吴茱萸碱对黑色素瘤细胞的耐药逆转作用

黑色素瘤是目前恶性程度最高的肿瘤之一。临床上,常采用维罗非尼（PLX4032）作为晚期患者的治疗药物,但患者很快就出现了耐药。因此,如何克服耐药、提高患者生存率成为急需解决的问题。本文选用中药吴茱萸碱（EVO）与黑色素瘤A375细胞、对PLX4032耐药的A375细胞（A375/R）进行相关研究。采用CCK-8法检测发现,用EVO的细胞非毒性浓度0.5 μmol/L处理A375/R,PLX4032对A375/R细胞的半数抑制浓度（IC50）降低,逆转倍数为3.85。显示EVO能够增强黑色素瘤A375/R细胞对PLX4032的敏感性。后续实验分为对照组、EVO组、PLX组、PLX + EVO组。流式细胞仪检测结果显示,EVO组细胞凋亡率为5.88%,PLX组细胞凋亡率为17.88%,PLX + EVO组细胞凋亡率为30.28%。细胞集落结果证明,PLX4032联合EVO能够抑制A375/R细胞的克隆形成。免疫印迹法结果证明,联合用药能够上调促凋亡蛋白质Bax、胱天蛋白酶3的表达量,下调p-Akt、p-NF-κB-p65及抗凋亡蛋白Bcl-2的蛋白质水平。以上结果均表明,EVO能够有效逆转黑色素瘤A375/R细胞耐药,并且诱导细胞凋亡,抑制细胞增殖。     

高三尖杉酯碱通过下调IRS4蛋白质的表达抑制黑色素瘤维罗非尼耐药细胞周期和增殖

黑色素瘤是一种死亡率极高的恶性肿瘤。近年来,其发病率逐年上升且耐药问题日渐突出。因此,如何克服耐药提高治疗效果是目前急需解决的问题。本文对高三尖杉酯碱对黑色素瘤维罗非尼耐药细胞A375R的作用及其机制进行初步探究。采用CCK-8法检测发现,维罗非尼对黑色素瘤细胞A375和A375R的半数抑制浓度(IC_(50))分别为0.94μmol/L和49.09μmol/L,其耐药倍数为52.22倍,证明A375R具有耐药性,可进行后续实验;高三尖杉酯碱作用于A375R细胞24、48、72 h后的IC_(50)分别为18.57 ng/mL、9.88 ng/mL、9.23 ng/mL,说明高三尖杉酯碱能有效抑制A375R细胞的增殖,且呈时间和浓度依赖性。克隆形成实验显示与对照组相比,给予高三尖杉酯碱5、10、20 ng/mL,作用24 h,克隆形成率分别为52.18%、29.72%、2.36%,表明高三尖杉酯碱能有效抑制A375R细胞的克隆形成。流式细胞术检测显示,G_0/G_1期细胞由正常对照组的38.94%增加至55.05%,细胞阻滞在G_0/G_1期。蛋白质印迹结果显示,与A375细胞相比,A375R细胞中胰岛素受体底物4(IRS4)、p-Akt蛋白质表达水平上调,使用高三尖杉酯碱能下调A375R中IRS4、p-Akt、细胞周期蛋白E1、CDK2蛋白质的表达水平。以上结果表明,高三尖杉酯碱可能通过下调IRS4蛋白表达抑制PI3K/Akt通路的激活,使细胞周期阻滞在G_0/G_1期,从而抑制黑色素瘤耐药细胞的增殖。     

复方浙贝母药物血清抑制L1210/CDDP细胞增殖与诱导凋亡研究

目的：观察复方浙贝药物血清抑制耐顺铂的小鼠淋巴细胞系（L1210/CDDP）增殖和诱导凋亡效应,并在此基础上探讨复方浙贝颗粒逆转白血病细胞多药耐药机制。方法：MTT法测定不同浓度组复方浙贝颗粒含药血清抑制L1210/CDDP增殖;Annex-in-V/PI双染经流式细胞仪（FCM）定量检测其含药血清诱导L1210/CDDP细胞早期凋亡率。结果：不同剂量复方浙贝颗粒含药血清组均具有抑制L1210/CDDP细胞增殖效应,对其凋亡抑制率分别为：29.27%（高剂量组）、31.78%（中剂量组）、21.20%（低剂量组）、25.31%（阿霉素组）;实验各组的A570nm与空白对照组比较明显下降（P〈0.01）。经FCM双荧光法检测其能够诱导L1210/CDDP细胞早期凋亡,实验各组凋亡率分别为：55.12%（中剂量血清组）、34.23%（低剂量组）、25.69%（高剂量组）,24.65%（空白血清组）。结论：复方浙贝颗粒含药血清能够明显抑制L1210/CDDP细胞增殖,并能诱导其凋亡。     

G6PD缺陷抑制人黑色素瘤细胞裸鼠移植瘤的生长

敲减葡糖6-磷酸脱氢酶(G6PD)表达的人黑色素瘤A375细胞(A375-G6PDΔ) 呈现生长增殖抑制和凋亡率升高. 为明确G6PD缺陷对裸鼠体内成瘤的影响及其可能机制,用A375-WT与A375-G6PDΔ细胞制作裸鼠荷瘤模型,观察体内瘤体生长,real-time PCR、免疫组织化学染色与紫外分光光度法分别检测瘤体组织G6PD mRNA、G6PD蛋白及酶活性,Western 印迹分析凋亡相关蛋白,分光光度法测定NADPH和GSH/GSSG水平. 结果显示,A375-G6PDΔ细胞注射组的裸鼠成瘤时间延长,瘤体生长明显减慢,瘤体的体积与质量显著低于A375 WT细胞注射组（P <0.01）;与A375-WT细胞注射组相比,A375 G6PDΔ细胞注射组的裸鼠瘤体组织中G6PD mRNA表达、G6PD阳性细胞数与G6PD活性分别降低了87.10%、77.20%与75.77%（P<0.01）,G6PD、p53和Bcl-2的表达分别降低了67.92%、65.54%和62.32%（P<0.01）,Fas升高了86.38%（P<0.01）,NADPH和GSH/GSSG分别降低了74.37%和86.02%（P<0.01）. 结果提示,G6PD缺陷可能通过减少核酸等合成的原料、改变细胞内氧化还原状态及凋亡相关蛋白表达抑制裸鼠瘤体生长与增殖,这为黑色素瘤发生和治疗研究提供了新的线索.     

新狼毒素A诱导人黑色素瘤A375细胞凋亡的机制研究

为探讨新狼毒素A抑制人黑色素瘤A375细胞增殖及诱导凋亡的作用。本实验采用噻唑蓝(MTT)法检测新狼毒素A对人黑色素瘤A375细胞增殖的影响,Hoechst33258法观察细胞形态,流式细胞仪检测细胞凋亡率和线粒体膜电位,Westernblot检测凋亡相关蛋白表达。结果显示新狼毒素A以时间剂量依赖性的方式显著抑制A375细胞的增殖;不同浓度新狼毒素A(0、15、30、45μmol/L)作用于A375细胞48h后细胞出现显著凋亡特征,新狼毒素A增加A375细胞的凋亡率且降低了线粒体膜电位,上调Bax、caspase-3和细胞色素C(CytochromeC)蛋白的表达,下调Bcl-2蛋白的表达。以上结果说明新狼毒素A抑制A375细胞增殖,诱导细胞凋亡,其机制可能与诱导线粒体途径的凋亡有关。     

臭椿酮诱导人黑色素瘤A375细胞凋亡及其机制研究

为研究臭椿酮(Ailanthone,AIL)诱导人黑色素瘤A375细胞凋亡的作用及作用机制,以人黑色素瘤A375细胞为研究对象,采用MTT法测定AIL对人黑色素瘤A375细胞生长增殖的抑制作用。用倒置相差显微镜观察AIL对A375细胞形态的影响,用荧光倒置显微镜观察Hoechst33258染色后AIL对A375细胞核的影响,用AnnexinV-FITC/PI双染法检测AIL诱导A375细胞凋亡的作用,用分光光度法检测caspase-3和caspase-9的活性,Westernblot检测p-PI3Kβ(Ser1070),PI3Kβ,p-Akt(Ser473)和Akt蛋白表达水平的变化,接着用PI3K抑制剂LY294002进行干预,进一步验证AIL对PI3K/Akt信号通路及细胞凋亡的影响。实验结果表明,AIL能够明显抑制A375细胞增殖,使A375细胞数目变少、附着力和透光性减弱,AIL能够诱导A375细胞凋亡,使其细胞核染色质发生固缩并呈现高亮,且使A375细胞早期及晚期凋亡率均增加,AIL作用后能够使caspase-3和caspase-9活性增加,AIL能够抑制PI3K和Akt蛋白磷酸化,从而使PI3K/Akt信号通路失活。较AIL单独作用,AIL和LY294002共同作用后对PI3K和Akt蛋白磷酸化的抑制作用增强且诱导凋亡作用增加,进一步说明AIL通过失活PI3K/Akt信号通路来诱导A375细胞凋亡。     

早孕因子单克隆抗体对黑色素瘤细胞A-375增殖、凋亡的影响

目的:研究早孕因子单克隆抗体(EPF-McAb)对黑色素瘤细胞A-375增殖、凋亡的影响。方法:体外培养黑色素瘤细胞A-375,通过CCK-8实验检测EPF-McAb对A-375细胞增殖的影响;通过流式细胞术检测EPF-McAb对A-375细胞凋亡的影响;Western Blot检测EPF-McAb对A-375细胞EPF蛋白表达的影响。结果:CCK-8结果显示EPF-McAb可以抑制黑色素瘤细胞A-375的增殖,且随着作用时间延长和药物浓度的增加,对A-375细胞增殖的抑制作用也增强;流式细胞术实验结果显示EPF-McAb可以促进黑色素瘤细胞A-375的凋亡,且调亡率随着药物浓度的增加而升高,0.2、0.4、0.8 mg/m L的EPF单抗作用于A-375细胞24 h后,细胞调亡率分别为:14.68%(P0.01)、19.81%(P0.01)、23.97%(P0.01);Western Blot实验结果显示EPF-McAb可以降低黑色素瘤细胞A-375 EPF蛋白的表达。结论:早孕因子单克隆抗体可以抑制黑色素瘤细胞A-375的增殖,并促进其凋亡。     

干扰素α1b对黑色素瘤细胞A375增殖、迁移和凋亡的影响

目的:研究干扰素α1b(IFN-α1b)对人恶性黑色素瘤细胞A375增殖、迁移和凋亡的影响。方法:采用体外培养的黑色素瘤细胞A375,通过MTT法检测和比较IFN-α1b和IFN-α2b对A375细胞的增殖的抑制作用;细胞划痕实验分析IFN-α1b对A375细胞迁移的影响;Annexin V-FITC/PI染色后采用荧光显微镜观察和流式细胞术分析IFN-α1b对A375细胞凋亡的影响。结果:MTT结果显示随着IFN-α1b和IFN-α2b浓度的增加,A375细胞的生长抑制率逐渐升高,药物作用48 h和72 h后,IFN-α1b作用的A375细胞生长抑制率明显高于IFN-α2b,IFN-α1b和IFN-α2b作用48 h的IC50分别为(22.69±1.52)μg/mL和(35.69±1.01)μg/mL(P0.01);IFN-α1b和IFN-α2b作用72 h的IC50分别为(10.00±0.98)μg/mL和(25.02±0.44)μg/mL(P0.01)。细胞划痕实验显示IFN-α1b能够使A375细胞的迁移指数和迁移能力降低,且随着IFN-α1b药物浓度的增加抑制细胞迁移作用增强。随着50μg/mL IFN-α1b作用的A375细胞的凋亡率为(29.31±0.45)%,较对照组(8.21±1.36)%相比明显上升(P0.01)。结论:IFN-α1b对人恶性黑色素瘤细胞A375具有生长抑制作用,与IFN-α2b相比,IFN-α1b的抑制作用更加显著;IFN-α1b能够降低细胞A375的迁移能力并诱导其凋亡。     

奥利司他逆转肺腺癌A549/DDP细胞株顺铂耐药及机制研究

本研究的目的是观察奥利司他(Orlistat)逆转肺腺癌耐顺铂细胞株A549/DDP的耐药作用并考察其可能的分子机制。应用CCK-8法检测并计算肺腺癌耐药细胞株A549/DDP对顺铂(cisplatin, DDP)的耐药指数(resistance index, RI),筛选奥利司他的最佳实验浓度并观察其对A549/DDP细胞的耐药逆转效果;倒置荧光显微镜下观察各实验组细胞的形态学变化及Hoechst 33258荧光染色后细胞凋亡形态学改变;采用流式细胞术检测不同药物处理对细胞凋亡率的影响;Western blotting检测各实验组细胞P-gp、FASN、PI3K、Akt、p-Akt、NF-κB、Bcl-2和cleved-caspase-3的表达情况。结果显示,奥利司他抑制了A549/DDP耐药细胞株的增殖,且具有浓度依赖性。奥利司他与DDP联用增加了耐药株A549/DDP细胞对DDP的敏感性,耐药逆转倍数为5.02。倒置荧光显微镜观察到联合用药组细胞出现了明显的凋亡形态学改变。流式细胞术结果表明,与对照组和单药组比较,两种药物联用后细胞的凋亡率显著提高(p0.05)。Western blotting检测结果显示,相较于其它3组,联合用药组中耐药相关蛋白P-gp表达下调(p0.01),PI3K、p-Akt、NF-κB表达水平均显著降低(p0.01),抗凋亡蛋白Bcl-2表达下降(p0.01),凋亡相关蛋白cleved-caspase-3表达上调(p0.01);虽然FASN的表达在单用奥利司他组及联合用药组中均降低,但这两组间的FASN表达水平并无统计学差异。以上结果表明,奥利司他能够提高A549/DDP耐药细胞株对化疗药物DDP的敏感性,具有逆转肺腺癌细胞耐药性的作用,其机制与降低耐药蛋白P-gp的表达、抑制PI3K/Akt/NF-κB信号通路以及其下游凋亡相关蛋白有关。     

干扰血红素加氧酶-1改善黑色素瘤细胞对顺铂耐药性的机制研究

目的:探讨血红素加氧酶-1(HO-1)对皮肤黑色素瘤细胞耐药性的影响并分析其分子机制。方法:采用慢病毒载体介导RNA干扰HO-1稳定低表达的恶性皮肤黑色素瘤细胞A375(KD组),用RT-PCR和Western印迹验证其干扰效率;CCK-8法检测HO-1敲减后细胞的增殖情况及对药物顺铂的敏感性变化;流式细胞术检测顺铂对细胞凋亡的影响;RT-PCR及Western印迹检测顺铂处理的HO-1稳定敲减细胞系中凋亡相关基因的变化。结果:构建了HO-1稳定低表达的A375细胞株,敲低HO-1明显抑制细胞增殖;敲低HO-1后细胞对顺铂的敏感性增加,在低浓度顺铂(0.25～6μg/m L)处理的情况下,A375-sh HO-1细胞抑制率比正常A375细胞高8倍以上;顺铂处理后,在RNA水平和蛋白水平上A375-sh HO-1细胞中凋亡相关基因Bax、active Caspase-3显著增加,并降低Bcl-2的表达。结论:RNA干扰抑制HO-1基因表达能够增强皮肤黑色素瘤细胞A375对顺铂的敏感性。     

BH3-only protein silencing contributes to acquired resistance to PLX4720 in human melanoma

B-RAF is mutated to a constitutively active form in 8% of human cancers including 50% of melanomas. In clinical trials, the RAF inhibitor, PLX4032 (vemurafenib), caused partial or complete responses in 48–81% of mutant B-RAF harboring melanoma patients. However, the average duration of response was 6–7 months before tumor regrowth, indicating the acquisition of resistance to PLX4032. To understand the mechanisms of resistance, we developed mutant B-RAF melanoma cells that displayed resistance to RAF inhibition through continuous culture with PLX4720 (the tool compound for PLX4032). Resistance was associated with a partial reactivation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, recovery of G1/S cell-cycle events, and suppression of the pro-apoptotic B-cell leukemia/lymphoma 2 (Bcl-2) homology domain 3 (BH3)-only proteins, Bcl-2-interacting mediator of cell death-extra large (Bim-EL) and Bcl-2 modifying factor (Bmf). Preventing ERK1/2 reactivation with MEK (mitogen-activated protein/extracellular signal-regulated kinase kinase) inhibitors blocked G1-S cell-cycle progression but failed to induce apoptosis or upregulate Bim-EL and Bmf. Treatment with the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid, led to de-repression of Bim-EL and enhanced cell death in the presence of PLX4720 or AZD6244 in resistant cells. These data indicate that acquired resistance to PLX4032/4720 likely involves ERK1/2 pathway reactivation as well as ERK1/2-independent silencing of BH3-only proteins. Furthermore, combined treatment of HDAC inhibitors and MEK inhibitors may contribute to overcoming PLX4032 resistance.     

PLX4032, a potent inhibitor of the B-Raf V600E oncogene, selectively inhibits V600E-positive melanomas

Targeted intervention of the B-Raf V600E gene product that is prominent in melanoma has been met with modest success. Here, we characterize the pharmacological properties of PLX4032, a next-generation inhibitor with exquisite specificity against the V600E oncogene and striking anti-melanoma activity. PLX4032 induces potent cell cycle arrest, inhibits proliferation, and initiates apoptosis exclusively in V600E-positive cells in a variety of in vitro experimental systems; follow-up xenograft studies demonstrate extreme selectivity and efficacy against melanoma tumors bearing the V600E oncoproduct. The collective data support further exploration of PLX4032 as a candidate drug for patients with metastatic melanoma; accordingly, validation of PLX4032 as a therapeutic tool for patients with melanoma is now underway in advanced human (Phase III) clinical trials.     

AEBP1 upregulation confers acquired resistance to BRAF (V600E) inhibition in melanoma

An activating BRAF (V600E) kinase mutation occurs in approximately half of melanomas. Recent clinical studies have demonstrated that vemurafenib (PLX4032) and dabrafenib, potent and selective inhibitors of mutant v-raf murine sarcoma viral oncogene homolog B1 (BRAF), exhibit remarkable activities in patients with V600 BRAF mutant melanomas. However, acquired drug resistance invariably develops after the initial treatment. Identification of acquired resistance mechanisms may inform the development of new therapies that elicit long-term responses of melanomas to BRAF inhibitors. Here we report that increased expression of AEBP1 (adipocyte enhancer-binding protein 1) confers acquired resistance to BRAF inhibition in melanoma. AEBP1 is shown to be highly upregulated in PLX4032-resistant melanoma cells because of the hyperactivation of the PI3K/Akt-cAMP response element-binding protein (CREB) signaling pathway. This upregulates AEBP1 expression and thus leads to the activation of NF-κB via accelerating IκBa degradation. In addition, inhibition of the PI3K/Akt-CREB-AEBP1-NF-κB pathway greatly reverses the PLX4032-resistant phenotype of melanoma cells. Furthermore, increased expression of AEBP1 is validated in post-treatment tumors in patients with acquired resistance to BRAF inhibitor. Therefore, these results reveal a novel PI3K/Akt-CREB-AEBP1-NF-κB pathway whose activation contributes to acquired resistance to BRAF inhibition, and suggest that this pathway, particularly AEBP1, may represent a novel therapeutic target for treating BRAF inhibitor-resistant melanoma.     

Identification of PLX4032‐resistance mechanisms and implications for novel RAF inhibitors

BRAF inhibitors improve melanoma patient survival, but resistance invariably develops. Here we report the discovery of a novel BRAF mutation that confers resistance to PLX4032 employing whole‐exome sequencing of drug‐resistant BRAF^V600K melanoma cells. We further describe a new screening approach, a genome‐wide piggyBac mutagenesis screen that revealed clinically relevant aberrations (N‐terminal BRAF truncations and CRAF overexpression). The novel BRAF mutation, a Leu505 to His substitution (BRAF^L505H), is the first resistance‐conferring second‐site mutation identified in BRAF mutant cells. The mutation replaces a small nonpolar amino acid at the BRAF‐PLX4032 interface with a larger polar residue. Moreover, we show that BRAF^L505H, found in human prostate cancer, is itself a MAPK‐activating, PLX4032‐resistant oncogenic mutation. Lastly, we demonstrate that the PLX4032‐resistant melanoma cells are sensitive to novel, next‐generation BRAF inhibitors, especially the ‘paradox‐blocker’ PLX8394, supporting its use in clinical trials for treatment of melanoma patients with BRAF‐mutations.     

PLX4032, a selective BRAFV600E kinase inhibitor,activates the ERK pathway and enhances cell migration and proliferation of BRAFWT melanoma cells

BRAF^V600E/K is a frequent mutationally active tumor-specific kinase in melanomas that is currently targeted for therapy by the specific inhibitor PLX4032. Our studies with melanoma tumor cells that are BRAF^V600E/K and BRAF^WT showed that, paradoxically, while PLX4032 inhibited ERK1/2 in the highly sensitive BRAF^V600E/K, it activated the pathway in the resistant BRAF^WT cells, via RAF1 activation, regardless of the status of mutations in NRAS or PTEN. The persistently active ERK1/2 triggered downstream effectors in BRAF^WT melanoma cells and induced changes in the expression of a wide-spectrum of genes associated with cell cycle control. Furthermore, PLX4032 increased the rate of proliferation of growth factor-dependent NRAS Q61L mutant primary melanoma cells, reduced cell adherence and increased mobility of cells from advanced lesions. The results suggest that the drug can confer an advantage to BRAF^WT primary and metastatic tumor cells in vivo and provide markers for monitoring clinical responses.     

BRAF silencing by short hairpin RNA or chemical blockade by PLX4032 leads to different responses in melanoma and thyroid carcinoma cells

BRAF-activating mutations have been reported in several types of cancer, including melanoma ( approximately 70% of cases), thyroid (30-70%), ovarian (15-30%), and colorectal cancer (5-20%). Mutant BRAF has constitutive kinase activity and causes hyperactivation of the mitogen-activated protein kinase pathway. BRAF silencing induces regression of melanoma xenografts, indicating the essential role of BRAF for cell survival. We set up an inducible short hairpin RNA system to compare the role of oncogenic BRAF in thyroid carcinoma versus melanoma cells. Although BRAF knockdown led to apoptosis in the melanoma cell line A375, the anaplastic thyroid carcinoma cell ARO underwent growth arrest upon silencing, with little or no cell death. Reexpression of the thyroid differentiation marker, sodium iodide symporter, was induced after long-term silencing. The different outcome of BRAF down-regulation in the two cell lines was associated with an opposite regulation of p21(CIP1/WAF1) expression levels in response to the block of the BRAF mitogenic signal. These results were confirmed using a specific BRAF small-molecule inhibitor, PLX4032. Restoration of p21(CIP1/WAF1) expression rescued melanoma cells from death. Altogether, our data indicate that oncogenic BRAF inhibition can have a different effect on cell fate depending on the cellular type. Furthermore, we suggest that a BRAF-independent mechanism of cell survival exists in anaplastic thyroid cancer cells.