Anti-apoptotic effects of caspase inhibitors on rat intervertebral disc cells

BACKGROUND: Apoptosis is thought to be a critical component of disc degeneration. Two main pathways of Fas-mediated apoptosis have been identified: Type I, which is the death-inducing signaling complex pathway, and Type II, which is the mitochondrial pathway. The apoptotic pathway for anulus fibrosus cells, which is phenotypically different from that of nucleus pulposus cells, has not been elucidated to our knowledge. The ultimate initiators or executioners of apoptosis are caspases. There are also inhibitors of caspases, which have the potential of being used as anti-apoptotic therapeutic agents. We therefore undertook this study to determine (1) the apoptotic pathway of anulus fibrosus cells and (2) the anti-apoptotic potential of caspase inhibitors. METHODS: Rat anulus fibrosus cells were isolated, cultured, and placed in either 0% (apoptosis-promoting condition) or 10% (normal control) fetal bovine serum. We identified and quantified the presence of apoptotic cell death, caspase activities, and loss of mitochondrial membrane potential. In addition, we examined the cells for the expression of Fas, procaspases, and cytochrome-c. Finally, we analyzed the degree of anti-apoptotic effects of caspase inhibitors on the cells in 1% fetal bovine serum. RESULTS: The percentage of apoptosis and Fas expression in the cells incubated in 0% fetal bovine serum were increased compared with those in the cells incubated in 10% fetal bovine serum (both p < 0.001). Caspase-8, 9, and 3 activities were increased and expression of procaspases was decreased in the 0% fetal bovine serum compared with those in the 10% fetal bovine serum (all p < 0.001). In contrast, the loss of mitochondrial membrane potential and cytochrome-c release into the cytosol were unchanged in the 0% fetal bovine serum. Pancaspase and caspase-8 inhibitors reduced apoptotic cell death (p < 0.001 and p < 0.05, respectively), but caspase-9 inhibitor did not reduce apoptotic cell death. CONCLUSIONS: Our results suggest that, unlike nucleus pulposus cells, anulus fibrosus cells are Fas Type-I cells, which undergo apoptosis through the death-inducing signaling complex. We also found that apoptosis of intervertebral disc cells can be attenuated by caspase inhibitors.     

High glucose evokes an intrinsic proapoptotic signaling pathway in mesangial cells

BACKGROUND: In response to chronic hyperglycemia, microvascular cells undergo stress and injury, which can lead to cell death. We characterized a proapoptotic signaling pathway whereby high glucose evokes an intrinsic, caspase-9-dependent mechanism of cell death in human mesangial cells. METHODS: Biochemical (caspase activity, cytochrome-c release, etc.) and morphologic (chromatin condensation and nuclear segmentation) features of apoptotic cell death were assessed in cultured human mesangial cells exposed to high glucose, a risk factor for mesangial cell injury and diabetic glomerulosclerosis. Proapoptotic signaling was also analyzed in the db/db murine model of kidney injury in diabetes. RESULTS: Incubation in high glucose caused cytotoxicity and apoptosis in mesangial cells. High glucose stimulated mitochondrial release of cytochrome-c, cleavage of procaspase-9, and caspase-9 enzyme activity, suggesting an intrinsic pathway of proapoptotic signaling. In contrast, caspase-8 was unaffected by high glucose. A cell-permeable, caspase-9-selective inhibitor blocked caspase-3 activation and prevented chromatin condensation and nuclear segmentation in cells treated with high glucose. To determine whether an intrinsic signaling pathway occurs in the diabetic kidney in vivo, apoptosis was investigated in diabetic 8- and 16-week db/db murine kidneys. Effector caspases-3 and -7 were activated in diabetic db/db kidneys but not in age-matched nondiabetic db/m controls. At 16 weeks, apoptotic cells in db/db glomeruli were identified on the basis of nuclear segmentation and DNA fragmentation. Apoptosis of glomerular cells correlated with expansion of the mesangial matrix and with worsening of albuminuria. Consistent with an intrinsic signaling pathway, caspase-9 cleavage was elevated only in db/db kidneys, whereas activation of caspase-8 and caspase-12 was undetectable. CONCLUSION: These findings support the hypothesis that hyperglycemia evokes an intrinsic pathway of proapoptotic signaling in mesangial cells. In addition, these results point to an important role for the intrinsic pathway in microvascular injury in the diabetic kidney in vivo.     

Targeted RNA interference of PI3K pathway components sensitizes colon cancer cells to TNF-related apoptosis-inducing ligand (TRAIL)

BACKGROUND: The phosphoinositide 3-kinase (PI3K/Akt) pathway transduces signals initiated from growth factors. Previously, we identified an important role for PI3K/Akt in colon cancer progression. The purpose of this study was to determine (1) whether short interfering RNA (siRNA) directed to PI3K/Akt components can render colon cancer cells sensitive to treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and (2) the cellular mechanisms contributing to the enhanced sensitivity. METHODS: Human colon cancer cells KM20 and KM12C (both TRAIL resistant) were transfected with siRNA directed against the PI3K p85alpha regulatory subunit Akt1 or nontargeting control sequence and then treated with TRAIL (100 ng/mL) or vehicle. A ribonuclease protection assay was performed to assess changes in TRAIL receptor expression. Protein was extracted and analyzed by Western blot for expression of cleavage of TRAIL receptors (death receptor (DR) 4 and 5), caspase-3, caspase-8, and BID. Apoptosis was measured by enzyme-linked immunosorbent assay of DNA fragmentation. RESULTS: Combination treatment with p85alpha or Akt1 siRNA and TRAIL increased apoptosis in KM20 and KM12C cells, compared with TRAIL alone; these results were corroborated further by complete inhibition of apoptosis by Z-acetyl-Asp-Glu-Val-Asp-(DEVD)-fmk, a caspase-3 inhibitor. Furthermore, siRNA-mediated PI3K pathway inhibition resulted in increased expression of the TRAIL death receptors 4 and 5. CONCLUSIONS: Inhibition of PI3K/Akt by RNA interference sensitizes resistant colon cancer cells to TRAIL-induced cell death through the induction of TRAIL receptors and activation of caspase-3 and caspase-8. Agents that selectively target the PI3K/Akt pathway may enhance the effects of chemotherapeutic agents and provide novel adjuvant treatment for selected colon cancers.     

Caspase-8 activation is necessary but not sufficient for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in the prostatic carcinoma cell line LNCaP

BACKGROUND: The differential sensitivity of tumor cells to TRAIL-induced apoptosis may be mediated by different intracellular inhibitors of apoptosis, and only a few reports have described the pathway(s) that are activated in response to TRAIL in prostate cells. METHODS: LNCaP was transfected with a dominant-negative form of FADD (FADD-DN) and cells were selected in the presence of hygromycin. Cell viability was estimated by calcein assay. Apoptosis was estimated by caspase activation using both fluorogenic substrates and Western blot analysis of activated caspases. To detect cytochrome c release, mitochondria-free cytosol was prepared and Western blot analysis was performed. RESULTS: LNCaP is resistant to TRAIL but TRAIL transiently induces DEVDase activity and activation of caspase-8; caspase-2, -3, -7, and -9 were not activated. Wortmannin, an inhibitor of the PI3K/Akt pathway, converted the phenotype of LNCaP from TRAIL-resistant to -sensitive. In the presence of wortmannin TRAIL induced activation of caspase-2, -3, -7, -8, and -9, as well as dissipation of mitochondrial transmembrane potential and release of cyto-chrome c from mitochondria into the cytosol. In addition, combined TRAIL and wortmannin treatment resulted in cleavage of several proteins: PARP, Akt, p21/WAF1, and MDM2 as well as dephosphorylation of Akt. The proteolysis of p21/WAFI and Akt, which are known survival factors, presumably amplify the apoptotic cascade in LNCaP. Transfection of FADD-DN in LNCaP resulted in inhibition of caspase activation as well as in resistance to combined treatment with TRAIL and wortmannin. CONCLUSIONS: These results suggest that caspase-8 activation is necessary but not sufficient for TRAIL-mediated apoptosis and is presumably blocked downstream of caspase-8 by the PI3K/Akt pathway.     

硫利达嗪对胃癌细胞生长的抑制作用与机制研究

目的:探讨硫利达嗪对胃癌细胞体外生长的抑制作用及其机制。方法:不同浓度的硫利达嗪作用于胃癌SGC-7901细胞24 h后,分别用MTT法与流式细胞仪检测胃癌细胞的增殖与凋亡情况,以及用Western blot检测凋亡相关蛋白bax、bcl-2、caspase-3的表达情况。结果:硫利达嗪作用后,SGC-7901细胞的增殖明显抑制,凋亡率明显增加,抗凋亡蛋白bcl-2表达下调、促凋亡蛋白bax表达上调、caspcase-3蛋白表达上调,且均呈浓度依赖性(均P0.05)。结论:硫利达嗪对人胃癌细胞体外的生长有明显抑制作用,该作用可能与其并活化caspase-3依赖的凋亡途径有关。     

Proteasome inhibition blocks caspase-8 degradation and sensitizes prostate cancer cells to death receptor-mediated apoptosis

BACKGROUND: Proteasome inhibition through the administration of Velcade is a viable chemotherapeutic strategy that is approved to treat multiple myeloma and is being evaluated for efficacy against prostate cancer. Currently, the apoptotic pathways that contribute to this anticancer response are poorly understood. Our goal is to test the extent to which proteasome inhibition modulates apoptosis through death receptor pathways. METHODS: Several prostate cancer cell lines and primary prostate epithelial cells (PrECs) were used as models. The death receptor pathway was activated by the expression of Fas ligand (FasL) or addition of TNF-related apoptosis-inducing ligand (TRAIL) in the presence or absence of proteasome inhibitors. The apoptotic response was quantified by annexin V, TUNEL and nuclear condensation assays. Western blot analysis was conducted to quantify protein levels and enzyme assays were used to measure caspase activity. RESULTS: Proteasome inhibition markedly sensitized prostate cancer cells to apoptosis initiated by Fas ligand (FasL) or TRAIL. In the presence of either death ligand, procaspase-8 processing occurred, but led to minimal amounts of active caspase-8. The addition of Velcade, however, led to robust active caspase-8 protein abundance and activity. In the presence of Velcade the caspase-8 p18 subunit half-life increased from 22 min to over 2 hr. CONCLUSIONS: These findings demonstrate that proteasome inhibition can sensitize cells to apoptosis elicited by tumor necrosis factor ligands and retarding caspase-8 degradation provides one explanation for this activity. This study suggests that the clinical efficacy of Velcade may result, at least in part, from the activity of death ligands.     

Radiotherapy response in oral squamous carcinoma cell lines: evaluation of apoptotic proteins as prognostic factors

BACKGROUND: In this study, we investigated the importance of apoptosis for cell death after radiotherapy, and whether the expression of pro- and anti-apoptotic proteins has any correlation to the radiosensitivity. METHODS: Three oral squamous cell carcinoma cell lines, UT-SCC-2, UT-SCC-9 and UT-SCC-24A, were subjected to radiotherapy. After irradiation, viable and dead cells were counted to determine radiation sensitivity and apoptosis was analyzed by measurement of caspase-3 activity. The expressions of pro- and anti-apoptotic proteins were assessed using western blot analyses. RESULTS AND CONCLUSION: After irradiation, apoptotic morphology and caspase-3 activity were only detected in cell lines exhibiting high or moderate radiosensitivity. Western blot analysis indicates that survivin, epidermal growth factor receptor, cyclooxygenase-2, and Bcl-x(L) are critical components in irradiation resistance of the investigated cell lines. Moreover, our results suggest that apoptotic cell death and the balance between pro- and anti-apoptotic proteins are of importance for the outcome of radiotherapy.     

Homocysteine enhances apoptosis in human bone marrow stromal cells

INTRODUCTION: High plasma homocysteine (Hcy) levels have been associated with increased risk of fracture. Since Hcy has been shown to induce apoptosis in many cell types, including vascular endothelial cells, we hypothesized that Hcy would have a similar apoptotic effect on osteoblasts, leading to osteoporosis by reducing bone formation. MATERIALS AND METHODS: Using primary human bone marrow stromal cells (hBMSC) and HS-5 cell line (human bone marrow stromal cell line), we investigated the effects of Hcy on these cells by cell viability assay and analysis of cytoplasmic histone-associated DNA fragments. Caspase activity assay, Western blots, and electrophoresis mobility shift assay (EMSA) were performed to find the mechanism of apoptosis. Intracellular reactive oxygen species (ROS) were measured by spectrometry using dichlorofluorescein diacetate, and cellular total glutathione level was determined by a commercially available kit. N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) were used as tools for investigating the role of ROS and nuclear factor-kappaB (NF-kappaB), respectively. RESULTS: Hcy induced apoptosis in primary human bone marrow stromal cells and the HS-5 cell line, and this apoptotic effect was caspase-dependent. In addition, Hcy increased cytochrome c release into the cytosol, and activated caspase-9 and caspase-3, but not caspase-8, indicating that Hcy induces apoptosis via the mitochondria pathway. Hcy increased ROS, and NAC inhibited the apoptotic effect of Hcy. Western blot and EMSA showed that Hcy activated the NF-kappaB pathway. PDTC blocked Hcy-induced caspase-3 activation and apoptosis. CONCLUSION: These results suggest that Hcy induces apoptosis via the ROS-mediated mitochondrial pathway and NF-kappaB activation in hBMSCs, and that Hcy may contribute to the development of osteoporosis by reducing bone formation. Antioxidants may have a role in preventing bone loss in individuals with hyperhomocysteinemia.     

Cyclic stretch-induced apoptosis in rat annulus fibrosus cells is mediated in part by endoplasmic reticulum stress through nitric oxide production

Various mechanical stresses in vivo induce disc cell apoptosis and intervertebral disc (IVD) degeneration, but the underlying molecular mechanism is not fully known. The aim of this study was to investigate the role of endoplasmic reticulum stress in cyclic stretch-induced apoptosis of rat annulus fibrosus (AF) cells. Flexercell Tension Plus system was used to apply cyclic stretch to rat annulus fibrosus cells at a frequency of 0.5 Hz with 20% elongation for 12, 24, 36, or 48 h. Apoptosis was detected by flow cytometry, and nuclei morphologic changes were visualized by Hoechst 33258 staining and caspase-8, 9 activity assays. The expression of the markers of endoplasmic reticulum stress including CHOP, GRP78, and caspase-12 were determined by RT-PCR and Western blot. Mitochondrial membrane potential change was observed by JC-1 staining in situ. In addition, the levels of the nitric oxide (NO) were determined with the Griess reaction and fluorescence staining. The results indicated that cyclic stretch at a frequency of 0.5 Hz with 20% elongation-induced apoptosis in rat AF cells. Prolonged exposure of the unphysiologically cyclic stretch to AF cells caused NO overproduction, up-regulation of endoplasmic reticulum stress markers including CHOP, GRP78, and caspase-12, depolarization of mitochondria and activation of caspase-9. However, cyclic stretch at this level had no effect on caspase-8 activity. In addition, specific inhibitor of caspase-12 (Z-ATAD-FMK) and caspase-9 (Z-LEHD-FMK) partly suppressed cyclic stretch-induced AF cell apoptosis and the anti-apoptotic effects of the caspase inhibitors were additive. Our data suggest that endoplasmic reticulum stress, likely mediated by NO, contributes to the AF cell apoptosis induced by cyclic stretch in addition to the mitochondrial pathway. These findings could be helpful to understand the mechanism of disc cell apoptosis, the root cause of IVD degeneration.     

Targeted induction of apoptosis via TRAIL and cryoablation: a novel strategy for the treatment of prostate cancer

Adjuvant therapies contribute to the successful treatment of cancer. Our previous reports have shown that combining cryoablation with cytotoxic agents enhances cell death. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic agent that preferentially induces apoptosis in a variety of human cancer cells. Human prostate cancer cells (PC-3) are resistant to many cytodestructive agents, including cryoablation and TRAIL. Here, we evaluated the effects of TRAIL combined with cryoablation on PC-3 and normal prostate (RWPE-1) cell death. Exposure of PC-3 cells to freezing (-10 degrees C) or TRAIL (500 ng/ml) results in minimal cell death, whereas a complete loss of viability is observed with the simultaneous combination. The synergistic effect was found to be due to a marked increase in apoptosis. Western blot analysis revealed a significant level of caspase-8 and -3 cleavage between 12 and 24 h post-exposure. Caspase activation assays provided similar results and also indicated a role for caspase-9. Inhibitors to caspase-8 and -9 along with a pan-caspase inhibitor were incorporated to determine which pathway was necessary for the combined efficacy. Inhibition of caspase-8 significantly blocked the combination-induced cell death compared to cells that did not receive the inhibitor (63% compared to 10% viable). The addition of the caspase-9 inhibitor resulted in only a minimal protection. Importantly, the combination was not effective when applied to normal prostate cells. The results describe a novel therapeutic model for the treatment of prostate cancer and provide support for future in vivo studies.     

Erratum

To evaluate the effect of estrogen and androgen levels on erythrocyte deformability in endocrinological erectile dysfunction patients. Methods: The estrogen level, androgen level, IR of 30 psychogenic and 15 endocriological ED were studied and the correlation between the estrogen and androgen levels and RI were analyzed. Results: There is a negative correlation between the androgen and estrogen levels and IR;     

E2F1 expression in LNCaP prostate cancer cells deregulates androgen dependent growth, suppresses differentiation, and enhances apoptosis

INTRODUCTION AND OBJECTIVES: To investigate the role of E2F/RB in androgen independent proliferation, differentiation, and sensitivity to apoptotic stimuli of LNCaP prostate cancer cells. METHODS: The effects of E2F1 overexpression on androgen independent proliferation, differentiation, and apoptotic responses was assessed by flow cytometry, Western blot analysis and staining of nuclei. RESULTS: Overexpression of E2F1 in LNCaP cells confers resistance to an androgen withdrawal-mediated growth arrest, prevents differentiation, and modifies apoptotic responses. Androgen independent proliferation is associated with a dose dependent elevation of cyclin E. Cells expressing high levels of E2F1 continue to express androgen receptor and have a diminished expression of neuronal specific enolase when cultured in androgen-depleted media. Additionally, E2F1-expressing cells are more sensitive to etoposide-induced apoptosis. Western blot analysis revealed that LNCaP-E2F1 cells have elevated expression of p73, Apaf-1, caspase-3, caspase-7, but expression of caspase-8 and -9, p14(ARF), and Mcl-1, is unaltered. CONCLUSION: This is the first study that describes E2F1-dependent modifications of androgen dependence, differentiation, and sensitivity to apoptotic stimuli in LNCaP cells. Our analysis also identifies a subset of E2F1 targets that are instrumental in altering proliferative, differentiation, and apoptotic properties. Deregulation of the E2F/RB pathway and subsequent modification of key regulatory proteins may promote the development of hormone-refractory prostate tumors.     

自噬抑制剂3-甲基腺嘌呤对结直肠腺癌细胞生长与Notch1蛋白表的影响

目的：探讨自噬抑制剂3-甲基腺嘌呤（3-MA）对人大肠癌SW480细胞生长与Notch1蛋白表达的影响。方法：将3-MA（5 mmol/L）作用于SW480细胞24 h后（以培养相同时间无处理的SW480细胞为对照）,分别免疫组化和Western blot法检测细胞Notch1蛋白的表达,用CCK-8法和Annexin/PI双染法检测细胞增殖与凋亡。结果：免疫组化与Western blot结果均显示,3-MA作用后,SW480细胞Notch1蛋白的表达明显下调（均P<0.05）;增殖与凋亡检测结果显示,3-MA作用后,SW480细胞增殖率明显降低,而凋亡率明显增加（均P<0.05）。结论： 3-MA能抑制结直肠癌细胞的增殖并促进其凋亡,该作用可能与3-MA抑制Notch1蛋白表达从而改变细胞自噬水平有关。

     

Trauma-hemorrhagic shock mesenteric lymph induces endothelial apoptosis that involves both caspase-dependent and caspase-independent mechanisms

OBJECTIVE: To determine the mechanism by which gut-derived factors present in mesenteric lymph from rats subjected to trauma-hemorrhagic shock (T/HS) induce endothelial cell death. SUMMARY BACKGROUND DATA: Intestinal ischemia after hemorrhagic shock results in gut barrier dysfunction and the subsequent production of biologically active and tissue injurious factors by the ischemic gut. These factors are carried in the mesenteric lymph and reach the systemic circulation via the mesenteric lymph, thereby ultimately resulting in distant organ injury. Although studies have established that trauma-hemorrhagic (T/HS) shock but not trauma-sham-shock (T/SS) mesenteric lymph is cytotoxic to endothelial cells, whether T/HS lymph-induced endothelial cell death occurs via an apoptotic or a necrotic pathway is unknown. The mechanisms underlying T/HS lymph-induced cytotoxicity are likewise unknown. METHODS: Human umbilical vein endothelial cell (HUVEC) monolayers were incubated with medium, sham-shock, or post shock mesenteric lymph (5%) for 4 hours, after which the mode of cell death (ie, apoptosis versus necrosis) was determined using morphologic (confocal microscopy), biochemical (nucleosomal release), and DNA-based (gel electrophoresis) assays. To clarify the cellular pathways involved in T/HS lymph-induced HUVEC cell death, caspase-3, caspase-9, caspase-8, and BID activity was measured as was the ability of the pan-caspase inhibitor z-VAD-fmk to prevent T/HS lymph-induced cell death. RESULTS: T/HS, but not T/SS, mesenteric lymph or medium was cytotoxic and caused the appearance of the classic morphologic signs of apoptosis, including membrane blebbing, cell shrinkage, and apoptotic body formation. Nucleosomal release and a DNA laddering pattern was also observed in the HUVECs incubated with T/HS lymph. These signs of apoptosis were associated with increased caspase activity as reflected in activation of the pro-apoptotic caspases, caspase-8, -9, and -3, as well as the pro-apoptotic bcl-2-related protein BID. However, since the broad-spectrum caspase inhibitor z-VAD-fmk delayed T/HS lymph-induced HUVEC cell death, but did not prevent it fully, it appears that other factors besides caspases are involved in the endothelial cell toxicity of T/HS lymph. CONCLUSIONS: Gut-derived factors in T/HS, but not T/SS, mesenteric lymph cause endothelial cell death via an apoptotic mechanism that involves both caspase-dependent and caspase-independent pathways.     

皮质酮诱导大鼠Leydig细胞凋亡是一经caspase-3激活的过程

目的 超生理剂量的皮质酮(大鼠体内的糖皮质激素)能诱导大鼠Leydig细胞凋亡。但有关皮质酮诱导Leydig细胞凋亡的细胞内机制尚不清楚。本研究旨在观察皮质酮是否经caspase-3激活的途径诱导大鼠Leydig细胞凋亡。方法 采用Western Blot方法检测不同时间点上经皮质酮处理的大鼠Leydig细胞中caspase-3酶原及裂解的caspase-3酶表达情况。运用荧光发光法检测不同时间点上经皮质酮处理的人鼠Leydig细胞中caspase-3酶活性。结果 caspase-3酶原表达水平在皮质酮处理6h时开始上升，12h及24h时表达量下降，而具生物活性的、裂解的caspase-3酶于12h时开始出现，24h时的表达水平最为显著。caspase-3酶活性在皮质酮处理12h时明显升高，以24h时最为显著。Caspase-3抑制剂DEVD-CHO对经皮质酮处理12、24及48h的Leydig细胞中的caspase-3酶活性均具有明显的抑制作用，加caspase-3抑制剂的处理组其细胞基因组DNA电泳未见有凋亡特征性的梯状条带。结论 皮质酮诱导的大鼠Leydig细胞凋亡是一经caspase-3激活的过程。     

Cordyceps sinensis mycelium induces MA-10 mouse Leydig tumor cell apoptosis by activating the caspase-8 pathway and suppressing the NF-kappaB pathway

Cordyceps sinensis has been used as nutritious food and medicine in Chinese society. CS can inhibit tumor growth and induce tumor cell apoptosis. CS induced MA-10 mouse Leydig tumor cell death, but the anti-tumor mechanisms are not fully understood. Thus, the aim of this study was to investigate the apoptotic effect of CS on MA-10 cells and determine the molecular mechanism. CS (2-10 mg/ml) was added to MA-10 cells at different time scales (0-24 h). The condensation of DNA chromatin and apoptotic nuclear fragmentation increased in CS-treated MA-10 cells. Western blot analysis showed that 3 hours of CS treatment caused an increase in caspase-3 and -8 expressions only, which provided further evidence for the involvement of caspase-3 and -8 in CS-induced MA-10-cell apoptosis. CS blocked NF-?B protein expression in a dose-dependent relationship. CS induces MA-10 cell apoptosis by activating caspase-8-dependent and caspase-9-independent pathways and downregulating NF-?B protein expression.     

DHA对人肝癌细胞增殖与凋亡的影响及其机制的研究

目的：探讨二十二碳六烯酸（DHA）对人肝癌细胞株Bel-7402增殖和凋亡的影响及其机制。方法：用不同浓度的DHA（0,25,50,100,200 μmol/L）分别作用人肝癌Bel-7402细胞24,48,72 h后,用MTT法检测细胞的增殖情况、Western blot检测Bcl-2和Bax蛋白的表达,并分别用流式细胞仪、real-time PCR和caspase-3活性检测试剂盒检测上述梯度浓度的DHA作用 Bel-7402细胞48 h后的凋亡情况、Bim基因表达和caspase-3活性。结果：不同浓度、不同时间DHA作用后,Bel-7402细胞增殖明显抑制,呈浓度和时间依赖性（均P<0.05）;细胞Bax蛋白表达增加、Bcl-2蛋白表达降低,呈明显浓度依赖性（均P<0.05）,但无明显时间依赖性（均P>0.05）。不同浓度DHA作用48 h后,Bel-7402细胞凋亡率、Bim基因表达和caspase-3的活性均明显增加,且均呈浓度依赖性（均P<0.05）。结论：DHA可抑制人肝癌Bel-7402细胞的增殖并诱导细胞凋亡,其机制可能与激活线粒体凋亡通路有关。     

Contribution of death receptor and mitochondrial pathways to Fas-mediated apoptosis in the prostatic carcinoma cell line PC3

BACKGROUND: Two main pathways of apoptosis in mammalian cells have been described: the death receptor pathway and the mitochondrial pathway. Two different cell types have been identified for Fas-mediated apoptosis, each using almost exclusively one of two different signaling pathways. Human prostatic carcinoma cell line, PC3 is sensitive to Fas-mediated apoptosis, but relation of receptor and mitochondrial pathways is not clear. METHODS: Cell viability was estimated by calcein assay. Apoptosis was determined by preparation of DNA ladder. Expression of Fas-associated death domain-dominant negative (FADD-DN) and Bcl-2, activation of caspases, PARP, DFF45, Bid cleavage, and cytochrome c release were assessed using Western blotting techniques. [(35)S] Methionine-labeled caspase-3 was transcribed in vitro and translated using the TNT kit (Promega). A vector containing caspase-3 was prepared by the ligation of EcoR I/BamHI flanked PCR fragment of full size caspase-3 cDNA into pBlusckript II SK(+/-) (Stratagen). RESULTS: Overexpression of both FADD-DN and Bcl-2 genes prevent Fas-mediated apoptosis in PC3. As predicted, overexpression of FADD-DN prevented activation of caspase-8 and Bid cleavage and attenuated the release of cytochrome c and activation of caspases -2, -7, and -9. Bcl-2 overexpression did not affect caspase-8 activation and cleavage of Bid but blocked the release of cytochrome c and activation of mitochondria localized caspases -2, -7, and-9. Overexpression of FADD-DN and Bcl-2 affected the activation of caspase-3 and PARP cleavage differently: FADD-DN attenuated the activation of caspase-3 and PARP cleavage whereas Bcl-2 overexpression prevented caspase-3 activation and completely blocked cleavage of PARP. CONCLUSIONS: These data suggest that activation of caspase-8 is necessary but not sufficient to complete Fas-mediated apoptosis in PC3 cells without activation of the mitochondrial pathway. In addition, caspase-3 activation after Fas-receptor ligation involves two steps and is dependent on mitochondrial activation.     

RNA干扰降低环氧化酶2基因表达对人胆管癌QBC939细胞的体外作用

目的研究小干扰RNA（siRNA）降低环氧化酶2（COX-2）基因对胆管癌QBC939细胞凋亡及凋亡蛋白caspase-3蛋白表达的影响。方法采用RNA干扰的方法,将胆管癌细胞株QBC939细胞分为4组：COX-2 siRNA干预组、阴性对照siRNA组、空脂质体、空白对照组。将COX-2 siRNA转染入QBC939细胞;流式细胞仪检测siRNA降低COX-2基因表达对凋亡的作用,WesternBlot法检测siRNA降低COX-2基因表达对caspase-3表达变化的影响。结果 RNA干扰后QBC939细胞COX-2表达下降至空白对照组的42%,流式细胞仪检测结果显示COX-2 siRNA干预组细胞的凋亡率以及凋亡蛋白caspase-3的表达明显高于其他3个对照组。结论 RNA干扰可抑制细胞COX-2蛋白的表达,促使胆管癌QBC939细胞的凋亡,caspase-3途径可能是其调控通路之     

Emodin induces apoptosis in human prostate cancer cell LNCaP

AIM: To elucidate effects and mechanisms of emodin in prostate cancer cells. METHODS: Viability of emodin-treated LNCaP cells and PC-3 cells was measured by MTT assay. Following emodin treatments, DNA fragmentation was assayed by agarose gel electrophoresis. Apoptosis rate and the expression of Fas and FasL were assayed by flow cytometric analysis. The mRNA expression levels of androgen receptor (AR), prostate-specific antigen (PSA), p53, p21, Bcl-2, Bax, caspase-3, -8, -9 and Fas were detected by RT-PCR, and the protein expression levels of AR, p53 and p21 were detected by Western blot analysis. RESULTS: In contrast to PC-3, emodin caused a marked increase in apoptosis and a decrease in cell proliferation in LNCaP cells. The expression of AR and PSA was decreased and the expression of p53 and p21 was increased as the emodin concentrations were increased. In the same time, emodin induced apoptosis of LNCaP cells through the upregulation of caspase-3 and -9, as well as the increase of Bax /Bcl-2 ratio. However, it did not involve modulation of Fas or caspase-8 protein expression. CONCLUSION: In prostate cancer cell line, LNCaP, emodin inhibites the proliferation by AR and p53-p21 pathways, and induces apoptosis via the mitochondrial pathway.