冬凌草甲素通过诱导人宫颈癌HeLa细胞自噬下调凋亡的机制

研究冬凌草甲素通过诱导人宫颈癌HeLa细胞自噬拮抗凋亡的机制。MTT法测定冬凌草甲素对HeLa细胞的细胞毒作用。通过相差显微镜观察细胞形态学变化，用琼脂糖凝胶电泳检测DNA片段化，用流式细胞仪检测细胞自噬和凋亡水平，用Western blotting检测分析药物对蛋白质表达的影响。冬凌草甲素明显抑制HeLa细胞的增殖，诱导HeLa细胞凋亡，同时诱导HeLa细胞发生自噬。Western blotting检测结果表明，冬凌草甲素作用24 h后，促凋亡蛋白Bax、细胞色素c和控制Bax活力的去乙酰化酶SIRT-1的表达明显改变。冬凌草甲素(64 μmol·L^-1)诱导的自噬通过影响SIRT-1和线粒体途径蛋白的表达下调凋亡。     

Betulinic acid inhibits autophagic flux and induces apoptosis in human multiple myeloma cells in vitro

Aim:

To investigate the effects of betulinic acid (BA) on apoptosis and autophagic flux in multiple myeloma cells and the relationship between the two processes.

Methods:

The proliferation of human multiple myeloma KM3 cells was measured with MTT assay. FITC/PI double-labeled flow cytometry (FCM) and Hoechst 33258 staining were used to analyze the cell apoptosis. Caspase 3, PARP, Beclin1, LC3-II, and P62 were detected using Western blotting.

Results:

Treatment of KM3 cells with BA (5−25 μg/mL) suppressed the cell proliferation in time- and dose-dependent manners. The IC₅₀ values at 12, 24, and 36 h were 22.29, 17.36, and 13.06 μg/mL, respectively. BA treatment dose-dependently induced apoptosis of KM3 cells, which was associated with the activation of caspase 3. However, Z-DEVD-FMK, a specific inhibitor of caspase 3, did not decrease, but rather sensitized the cells to BA-induced apoptosis, suggesting an alternative mechanism involved. On other hand, BA treatment dose-dependently increased the accumulation of LC3-II and P62 in KM3 cells, representing the inhibition of autophagic flux. Furthermore, BA treatment dose-dependently downregulated the expression of Beclin 1, an important inducer of autophagy, in KM3 cells. In the presence of BA, Z-DEVD-FMK induced autophagy and increased the amount of LC3-II in KM3 cells, which may occur via attenuating BA-induced decrease in the level of Beclin 1. Similarly, rapamycin, an autophagy inducer, increased the amount of LC3-II in KM3 cells. In the presence of BA, rapamycin caused further increase in the amount of LC3-II. Furthermore, rapamycin sensitized BA-treated KM3 cells to apoptosis.

Conclusion:

The results demonstrate that BA induces apoptosis and blocks autophagic flux in KM3 cells. Furthermore, in addition to activation of caspase 3, the inhibition of autophagic flux also contributes to the BA-mediated apoptosis of KM3 cells.     

ROS在小白菊内酯诱导MM细胞凋亡中的作用

目的研究倍半萜内酯小白菊内酯(parthenolide,PL)对多发性骨髓瘤原代细胞和细胞株诱导的凋亡作用及其机制。方法以多发性骨髓瘤原代细胞及细胞株为研究对象,利用台盼蓝染色检测细胞存活率,用DAPI染色法检测细胞凋亡率,流式细胞仪检测反应氧(ROS)和线粒体膜电位水平变化。结果2.5μmol.L-1PL引起多发性骨髓瘤(multi-ple myeloma,MM)细胞明显凋亡及线粒体膜电位下降,而正常淋巴细胞在同等条件下不受影响。用2.5μmol.L-1PL作用10 h后,KMM-1和MM1S细胞ROS水平明显增加。用自由基清除剂L-N-乙酰半胱氨酸(L-NAC)预处理可抑制ROS的产生,对两种细胞存活率、凋亡率无影响。用还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶抑制剂二亚苯基烟碱氯化物(diphenylene iodonium chloride,DPI)预处理KMM-1和MM1S,可明显抑制PL介导的ROS的产生,并对两种细胞凋亡率无影响。结论PL诱导多发性骨髓瘤细胞凋亡,而正常淋巴细胞在同等条件下不受影响。PL的凋亡活性是通过引起MM细胞氧化应激而介导的,并进一步推测NADPH氧化活性与PL介导的MM细胞ROS的产生相关。     

Triptolide induces cell-cycle arrest and apoptosis of human multiple myeloma cells in vitro via altering expression of histone demethylase LSD1 and JMJD2B

Aim:

To elucidate the relationship between triptolide-induced changes in histone methylation and its antitumor effect on human multiple myeloma (MM) cells in vitro.

Methods:

Human multiple myeloma cell line RPMI8226 was used. Apoptosis was evaluated using Annexin-V-FITC/PI-labeled flow cytometry, Hoechst 33258 staining, and transmission electron microscopy. Flow cytometry was used to detect the cell cycle distribution of the apoptotic cells. The presence of the LSD1, JMJD2B, H3K4me2, H3K9me2, and H3K36me2 proteins was verified by Western blot analysis. Semi-quantitative real-time PCR was performed to examine the expression of LSD1 and JMJD2B.

Results:

Triptolide (10–160 nmol/L) suppressed the proliferation of MM cells in a dose- and time-dependent manner with an IC₅₀ value of 99.2±9.0 nmol/L at 24 h. Triptolide (50 nmol/L) induced G₀/G₁ cell cycle arrest in MM cells. The agent (50–150 nmol/L) induced apoptosis of MM cells in a dose-dependent manner. The same concentrations of triptolide suppressed the expression of dimethylated H3K4, dimethylated H3K9 and dimethylated H3K36 by altering the expression of histone demethylase LSD1 and JMJD2B without affecting the expression of histone demethylase LSD1.

Conclusion:

Triptolide potently inhibits the growth of MM cells via regulating the expression of histone demethylase LSD1 and JMJD2B, which lead to abnormal histone methylation.     

Isoliquiritigenin treatment induces apoptosis by increasing intracellular ROS levels in HeLa cells

This study focuses on the relationship between the apoptosis induced by isoliquiritigenin (ISL) and the production of reactive oxygen species (ROS). Cell viability was evaluated using sulforhodamine B assay. The apoptotic rate was determined via flow cytometry. Intracellular ROS level was assessed using the 2,7-dichlorofluorescein probe assay. Poly-ADP-ribose polymerase (PARP) protein expression was examined using Western blot analysis. The results showed that ISL treatment inhibited cell proliferation by inducing apoptosis. The increased apoptotic rate and ROS production induced by ISL were inhibited by the co-treatment of ISL and free radical scavenger N-acetyl-cysteine (NAC), catalase (CAT), and 4,5-dihydroxyl-1,3-benzededisulfonic acid (Tiron). On the contrary, the increased apoptotic rate and the ROS production were compensated by the co-treatment of ISL and l-buthionine-(S,R)-sulfoximine (BSO). ISL treatment increased the degradation of PARP, which was counteracted by antioxidants (NAC or CAT), whereas the combination treatment of ISL and pro-oxidant (BSO) enhanced the PARP degradation induced by ISL. Our findings suggested that ISL treatment induced apoptosis by increasing intracellular ROS levels in HeLa cells.     

S1P4受体通过Akt调节骨髓瘤细胞凋亡的机制研究

目的探讨S1P受体对多发性骨髓瘤(MM)细胞系LP-1生存及凋亡的影响及调控机制。方法应用芬戈莫德(FTY720)抑制LP-1细胞S1P受体,CCK-8法测定细胞生存率,流式细胞仪检测凋亡率。应用qRT-PCR方法检测LP-1细胞中S1P受体的表达情况。应用S1P4R-shRNA敲除S1P4受体,采用荧光显微镜及qRT-PCR法测定病毒感染率及感染后S1P4受体表达。Western blot方法检测S1P4受体敲除后抗凋亡蛋白Akt、Survivin、Birc4、Bag-1表达情况。结果 FTY720处理后,LP-1细胞生存率明显下降,凋亡率明显上升,与对照组相比差异均具有统计学意义(P<0.05)。LP-1细胞中S1P4受体呈高表达,敲除S1P4受体后,抗凋亡蛋白Akt、Survivin、Birc4、Bag-1表达明显下降(P<0.05)。结论 S1P受体对LP-1细胞生存具有重要作用。其中S1P4受体在LP-1细胞凋亡中起主要作用。S1P4受体可通过Akt调节下游抗凋亡蛋白Survivin、Birc4、Bag-1的表达,从而影响LP-1细胞凋亡。     

Effects of triptolide on RIZ1 expression, proliferation, and apoptosis in multiple myeloma U266 cells

Aim:

To investigate the effects of triptolide on proliferation and apoptosis as well as on the expression of RIZ1 in the human multiple myeloma cell line U266 in vitro.

Methods:

The effect of triptolide on the growth of U266 cells was studied by MTT assay. Apoptosis was detected by Hoechst 33258 staining and Annexin V/PI double-labeled flow cytometry, and caspase-3 mRNA was measured by RT-PCR. Western blotting, flow cytometry and RT-PCR were used to assess the expression of RIZ1, and the location and expression of H3K9me1 were detected by confocal microscopy and Western blotting.

Results:

Triptolide significantly inhibited the proliferation of U266 cells in a time- and concentration-dependent manner (the IC₅₀ value for a 24-h exposure was 157.19±0.38 nmol/L). Triptolide induced typical apoptotic morphological changes. Triptolide 40, 80, and 160 nmol/L treatment induced significant caspase-3-dependent apoptosis compared with control group (10.5%±1.23%, 37.9%±2.45%, and 40.5%±2.30% vs 3.8%±1.98%, P<0.05). Compared with peripheral blood monocular cells (PBMC) from healthy donors, the protein expression of RIZ1 in U266 cells was relatively low, but the mRNA and protein expression of RIZ1 were strikingly increased by triptolide in a concentration-dependent manner. Triptolide increased the protein expression of RIZ1 and RIZ1 methylates histone H3 lysine 9 in U266 cells.

Conclusion:

Triptolide increased the protein expression of RIZ1, inhibited the proliferation, and induced caspase-dependent apoptosis in U266 cells.     

Quercetin attenuates NLRP3 inflammasome activation and apoptosis to protect INH-induced liver injury via regulating SIRT1 pathway

Severe hepatotoxicity greatly limits the clinical application of the first-line anti-tuberculosis drug isoniazid(INH). Quercetin(Que) has multiple pharmacological properties, and is regarded as a potential protective agent against a variety of organ injuries. However, the exact effect of quercetin on INH-induced hepatotoxicity and the underlying mechanisms are not yet completely understood. In this study, liver injury models were established in rats and L02 cells to reveal the protective effect of Que on INH-induced hepatotoxicity and the relevant mechanism. The in vivo results indicated that Que pretreatment reduced the level of ALT/AST, improved the liver histopathological changes and substantially mitigated apoptosis in rats. In vitro, it evidently relieved INH-induced cell viability loss and apoptosis in L02 cells. Furthermore, the studies on mechanisms elucidated that Que remarkably elevated the expression of SIRT1 and suppressed NLRP3 inflammasome activation. Meanwhile, Que significantly inhibited the level of tumor suppressor P53, Bax, cleaved-cas3 expressionl and increased Bcl-2 expression to reduce apoptosis in vivo and in vitro. However, SIRT1 inhibitor EX527 reversed the suppression of Que on NLRP3 inflammasome activation and the protection of Que on rat liver injury and cell apoptosis. In short, our findings showed that Que exhibited protective effects against INH-induced liver damage via inhibiting the activation of NLRP3 inflammasome and apoptosis in a SIRT-dependent manner.     

ROS activates JNK-mediated autophagy to counteract apoptosis in mouse mesenchymal stem cells in vitro

Aim:

Transplantation of mesenchymal stem cells (MSCs) for the treatment of diabetic erectile dysfunction (ED) is hampered by apoptosis of the transplanted cells. In diabetic ED, there is increased oxidative stress and decreased NO in the corpora cavernosa, and reactive oxygen species (ROS) induce apoptosis of the transplanted cells. In this study we examined whether and how autophagy was involved in ROS-induced apoptosis of MSCs.

Methods:

Mouse C3H10 MSCs were treated with H₂O₂ to simulate the high oxidative condition in diabetic ED. Cell viability was measured using MTT assay. Apoptosis was analyzed by flow cytometry. Apoptosis- and autophagy-related proteins were detected with Western blot assays. Intracellular autophagosome accumulation was studied using transmission electron microscopy.

Results:

Treatment of MSCs with H₂O₂ (50–400 μmol/L) inhibited the cell viability in concentration- and time-dependent manners. Furthermore, H₂O₂ (300 μmol/L) induced apoptosis, as well as activated autophagy in MSCs. Pretreatment with lysosome inhibitor chloroquine (10 μmol/L) or PI3K inhibitor 3-methyladenine (5 mmol/L) significantly enhanced H₂O₂-induced cell death. Pretreatment with JNK inhibitor SP600125 (10 μmol/L) abrogated H₂O₂-induced accumulation of LC3-II, and attenuated H₂O₂-induced reduction of Bcl-2 levels in MSCs.

Conclusion:

ROS induce autophagy to counteract apoptosis in MSCs by activation of JNK. Thus, augmentation of autophagy may reduce apoptosis, prolonging MSC survival and improving MSC-based therapeutic efficacy for diabetic ED.     

RIP3 overexpression sensitizes human breast cancer cells to parthenolide in vitro via intracellular ROS accumulation

Aim： Receptor-interacting protein 3 （RIP3） is involved in tumor necrosis factor receptor signaling, and results in NF-KB-mediated prosurvival signaling and programmed cell death. The aim of this study was to determine whether overexpression of the RIP3 gene could sensitize human breast cancer cells to parthenolide in vitro. Methods： The expression of RIP3 mRNA in human breast cancer cell lines （MCF-7, MDA-MB-231, MDA-MB-435 and T47D） was detected using RT-PCR. Both MDA-MB-231 and MCF-7 cells were transfected with RIP3 expression or blank vectors via lentivirus. Cell viability was measured with MTT assay; intracellular ROS level and cell apoptosis were analyzed using flow cytometry. Results： RIP3 mRNA expression was not detected in the four human breast cancer cell lines tested. However, the transfection induced higher levels of RIP3 protein in MCF-7 and MDA-MB-231 cells. Furthermore, overexpression of RIP3 decreased the IC50 values of parthenolide from 17.6 to 12.6 μmol/L in MCF-7 cells, and from 16.6 to 9.9 μmol/L in MDA-MB-231 cells. Moreover, overexpression of RIP3 significantly increased parthenolide-induced apoptosis and ROS accumulation in MCF-7 and MDA-MB-231 cells. Pretreatment with N-acetyl-cysteine abrogated the increased sensitivity of RIP3-transfected MCF-7 and MDA-MB-231 cells to parthenolide. Conclusion： Overexpression of RIP3 sensitizes MCF-7 and MDA-MB-231 breast cancer cells to parthenolide in vitro via intracellular ROS accumulation.     

补肾养骨汤诱导多发性骨髓瘤细胞株 KM3 的 凋亡及机制探讨

目的 探讨补肾养骨汤对人多发性骨髓瘤细胞株 KM3 细胞增殖、凋亡的影响及其机制。方法 取对数生 长期 KM3 细胞,分别加入 5%、10%、20% 的补肾养骨汤含药血清培养,培养 12、24、48 和 72 h 后,采用 Cell Counting Kit-8（CCK-8）法检测补肾养骨汤对 KM3 细胞增殖的影响;KM3 细胞经过不同浓度含药血清培养 72 h 后,采用流式 细胞术检测补肾养骨汤对 KM3 细胞周期和凋亡作用;RT-qPCR 和 Western blot 检测 B 淋巴细胞瘤-2 基因（Bcl-2）、 Bcl-2 相关蛋白（Bax）和核转录因子（NF）-κB 的表达水平。结果 补肾养骨汤抑制 KM3 细胞株的增殖,诱导 KM3 细胞株呈剂量依赖性凋亡,使 KM3 细胞周期停滞在 G0/G1 期,导致 KM3 细胞株 Bcl-2、NF-κB 表达下调,Bax 表达 上调。结论 补肾养骨汤抑制 KM3 细胞的增殖,影响细胞周期,诱导其凋亡,此作用机制可能与 Bcl-2、Bax 和 NF- κB 表达异常有关。     

PARP-1抑制剂下调SIRT1基因表达诱导非小细胞肺癌A549细胞凋亡的机制

目的 观察PARP-1抑制剂Olaparib对非小细胞肺癌A549细胞凋亡的影响及可能机制.方法 常规培养A549细胞,分别给予不同剂量Olaparib(0、5、10、20、50μmol/L)和(或)SIRT1激动剂SRT1720(1μmol/L)、SIRT1抑制剂EX527(1μmol/L)处理24 h,MTT法检测细胞增殖,流式细胞术检测细胞凋亡,Western-blot检测细胞SIRT1及凋亡相关基因表达.结果 Olaparib处理A549细胞24 h后,细胞存活率、SIRT1及抗凋亡基因Bcl-2蛋白表达逐渐降低,凋亡率及促凋亡基因Bax、Caspase-3蛋白表达逐渐增加,呈剂量依赖性(P<0.05).单独给予EX527处理A549细胞能够模拟Olaparib的上述作用(P<0.05),但SRT1720和Olaparib联合组能够拮抗Olaparib的上述作用(P<0.05).结论 PARP-1抑制剂Olaparib通过下调SIRT1基因表达抑制A549细胞增殖,并诱导其凋亡.     

吴茱萸碱在诱导人黑色素瘤A375-S2细胞凋亡过程中对SIRT1和p53蛋白的影响

目的研究吴茱萸碱(evodiamine)在诱导A375-S2细胞凋亡过程中对SIRT1和p53蛋白表达的调控。方法电镜观察吴茱萸碱诱导细胞凋亡过程中细胞形态的变化。Western blot方法分析药物作用后对SIRT1蛋白和p53及其下游p21蛋白表达的影响。结果经15μmol.L-1吴茱萸碱处理24 h的A375-S2细胞表现出典型的凋亡特征,即细胞表面微绒毛消失、胞质空泡化、染色质浓集、边聚。在吴茱萸碱诱导A375-S2细胞凋亡过程中,SIRT1蛋白的表达下降,p53和p-p53的表达有所上升,其下游p21蛋白也被激活。结论在吴茱萸碱诱导的A375-S2细胞凋亡中,p53及其下游p21蛋白被活化,SIRT1蛋白表达被下调。     

G226, a new epipolythiodioxopiperazine derivative,triggers DNA damage and apoptosis in human cancer cells in vitro via ROS generation

Aim:

G226 is a novel derivative of epipolythiodioxopiperazines with potent inhibitory activity against cancer cells. Here, we sought to identify potential targets involved in the anti-cancer activity of G226.

Methods:

Cell proliferation assay was conducted in a panel of 12 human cancer cell lines. The activities of topoisomerase I (Topo I) and Topo II were studied using supercoiled pBR322 DNA relaxation and kDNA decatenation assays. ROS production was assessed with probes DCFH-DA and H&E. Western blot analysis and flow cytometry were used to examine DNA damage, apoptosis and cell cycle changes.

Results:

G226 displayed potent cytotoxicity in the 12 human cancer cell lines with a mean IC₅₀ value of 92.7 nmol/L. This compound (1–100 μmol/L) selectively inhibited the activity of Topo II, and elevated the expression of phosphorylated-H2AX in a dose-dependent manner. In Topo II-deficient HL60/MX2 cells, however, G226-induced DNA damage, apoptosis and cytotoxicity were only partially reduced, suggesting that Topo II was not essential for the anti-tumor effects of G226. Furthermore, G226 (0.125–2 μmol/L) dose-dependently elevated the intracellular levels of H₂O₂ and in the cancer cells, and pretreatment with GSH, NAC or DTT not only blocked G226-induced intracellular accumulation of ROS, but also abrogated G226-mediated phosphorylation of H2AX, apoptosis and cytotoxicity.

Conclusion:

G226-mediated ROS production contributes to the anti-cancer activity of this compound.     

Autophagy potentially protects against 2,3,7,8‐tetrachlorodibenzo‐p‐Dioxin induced apoptosis in SH‐SY5Y cells

The environmental toxicant TCDD may elicit cytotoxic effects by inducing reactive oxygen species (ROS) generation. Autophagy is one of the first lines of defense against oxidative stress damage. Herein, we investigated whether autophagy played a regulatory role in TCDD‐induced neurotoxicity. Here, we showed that TCDD exposure caused marked autophagy in SH‐SY5Y cells, whose dose range was close to that inducing apoptosis. Electron microscopic and Western blot analyses revealed that TCDD induced autophagy at a starting dose of approximate 100 nM. Interestingly, 100–200 nM TCDD exposure resulted in obviously decreased cell viability and evident apoptotic phenotype. Furthermore, the levels of pro‐apoptotic molecules, Bax and cleaved‐PARP, increased significantly, whereas Bcl2 declined after exposed to 100 nM TCDD. In addition, the apoptosis was verified using flow cytometrical analysis. These data strongly suggested that TCDD induced both autophagy and apoptosis at a similar dose range in SH‐SY5Y cells. Interestingly, pretreatment with ROS scavenger, N‐acetyl‐cysteine (NAC), could effectively block both TCDD‐induced apoptosis and autophagy. More surprisingly, inhibition of autophagy with 3‐methyladenine (3MA), remarkably augmented TCDD‐induced apoptosis. The findings implicated that the onset of autophagy might serve as a protective mechanism to ameliorate ROS‐triggered cytotoxic effects in human SH‐SY5Y neuronal cells under TCDD exposure. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1068–1079, 2016.     

Reactive oxygen species contribute to oridonin-induced apoptosis and autophagy in human cervical carcinoma HeLa cells

Aim:

To investigate the role of reactive oxygen species (ROS) in oridonin-induced apoptosis and autophagy in HeLa cells.

Methods:

The cell viability was measured using MTT assay. Morphological changes of apoptosis and autophagy were examined using Hoechst 33258 staining and monodansylcadaverine (MDC) staining, respectively. The mitochondrial membrane potential (ΔΨm) was measured using fluorescent dye rhodamine 123. DCF-induced fluorescence was used to measure the intracellular ROS level. Protein expression was examined using Western blot.

Results:

Treatment of HeLa cells with oridonin (20–160 μmol/L) inhibited the cell growth in time- and concentration-dependent manners. The cells treated with oridonin (80 μmol/L) for 24 h displayed marked DNA fragmentation and MDC-positive autophagosomes. In the presence of the specific autophagy inhibitor 3-MA (2 mmol/L), the oridonin-induced apoptosis was significantly enhanced. Treatment of HeLa cells with oridonin (20–120 μmol/L) induced intracellular ROS generation in a concentration-dependent manner. In the presence of the ROS scavenger NAC (5 mmol/L), the oridinin-induced ROS generation was markedly reduced. NAC (5 mmol/L) or non-thiol antioxidant catalase (1000 U/mL) significantly reduced the oridonin-induced inhibition of cell growth and apoptosis. Furthermore, oridonin significantly reduced ΔΨm, which was blocked by NAC. Oridonin markedly increased Bax expression in mitochondria, and decreased Bcl-2 expression in both the cytosol and mitochondria. Oridonin also markedly increased the phosphorylation of Bcl-2 in the cytosol. All the effects were blocked by NAC. Oridonin increased the levels of caspase-3 and caspase-8, and decreased the expression of pro-caspase 3 and pro-caspase 9, which were blocked by NAC.

Conclusion:

ROS plays a critical role in oridonin-induced apoptosis and autophagy.     

米非司酮对体外培养子宫肌瘤细胞增殖与凋亡的作用

目的研究米非司酮(mifepristone,MIF)对体外培养子宫肌瘤细胞(LSMCs)增殖与凋亡的作用。方法应用MTT比色法观察MIF对LSMCs生长的影响;采用DNA缺口原位末端标记法(TUNEL)观察MIF作用前后LSMCs细胞增殖与凋亡变化;实时荧光定量PCR(qRT-PCR)法检测细胞Bcl-2、Bax的mRNA水平。结果不同浓度MIF均能抑制LSMCs细胞生长,MIF≥1×10-7mol.L-1对细胞生长产生剂量依赖性抑制作用。用1×10-5mol·L-1的MIF处理72 h后,LSMCs细胞的增殖降低、凋亡率增加,P<0.05;且伴随Bax/Bcl-2表达的上升。结论 MIF具有抑制LSMCs增殖、诱导LSMCs凋亡的作用。