased the oxidative stress. Hepatocytes exposed for 24 h to AA (5 mM) plus DOP (100 microM), showed atrophy of nuclei, including chromatin condensation and a ladder-like DNA fragmentation pattern, characteristic of apoptosis. Benzyl-oxycarbonyl-Asp-CH2OC (O)-2,6-dichlorobenzene (Z-Asp-CH2-DCB, 50 microM), an inhibitor for caspases, improved the viability and ladder-like DNA fragmentation in cells exposed to DOP (200 or 500 microM) alone or AA ( 5 mM) plus DOP (100 microM). However, loss of viability on exposure to a high concentration of AA (10 mM) and ladder-like DNA fragmentation were not affected by Z-Asp-CH2-DCB. These results indicated that the synergistic increase in oxidative stress, activation of caspases and DNA fragmentation induced by DOP potentiated the hepatotoxicity of AA.     

Combination acetaminophen and doxapram potentiated hepatotoxicity in mouse primary cultured hepatocytes

Doxapram-induced potentiation of acetaminophen-induced reductions in cell viability and apoptosis was examined in mouse primary cultured hepatocytes. Loss of viability following exposure of acetaminophen and/or doxapram in cultured hepatocytes was assessed by monitoring [3H]-thymidine incorporation and mitochondrial activity, and apoptosis of hepatocytes was determined by nuclear microscopic observation and from detection of a ladder-like DNA fragmentation pattern in agarose gel electrophoresis. The combination of acetaminophen (5 mM) and doxapram (10, 20, 50 or 100 microM) potentiated the reduction in cell viability and increased lipid peroxide levels of hepatocytes. Hepatocytes exposed for 24 h to acetaminophen (5 mM) plus doxapram (100 microM) showed atrophy of nuclei including chromatin condensation and a ladder-like DNA fragmentation pattern characteristic of apoptosis. Antioxidant (N-acetylcysteine), iron-chelator (deferoxamine), intracellular calcium ion chelator (quin 2-AM), endonuclease inhibitor (aurintricarboxylic acid) and poly (ADP-ribose) polymerase inhibitor (3-aminobenzamide) all improved the viability of cells and eliminated the ladder-like DNA fragmentation in cells exposed to acetaminophen plus doxapram. In conclusion, the combination acetaminophen and doxapram potentiated the reduction in cell viability and apoptosis in mouse primary cultured hepatocytes. We suggest that careful observation for hepatotoxicity is recommended when acetaminophen and doxapram are prescribed simultaneously.     

Effects of cisplatin on mitochondrial function in Jurkat cells

In this work, we measured the effects of pharmacological concentrations of cisplatin (cis-diaminedichloroplatinum II) on mitochondrial function, cell viability, and DNA fragmentation in Jurkat cells. The exposure of cells to 0-25 microM cisplatin for 3 h had no immediate effect on cellular mitochondrial oxygen consumption, measured using a palladium-porphyrin oxygen sensing phosphor. Similarly, the cell viability as measured by trypan blue staining was unchanged immediately following exposure to the drug, and no small DNA fragments, characteristic of drug-induced apoptosis, appeared. At 24 h after exposure to cisplatin, cellular respiration and viability decreased relative to controls and the amount of small DNA fragments, measured using quantitative agarose gel electrophoresis, was proportional to the concentration of cisplatin present during the drug exposure period. The small DNA fragments showed the banding pattern (with a spacing of approximately 300 bp) characteristic of drug-induced cell death by apoptosis. The changes in respiration and DNA fragmentation correlated linearly with the amount of platinum bound to DNA, determined by atomic absorption spectroscopy immediately following drug exposure. The oxygen consumption by beef heart mitochondria was not affected 0-24 h after exposure to 25 microM cisplatin or to solutions containing the monoaquated form of the drug, suggesting that the drug does not attack the mitochondrial respiratory chain directly. Cells exposed to the peptide benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone, which blocks apoptosis by the caspase pathway, showed a decrease in cisplatin-induced DNA fragmentation but not in the impairment of cellular respiration. Thus, although apoptosis is caspase-dependent, the impairment of cellular respiration is independent of the caspase system. Collectively, these results suggest that alteration in mitochondrial function is a secondary effect of cisplatin cytotoxicity in Jurkat cells.     

Aloe emodin-induced apoptosis in t-HSC/Cl-6 cells involves a mitochondria-mediated pathway

The aim of our study was to clarify the apoptosis pathway induced by aloe emodin, an hydroxyanthraquinone present in aloe vera leaves, in rat hepatic stellate cells transformed by simian virus 40 (t-HSC/Cl-6), which retain the features of activated rat stellate cells. Apoptosis was determined by DNA fragmentation, caspase activity assay and western blotting analysis. Treatment of t-HSC/Cl-6 cells with 12.5, 25, or 50 microM aloe emodin inhibited t-HSC/Cl-6 cell viability in a dose- and time-dependent manner. The induction of apoptosis by aloe emodin was confirmed by typical DNA ladder formation and annexin v-propidium iodide flow-cytometric analysis. Aloe emodin treatment of t-HSC/Cl-6 cells caused activation of caspase-3 and caspase-9, detected with a caspase activity assay, although no change was observed in caspase-8 activity. Western blotting showed caspase-3 and caspase-9 active forms and the subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. Aloe emodin induced mitochondrial membrane depolarization. Our data also show that cytochrome c increased in the cytosol but decreased in the mitochondria in a time-dependent manner. Increased Bax and unchanged Bcl-2 levels resulted in an increased Bax/Bcl-2 ratio. Thus, our research provides evidence that aloe emodin-induced apoptosis involves a mitochondria-associated apoptosis pathway.     

Apoptosis, necrosis and cell proliferation-inhibition by cyclosporine A in U937 cells (a human monocytic cell line).

The immunosuppressive drug cyclosporine A (CsA) has been used in both organ transplantation and the treatment of autoimmune disorders. However, the drug causes adverse effects in the kidney, liver and nervous system, characterized by cellular loss in the affected area. Apoptosis has been shown to play a role in CsA-induced cytotoxicity. Because permeabilization of the mitochondrial membrane is a common criterion in most apoptotic settings in vertebrate cells, here we evaluated whether CsA causes loss of mitochondrial function in the pathway leading to cellular cytotoxicity. We found that CsA caused a concentration- and time-dependent loss of cell viability in the U937 cell line. Treatment of cells at a dose of 10 microM CsA resulted in G0/G1 arrest with a concurrent decrease in the number of cells in the S and G2/M phases of the cell cycle. In mechanistic studies related to the loss of viability, treating cells with 10 microM CsA for 24 h resulted in both DNA fragmentation and an increase of annexin-V-positive cells. CsA treatment also increased activity of the cysteine protease caspase-3, decreased the mitochondrial membrane potential and induced the release of cytochrome c into the cytosol. Furthermore, CsA treatment increased the number of cells in the sub-G0/G1 peak, indicative of a reduction in DNA, although this increase was not observed when cells were pre-treated with a broad caspase inhibitor. In the study, we also found that a higher dose of CsA induces LDH release when the cells were incubated for a longer period. Taken together, these data suggest that the mode of cell death induced by CsA is dose- and time-dependent. Short-term incubation with lower doses of CsA arrests cell growth; this arrest overlaps with the occurrence of apoptosis and then with necrosis after longer treatment periods with higher doses of CsA.     

The effect of ursodeoxycholic acid on glycochenodeoxycholic acid-induced apoptosis in rat hepatocytes

Ursodeoxycholic acid (UDCA) has been widely used for treating cholestatic liver diseases. However, in a recent review of clinical trial articles, its therapeutic benefits were not proven. Therefore, we investigated whether UDCA prevents or potentiates glycochenodeoxycholic acid (GCDCA)-induced apoptosis in isolated rat hepatocytes. Hepatocellular cytotoxicity was assessed by lactate dehydrogenase (LDH) release, and apoptosis evaluated by DNA fragmentation, caspase activities, release of cytochrome C from mitochondria, and mitochondrial membrane potential change (Deltapsi). When hepatocytes were co-incubated with GCDCA and UDCA for a short time (2-6 h), GCDCA-induced LDH release was significantly reduced, while prolonged co-incubation (12-20 h) increased it. Similarly, the same co-incubation for a short time resulted in the inhibition of caspase activities and cytochrome C release, while prolonged incubation enhanced them compared with the incubation with GCDCA alone. Furthermore, UDCA significantly promoted the GCDCA-induced Deltapsi decline after 4h of incubation. These results demonstrated that UDCA reduced GCDCA-induced apoptosis in short incubation, but potentiated it in prolonged incubation. Based on these, we propose a hypothesis that induction of Deltapsi decrease from earlier stage of incubation may be responsible for the aggravation of GCDCA-induced apoptosis in long-term exposure, and would like to raise caution about clinical long-term use of UDCA.     

2,4-dinitrophenol induces G1 phase arrest and apoptosis in human pulmonary adenocarcinoma Calu-6 cells.

2,4-dinitrophenol (DNP) is an uncoupler of oxidative phosphorylation in the mitochondria. Here, we investigated the effect of DNP on the growth of Calu-6 lung cancer cells in view of cell cycle, apoptosis, ROS production and GSH content. DNP dose-dependently decreased cell viability at 72 h (EC50 of about 200 microM) as measured by a MTT assay. The lower doses of DNP induced a G1 arrest of the cell cycle in Calu-6 cells. Analysis of the cell cycle regulatory proteins demonstrated that DNP decreased the steady-state levels of cyclin proteins and cyclin dependent kinase (CDK), but increased the protein levels of cyclin dependent kinase inhibitor (CDKI) p27. DNP also caused a marked increase in apoptosis, as evidenced by DNA fragmentation (sub-G1 DNA content), DAPI staining, the loss of mitochondrial membrane potential (DeltaPsim), externalization of phosphatidylserine (PS). In addition, DNP-treated cells significantly increased the intracellular H2O2 and O2.- levels. All of caspase inhibitors could markedly rescue Calu-6 cells from DNP-induced cell death and only pan-caspase inhibitor, Z-VAD-FMK, could slightly prevent the loss of mitochondrial membrane potential (DeltaPsim). However, none of the caspase inhibitors reduced the increased H2O2 levels, but the increased O2.- levels was slightly attenuated by pan-caspase inhibitor. In addition, the depletion of GSH content in DNP-treated cells was prevented by all of caspase inhibitors. In conclusion, DNP, which induced ROS and reduced GSH content, inhibited the growth of Calu-6 cells via cell cycle arrest at G1 phase and apoptosis.     

Curcumin protects mitochondria from oxidative damage and attenuates apoptosis in cortical neurons

AIM: To investigate the effect of curcumin on tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in rat cortical neurons and to explore the possible mechanism. METHODS: Primary cultured rat cortical neurons wereperformed in vitro and cell viability was measured by MTT assay. DNA fragmentation was used to evaluate cellapoptosis. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (Aψm) was determined by flow cytometric assay. Cellular glutathione (GSH) content was measured by spectrophotometer.‘ Bcl-2family proteins, cytochrome c, cleaved caspase-3, and poly (ADP-ribose) polymerase (PARP) were detected byWestern blot. RESULTS: Exposure of tBHP 100 μmol/L to neurons for 60 rain resulted in △ψm loss and cyto-chrome c release from mitochondria and subsequent activation of caspase-3 and PARP cleavation, and cell apoptosis.After removal of tBHP and then further treatment with curcumin (2.5-20 μmol/L) for 18 h, curcumin abrogated △ψm loss and cytochrome c release, blocked activation of caspase 3, and altered the expression of Bcl-2 family.Further curcumin treatment also prevented cellular GSH and decreased intracellular ROS generation markedly.Curcumin eventually attenuated tBHP-induced apoptosis in cortical neurons. CONCLUSION: Curcumin mayattenuate oxidative damages in cortical neurons by reducing intracellular production of ROS and protecting mito-chondria from oxidative damage.     

Beta2-adrenoceptor activation inhibits Shiga toxin2-induced apoptosis of renal tubular epithelial cells

Apoptosis is regulated by several pathways, such as caspases, mitogen activated protein kinase (MAPK) and cAMP/cAMP-dependent protein kinase A (PKA) cascade. This study investigated the effect of beta(2)-adrenoceptor activation on Shiga toxin (Stx)2-induced apoptosis in renal tubular cells and the contribution of these signalling pathways. Cultured human adenocarcinoma-derived tubular cells were exposed to Stx2 (64 pg/mL) for 2-24hr following the addition of the beta(2)-adrenoceptor agonist (terbutaline) to the incubation medium. Stx2-induced apoptosis and its amelioration by beta(2)-adrenoceptor activation was confirmed using DNA degradation assays and by flow cytometry for annexin V, mitochondrial membrane potential and caspase(-3 and -7) activity. Exposure of cells to Stx2 for 24hr increased the DNA fragmentation to 11.6+/-0.9%, compared to 3.3+/-0.2% in control cells (P<0.05) but was decreased to approximately 5-7% (P<0.05) in the presence of terbutaline. Furthermore, Stx2-stimulated apoptosis, detected by TUNEL, annexin V and mitochondrial potential, was inhibited by terbutaline (P<0.05) which was prevented by cAMP-PKA inhibitors and a beta(2)-adrenoceptor antagonist. However, inhibition of Stx2-mediated caspase activity by terbutaline was partially blocked by cAMP-PKA inhibitors. On the other hand, p38MAPK inhibition by terbutaline prevented Stx2-induced apoptosis and caspase activity through a cAMP-independent pathway via beta(2)-adrenoceptor. These data indicate that beta(2)-adrenoceptor activation can inhibit Stx2-induced apoptosis of the cells, which may be caused by a reduction in caspase activity through cAMP-PKA activation and the p38MAPK pathway.     

Aspirin and sodium salicylate inhibit proliferation and induce apoptosis in rheumatoid synovial cells

Aspirin has been reported to induce apoptosis in a variety of cell lines. In this study, we examined whether aspirin and sodium salicylate inhibit cell growth and induce apoptosis in rheumatoid synovial cells. Synovial cells were obtained from patients with rheumatoid arthritis, and the cells were treated with aspirin or sodium salicylate (0.1-10 mM) for 24 h. Cell proliferation and viability were assessed by 5-bromo-2'-deoxyuridine incorporation and by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) assay, respectively. The apoptosis of synovial cells was identified by DNA fragmentation assay and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. Aspirin and sodium salicylate suppressed the proliferation (IC50 (concentration causing 50% inhibition of cell proliferation): 2.1 and 1.2 mM, respectively) and reduced the viability (IC50: 2.0 and 1.4 mM, respectively) of synovial cells in a concentration-dependent manner at 0.3-10 mM. Furthermore, they induced DNA fragmentation and increased the number of TUNEL-positive synovial cells. These results suggest that aspirin and sodium salicylate can inhibit the proliferation of rheumatoid synovial cells through induction of apoptosis.     

Evaluation of cultured, precision-cut rat liver slices as a model to study drug-induced liver apoptosis

The precision-cut liver slice culture model has been used widely to investigate drug metabolism and drug-induced necrosis. However, apoptosis, a key mediator of liver toxicity remains to be studied in this model. We evaluated apoptosis induced by thioacetamide (TAA) in rat liver slices, and in livers taken from TAA-treated rats as a control. Rat liver slices were treated with 50, 75 and 100 mM of TAA for 15 h. Histopathological examination of the liver slices revealed specific centrilobular localization of apoptotic hepatocytes at 75 mM but randomly distributed at 100 mM. Apoptosis in centrilobular hepatocytes was confirmed by appearance of cleavage products of caspase-3 and DNA fragmentation studied by TUNEL method. A concentration-dependent release of cytochrome c was observed in the slices, suggesting a role for mitochondria in the apoptosis triggered by TAA. The in vitro results were compared to the data obtained in male Sprague-Dawley rats given a single ip injection of 40 mg/kg TAA and sacrificed 1, 2, 3 and 6 h after dosing. Histopathological analyses showed specific centrilobular localization of apoptosis after 6 h treatment. Caspase-3 activation, DNA fragmentation and cytochrome c release were also observed in the liver of rats treated with TAA. Overall these data indicated that precision-cut liver slices provide a valuable in vitro system to study drug-induced liver apoptosis.     

Chelerythrine and dihydrochelerythrine induce G1 phase arrest and bimodal cell death in human leukemia HL-60 cells

A quaternary benzo[c]phenanthridine alkaloid chelerythrine displays a wide range of biological activities including cytotoxicity to normal and cancer cells. In contrast, less is known about the biological activity of dihydrochelerythrine, a product of chelerythrine reduction. We examined the cytotoxicity of chelerythrine and dihydrochelerythrine in human promyelocytic leukemia HL-60 cells. After 4 h of treatment, chelerythrine induced a dose-dependent decrease in the cell viability with IC₅₀ of 2.6 μM as shown by MTT reduction assay. Dihydrochelerythrine appeared to be less cytotoxic since the viability of cells exposed to 20 μM dihydrochelerythrine for 24 h was reduced only to 53%. Decrease in the viability induced by both alkaloids was accompanied by apoptotic events including the dissipation of mitochondrial membrane potential, activation of caspase-9 and -3, and appearance of cells with sub-G1 DNA. Moreover, chelerythrine, but not dihydrochelerythrine, elevated the activity of caspase-8. A dose-dependent induction of apoptosis and necrosis by chelerythrine and dihydrochelerythrine was confirmed by annexin V/propidium iodide dual staining flow cytometry. Besides, both alkaloids were found to induce accumulation of HL-60 cells in G1 phase of the cell cycle. We conclude that both chelerythrine and dihydrochelerythrine affect cell cycle distribution, activate mitochondrial apoptotic pathway, and induce apoptosis and necrosis in HL-60 cells.     

Mechanism of ricin-induced apoptosis in human cervical cancer cells

The mechanism of ricin-induced apoptosis in human cervical cancer cell line HeLa was studied. The present study demonstrated that ricin induces apoptosis of human cervical cancer cells (HeLa) in a time dependent manner with an IC(50) for cell viability of 1 microg/ml. Ricin treatment resulted in a time dependent increase in LDH leakage, DNA fragmentation, percent apoptotic cells, generation of reactive oxygen species and depletion of intracellular glutathione levels. DNA agarose gel electrophoresis showed typical oligonucleosomal length DNA fragmentation. Additionally, DNA diffusion assay was performed to confirm DNA damage and apoptosis. Ricin activated caspase-3 as evidenced by both proteolytic cleavage of procaspase-3 into 20 and 18 kDa subunits, and increased protease activity. Caspase activity was maximum at 4h and led to the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP), resulting in the 85 kDa cleavage product. Ricin-induced caspase-3 activation also resulted in cleavage of DNA fragmentation factor-45 (DFF45/ICAD) and DFF40 or caspase-activated DNase in HeLa cells. Activation of caspase-3, cleavage of PARP and DNA fragmentation was blocked by pre-treatment with caspase-3 specific inhibitor Ac-DEVD-CHO (100 microM) and broad-spectrum caspase inhibitor Z-VAD-FMK (40 microM). Ricin-induced DNA fragmentation was inhibited by pre-treatment with PARP inhibitors 3-aminobenzamide (100 microM) and DPQ (10 microM). Our results indicate that ricin-induced cell death was mediated by generation of reactive oxygen species and subsequent activation of caspase-3 cascade followed by down stream events leading to apoptotic mode of cell death.     

Oleanolic acid induces apoptosis of MKN28 cells via AKT and JNK signaling pathways

Context: Oleanolic acid (OA) belongs to the triterpenoid compound group existing widely in food, medicinal herbs and other plants. Its effects on gastric cancer cells and the mechanisms involved have not been investigated.

Objective: This study aimed to substantiate whether OA induces apoptosis of gastric cancer cell line (MKN28) and to elucidate the molecular mechanism involved.

Materials and methods: Cell viability was assessed by MTT assay within the range of 0–160?μg/mL. The effects of OA (5, 10 and 20?μg/mL) on apoptosis of MKN28 cells were evaluated by flow cytometry, DNA fragmentation and mitochondrial membrane potential assays. Western blot and FQRT-PCR assays were used to investigate the mechanism of cell apoptosis induced by OA (5 and 10?μg/mL).

Results: OA evidently inhibited cell viability with IC₅₀ of 44.8 and 15.9?μg/mL at 12 and 24?h, respectively. Furthermore, OA increased JNK phosphorylation, decreased AKT phosphorylation, but did not affect p38 and ERK phosphorylation in MKN28 cells. In contrast, OA also significantly enhanced the mRNA expression levels of caspase 3, caspase 9 and Apaf-1 in MKN28 cells.

Conclusion: OA induces apoptosis of MKN28 cells via the mitochondrial pathway regulated by AKT and JNK signaling pathways.     

杨梅素诱导人肝癌HepG2凋亡机制研究

目的对杨梅素诱导人肝癌HepG2细胞凋亡机制进行研究。方法采用MTT法测定细胞死亡率;采用倒置显微镜观察细胞形态学变化;用琼脂糖电泳观察DNA片段化。用Western印迹法分析相关蛋白的变化。结果杨梅素明显抑制HepG2细胞的增殖,诱导HepG2细胞调亡。Caspase-3,caspase-9抑制剂可明显抑制100μg/ml杨梅素诱导的凋亡。Western印迹结果显示100μg／ml杨梅素作用于细胞后,细胞色素c水平明显升高。结论杨梅素（100μg／ml）通过激活线粒体途径诱导HepG2细胞凋亡。     

Cellular toxicity of hydrazine in primary rat hepatocytes.

Hydrazine (HzN) is an aircraft fuel and propellant used by the U.S. Air Force. The current study was undertaken to evaluate the acute toxicity of HzN in primary rat hepatocytes in vitro with reference to oxidative stress. The effects of short-term exposure (4 h) of hepatocytes to HzN were investigated with reference to viability, mitochondrial function, and biomarkers of oxidative stress. The viability data showed an increase in lactate dehydrogenase leakage and a decrease in mitochondrial activity with increasing concentration of HzN. The results of studies of oxidative stress biomarkers showed a depletion of reduced glutathione (GSH) and an increase in oxidized GSH, increased reactive oxygen species generation, lipid peroxidation, and reduced catalase activity. Furthermore, depletion of GSH and catalase activity in hepatocytes by buthionine sulfoximine and 3-amino triazole, respectively, prior to exposure to HzN, increased its toxicity. The results suggest that acute HzN-induced cytotoxicity in rat hepatocytes is likely to be mediated through oxidative stress.     

Participation of various kinases in staurosporine induced apoptosis of RAW 264.7 cells

Staurosporine induced apoptosis of RAW 264.7 cells, a mouse macrophage-like cell line, as determined by DNA fragmentation, the increase of annexin V-stained cells, and the cleavage of poly(ADP-ribose)polymerase (PARP), a substrate of caspase. Analysis of the increase in the percentage of sub-G(1) cells revealed that the DNA fragmentation occurred in a time- and concentration-dependent manner at 0.021-2.1 microM of staurosporine. Staurosporine induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) but suppressed spontaneous phosphorylation of p44/42 MAPK. The p38 MAPK inhibitor SB203580, the MAPK/extracellular signal-regulated kinase kinase inhibitor PD98059 and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 potentiated the staurosporine-induced PARP cleavage and DNA fragmentation. The protein kinase A (PKA) inhibitor H-89 potentiated the staurosporine-induced DNA fragmentation without potentiating the PARP cleavage. In contrast, the protein kinase C (PKC) inhibitor Ro-31-8425 suppressed the PARP cleavage and DNA fragmentation. These findings suggested that staurosporine induces apoptosis via the caspase cascade in RAW 264.7 cells. The staurosporine-induced apoptosis is positively regulated by PKC, negatively regulated by p38 MAPK, p44/42 MAPK and PI3K via the caspase cascade, and negatively regulated by PKA without regulation of caspase activation.