STAT3抑制剂及烟酰胺联合用药对肝癌HepG2细胞增殖的抑制作用及机制研究

目的　探究STAT3抑制剂（Stattic和S3I-201）以及烟酰胺腺嘌呤二核苷酸（NAD）前体烟酰胺联合用药对肝癌HepG2细胞增殖的影响及机制。方法　以肝癌细胞株HepG2为模型,使用STAT3抑制剂、烟酰胺以及两者联合处理细胞,通过细胞计数、Western blot和qPCR等检测药物对细胞增殖、STAT3表达、STAT3下游靶基因［锌指转录因子1（SNAIL1）、血管内皮生长因子A（VEGFA）、锌指结合蛋白1（ZEB1）］表达和与细胞增殖［微小染色体维持蛋白7（MCM7）、增殖标志物Ki-67（MKI67）、Myc原癌基因蛋白（MYC）］、细胞凋亡［B淋巴细胞瘤2相关X蛋白（BAX）、B淋巴细胞瘤-2（BCL-2）、髓样细胞白血病蛋白1（MCL-1）］、上皮-间充质转化［紧密连接蛋白-1（TJP1）、母亲DPP同源物3（SMAD3）］以及糖代谢［己糖激酶2（HK2）、磷酸果糖激酶（PFKL）、M型丙酮酸激酶（PKM）、葡萄糖转运蛋白1（GLUT1）、乳酸脱氢酶A（LDHA）］相关基因的mRNA表达水平的影响。结果　STAT3抑制剂和烟酰胺处理均能降低STAT3磷酸化水平,抑制细胞增殖,降低细胞增殖、抗凋亡、上皮-间充质转化以及糖代谢相关基因表达水平,上调促凋亡相关基因表达水平。并且,联合用药对STAT3磷酸化水平、细胞增殖以及上述生物过程相关基因的表达水平影响更显著。结论　STAT3抑制剂和烟酰胺可抑制肝癌细胞增殖,并且联合用药效果更显著。这种抑制作用可能与促进凋亡和抑制上皮-间充质转化及糖酵解有关。     

灯盏花乙素对非小细胞肺癌细胞JAK/STAT信号通路的影响

目的 研究灯盏花乙素对非小细胞肺癌细胞JAK/STAT信号通路及其凋亡机制的影响。方法 培养非小细胞肺癌A549细胞,经不同浓度灯盏花乙素处理24 h后收集细胞。采用MTT法观察细胞增殖,流式细胞术检测细胞凋亡;RT-qPCR法检测JAK2,STAT3,Pim1和Bcl-2的mRNA表达情况;Western blot检测JAK2和STAT3蛋白表达水平。结果 MTT法和流式细胞术结果显示,灯盏花乙素能降低A549细胞的存活率,促进其凋亡,并呈剂量依赖效应;RT-qPCR和Western blot结果显示,与对照组比较,灯盏花乙素各组JAK2,STAT3,Pim1和Bcl-2的mRNA表达降低,JAK2和STAT3蛋白水平下降,且与加入的灯盏花乙素浓度成反比。结论 灯盏花乙素可以促进非小细胞肺癌A549细胞凋亡,其机制可能是通过抑制JAK/STAT信号转导通路的激活,减少JAK2和STAT3蛋白的表达。     

LZ-106诱导人肺癌NCI-H2228细胞凋亡的研究

目的 研究LZ-106对间变性淋巴瘤激酶（ALK）激酶活性的抑制作用,及对ALK阳性细胞的生长抑制作用。方法 采用分子对接模拟LZ-106与ALK的结合,通过体外激酶活性实验检测其对ALK激酶活性的影响,通过CCK-8实验检测其对H2228细胞的生长抑制作用,通过Annexin V/PI细胞凋亡实验检测其对H2228细胞的凋亡作用,通过Western blotting检测其对H2228细胞p-ALK及下游信号通路关键激酶表达的影响。结果 LZ-106能抑制ALK激酶活性,半数抑制浓度（IC₅₀）为（15.51±2.09）nmol/L,其能显著抑制H2228细胞存活率,并能浓度相关性地诱导细胞凋亡。Western blotting实验显示LZ-106可以显著抑制p-ALK、p-蛋白激酶B（Akt）及p-信号转导因子和转录激活因子3（STAT3）的表达,且Akt及STAT3激活剂可以明显削弱LZ-106的凋亡诱导作用。结论 LZ-106显著抑制ALK激酶活性,诱导ALK阳性细胞凋亡,激活Akt及STAT3可以逆转LZ-106所致的细胞凋亡。     

The inhibitory effect of CIL-102 on the growth of human astrocytoma cells is mediated by the generation of reactive oxygen species and induction of ERK1/2 MAPK

CIL-102 (1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone) is the major active agent of the alkaloid derivative of Camptotheca acuminata, with multiple pharmacological activities, including anticancer effects and promotion of apoptosis. The mechanism by which CIL-102 inhibits growth remains poorly understood in human astrocytoma cells. Herein, we investigated the molecular mechanisms by which CIL-102 affects the generation of reactive oxygen species (ROS) and cell cycle G2/M arrest in glioma cells. Treatment of U87 cells with 1.0 μM CIL-102 resulted in phosphorylation of extracellular signal-related kinase (ERK1/2), downregulation of cell cycle-related proteins (cyclin A, cyclin B, cyclin D1, and cdk1), and phosphorylation of cdk1Tyr¹⁵ and Cdc25cSer²¹⁶. Furthermore, treatment with the ERK1/2 inhibitor PD98059 abolished CIL-102-induced Cdc25cSer²¹⁶ expression and reversed CIL-102-inhibited cdk1 activation. In addition, N-acetyl cysteine (NAC), an ROS scavenger, blocked cell cycle G2/M arrest and phosphorylation of ERK1/2 and Cdc25cSer²¹⁶ in U87 cells. CIL-102-mediated ERK1/2 and ROS production, and cell cycle arrest were blocked by treatment with specific inhibitors. In conclusion, we have identified a novel CIL-102-inhibited proliferation in U87 cells by activating the ERK1/2 and Cdc25cSer²¹⁶ cell cycle-related proteins and inducing ROS production; this might be a new mechanism in human astrocytoma cells.     

Nickel sulfate induced apoptosis via activating ROS‐dependent mitochondria and endoplasmic reticulum stress pathways in rat Leydig cells

Nickel can induce apoptosis of testicular Leydig cells in mice, whereas the mechanisms remain unclear. In this study, we investigated the role of nickel‐induced reactive oxygen species (ROS) generation in mitochondria and endoplasmic reticulum stress (ERS) mediated apoptosis pathways in rat Leydig cells. Fluorescent DCF and Annexin‐V FITC/PI staining were performed to measure the production of ROS and apoptosis in Leydig cells. RT‐qPCR and Western blot were conducted to analyze the key genes and proteins involved in mitochondria and ERS apoptotic pathways. The results showed that nickel sulfate induced ROS generation, consequently resulted in nucleolus deformation and apoptosis in testicular Leydig cells, which were then attenuated by ROS inhibitors of N‐acetylcysteine (NAC) and 2,2,6,6‐tetramethyl‐1‐piperidinyloxy (TEMPO). Nickel sulfate‐triggered Leydig cells apoptosis via mitochondria and ERS pathways was characterized by the upregulated mRNA and proteins expression of Bak, cytochrome c, caspase 9, caspase 3, GRP78, GADD153, and caspase 12, which were inhibited by NAC and TEMPO respectively. The findings indicated that nickel‐induced ROS generation was involved in apoptosis via mitochondria and ERS pathways in rat Leydig cells.     

Cryptotanshinone enhances the efficacy of Bcr-Abl tyrosine kinase inhibitors via inhibiting STAT3 and eIF4E signalling pathways in chronic myeloid leukaemia

ContextA portion of patients with chronic myeloid leukaemia (CML) develop resistance to the Bcr-Abl tyrosine kinase inhibitors (TKIs), limiting the clinical applications. Previous results have demonstrated the synergistic effects between cryptotanshinone (CPT) and imatinib on apoptosis of CML cells in vitro.ObjectiveTo determine the antileukemia effects of CPT and TKIs on the resistant CML cells, and further investigate the effect of combined treatment of CPT and imatinib on tumour growth and apoptosis in the xenograft model and clarify its regulatory mechanisms.Materials and methodsThe combination effects of CPT and second-generation TKIs were evaluated in resistant CML cells K562-R. CPT and imatinib were orally administered once daily for 21 days on K562-R xenografts in nude mice (6 per group). Tumour proliferation and apoptosis were examined by Ki-67, PCNA and TUNEL staining. The expression levels of apoptotic markers and activities of STAT3 and eIF4E pathways were determined via immunohistochemistry staining and western blotting analysis.ResultsCPT significantly enhanced the antiproliferative effects of TKIs, via triggering cleavages of caspase proteins, and inhibiting activities of STAT3 and eIF4E pathways. The administration of CPT and imatinib dramatically inhibited the tumour growth of xenografts and achieved a suppression of 60.2%, which is 2.6-fold higher than that of single imatinib group. Furthermore, CPT and imatinib increased the apoptotic rates and markedly decreased the phosphorylation levels of STAT3 and eIF4E.ConclusionsOur results demonstrated that CPT could significantly enhance the antileukemia efficacy of TKIs, suggesting the therapeutic potential of CPT to overcome CML resistance.     

Activation of a nuclear factor of activated T-lymphocyte-3 (NFAT3) by oxidative stress in carboplatin-mediated renal apoptosis

BACKGROUND AND PURPOSE

Although carboplatin is currently used as a therapeutic drug for ovarian, breast, and non-small cell lung cancers, it has serious side effects including renal and cardiac toxicity. Herein, we examined the effect of carboplatin on murine renal tubular cell (RTC) apoptosis both in vivo and in vitro and the underlying molecular mechanisms associated with its activation of the nuclear factor of activated T-lymphocytes-3 (NFAT3).

EXPERIMENTAL APPROACH

Mechanisms of carboplatin-mediated renal apoptosis were examined using NFAT-reporter transgenic mice and RTCs with NFAT3 overexpression or knockdown.

KEY RESULTS

We demonstrated that carboplatin initiated an intrinsic apoptotic pathway of activating caspase-3 and -9, accompanied by a decrease in the ratio of Bcl-XL/Bax and a significant increase in Bcl-XS. Carboplatin increased NFAT activation in NFAT-luciferase reporter transgenic mice, RTCs and cells exogenously overexpressing NFAT3 that exacerbated cell death. Furthermore, the addition of either N-acetylcysteine (NAC, an antioxidant) or NFAT inhibitors, including FK-506 (tacrolimus), cyclosporin A (CsA, a calcineurin inhibitor), and BAPTA-AM (a calcium chelator) successfully reversed carboplatin-mediated cell apoptosis, which was further confirmed using siNFAT3. Additionally, NAC blocked NFAT3 activation by inhibition of NADPH oxidase activation, and ERK/JNK and PKC pathways, resulting in a decrease in cell apoptosis; the therapeutic effect of NAC was verified in vivo.

CONCLUSION AND IMPLICATIONS

The results presented herein show that carboplatin-mediated reactive oxygen species might signal calcineurin and NFAT3 activation in RTCs, whereas NAC and NFAT inhibitors reversed carboplatin-mediated RTC apoptosis, suggesting that oxidative stress-mediated NFAT3 activation is essential for carboplatin-mediated RTC apoptosis.     

LW-213 induces cell apoptosis in human cutaneous T-cell lymphomas by activating PERK–eIF2α–ATF4–CHOP axis

Cutaneous T-cell lymphoma (CTCL) is characterized by a heterogeneous group of extranodal non-Hodgkin lymphomas, in which monoclonal T lymphocytes infiltrate the skin. LW-213, a derivative of wogonin, was found to induce cell apoptosis in chronic myeloid leukemia (CML). In this study, we investigated the effects of LW-213 on CTCL cells and the underlying mechanisms. We showed that LW-213 (1–25 μM) dose-dependently inhibited human CTCL cell lines (Hut-102, Hut-78, MyLa, and HH) with IC₅₀ values of around 10 μM, meanwhile it potently inhibited primary leukemia cells derived from peripheral blood of T-cell lymphoma patients. We revealed that LW-213-induced apoptosis was accompanied by ROS formation and the release of calcium from endoplasmic reticulum (ER) through IP₃R-1channel. LW-213 selectively activated CHOP and induced apoptosis in Hut-102 cells via activating PERK–eIF2α–ATF4 pathway. Interestingly, the degree of apoptosis and expression of ER stress-related proteins were alleviated in the presence of either N-acetyl cysteine (NAC), an ROS scavenger, or 2-aminoethyl diphenylborinate (2-APB), an IP₃R-1 inhibitor, implicating ROS/calcium-dependent ER stress in LW-213-induced apoptosis. In NOD/SCID mice bearing Hut-102 cell line xenografts, administration of LW-213 (10 mg/kg, ip, every other day for 4 weeks) markedly inhibited the growth of Hut-102 derived xenografts and prolonged survival. In conclusion, our study provides a new insight into the mechanism of LW-213-induced apoptosis, suggesting the potential of LW-213 as a promising agent against CTCL.     

N-乙酰半胱氨酸对糖氧剥夺诱导星形胶质细胞损伤的抑制作用及机制研究

目的 探讨N-乙酰半胱氨酸（N-acetyl-L-cysteine,NAC）对糖氧剥夺（oxygen and glucose deprivation,OGD）诱导的星形胶质细胞损伤过程中AKT信号通路的影响及其意义。方法 以原代培养的星形胶质细胞建立OGD模型,并分为4组：对照组,OGD组,NAC组和NAC+AKT特异性抑制剂MK-2206组（NAC+MK-2206组）;细胞培养24 h后,采用噻唑蓝（MTT）法检测细胞存活率、流式细胞仪技术分析细胞凋亡比例,试剂盒检测细胞内SOD活性和丙二醛（MDA）含量,蛋白免疫印迹法检测细胞内AKT、磷酸化AKT（p-AKT）、mTORC1、磷酸化mTORC1（p-mTORC1）、胞浆型磷脂酶A2（cLPA2）、caspase3及Bcl-2的表达水平。结果 与OGD组相比,NAC组的细胞存活率,p-AKT、p-mTORC1及Bcl-2表达和SOD活性增加,细胞凋亡比例,cPLA2、caspase3表达,MDA含量降低。与NAC组相比,NAC+MK-2206组的细胞存活率,p-AKT、p-mTORC1及Bcl-2表达和SOD活性降低,细胞凋亡比例、cPLA2、MDA含量、caspase3表达增加。结论 NAC缓解OGD诱导AKT信号通路抑制作用,降低cPLA2诱导细胞凋亡作用。     

Curcumin synergistically increases effects of β-interferon and retinoic acid on breast cancer cells in vitro and in vivo by up-regulation of GRIM-19 through STAT3-dependent and STAT3-independent pathways

Objective: The study aimed to investigate the effects of combination treatment of curcumin and β-interferon (IFN-β)/retinoic acid (RA) on breast cancer cells, including cell viability, apoptosis and migration, and to determine the mechanisms related to GRIM-19 through STAT3-dependent and STAT3-independent pathways.

Methods: The following groups were used for the in vitro experiment: control siRNA, GRIM-19 siRNA, IFN-β/RA and IFN-β/RA?+?curcumin. Cell viability is by the MTT method, cell apoptosis by flow cytometry and cell migration by wound healing experiment; GRIM-19, STAT3, survivin, Bcl-2, GADD153 and COX-2 expression was measured by Western blot. In vivo experiment, MCF-7 cells were subcutaneously injected into nude mice.

Results: GRIM-19 siRNA promoted MCF-7 cell proliferation and migration; inhibited cell apoptosis; and promoted the expression of STAT3, survivin, Bcl-2 and MMP-9. IFN-β/RA inhibited cell proliferation and migration; promoted cell apoptosis; up-regulated GRIM-19; and inhibited the expression of STAT3, survivin, Bcl-2 and MMP-9. Combination treatment of curcumin and IFN-β/RA had a stronger effect than that of the IFN-β/RA group. In addition, curcumin and IFN-β/RA combination inhibited the expression of COX-2 and up-regulated GADD153.

Conclusion: Curcumin synergistically increases the effects of IFN-β/RA on breast cancer cells. The mechanism may be related to the up-regulation of GRIM-19 through STAT3-dependent and STAT3-independent pathways.