Cadmium induces apoptotic cell death in WI 38 cells via caspase-dependent Bid cleavage and calpain-mediated mitochondrial Bax cleavage by Bcl-2-independent pathway

Previous reports have demonstrated that cadmium (Cd) may induce cell death via apoptosis, but the mechanism responsible for cellular death is not clear. In this study, we investigated the signaling pathways implicated in Cd-induced apoptosis in lung epithelial fibroblast (WI 38) cells. Apoptotic features were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, propidium iodide staining and DNA laddering. A treatment of cadmium caused the caspase-8-dependent Bid cleavage, the release of cytochrome c (Cyt c), activation of caspase-9 and -3, and PARP cleavage. A caspase-8 specific inhibitor prevented the Bid cleavage, caspase-3 activation and cell death. Alternatively, we observed that full-length Bax was cleaved into 18-kDa fragment (p18/Bax); this was initiated after 12 h and by 36 h the full-length Bax protein was totally cleaved to the p18/Bax, which caused a drastic release of Cyt c from mitochondria. The p18/Bax was detected exclusively in the mitochondrial fraction, and it originated from mitochondrial full-length Bax, but not from the cytosol full-length Bax. Cd also induced the activation of the mitochondrial 30-kDa small subunit of calpain that was preceded by Bax cleavage. Cd induced the upregulation of Bcl-2 and the degradation of p53 protein. N-acetyl cysteine effectively inhibited the Cd-induced DeltaPsim reduction, indicating ROS acts upstream of mitochondrial membrane depolarization. Taken together, our results suggest that Cd-induced apoptosis was thought to be mediated at least two pathways; caspase-dependent Bid cleavage, and the other is calpain-mediated mitochondrial Bax cleavage. Moreover, we found that the function of Bid and Bax was not dependent of Bcl-2, and that ROS can also contribute in the Cd-induced cell death.     

Nickel(II)-induced nasal epithelial toxicity and oxidative mitochondrial damage

In probing the underlying mechanisms of nickel(II)-induced cytotoxicity on nasal epithelium, we investigated the effects of nickel(II) acetate on nasal epithelial RPMI-2650 cells. Nickel(II) elicited apoptosis, as signified by pyknotic and fragmented nuclei, increased caspase-3/7 activity, and an increase in annexin V binding, hypodiploid DNA, and Bax/Bcl-2 protein ratio. Nickel(II)-induced G₂/M arrest was associated with up-regulation of p21^WAF1/CIP1 expression, decrease in phosphorylation at Thr¹⁶¹ of Cdc2, and down-regulation of cyclin B1. Associated with these responses, ROS generation and mitochondrial depolarization increased in a nickel(II) concentration-dependent fashion. Pretreatment with N-acetylcysteine (NAC) attenuated these changes. p53 reporter gene assay and analyses of p53, Puma, Bax, and Bcl-2 protein levels indicated that NAC inhibited nickel(II)-induced activation of p53-mediated mitochondrial apoptotic pathway. Collectively, our study provides evidences that nickel(II) may induce oxidative damage on nasal epithelium in which antioxidant NAC protects cells against nickel(II)-induced apoptosis through the prevention of oxidative stress-mediated mitochondrial damage.     

Catalpol inhibits apoptosis in hydrogen peroxide-induced PC12 cells by preventing cytochrome c release and inactivating of caspase cascade.

In the present study, using a rat pheochromocytoma (PC12) cell line, the effect of catalpol on H2O2-induced apoptosis was studied. The apoptosis in H2O2-induced PC12 cells was accompanied by down-regulation of Bcl-2, up-regulation of Bax, the release of mitochondrial cytochrome c to cytosol and sequential activation of caspase-1 and caspase-3 then leading to cleavage of poly-ADP-ribose polymerase (PARP). Catalpol not only suppressed the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol, but also attenuated caspase-3 activation, PARP cleavage, and eventually protected against H2O2-induced apoptosis. Taken together, these results suggest that treatment of PC12 cells with catalpol can block H2O2-induced apoptosis by the regulation of Bcl-2 family members, as well as suppression of cytochrome c release and caspase cascade activation.     

P53-mediated cell cycle arrest and apoptosis through a caspase-3- independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells

Aim： To study the caspase-3-independent mechanisms in oridonin-induced MCF-7 human breast cancer cell apoptosis in vitro. Methods： The viability of oridonin- treated MCF-7 cells was measured by MTT （thiazole blue） assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Bax, Bcl-2, caspase-3, caspase-9, heat shock protein （Hsp）90, p53, p-p53, p21, Poly （ADP-ribose） polymerase （PARP）, and the inhibitor of caspase-activated DNase （ICAD） protein expressions were detected by Western blot analysis. Results： Oridonin inhibited cell growth in a time- and dose-dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pancaspase inhibitor Z-VAD-fmk and calpain inhibitor II both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of △ψmit were detected in oridonin-induced MCF-7 cell apoptosis, which was involved in a postmitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by capase-3. Condusion： DNA damage provoked alternations in the mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-induced MCF-7 cells.     

大萼香茶菜甲素通过线粒体途径诱导HL-60白血病细胞凋亡

目的：观察大萼香茶菜甲素（macrocalyxinA,MA）体外诱导HL-60细胞凋亡,并探讨其作用机制。方法：不同浓度的MA与HL-60细胞进行培养,采用MTT比色法观察其对HL-60细胞增殖的抑制作用;细胞形态学、DNA含量、DNA梯度电泳及细胞周期分析、Annexin—V／PI双标记和Hoechst 33258荧光染色等分析其促细胞凋亡效应;流式细胞术和RT-PCR分别检测Bcl-2、Bax、P53、Fas、线粒体膜蛋白（Ap02．7）、线粒体跨膜电位（AWm）与Bcl-2、Bax、P53、caspase-3 mRNA变化水平,研究其促凋亡机制。结果：MA呈现作用时间和剂量依赖性地抑制HL-60细胞增殖和活力;HL-60细胞经MA作用后,Wright—Giemsa染色和Hoechst荧光染色后细胞出现典型的凋亡小体,细胞阻滞于G0／G1期,DNA片段化,亚二倍体明显增高,Annexin—V／PI标记升高;MA诱导HL-60细胞凋亡过程中,Bcl-2、Fas、P53表达无明显变化,Bax、线粒体膜蛋白（Apo2．7）、caspase-3表达显著增加,Bax／Bcl-2比值升高,龇下降。结论：MA能抑制HL-60细胞增殖和细胞活力、诱导细胞凋亡,其机制通过上调Bax基因和Bax／Bcl-2比值,使线粒体膜电位下降、膜通透性增高,最终使caspase-3激活而促进凋亡。     

Olaquindox induces apoptosis through the mitochondrial pathway in HepG2 cells

Olaquindox is used in China as feed additive for growth promotion in pigs. Recently, we have demonstrated that olaquindox induced genome DNA damage and oxidative stress in HepG2 cells. The aim of this study was to explore the molecular mechanism of cell cycle arrest and apoptosis induced by olaquindox in HepG2 cells. In the present study olaquindox induced cell cycle arrest to the S phase and dose-dependent apoptotic cell death in HepG2 cells, indicated by accumulation of sub-G1 cell population, nuclear condenstion, DNA fragmentation, caspases activation and PARP cleavage. Meanwhile, the data showed that olaquindox triggered ROS-mediated apoptosis in HepG2 cells correlated with both the mitochondrial DNA damage and nuclear DNA damage, collapse of Δψ_m, opening of mPTP, down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we also found that olaquindox increased the expression of p53 protein and induced the release of cytochrome C from mitochondria to cytosol. In conclusion, olaquindox induced apoptosis of HepG2 cells through a caspase-9 and -3 dependent mitochondrial pathway, involving p53, Bcl-2 family protein expression, Δψ_m disruption and mPTP opening.     

A decisive role of mitochondria in defining rate and intensity of apoptosis induction by different alkaloids

Sanguinarine, chelerythrine and chelidonine possess prominent apoptotic effects towards cancer cells. In this study, we found that sanguinarine and chelerythrine induce apoptosis in human CEM T-leukemia cells, and that is accompanied by an early increase in cytosolic cytochrome c that precedes caspases-8, -9 and -3 processing. During apoptosis induction by sanguinarine and chelerythrine, reactive oxygen species (ROS) was rapidly generated and DeltaPsi(mt) dissipated, while Bax, Bcl-2 and Bcl-X((L/S)) proteins' content in the mitochondrial fraction did not change significantly. Caspase-3 activation and DNA fragmentation were considerably inhibited by N-acetyl-cysteine (NAC). Chelidonine induced only a slight release of cytochrome c (12h), parallel to caspase-3 activation. Effect of sanguinarine or chelerythrine towards mitochondria was confirmed by marked changes in morphology of this organelle (3h), while chelidonine did not affect mitochondria intactness. Sanguinarine or chelerythrine also caused an intensive DNA damage in cells in 1h, however a massive increase in number of such impaired cells occurred in 6h, while chelidonine induced intensive DNA damage in 15-20% cells only in 24h. Thus, our results demonstrated that rapid cytochrome c release in CEM T-leukemia cells exposed to sanguinarine or chelerythrine was not accompanied by changes in Bax, Bcl-2 and Bcl-X((L/S)) proteins in the mitochondrial fraction, and preceded activation of the initiator caspase-8.     

Sulforaphane induces cytotoxicity and lysosome- and mitochondria-dependent cell death in colon cancer cells with deleted p53

Mechanisms and pathways responsible for cytotoxicity of sulforaphane (SF) in colon cancer cells with deleted p53 were investigated during 48 h of exposure. SF showed dose-dependent cytotoxicity and proapoptotic activity in the present model. In addition, in HCT-116 p53KO cells SF induced DNA damage with the subsequent cellular response and signaling not including p53 and caspase-2 pathways. Conversely, in SF-treated cells JNK was activated which led to an early lysosomal membrane permeabilization, release of cathepsin B and D and activation of Bid by specific cleavage. Concomitantly, the expression of Bax increased in the presence of JNK-mediated Bcl-2 inhibition which was followed by mitochondrial release of cytochrome c and activation of apoptosis. These results suggest that SF may be useful as a chemopreventive agent in colon cancer with inactivated or lost p53.     

Involvement of BH3-only proapoptotic proteins in mitochondrial-dependent Phenoxodiol-induced apoptosis of human melanoma cells

Phenoxodiol is a chemically modified analogue of the plant hormone isoflavone with antitumour activities. In the present study, we have examined its ability to induce apoptosis in human melanoma cells and the mechanisms involved. Apoptosis was observed in Phenoxodiol-treated cells by using annexin V/propidium iodide staining and determining mitochondrial membrane potential. To determine which caspase pathways were involved in Phenoxodiol-induced apoptosis, studies were performed using specific caspase inhibitors. Western studies were performed to ascertain which proteins of the apoptosis cascade were affected to cause Phenoxodiol-induced apoptosis. We found that induction of apoptosis by Phenoxodiol was maximal at 48 h with a range of apoptosis of 12+/-4 to 48+/-5% in different melanoma lines. This apoptosis was mainly dependent on activation of caspase-3 and caspase-9. Apoptosis was associated with induction of changes in mitochondrial membrane potential and was inhibited by over-expression of Bcl-2. Variation in sensitivity to Phenoxodiol appeared related to events upstream of the mitochondria and the degree of conformational change in Bax. The p53-regulated BH3-only proteins (Bad, PUMA and Noxa) were increased in the sensitive, but not in the resistant lines, whereas Bim was increased in all the lines tested. Bim appeared, however, to be partially involved because reduction of Bim by RNA interference resulted in decreased levels of apoptosis. Together, these studies suggest that Phenoxodiol induces apoptosis of melanoma cells by induction of p53-dependent BH3 proteins (Bad, PUMA and Noxa) and the p53-independent Bim protein, resulting in activation of Bax and its downstream events.     

Apoptotic effects of satratoxin H is mediated through DNA double-stranded break in PC12 cells

Satratoxin H is an important air- and food-borne mycotoxin, which has been implicated in human health damage. Satratoxin H is known to induce apoptosis as well as genotoxicity in PC12 cells. In the present study, we further investigated the mechanism of apoptotic effects of satratoxin H with focus on caspase-3 and poly-ADP-ribose polymerase (PARP) pathway. We also examined whether it induces DNA damage in PC12 cells. In the cells treated with satratoxin H, caspase-3 was cleaved in a time-dependent manner. Furthermore, satratoxin H induced cleavage of PARP, one of the downstream molecules of caspase-3. The cleavage was inhibited by SB203580, a p38 MAPK inhibitor, or SP600125, a JNK inhibitor. Satratoxin H, however, had no effect on expression levels of Bax and Bcl-2. Furthermore, the micronucleus assay revealed that satratoxin H induced chromosome break. Also, satratoxin H increased the level of phosphorylation of histone H2A, indicating that it caused DNA double-stranded breaks in PC12 cells. Meanwhile, no genotoxicity was detected with any of treatments carried out in the alkaline comet assay. These results imply that satratoxin H induces genotoxicity by DNA double-stranded break. Our results suggest a considerable potential for the genotoxic risk associated with the presence of satratoxin H.     

Butein, a plant polyphenol, induces apoptosis concomitant with increased caspase-3 activity, decreased Bcl-2 expression and increased Bax expression in HL-60 cells

In the present study we have investigated whether butein could induce apoptosis in human leukaemic HL-60 cells. The treatment of HL-60 cells with butein induced apoptotic cell death as determined by morphological and biochemical changes. Apoptotic DNA fragments in the butein-treated HL-60 cells were increased gradually as determined by flow cytometric analysis. The caspase-3 activity was increased during butein-induced apoptosis. However, caspase-3 inhibitor abrogated the butein-induced DNA fragmentation. Furthermore, the treatment of HL-60 cells with butein decreased the expression of Bcl-2 protein, but increased the expression of Bax protein. These results suggest that butein-induced apoptosis is mediated through the activation of caspase-3 and it is associated with changed expression of Bcl-2 and Bax proteins.     

人参皂甙Rg1对抗多巴胺诱导的PC12细胞凋亡

目的:探讨外源性多巴胺诱导PC12细胞凋亡以及人参皂甙Rg1保护作用的分子机制。方法:流式细胞仪定量测定PC12细胞的凋亡和Bcl-2、Bax蛋白的表达;电子显微镜观察PC12细胞的形态;凝胶电泳评价DNA的断裂;荧光分光光度计法测定caspase-3的活力;半定量RT-PCR分析bcl-2和bax mRNA的表达。结果:多巴胺(浓度为0.15、0.30、0.45和0.60mmol/L)诱导PC12细胞凋亡,各剂量组细胞凋亡率分别从对照组1.1％±0.4％增加到41％±3％,46.4％±2.7％,53％±3％和64.5％±2.7％;人参皂甙Rg1 10μmol/L预处理24h后,较多巴胺0.45mmol/L单独处理时,PC12细胞的凋亡率和caspase-3的活力分别从53％±3％和683±8(平均荧光强度)下降到1.9％±0.6％和325±5,Bcl-2蛋白阳性率从14.3％±1.1％增加到25.9％±1.6％,Bax蛋白阳性率从48％±3％下降到35％±3％。结论:人参皂甙Rg1通过抑制cas-pase-3的激活并调节Bcl-2和Bax两者间蛋白的比值对抗多巴胺对PC12细胞凋亡的诱导作用。     

Humic acid induces G1 phase arrest and apoptosis in cultured vascular smooth muscle cells

Humic acid (HA) in well water used by the inhabitants for drinking is one of the possible etiological factors for Blackfoot disease (BFD). In this study, the ability of HA to inhibit cell cycle progression and induce apoptosis in cultured smooth muscle cells (SMCs; A7r5) was investigated. Treatment of the SMCs at various HA concentrations (25-200 microg/mL) resulted in sequences of events marked by apoptosis, as shown by loss of cell viability, morphology change, and internucleosomal DNA fragmentation. HA-induced apoptotic cell death that is associated with loss of mitochondrial membrane potential (Delta Psi m), cytochrome c translocation, caspase-3, -8, and -9 activation, poly ADP-ribose polymerase (PARP) degradation, dysregulation of Bcl-2 and Bax, and upregulation of p53 and phospholyrated p53 (p-p53) in SMCs. Flow cytometry analysis demonstrated that HA blocked cell cycle progress in the G1 phase in SMCs. This blockade of cell cycle was associated with reduced amounts of cyclin D1, CDK4, cyclin E, CDK2, and hyperphosphorylated retinoblastoma protein (pRb) in a time-dependent manner. Apparent DNA strand breaks (DNA damage) were also detected in a dose-dependent manner using Single-cell gel electrophoresis assay (comet assay). Furthermore, HA induced dose-dependent elevation of reactive oxygen species (ROS) level in SMCs, and antioxidant vitamin C and Trolox effectively suppressed HA-induced DNA damage and dysregulation of Bcl-2/Bax. Our findings suggest that HA-induced DNA damage, cell cycle arrest, and apoptosis in SMCs may be an underlying mechanisms for the atherosclerosis and thrombosis observed in the BFD endemic region.     

Pyranocoumarin(+/-)-4'-O-acetyl-3'-O-angeloyl-cis-khellactone induces mitochondrial-dependent apoptosis in HL-60 cells

The pyranocoumarin (+)-4'-O-acetyl-3 'O-angeloyl-cis-khellactone (PC) isolated from Radix Peucedani (root of Peucedanum praeruptorum Dunn) showed a dose-dependent effect at 10 -30 pg/mL on causing apoptotic DNA and nuclear fragmentations in HL-60 cells. After 24 h of PC treatment there were losses of mitochondrial membrane potential and cytochrome c. PC also increased total cellular and mitochondrial Bax protein, stimulated an increase in caspase-dependent Bcl-2 cleavage but showed no effect on Bcl-Xv. These observations strongly suggest activation of the mitochondria apoptotic pathway. The pan-specific caspase inhibitor, ZVAD-fmk, abolished the PC-induced apoptosis,whereas the caspase-8 inhibitor IETD-fmk showed no effect, implying the involvement of the caspase 9 pathway. PC caused a 2 to 12 hour transient increase in phospho-ERK, and a 72 h-long activation of JNK. Pre-treatment with the MEK inhibitor PD98059, which suppresses ERK activation, paradoxically promoted PC-induced mitochondrial cytochrome c release, procaspase-3 and -8 cleavage, and enhanced apoptosis. Our results show that PC triggers mitochondria-mediated apoptosis in HL-60 cells, and the involvement of ERK and JNK signal pathways in the process.     

A novel homospermidine conjugate inhibits growth and induces apoptosis in human hepatoma cells

Aim： To elucidate the mechanism responsible for the antiproliferative effects of a novel homospermidine conjugate, anthracenylmethyl homospermidine （ANTMHspd）, in the human hepatoma BEL-7402 cell line. Methods： The viability of the cells was assessed by MTT assay and the trypan blue dye exclusion method. Morphological changes were observed by fluorescence microscopy with Hoechst 33258 staining. Cell cycle distribution, apoptosis, and mitochondrial membrane potential were measured by flow cytometry. Protein expression was detected by Western blot analysis. Results： ANTMHspd strongly decreased BEL-7402 cell proliferation in a dose- and time-dependent manner. Hoechst 33258 staining and the flow cytometry assay showed that ANTMHspd induced cell apoptosis and cell cycle perturbation. Furthermore, ANTMHspd could induce mitochondrial membrane potential loss and cytochrome c release and enhance cleaved caspase-3, cleaved caspase-9, and Bax protein expression without caspase-8 activation. ANTMHspd could also decrease the expression of Bcl-2 and cytochrome c in mitochondria. In addition, the specific inhibitors of caspase-9 and caspase-3 almost abolished the ANTMHspd-induced caspase-9 and caspase-3 activation, respectively. Conclusion： ANTMHspd could induce BEL-7402 cell apoptosis via the mitochondrial/caspase-dependent pathway and the Bcl-2 family was involved in the control of apoptosis.     

Activation of protein kinase C delta by proteolytic cleavage contributes to manganese-induced apoptosis in dopaminergic cells: protective role of Bcl-2

Chronic inorganic manganese exposure causes selective toxicity to the nigrostriatal dopaminergic system, resulting in a Parkinsonian-like neurological condition known as Manganism. Apoptosis has been shown to occur in manganese-induced neurotoxicity; however, the down-stream cellular target of caspase-3 that contributes to DNA fragmentation is not established. Herein, we demonstrate that proteolytic activation of protein kinase Cdelta (PKCdelta) by caspase-3 plays a critical role in manganese-induced apoptotic cell death. Treatment of PC12 cells with manganese caused a sequential activation of mitochondrial-dependent pro-apoptotic events, including mitochondrial membrane depolarization, cytochrome c release, caspase-3 activation, and DNA fragmentation. Overexpression of Bcl-2 in PC12 cells remarkably attenuated each of these events, indicating that the mitochondrial-dependent apoptotic cascade contributes to manganese-induced apoptosis. Furthermore, PKCdelta was proteolytically cleaved by caspase-3, causing a persistent activation of the kinase. The manganese-induced proteolytic cleavage of PKCdelta was significantly blocked by Bcl-2-overexpression. Administration of active recombinant PKCdelta induced DNA fragmentation in PC12 cells, suggesting a pro-apoptotic role of PKCdelta. Furthermore, expression of catalytically inactive mutant PKCdelta(K376R) via a lentiviral gene delivery system effectively attenuated manganese-induced apoptosis. Together, these results suggest that the mitochondrial-dependent caspase cascade mediates apoptosis via proteolytic activation of PKCdelta in manganese-induced neurotoxicity.     

p53-mediated cell cycle arrest and apoptosis induced by shikonin via a caspase-9-dependent mechanism in human malignant melanoma A375-S2 cells

Natural products regulate cell growth in response to oncogene activation that induces cell cycle arrest and apoptosis in tumor cell lines. We investigated the mechanisms of caspase activation in human malignant melanoma, A375-S2 cells, by the natural product shikonin, which was isolated from the plant Lithospermum erythrorhizon SIEB. et ZUCC. Shikonin inhibited cell growth in a time- and dose-dependent manner, which might be mediated through up-regulation of p53 and down-regulation of cyclin-dependent protein kinase 4. Caspase activation was detected in shikonin-induced cell apoptosis, which involved in a post-mitochondrial caspase-9-dependent pathway. Decreased Bcl-2 protein levels and increased Bax protein levels were positively correlated with elevated expression of p53 protein. Apoptosis-inducing factor, another apoptotic protein of mitochondria, partially contributed to shikonin-induced release of cytochrome c. Taken together, shikonin-induced DNA damage activates p53 and caspase-9 pathways.     

DNA引物酶抑制剂碘化-3,3′-二乙基-9-甲基-硫杂羰花青诱导人白血病HL-60细胞凋亡

目的研究DNA引物酶抑制剂碘化-3,3′-二乙基-9-甲基-硫杂羰花青(DMTCCI)诱导人粒细胞性白血病HL-60细胞凋亡并探索其机制。方法分别采用不同浓度的DMTCCI处理培养于RPMI-1640培养基的HL-60细胞。采用MTT法检测DMTCCI对HL-60细胞的生长抑制作用。采用流式细胞仪和DNA琼脂糖凝胶电泳方法检测细胞凋亡。采用蛋白免疫印迹(Western blotting)法观察凋亡相关蛋白survivin， Bcl-xL， Bad， Bax， Bcl-2， caspase-9， caspase-3， caspase-6， PARP， DFF45和lamin B的表达。采用ApoAlert Caspase-3分析试剂盒检测caspase-3的活性。结果DMTCCI具有抑制人白血病HL-60细胞增殖的作用，其IC₅₀值为0.24 μmol·L^-1。流式细胞仪和DNA琼脂糖凝胶电泳结果显示，DMTCCI可诱导HL-60细胞凋亡。在经DMTCCI处理的HL-60细胞中，survivin和Bcl-xL蛋白的表达水平下调，Bad和Bax蛋白的表达水平上调，Bcl-2蛋白的表达水平无变化，caspase-9，caspase-3，caspase-6，PARP，DFF45和lamin B被分别裂解，产生相应裂解产物。在HL-60细胞中，caspase-3的活性在1 μmol·L^-1 DMTCCI处理3 h时明显升高，在处理12 h时达到最高峰。结论DMTCCI可抑制人白血病HL-60细胞的增殖并诱导其发生细胞凋亡。Bcl-2家族蛋白、survivin和caspases家族蛋白可能参与了上述诱导HL-60细胞凋亡的过程。     

A novel beta-lactam antibiotic activates tumor cell apoptotic program by inducing DNA damage

Many of the anticancer drugs in current use are toxic and thus limited in their efficacy. It therefore becomes essential to develop novel chemotherapeutic agents with lower levels of toxicity. The beta-lactam antibiotics have been used for many years to treat bacterial infections with limited or no toxicity. Until now, it has never been shown that beta-lactams could kill tumor cells. Here, for the first time, we have discovered and characterized the apoptosis-inducing properties of a family of novel beta-lactam antibiotics against human leukemia, breast, prostate, and head-and-neck cancer cells. We found that one particular lead compound (lactam 1) with an N-methylthio group was able to induce DNA damage and inhibit DNA replication in Jurkat T cells within a 2-h treatment. This was followed by p38 mitogen-activated protein kinase activation, S phase arrest, and apoptotic cell death. p38 was found to be a central player in beta-lactam-induced apoptosis and resided downstream of DNA damage but upstream of caspase activation. Accompanying caspase-8 activation was cleavage of the pro-apoptotic Bcl-2 family protein Bid, and release of the mitochondrial cytochrome c. This was also associated with activation of caspase-9 and -3. Analogs of lactam 1 in which the N-methylthio group was replaced with other organothio chains exhibited progressive decreased potencies to induce DNA damage, p38 kinase activation, S phase arrest, and apoptosis, demonstrating requirement of the N-methylthio group. Because of the ease of synthesis and structural manipulation, we believe these beta-lactams may have the potential to be developed into anticancer agents.