阿霉素诱导PI3'K/Akt/FKHRL1通路激活与胃癌细胞化疗耐药性的关系

目的 探讨阿霉素诱导胃癌细胞磷脂酰肌醇3’-激酶（PI3’K）／Akt／FKHRL1通路的激活对胃癌细胞SGC-7901化疗效果的影响及二者的关系。方法 阿霉素及PI3’K／Akt抑制剂Wortmannin分别作用于胃癌细胞SGC-7901，MTT比色法检测胃癌细胞的生存率，Western印迹法检测FKHRL1磷酸化表达水平。结果 阿霉素抑制胃癌细胞SGC-7901的生长，呈时间依赖性诱导FKHRL1磷酸化。Wortmannin可明显增强阿霉索的细胞生长抑制作用，同时下调阿霉素诱导的磷酸化FKHRL1表达。结论 阿霉素可能通过激活SGC-7901细胞的PI3’K／Akt通路诱导FKHRL1磷酸化，从而影响胃癌细胞的化疗耐药性。Wortmannin可以阻断PI3’K／Akt／FKHRL1通路而提高胃癌的化疗敏感性。     

PI-103在体外抗急性髓细胞白血病效应的实验研究

目的研究急性髓细胞白血病（AML）细胞P13K-Akt—mTOR信号转导通路各基因的表达及PI-103在体外对AML细胞增殖、凋亡及细胞周期的影响。方法RT-PCR法检测AML细胞P13K、Akt、mTORmRNA的表达;四甲基偶氮唑蓝法检测PI-103对AML细胞的增殖作用;流式细胞仪检测PI-103对AML细胞的凋亡率和细胞周期的影响。结果①81．25％的AML患者表达P13K基因,87．5％的患者表达mTOR基因,50％的患者同时表达此两种基因。②PI-103能明显提高PI3K—Akt-mTOR信号通路持续活化的AML细胞的增殖抑制率和凋亡率、阻滞细胞于G0／G1期（P均〈0．05）,且与剂量显著相关（P均〈0．05）。结论大部分AML患者存在PI3K-Akt-mTOR信号转导通路的异常活化;PI-103可通过PI3K、mTOR信号通路抑制AML细胞增殖、促进其凋亡。     

阿霉素诱导磷脂酰肌醇3′-激酶 丝氨酸／苏氨酸蛋白激酶FKHRL1通路对胃癌细胞化疗耐药性的影响

目的探讨阿霉素在胃癌细胞中诱导PI3′K／Akt／FKHRL1通路的激活，对胃癌细胞SGC-7901化疗效果的影响及两者的关系。方法2004-01-2005-09武汉大学人民医院消化内科采用MTT比色法检测胃癌细胞的生存率，Western印迹法检测FKHRL1磷酸化表达水平，同时观察PI3′K／Akt抑制剂wortmannin对阿霉素诱导的上述变化的影响。结果阿霉素抑制胃癌细胞SGC-7901的生长，呈时间依赖性的诱导FKHRL1磷酸化。wortmannin可明显增强阿霉素的细胞生长抑制作用，同时下调阿霉素诱导的磷酸化FKHRL1表达。结论阿霉素可能是通过激活SGC-7901细胞的PI3′K／Akt通路，呈时间依赖性诱导FKHRL1磷酸化。从而影响胃癌细胞的化疗耐药性。wortmannin可以阻断PI3′K／Akt／FKHRL1通路而提高胃癌的化疗敏感性。     

PI3K/Akt信号通路在Irisin抵抗阿霉素心肌毒性中的作用

目的 探究鸢尾素（Irisin）能否通过调控PI3K/Akt信号通路减轻阿霉素（Doxorubicin, Dox）心肌细胞毒性及其分子机制。 方法 将培养的H₉C₂细胞随机分为:对照（Con）组、Irisin处理（Irisin）组、Dox损伤（Dox）组、Dox + Akt抑制剂（Dox + LY294002）组、Irisin保护（Dox + Irisin）组、Dox + Irisin+Akt抑制剂（Dox + Irisin + LY294002）组。分别采用原位切口末端标记法（TUNEL）检测细胞凋亡率、Western Blot检测凋亡相关蛋白表达水平以及CCK-8试剂检测细胞活力,明确Irisin处理对Dox诱导的心肌凋亡的作用。 结果 体外实验证明,与Con组细胞相比,Dox处理后H₉C₂细胞凋亡率显著增加,并且凋亡相关蛋白Bax、Cleaved-caspase 3的表达显著增加,同时抗凋亡蛋白Bcl-2的表达量显著降低（P < 0.05）,而加入Irisin可显著逆转Dox导致的心肌细胞凋亡率增加和凋亡相关蛋白质的表达趋势。进一步的研究证实,Irisin通过促进Akt的磷酸化而上调PI3K/Akt信号通路,从而降低H₉C₂细胞的凋亡,而加入Akt抑制剂LY294002后,Irisin对Dox诱导的心肌细胞毒性的缓解作用被削弱,并且促凋亡相关蛋白的表达量也显著升高。 结论 Irisin可能通过上调PI3K/Akt信号通路抑制细胞凋亡,降低Dox诱导的心肌细胞毒性,为临床治疗Dox心肌损伤提供潜在的药物靶点和治疗策略。     

PI3K/Akt/mTOR信号转导通路在肝细胞癌发生发展中的作用

PI3K/Akt/mTOR信号转导通路可以通过调控基因表达,在肝细胞癌肿瘤细胞生成、增殖、转移和凋亡等过程中发挥重要作用。简述了PI3K/Akt/mTOR信号通路的组成及功能,对PI3K/Akt/mTOR通路在肝细胞癌进程中的作用机制及相关抑制剂的研究进展进行了综述,揭示阻断PI3K/Akt/mTOR通路可以成为肝细胞癌治疗新的靶点方向。     

LY294002在外源性H2S预处理肝纤维化大鼠肝星状细胞增殖、凋亡中的作用

目的探讨硫化氢(hydrogen sulfide,H2S)通过PI3K/Akt信号通路对大鼠肝星状细胞增殖、凋亡的调节作用。方法 MTT法分别检测NaHS(H2S的供体)和PI3K/Akt信号通路的特异性抑制剂LY294002对大鼠肝星状细胞(HSC-T6)的增殖、增殖抑制率的影响;采用流式细胞技术检测药物的有效浓度对HSC-T6凋亡的调节。结果低浓度(50μmol/L)的NaHS能够明显促进HSC-T6的增殖(F=955.949,P=0.000),LY294002可抑制HSC-T6的增殖,伴随药物浓度的升高细胞存活率降低(P<0.05),并诱导HSC-T6的凋亡,而二者联合应用诱导HSC-T6凋亡作用增强。结论 H2S可能通过PI3K/Akt信号通路促进HSC-T6的增殖,但对肝星状细胞的凋亡抑制作用并不显著,H2S可协同LY294002共同诱导HSC-T6细胞的凋亡。     

PI3K/Akt/mTOR信号通路抑制剂在肾癌治疗中的应用进展

磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白（PI3K/Akt/mTOR）信号通路是细胞内重要的信号转导通路之一，可以调节细胞生长、分化、迁移、存活、血管生成和代谢，并参与多种恶性肿瘤的发生发展。肾癌（RCC）作为泌尿系统常见的恶性肿瘤，有较高的转移率，且对放化疗具有明显的耐受性，随着肿瘤分子生物学的发展，肾癌靶向生物治疗逐渐成为研究的热点。PI3K/Akt/mTOR信号通路抑制剂对RCC的疗效显著，以该信号通路为靶点的RCC药物主要为PI3K抑制剂，Akt抑制剂和mTOR抑制剂，这些药物可通过抑制信号通路的异常激活，阻断肿瘤细胞的增殖和生长，诱导细胞凋亡，并抑制肿瘤的侵袭和血管生成。而PI3K、mTOR双重抑制剂虽然处于临床试验阶段，但是其在RCC治疗的研究中表现出更可靠的前景性。     

川芎嗪抗β淀粉样蛋白25～35诱导的SH-SY5Y细胞凋亡作用的机制

目的探讨川芎嗪是否作用PI3K/Akt通路抗β淀粉样蛋白(Aβ)25~35诱导的细胞凋亡。方法 Aβ25~35作用SH-SY5Y细胞建立AD细胞模型,MTT法检测细胞的生存率,流式细胞术检测细胞凋亡率;Western印迹检测p-Akt、Akt、Bax、Bcl-2、caspase-3蛋白表达。结果川芎嗪能够抑制Aβ25~35诱导的SH-SY5Y细胞凋亡和促进其存活;川芎嗪可抑制Aβ25~35引起的p-Akt/Akt、Bcl-2表达降低和Bax、caspase-3的表达增加。PI3K/Akt通路抑制剂LY294002能够抑制川芎嗪对Aβ25~35诱导的细胞凋亡的保护作用,抑制川芎嗪诱导的p-Akt/Akt、Bcl-2表达上调及Bax、caspase-3表达下调。结论川芎嗪拮抗Aβ25~35诱导的细胞凋亡与作用PI3K/Akt通路,调节Bcl-2、Bax、caspase-3的表达有关。     

硼替佐米抑制PI3K/Akt通路增强胃癌细胞对TRAIL诱导凋亡的敏感性

目的:研究硼替佐米对TRAIL诱导胃癌细胞凋亡的影响, 探讨PI3K/Akt通路在TRAIL诱导凋亡中的作用.方法:不同浓度的TRAIL和/或硼替佐米作用于人胃癌细胞系MGC803细胞, MTT法检测细胞存活率, 流式细胞术PI染色检测细胞凋亡.Western blot法检测caspase裂解及p-Akt表达水平的变化.结果:50 nmol/L硼替佐米预处理细胞2 h, 之后予100 μg/L TRAIL继续作用24 h, 细胞存活率明显低于TRAIL单独处理组(35.1%±2.7% vs71.0%±4.3%, P <0.01); 细胞凋亡率明显高于TRAIL单药组(31.3%±2.0% vs 8.2%±0.8%,P <0.01). 20 nmol/L硼替佐米预处理未能增强细胞对TRAIL的敏感性. 进一步研究发现,TRAIL可活化PI3K/Akt通路, 硼替佐米预处理可阻止PI3K/Akt通路的活化, 进而增强细胞对TRAIL诱导凋亡的敏感性.结论:硼替佐米通过抑制TRAIL诱导的PI3K/Ak t通路活化, 增强胃癌MGC803细胞对TRAIL诱导凋亡的敏感性.     

PI3K／Akt信号转导通路阻断剂对卵巢癌细胞顺铂敏感性的影响

目的 探讨磷脂酰肌醇3-激酶／丝氨酸-苏氨酸蛋白激酶（P13K／Akt）信号转导通路阻断剂LY294002对卵巢癌细胞顺铂（DDP）敏感性的影响。方法DDP和LY294002单独或联合作用人卵巢癌细胞,MTT法检测细胞生长抑制率,倒置显微镜观察凋亡细胞形态,流式细胞术检测凋亡细胞。结果加用LY294002后,DDP对卵巢癌细胞的抑制作用增强,细胞凋亡率上升。结论PI3K／AKt信号转导通路阻断剂可有效提高卵巢癌细胞对DDP的敏感性,在卵巢癌治疗中与DDP有一定协同作用。     

Murine cirrhosis induces hepatocyte epithelial mesenchymal transition and alterations in survival signaling pathways

Hepatocytes that reside in a chronically-injured liver have altered growth responses compared to hepatocytes in normal liver. Transforming growth factor beta (TGFbeta) is upregulated in the cirrhotic liver, and cirrhotic hepatocytes, unlike normal hepatocytes exposed to this cytokine, exhibit decreased apoptosis. In fetal hepatocytes, TGFbeta also induces epithelial-mesenchymal transition (EMT) and signaling changes in cell survival pathways. Here, chronic murine liver injury was induced by twice-weekly carbon tetrachloride administration for 8 weeks. Normal liver-derived hepatocytes (NLDH) and cirrhotic liver-derived hepatocytes (CLDH) were examined for EMT and the small mothers against decapentaplegic homolog (Smad), phosphatidylinositol-3-kinase (PI3K/Akt), and mitogen activated protein kinase (MAPK) pathways were investigated. Immunofluorescence imaging of cirrhotic livers demonstrated increased vimentin expression, which was confirmed by immunoblot analysis. In vitro, CLDH exhibited increased vimentin and type 1 collagen expression within cellular extensions consistent with EMT. Treatment with TGFbeta augmented the EMT response in CLDH. In contrast, untreated NLDH did not display features of EMT but responded to TGFbeta with increased vimentin expression and EMT characteristics. In response to PI3K/Akt inhibition, CLDH had decreased basal and insulin-stimulated p-Akt expression and decreased apoptosis compared to NLDH. In both NLDH and CLDH, vimentin expression was dependent on PI3K/Akt activity. CLDH demonstrated increased basal p-extracellular signal-regulated kinase expression that was independent of Smad and PI3K/Akt signaling. Inhibition of the MAPK pathway produced a marked increase in CLDH apoptosis. CONCLUSION: CLDH have increased vimentin and type 1 collagen expression and morphologic features consistent with EMT. In addition, compared to NLDH, the cellular signaling phenotype of CLDH changes from a MAPK-independent pathway to a MAPK-dependent cell survival pathway. These findings may have clinical implications for chemoprevention of hepatocellular carcinoma in the cirrhotic liver.     

Impaired PI3K/Akt signal pathway and hepatocellular injury in high-fat fed rats

AIM:To determine whether mitochondrial dysfunction resulting from high-fat diet is related to impairment of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt,also known as PKB) pathway. METHODS:Rat models of nonalcoholic fatty liver were established by high-fat diet feeding. The expression of total and phosphorylated P13K and Akt proteins in hepatocytes was determined by Western blotting. Degree of fat accumulation in liver was measured by hepatic triglyceride. Mitochondrial number and size were determined using quantitative morphometric analysis under transmission electron microscope. The permeability of the outer mitochondrial membrane was assessed by determining the potential gradient across this membrane.RESULTS:After Wistar rats were fed with high-fat diet for 16 wk,their hepatocytes displayed an accumulation of fat (103.1 ± 12.6 vs 421.5 ± 19.7,P < 0.01),deformed mitochondria (9.0% ± 4.3% vs 83.0% ± 10.9%,P < 0.05),and a reduction in the mitochondrial membrane potential (389.385% ± 18.612% vs 249.121% ± 13.526%,P < 0.05). In addition,the expression of the phosphorylated P13K and Akt proteins in hepatocytes was reduced,as was the expression of the anti-apoptotic protein Bcl-2,while expression of the pro-apoptotic protein caspase-3 was increased. When animals were treated with pharmacological inhibitors of P13K or Akt,instead of high-fat diet,a similar pattern of hepatocellular fat accumulation,mitochondrial impairment,and change in the levels of PI3K,Akt,Bcl-2 was observed. CONCLUSION:High-fat diet appears to inhibit the PI3K/Akt signaling pathway,which may lead to hepa-tocellular injury through activation of the mitochondrial membrane pathway of apoptosis.     

间充质干细胞分泌的血管内皮生长因子对心肌细胞的保护作用

目的:探索间充质干细胞通过其分泌的血管内皮生长因子对H9C2细胞的保护作用及机制。方法: 收集骨髓源间充质干细胞的条件培养基,分别处理培养,将其置于缺氧小室缺氧处理21 h,复氧处理6 h。阻断实验利用VEGFR阻断剂SU5416与间充质干细胞的条件培养基共处理。随后,利用流式细胞术ANNEXIN/PI双标法分析其凋亡变化。Western blotting分析H9C2细胞pAkt的表达。结果: RT PCR结果显示间充质干细胞表达血管内皮生长因子,Western blotting结果显示间充质干细胞条件培养基增加了H9C2细胞pAkt的水平。AnnexinV/PI分析发现间充质干细胞条件培养基明显降低了H9C2细胞缺氧复氧的凋亡,且这种抗凋亡作用能被VEGFR阻断剂SU5416或PI3 K/Akt途径阻断剂LY294002所阻断。结论: 间充质干细胞通过其分泌的血管内皮生长因子通过激活PI3 K/Akt途径保护H9C2细胞。     

DIDS通过PI3-K/Akt途径减弱缺血/再灌注损伤诱导心肌细胞凋亡

目的 探讨磷脂酰肌醇3激酶(PI3-K)/蛋白激酶B(Akt)信号通路在DIDS(4,4′-diisothiocyanostilbene-2,2′-disulfonic acid)减弱缺血/再灌注损伤（I/RI）诱导心肌细胞凋亡中的作用。方法 以I/RI诱导心肌细胞凋亡,然后用PI3-K特异性的抑制剂LY294002［22(42吗啉基)282苯基24氢212苯并吡喃242酮］进行干预。实验分为正常对照组、I/RI组、I/RI+DIDS组和I/RI+DIDS+LY294002组。通过噻唑蓝(MTT)比色法、Hoechest-33258染色和半胱天冬蛋白酶（Apo-ONETM Homogeneous Caspase）-3试剂盒以及Western blot分别检测:心肌细胞的存活率（%）、细胞核的形态变化和caspase-3活性、Akt的磷酸化。结果 ①DIDS能够显著抑制I/RI诱导的细胞存活率的下降,抑制凋亡小体的出现,抑制caspase-3的活性的增加(P<0.01)。②用LY294002预处理后,DIDS保护I/RI心肌细胞存活的作用减弱、凋亡小体增加和caspase-3活性明显升高(P<0.01)。③I/RI组与正常对照组Akt磷酸化无明显差异,DIDS能够显著增加I/RI组中Akt蛋白的磷酸化(P<0.01)。用LY294002预处理后,DIDS对I/RI组Akt蛋白磷酸化的影响明显减弱(P<0.01)。结论 DIDS可通过激活PI3-K/Akt信号通路减弱I/RI 诱导的心肌细胞的凋亡。     

Growth factor-induced phosphoinositide 3-OH kinase/Akt phosphorylation in smooth muscle cells: induction of cell proliferation and inhibition of cell death

OBJECTIVE: The signaling pathways mediating proliferation and apoptosis in vascular smooth muscle cells (VSMC) are not well established. It has previously been shown that activation of the phosphoinositide 3-OH kinase (PI3K)/Akt pathway or the ERK 1/2 pathway can mediate anti-apoptotic function in different cell types. This study determined the specific contribution of the PI3K/Akt and ERK pathway in the regulation of apoptosis and proliferation of VSMC. METHODS AND RESULTS: Incubation of rat VSMC with FCS, insulin or IGF-1 time-dependently stimulated the phosphorylation of Akt, however FCS but not insulin or IGF-1 activated the MAP-kinase ERK 1/2. Moreover, insulin inhibited H(2)O(2)-induced apoptosis via the Akt pathway as demonstrated by pharmacological inhibition of the PI3K or overexpression of a dominant negative Akt mutant. In contrast, FCS inhibited H(2)O(2)-induced apoptosis via the Akt and also the ERK pathway. FCS, but not insulin or IGF-1 induced VSMC proliferation, suggesting that Akt activation is necessary but not sufficient for VSMC proliferation. FCS-induced proliferation of VSMC was only mediated via the Akt pathway and not the ERK pathway. CONCLUSIONS: These results define a link between cell proliferation and programmed cell death in VSMC via the same signal transduction pathway, namely activation of the serine/threonine kinase Akt, which may have significant implication for the development of vascular diseases or remodeling.     

PI3K/Akt信号通路调节心脏功能的研究进展

PI3K/Akt是调节心脏功能的一条重要的信号转导通路,主要通过血管内皮生长因子介导血管生成、抑制心肌细胞凋亡、重构心室、促进细胞能量代谢等方面调节心脏功能。研究发现PI3K/Akt信号通路在内分泌疾病、肾病、肝纤维化、肿瘤及心血管疾病的发生、发展中起着重要作用。本文就PI3K/Akt信号通路调节心脏功能的分子机制作一阐述。     

Sulfatase 2 protects hepatocellular carcinoma cells against apoptosis induced by the PI3K inhibitor LY294002 and ERK and JNK kinase inhibitors

Background: Sulfatase 2 (SULF2), an extracellular heparan sulphate 6‐O‐endosulphatase, has an oncogenic effect in hepatocellular carcinoma (HCC) that is partially mediated through glypican 3, which promotes heparin‐binding growth factor signalling and HCC cell growth. SULF2 also increases phosphorylation of the anti‐apoptotic Akt kinase substrate GSK3β and SULF2 expression is associated with a decreased apoptotic index in human HCCs. Methods: We investigated the functional and mechanistic effects of SULF2 on drug‐induced apoptosis of HCC cells using immunohistochemistry, Western immunoblotting, gene transfection, real‐time quantitative polymerase chain reaction, MTT and apoptosis assays and immunocytochemistry. Results: The increased expression of SULF2 in human HCCs was confirmed by immunohistochemistry and immunoblotting. Treatment with inhibitors of MEK, JNK and PI3 kinases decreased the viability of SULF2‐negative Hep3B HCC cells and induced apoptotic caspase 3 and 7 activity, which was most strongly induced by the PI3K inhibitor LY294002. Forced expression of SULF2 in Hep3B cells significantly decreased activity of the apoptotic caspases 3 and 7 and induced resistance to LY294002‐induced apoptosis. As expected, LY294002 inhibited activation of Akt kinase by PI3K. Conversely, knockdown of SULF2 using an shRNA construct targeting the SULF2 mRNA induced profound cell growth arrest and sensitized the endogenously SULF2‐expressing HCC cell lines Huh7 and SNU182 to drug‐induced apoptosis. The effects of knockdown of SULF2 on HCC cells were mediated by decreased Akt phosphorylation, downregulation of cyclin D1 and the anti‐apoptotic molecule Bcl‐2, and upregulation of the pro‐apoptotic molecule BAD. Conclusion: The prosurvival, anti‐apoptotic effect of SULF2 in HCC is mediated through activation of the PI3K/Akt pathway.     

Inhibition of nuclear factor κB and phosphatidylinositol 3‐kinase/Akt is essential for massive hepatocyte apoptosis induced by tumor necrosis factor α in mice

Background/aims: Tumor necrosis factor (TNF)‐α itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro, is explained by the fact that the TNF‐α/TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through the activation of nuclear factor (NF)‐κB. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF‐α. Here, we focused on the roles of NF‐κB and phosphatidylinositol 3‐kinase (PI3K)‐regulated serine/threonine kinase Akt. Methods: TNF‐α was administered to BALB/c mice after treatment with an adenovirus expressing a mutant form IκBα (Ad5IκB), the PI3K inhibitor wortmannin, or both. Liver injury was assessed biochemically and histologically. The expression of Bcl‐2 family members and caspase activity were examined. Results: In the mice livers, treatment with Ad5IκB or the wortmannin suppressed the activation of NF‐κB or Akt, respectively. Suppression of either NF‐κB or Akt showed a slight increase in transaminase levels and focal liver cell death after TNF‐α administration. However, in mice treated with both Ad5IκB and wortmannin, TNF‐α administration resulted in massive hepatocyte apoptosis and hemorrhagic liver destruction in mice. The combination of Ad5IκB, wortmannin, and TNF‐α markedly increased the activation of caspase‐3 and ‐9, and activated caspase‐8 to a lesser degree, suggesting that TNF‐α‐induced hepatocyte apoptosis is dependent on type II cell death signaling pathway, probably through the mitochondria. Inhibition of the NF‐κB and PI3K/Akt pathways had no effect on expression of Bcl‐2 families. Conclusion: The inducible activation of NF‐κB and constitutive activation of Akt regulate hepatocyte survival against TNF‐α, which occurs independent of Bcl‐2 families.