Caspase‐3‐mediated cyclic stretch‐induced myoblast apoptosis via a Fas/FasL‐independent signaling pathway during myogenesis

Skeletal muscle cells are exposed to mechanical stretch during embryogenesis. Increased stretch may contribute to cell death, and the molecular regulation by stretch remains incompletely understood. The aim of this study was to investigate the effects of cyclic stretch on cell death and apoptosis in myoblast using a Flexercell Strain Unit. Apoptosis was studied by annexin V binding and PI staining, DNA size analysis, electron microphotograph, and caspase assays. Fas/FasL expression was determined by Western blot. When myoblasts were cultured on a flexible membrane and subjected to cyclic strain stress, apoptosis was observed in a time‐dependent manner. We also determined that stretch induced cleavage of caspase‐3 and increased caspase‐3 activity. Caspase‐3 inhibition reduced stretch‐induced apoptosis. Protein levels of Fas and FasL remained unchanged. Our findings implicated that stretch‐induced cell death is an apoptotic event, and that the activation of caspase cascades is required in stretch‐induced cell apoptosis. Furthermore, we had provided evidence that caspase‐3 mediated cyclic stretch‐induced myoblast apoptosis. Mechanical forces induced activation of caspase‐3 via signaling pathways independent of Fas/FasL system. J. Cell. Biochem. 107: 834–844, 2009. © 2009 Wiley‐Liss, Inc.     

自噬在应力诱导成肌细胞凋亡中的作用初探

目的:探讨自噬在周期性张应力介导的成肌细胞凋亡中的作用,以明确应力诱导内质网应激引起自噬与凋亡之间的关系。方法:在成功构建L6大鼠体外培养--力学刺激模型的基础上,采用Western Blot法分析周期性张应力对自噬相关蛋白LC3蛋白表达的影响,并通过Annexin V-FITC/PI流式细胞术检测细胞凋亡情况。加力组分别给予1,6,12,24 h的力学刺激(拉伸变形率为15%,频率为10循环/min),3-MA组和Rapamycin组在加力2 h前分别加入自噬抑制剂3-甲基腺嘌呤和自噬激活剂雷帕霉素并且加力24 h,0 h组与实验组在同时种板但是不给予力刺激。采用SPSS17.0统计软件对以上数据进行统计分析。结果:成肌细胞中的LC3II/LC3I值随加力时间延长呈上升趋势,24 h达最高(P0.05);抑制组的细胞凋亡率(18.75±1.06%)相对于0 h组(0.726±0.13%)和加力24 h组(14.84±1.14%)的明显升高(P0.05);Rapamycin组相对于加力24 h组的细胞凋亡率明显下降(8.88±1.08%vs 14.84±1.14%),但是细胞凋亡率仍然高于0 h组的(8.88±1.08%vs 0.726±0.13%)。结论:在一定时间范围内,周期性张应力可诱导成肌细胞发生自噬,并且自噬活性与作用时间成正比;自噬可以降低应力介导的成肌细胞凋亡的活性。     

PEG-liposomal oxaliplatin induces apoptosis in human colorectal cancer cells via Fas/FasL and caspase-8

Since cellular uptake of PEG [poly(ethylene glycol)]-liposomal L-OHP (oxaliplatin) induces bioactive changes in CRC (colorectal cancer), we have investigated its apoptotic effect and anticancer mechanism. Human CRC SW480 cells were treated with PEG-liposomal L-OHP and a caspase-8 inhibitor [Z-IETD-FMK (benzyloxycarbonyl-Ile-Glu-Thr-dl-Asp-fluoromethylketone)]. Apoptosis was measured by FCM (flow cytometry) and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay. Expression of Fas/FasL and cytochrome c was detected using FCM and an immunofluorescence assay. Expression of caspase-8, Bid, caspase-9, caspase-7 and activated caspase-3 (P17) was examined by Western blot analyses. The results indicated that PEG-liposomal L-OHP (28 μg/ml L-OHP) induced marked apoptosis in SW480 cells compared with 28 μg/ml free L-OHP. The expression levels of Fas, FasL, cytochrome c, caspase-9, caspase-7 and activated caspase-3 proteins were up-regulated, with a corresponding increase in apoptosis; however, expression of caspase-8 and Bid were down-regulated as apoptosis increased. When cells were treated with Z-IETD-FMK, apoptosis was inhibited, but there was little impact on the expression of Fas, FasL, cytochrome c, Bid, caspase-9, caspase-7 and activated caspase-3. These findings indicate that PEG-liposomal L-OHP enhances the anticancer potency of the chemotherapeutic agent; moreover, Fas/FasL and caspase-8 signalling pathways play a key role in mediating PEG-liposomal L-OHP-induced apoptosis.     

Effects of different magnitudes of mechanical strain on Osteoblasts in vitro

In addition to systemic and local factors, mechanical strain plays a crucial role in bone remodeling during growth, development, and fracture healing, and especially in orthodontic tooth movement. Although many papers have been published on the effects of mechanical stress on osteoblasts or osteoblastic cells, little is known about the effects of different magnitudes of mechanical strain on such cells. In the present study, we investigated how different magnitudes of cyclic tensile strain affected osteoblasts. MC3T3-E1 osteoblastic cells were subjected to 0%, 6%, 12% or 18% elongation for 24h using a Flexercell Strain Unit, and then the mRNA and protein expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) were examined. The results showed that cyclic tensile strain induced a magnitude-dependent increase (0%, 6%, 12%, and 18%) in OPG synthesis and a concomitant decrease in RANKL mRNA expression and sRANKL release from the osteoblasts. Furthermore, the induction of OPG mRNA expression by stretching was inhibited by indomethacin or genistein, and the stretch-induced reduction of RANKL mRNA was inhibited by PD098059. These results indicate that different magnitudes of cyclic tensile strain influence the biological behavior of osteoblasts, which profoundly affects bone remodeling.     

Programmed cell death of embryonic motoneurons triggered through the Fas death receptor

About 50% of spinal motoneurons undergo programmed cell death (PCD) after target contact, but little is known about how this process is initiated. Embryonic motoneurons coexpress the death receptor Fas and its ligand FasL at the stage at which PCD is about to begin. In the absence of trophic factors, many motoneurons die in culture within 2 d. Most (75%) of these were saved by Fas-Fc receptor body, which blocks interactions between Fas and FasL, or by the caspase-8 inhibitor tetrapeptide IETD. Therefore, activation of Fas by endogenous FasL underlies cell death induced by trophic deprivation. In the presence of neurotrophic factors, exogenous Fas activators such as soluble FasL or anti-Fas antibodies triggered PCD of 40-50% of purified motoneurons over the following 3-5 d; this treatment led to activation of caspase-3, and was blocked by IETD. Sensitivity to Fas activation is regulated: motoneurons cultured for 3 d with neurotrophic factors became completely resistant. Levels of Fas expressed by motoneurons varied little, but FasL was upregulated in the absence of neurotrophic factors. Motoneurons resistant to Fas activation expressed high levels of FLICE-inhibitory protein (FLIP), an endogenous inhibitor of caspase-8 activation. Our results suggest that Fas can act as a driving force for motoneuron PCD, and raise the possibility that active triggering of PCD may contribute to motoneuron loss during normal development and/or in pathological situations.     

Alveolar epithelial type II cell apoptosis in vivo during resolution of keratinocyte growth factor-induced hyperplasia in the rat

Keratinocyte growth factor (KGF) induces rapid and transient hyperplasia of alveolar epithelial type II cells. We sought to determine components of the apoptotic process involved in the resolution of this hyperplasia and the fate of the apoptotic cells. Rats received intrabronchial instillation of 5 mg KGF/kg body weight or diluent. Lungs were fixed 1, 2, 3, 5, and 7 days later. Apoptosis was identified by TdT-mediated dUTP nick-end labeling (TUNEL), double-labeling for TUNEL and the type II cell marker MNF116, and electron microscopy. Fas, FasL, Bax, Bcl-2, and pro- and active caspase-3 were studied by immunohistochemistry. Changes were quantified by stereology. Cell type specificity was investigated by immunofluorescence double staining. Type II cells exhibited Fas, FasL, Bcl-2, and procaspase-3 irrespective of treatment and time. Immunoelectron microscopy revealed Fas at the apical type II cell membrane. Bax staining was prominent in controls (45-95% of type II cell surface fraction), markedly decreased during hyperplasia at days 2 (20-40%) and 3 (0-10%), and reappeared at day 7 (25-45%) when apoptosis was prominent. Remnants of apoptotic type II cells were incorporated in membrane-bound vacuoles of type II cell neighbors as well as alveolar macrophages. The results indicate that type II cells can enter the Fas/FasL/caspase-3 pathway regulated by Bax and Bcl-2. High Bcl-2:Bax levels favor type II cell survival and a low rate of apoptosis during hyperplasia. Low Bcl-2:Bax levels favor type II cell apoptosis during resolution. Because of time-dependent changes that occur within a short time, the KGF-treated rat lung provides a useful in vivo model to investigate apoptosis in the context of tissue remodeling and repair.     

Inhibition of histone deacetylase activity enhances Fas receptor-mediated apoptosis in leukemic lymphoblasts

We recently reported that butyrate, an inhibitor of histone deacetylases, is capable of inducing Fas-independent apoptosis in the acute lymphoblastic leukemia cell line CCRF-CEM. Here we demonstrate that butyrate enhances Fas-induced apoptosis in this cell line. The application of different histone deacetylase inhibitors revealed that tetra-acetylated histone H4 is associated with the amplifying effect of butyrate on Fas-induced cell death. FasL, Fas, FADD, RIP, caspase-8, caspase-3, Bid, FLIP(S+L), FLASH and FAP-1, proteins known to act within the Fas-apoptosis cascade, showed no changes in their expression levels in cells treated with butyrate compared with untreated cells. Analyses of Fas-oligomerization and Western blotting as well as enzyme activity assays of caspase-2, caspase-3 and caspase-8 suggest that butyrate enhances Fas-induced apoptosis downstream of Fas but upstream of caspase-8 activation. In immunoprecipitation experiments a 37 kD butyrate-regulated protein was detected which specifically interacts with caspase-8.     

Glucocorticoid-induced, caspase-dependent organ apoptosis early after burn injury

Immune suppression and increased apoptotic loss of circulating lymphocytes have been reported after burn injury. However, little is known about the underlying mechanisms responsible for the increased apoptosis of lymphoid and parenchymal cells in solid organs and the role played by inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha) and Fas ligand (FasL), as well as by glucocorticoids. To evaluate the role of endogenously produced glucocorticoids and FasL, mice subjected to a 20% steam burn were pretreated with a glucocorticoid receptor antagonist (mifepristone) or a neutralizing murine Fas fusion protein. Three and twenty-four hours after burn injury, histological analysis, caspase-3 activity, and in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and phenotyping of lymphocyte populations for apoptosis were evaluated. Burn injury increased the number of apoptotic cells and caspase-3 activity in thymus and spleen, but not in other solid organs. Increased apoptosis was seen in several T and B cell populations from both thymus and spleen. Mifepristone pretreatment significantly reduced the apoptosis and caspase-3 activity after burn injury, whereas blocking FasL activity had only minimal effects. We conclude that corticosteroids, and not FasL, are primarily responsible for the increased caspase-3 activity and apoptosis in thymus and spleen cell populations early after burn injury.     

Effect of combined cyclic stretch and fluid shear stress on endothelial cell morphological responses

Endothelial cells in vivo are normally subjected to multiple mechanical stimuli such as stretch and fluid shear stress (FSS) but because each stimulus induces magnitude-dependent morphologic responses, the relative importance of each stimulus in producing the normal in vivo state is not clear Using cultured human aortic endothelial cells, this study first determined equipotent levels of cyclic stretch, steady FSS, and oscillatory FSS with respect to the time course of cell orientation. We then tested whether these levels of stimuli were equipotent in combination with each other by imposing simultaneous cyclic stretch and steady FSS or cyclic stretch and oscillatory FSS so as to reinforce or counteract the cells' orientation responses. Equipotent levels of the three stimuli were 2% cyclic stretch at 2%/s, 80 dynes/cm2 steady FSS and 20 +/- 10 dynes/cm2 oscillatory FSS at 20 dyne/cm2-s. When applied in reinforcing fashion, cyclic stretch and oscillatory, but not steady, FSS were additive. Both pairs of stimuli canceled when applied in counteracting fashion. These results indicate that this level of cyclic stretch and oscillatory FSS sum algebraically so that they are indeed equipotent. In addition, oscillatory FSS is a stronger stimulus than steady FSS for inducing cell orientation. Moreover, arterial endothelial cells in vivo are likely receiving a stronger stretch than FSS stimulus.     

Tocotrienol-induced caspase-8 activation is unrelated to death receptor apoptotic signaling in neoplastic mammary epithelial cells

Tocotrienols, a subclass in the vitamin E family of compounds, have been shown to induce apoptosis by activating caspase-8 and caspase-3 in neoplastic mammary epithelial cells. Since caspase-8 activation is associated with death receptor apoptotic signaling, studies were conducted to determine the exact death receptor/ligand involved in tocotrienol-induced apoptosis. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained in serum-free media. Treatment with 20 microM gamma-tocotrienol decreased+SA cell viability by inducing apoptosis, as determined by positive terminal dUTP nick end labeling (TUNEL) immunocytochemical staining. Western blot analysis showed that gamma-tocotrienol treatment increased the levels of cleaved (active) caspase-8 and caspase-3. Combined treatment with caspase inhibitors completely blocked tocotrienol-induced apoptosis. Additional studies showed that treatment with 100 ng/ml tumor necrosis factor-alpha (TNF-alpha), 100 ng/ml FasL, 100 ng/ml TNF-related apoptosis-inducing ligand (TRAIL), or 1 microg/ml apoptosis-inducing Fas antibody failed to induce death in +SA cells, indicating that this mammary tumor cell line is resistant to death receptor-induced apoptosis. Furthermore, treatment with 20 microM gamma-tocotrienol had no effect on total, membrane, or cytosolic levels of Fas, Fas ligand (FasL), or Fas-associated via death domain (FADD) and did not induce translocation of Fas, FasL, or FADD from the cytosolic to the membrane fraction, providing additional evidence that tocotrienol-induced caspase-8 activation is not associated with death receptor apoptotic signaling. Other studies showed that treatment with 20 microM gamma-tocotrienol induced a large decrease in the relative intracellular levels of phospho-phosphatidylinositol 3-kinase (PI3K)-dependent kinase 1 (phospho-PDK-1 active), phospho-Akt (active), and phospho-glycogen synthase kinase3, as well as decreasing intracellular levels of FLICE-inhibitory protein (FLIP), an antiapoptotic protein that inhibits caspase-8 activation, in these cells. Since stimulation of the PI3K/PDK/Akt mitogenic pathway is associated with increased FLIP expression, enhanced cellular proliferation, and survival, these results indicate that tocotrienol-induced caspase-8 activation and apoptosis in malignant +SA mammary epithelial cells is associated with a suppression in PI3K/PDK-1/Akt mitogenic signaling and subsequent reduction in intracellular FLIP levels.     

Escherichia coli induces apoptosis in human monocytic U937 cells through the Fas/FasL signaling pathway

Apoptosis is a genetically regulated cellular suicide mechanism that plays an essential role in development and in defense of multicellular organism. Escherichia coli (E. coli) can induce monocyte apoptosis; however, the mechanism is not clear. This study determines if Fas/FasL regulates E. coli-induced human monocyte line U937 cell apoptosis. We found that infection of U937 cells with E. coli induced rapid cell death in a dose- and time-dependent manner displaying the characteristic features of apoptosis. Moreover, opsonized E. coli induced U937 apoptosis with a higher apoptotic rate (53.29 ± 5.83%) than non-opsonized E. coli (19.37 ± 2.56%). Studying the underlying mechanisms we found that the E. coli-induced apoptosis was associated with a more prominent induction expression of Fas/FasL in a time- and dose-dependent manner. Furthermore, E. coli treatment resulted in a significant increase in the levels of DR5, TRAIL, and FADD, but exerted no statistically significant effects on the levels of DR4. The activity of caspase-8 enzyme increased in infection groups, positively correlated with apoptosis rate. Taken together, these results clearly indicate that receptor-mediated phagocytosis of E. coli induces apoptosis. Moreover, our findings suggest a possible regulatory role of Fas/FasL in the pathway of E. coli infection.     

Apoptosis and related proteins during parturition in prostaglandin F receptor-deficient mice

This study investigated whether apoptosis and related proteins are involved in parturition by comparative observation of FP-deficient mice without labor and wild type mice with vaginal delivery. We examined the expression of apoptosis, Fas, FasL, active caspase-3 and bcl-2 proteins in the amnion, placenta and decidua. DNA laddering in the amnion, placenta and decidua tissue did not significantly differ between FP-deficient and wild type mice on day 18 of pregnancy. Similar TUNEL staining results were found in all tissues of FP-deficient mice compared with those of wild type mice. A higher intensity of apoptotic cells was found in the decidua basalis. The index of TUNEL-positive cells were not significantly different in the amnion, placenta and decidua of FP-deficient mice compared with that of wild type mice on day 18 of pregnancy. Specific bands for Fas were clearly observed in the amnion, placenta and decidua tissue. FasL specific bands were observed in the placenta and decidua, but a few in amnion tissue. A great number of active caspase-3 specific bands were detected in decidua, while a few such bands were detected in the placenta and few bands in the amniotic tissue. Bands for bcl-2 were detected in the amnion, placenta and decidua tissue. The weakest band was in decidual tissue. Fas, FasL, active caspase-3, and bcl-2 specific bands did not show any significant differences between the two groups. These findings demonstrate that apoptosis, Fas, FasL, caspase-3, and Bcl-2 occur in mouse term placenta that is not involved in parturition.     

Silencing of death receptor and caspase-8 expression in small cell lung carcinoma cell lines and tumors by DNA methylation

Small cell lung cancer cell lines were resistant to FasL and TRAIL-induced apoptosis, which could be explained by an absence of Fas and TRAIL-R1 mRNA expression and a deficiency of surface TRAIL-R2 protein. In addition, caspase-8 expression was absent, whereas FADD, FLIP and caspases-3, -7, -9 and -10 could be detected. Analysis of SCLC tumors revealed reduced levels of Fas, TRAIL-R1 and caspase-8 mRNA compared to non-small cell lung cancer (NSCLC) tumors. Methylation-specific PCR demonstrated methylation of CpG islands of the Fas, TRAIL-R1 and caspase-8 genes in SCLC cell lines and tumor samples, whereas NSCLC samples were not methylated. Cotreatment of SCLC cells with the demethylating agent 5'-aza-2-deoxycytidine and IFNgamma partially restored Fas, TRAIL-R1 and caspase-8 expression and increased sensitivity to FasL and TRAIL-induced death. These results suggest that SCLC cells are highly resistant to apoptosis mediated by death receptors and that this resistance can be reduced by a combination of demethylation and treatment with IFNgamma.     

Interleukin-11 antagonizes Fas ligand-mediated apoptosis in IEC-18 intestinal epithelial crypt cells: role of MEK and Akt-dependent signaling

Interleukin-11 (IL-11) displays epithelial cytoprotective effects during intestinal injury. Antiapoptotic effects of IL-11 have been described, yet mechanisms remain unclear. Fas/CD95 death receptor signaling is upregulated in ulcerative colitis, leading to mucosal breakdown. We hypothesized that IL-11 inhibits Fas ligand (FasL)-mediated apoptosis in intestinal epithelia. Cell death was monitored in IEC-18 cells by microscopy, caspase and poly(ADP-ribose) polymerase cleavage, mitochondrial release of cytochrome c, and abundance of cytoplasmic oligonucleosomal DNA. RT-PCR was used to monitor Fas, cIAP1, cIAP2, XIAP, cFLIP, survivin, and Bcl-2 family members. Fas membrane expression was detected by immunoblot. Inhibitors of JAK2, phosphatidylinositol 3-kinase (PI3-kinase), Akt 1, MEK1 and MEK2, and p38 MAPK were used to delineate IL-11's antiapoptotic mechanisms. IL-11 did not alter Fas expression. Pretreatment with IL-11 for 24 h before FasL reduced cytoplasmic oligonucleosomal DNA by 63.2%. IL-11 also attenuated caspase-3, caspase-9, and poly(ADP-ribose) polymerase cleavage without affecting expression of activated caspase-8 p20 or cytochrome c release. IL-11 did not affect mRNA expression of the candidate antiapoptotic genes. The MEK1 and MEK2 inhibitors U-0126 and PD-98059 significantly attenuated the protection of IL-11 against caspase-3 and caspase-9 cleavage and cytoplasmic oligonucleosomal DNA accumulation. Although Akt inhibition reversed IL-11-mediated effects on caspase cleavage, it did not reverse the protective effects of IL-11 by DNA ELISA. We conclude that IL-11-dependent MEK1 and MEK2 signaling inhibits FasL-induced apoptosis. The lack of reversal of the IL-11 effect on DNA cleavage by Akt inhibition, despite antagonism of caspase cleavage, suggests that IL-11 inhibits caspase-independent cell death signaling by FasL in a MEK-dependent manner.     

The mycotoxin penicillic acid inhibits Fas ligand-induced apoptosis by blocking self-processing of caspase-8 in death-inducing signaling complex

Upon engagement with Fas ligand (FasL), Fas rapidly induces recruitment and self-processing of caspase-8 via the adaptor protein Fas-associated death domain (FADD), and activated caspase-8 cleaves downstream substrates such as caspase-3. We have found that penicillic acid (PCA) inhibits FasL-induced apoptosis and concomitant loss of cell viability in Burkitt's lymphoma Raji cells. PCA prevented activation of caspase-8 and caspase-3 upon treatment with FasL. However, PCA did not affect active caspase-3 in FasL-treated cells, suggesting that PCA primarily blocks early signaling events upstream of caspase-8 activation. FasL-induced processing of caspase-8 was severely impaired in the death-inducing signaling complex, although FasL-induced recruitment of FADD and caspase-8 occurred normally in PCA-treated cells. Although PCA inhibited the enzymatic activities of active recombinant caspase-3, caspase-8, and caspase-9 at similar concentrations, PCA exerted weak inhibitory effects on activation of caspase-9 and caspase-3 in staurosporine-treated cells but strongly inhibited caspase-8 activation in FasL-treated cells. Glutathione and cysteine neutralized an inhibitory effect of PCA on caspase-8, and PCA bound directly to the active center cysteine in the large subunit of caspase-8. Thus, our present results demonstrate that PCA inhibits FasL-induced apoptosis by targeting self-processing of caspase-8.     

Involvement of death receptor signaling in mechanical stretch-induced cardiomyocyte apoptosis

Recent evidences suggest that mechanical overload associated with abnormal blood pressure causes apoptosis in cardiovascular system. Still, the intracellular signaling leading to cardiomyocyte apoptosis has not been fully defined. Previous reports ascribed stretch-induced cardiomyocyte apoptosis to rennin-angiotensin-system (RAS) signaling and/or mitochondria-dependent apoptosis pathway. The present study shows the involvement of death receptor signaling in mechanical stretch-induced cardiomyocyte apoptosis. By employing a well-described in vitro stretch model, we studied stretch-induced apoptosis and found that the death receptor-mediated apoptotic signaling was activated in stretch-induced apoptosis in neonatal rat cardiomyocytes. The major finding are as following: (1) The mechanical stretch activated death receptor-mediated apoptotic signaling in cardiomyocytes, including activation of caspases 8, 9 and 3, up-regulation of Fas, FasL expression and cell surface trafficking of death ligands (FasL and TRAIL); (2) That exogenous death ligand (TRAIL) enhanced, while soluble death receptor (sDR5) neutralized, stretch-induced apoptosis; (3) Adenovirus-delivered dominant negative FADD (FADD-DN) significantly reduced apoptosis, caspases 8, 9, and 3 activation, and stretch-induced cyt c release from mitochondria. These data clearly suggested mechanical stretch activated death receptor-mediated apoptotic signaling in cardiomyocytes. In conclusion, our data suggest that the FADD-linked death receptor signaling may contribute to stretch-induced cardiomyocyte apoptosis, probably through activating mitochondria-dependent apoptotic signaling.     

重组荞麦胰蛋白酶抑制剂对人肝癌细胞的凋亡及半胱氨酸天冬酶活性的影响

先前的研究表明,基因重组荞麦胰蛋白酶抑制剂 (rBTI) 具有诱导不同肿瘤细胞凋亡的作用.为了揭示其诱导肿瘤细胞凋亡的可能机理,从基因水平上探讨与凋亡有关的分子事件,本研究用不同浓度的 rBTI 体外作用于人肝癌细胞 HepG2 后,采用 MTT 比色法检测抑制剂对epG2 细胞的抑制率,用 DNA 凝胶电泳和细胞核的形态学观察检测 HepG2 细胞的凋亡.结果表明,rBTI 在体外能够明显抑制 HepG2 细胞的增长,并诱导细胞凋亡.另外,细胞凋亡与Bcl-2/Bax mRNA 水平有关.通过 RT-PCR 检测发现,细胞经过rBTI处理后,抗凋亡基因Bcl-2 mRNA 水平下调,促凋亡基因 Bax mRNA 有所上调,而对照 GAPDH 无变化.对 HepG2细胞中 Fas/Fas 配体及半胱氨酸天冬酶（caspase）的研究证明,细胞经过 rBTI 处理后,对死亡受体 Fas mRNA没有影响; rBTI 可明显激活caspase-3 和 caspase-9 酶活性, 对caspase-8 活性几乎无影响.上述结果表明,rBTI 对HepG2 细胞具有明显的诱导凋亡作用,其诱导细胞凋亡的机制与 caspase-3 依赖性凋亡调节信号通路有关,未涉及 Fas/Fas 配体途径.