The novel synthetic oleanane triterpenoid CDDO (2-cyano-3, 12-dioxoolean-1, 9-dien-28-oic acid) induces apoptosis in Mycosis fungoides/Sézary syndrome cells

The novel synthetic oleanane triterpenoid CDDO (2-cyano-3, 12-dioxoolean-1, 9-dien-28-oic acid) can serve as a ligand for the peroxisome proliferator activator receptor-gamma (PPAR-gamma) and has been shown to inhibit cell proliferation, and to induce differentiation and apoptosis in tumor cell lines. Bexarotene is an RXR-selective retinoid that can induce apoptosis of mycosis fungoides (MF) and Sézary syndrome (SS) cells. Since the PPAR-gamma and RXR receptors can form heterodimers, we studied the effects of CDDO and its synergism with bexarotene on apoptosis in MF/SS cell lines (MJ, Hut78, and HH) and freshly isolated peripheral blood lymphocytes (PBL) from SS patients with circulating atypical T cells (CD4+CD26-). CDDO treatment at 1-5 microM for 48 h caused a concentration-dependent apoptosis in three MF/SS cell lines and patients' PBL compared to vehicle controls. Bexarotene augmented CDDO-induced apoptosis in these cells. PPAR-gamma was expressed but decreased by 47% in MJ, 42% in Hut78, and 77% in HH cells following CDDO treatment. The anti-apoptotic protein bcl-xL, but not bcl-2, was decreased by 69% in MJ, 31% in Hut78, and 59% in HH cells and caspase-3 was activated following CDDO treatment. Interestingly, the PPAR-gamma antagonist T007 did not block CDDO-induced apoptosis, and the more potent PPAR-gamma agonist rosiglitazone required much higher concentrations (>50 microM) than CDDO to induce apoptosis in MF/SS cells. In summary, CDDO induces apoptosis that is further enhanced by bexarotene and decreases the PPAR-gamma and bcl-xL proteins in MF/SS cells. CDDO's effects on MF/SS cells may be at least partly mediated through a PPAR-gamma-independent mechanism. Our findings suggest the rationale for further investigation of the clinical potential of CDDO, either alone or in combination with bexarotene for MF/SS patients.     

Kinome profiling analysis identified Src pathway as a novel therapeutic target in combination with histone deacetylase inhibitors for cutaneous T-cell lymphoma

BackgroundHistone deacetylase inhibitors (HDACi) are used to treat patients with cutaneous T-cell lymphoma (CTCL), but they show limited efficacy. Hence, combination therapies should be explored to enhance the effectiveness of HDACis.ObjectiveThis study was conducted to identify novel therapeutic targets that can be combined with HDACis for treating CTCL.MethodsWe performed a global kinome profiling assay of three CTCL cell lines (HH, MJ, and Hut78) with three HDACis (romidepsin, vorinostat, and belinostat) using the PamChip® microarray. The three cell lines were co-treated with romidepsin and an inhibitor against the tyrosine kinase pathway.ResultsPrincipal component analysis revealed that kinome expression patterns were mainly related to the cell origin and were not affected by the drugs. Few kinases were commonly activated by the HDACis. Most identified kinases were Src-associated molecules, such as annexin A2, embryonal Fyn-associated substrate, and progesterone receptor. Phosphorylated Src was not observed in any untreated cell lines, whereas Src phosphorylation was detected in two of the three cell lines after HDACi treatment. Ponatinib, a Src inhibitor, significantly enhanced romidepsin-induced apoptosis not only in HH, MJ, and Hut78 cells, but also in Myla and SeAx CTCL cell lines.ConclusionThe Src pathway is a possible target for combination therapy involving HDACis for CTCL.     

西达本胺联合苦参碱对皮肤T细胞淋巴瘤细胞系增殖、凋亡的影响及可能的凋亡机制

【摘要】 目的 研究西达本胺联合苦参碱对皮肤T细胞淋巴瘤（CTCL）细胞系的增殖抑制和凋亡诱导作用,探讨其凋亡机制。方法 采用0.4 μmol/L西达本胺、0.6 g/L苦参碱单药或联合分别作用HH、Hut78细胞24、48、72 h后,MTS法检测HH、Hut78细胞增殖率。二甲基亚砜（DMSO）处理HH、Hut78细胞作为对照组。西达本胺、苦参碱单药或联合作用两细胞系48 h后,流式细胞仪检测HH和Hut78细胞凋亡率,Western免疫印迹法检测各组细胞凋亡相关蛋白的表达。统计分析采用重复测量、单因素方差分析,组间两两比较采用LSD-t检验。结果 与对照组相比,西达本胺、苦参碱单药或联合均能一定程度抑制HH、Hut78细胞增殖（F = 15.88、558.26,P < 0.05、 < 0.001）。48 h时,Hut78细胞系苦参碱组（20.98% ± 1.53%）、西达本胺组（22.44% ± 7.74%）和联合组细胞凋亡率（44.53% ± 1.85%）均显著高于对照组（8.42% ± 4.23%;LSD-t = 4.76、5.31、13.69,均P < 0.05）,且联合组显著高于苦参碱组和西达本胺组（LSD-t = 8.93、8.37,均P < 0.01）。HH细胞系苦参碱组（13.98% ± 3.86%）、西达本胺组细胞凋亡率（13.61% ± 1.62%）与对照组（11.44% ± 1.43%）相比差异无统计学意义（均P > 0.05）,但联合组（20.94% ± 0.64%）显著高于对照组、苦参碱组和西达本胺组（LSD-t = 7.37、5.40、5.69,均P < 0.05）。HH细胞系中,联合组caspase-3剪切体蛋白表达显著高于对照组、苦参碱组和西达本胺组（均P < 0.05）,而E-cadherin、p65、p-Bad及Bcl-2蛋白表达显著低于其他3组（均P < 0.05）。Hut78细胞系中,苦参碱组、西达本胺组和联合组E-cadherin、p65、p-Bad、Bcl-2蛋白表达均显著低于对照组（均P < 0.05）,仅西达本胺组和联合组caspase-3剪切体蛋白表达显著高于对照组（均P < 0.05）。HH及Hut78细胞4组Bad蛋白表达差异均无统计学意义（均P > 0.05）。结论 西达本胺联合苦参碱可抑制HH、Hut78细胞增殖,诱导其凋亡,其机制可能与调控细胞凋亡相关蛋白E-cadherin、p65、p-Bad、Bcl-2及caspase-3剪切体蛋白表达有关。     

Bexarotene activates the p53/p73 pathway in human cutaneous T-cell lymphoma

Background Bexarotene is the first synthetic retinoid X receptor‐selective retinoid (rexinoid) approved for the treatment of cutaneous T‐cell lymphoma (CTCL). However, little is known about the signalling pathways by which it exerts its anticarcinogenic effect. Objectives To characterize the effects of bexarotene in CTCL cell lines and elucidate the underlying molecular pathways of its antineoplastic effect. Methods The cell lines Hut‐78, HH and MJ were used. Cell viability was assessed with the XTT assay. The self‐renewal potential of cells after bexarotene treatment was studied with the methylcellulose clonogenic assay. Flow cytometry was used to analyse the effects on cell cycle, Ki‐67 expression and apoptosis induction. Cell cycle and apoptosis‐related protein expression were determined by Western blot and immunofluorescence. Results Bexarotene induced a loss of viability and more pronounced inhibition of clonogenic proliferation in HH and Hut‐78 cells, whereas the MJ line exhibited resistance. Bexarotene upregulated and activated Bax in sensitive lines, although not enough to signal significant apoptosis. Instead, all data point to the inhibition of proliferation, rather than apoptosis, as the main mechanistic action of the rexinoid. Bexarotene signals both G₁ and G₂/M arrest by the modulation of critical checkpoint proteins. We further found that bexarotene activates p53 by phosphorylation at Ser15, which influences the binding of p53 to promoters for cell cycle arrest, induces p73 upregulation, and, in concordance, also modulates some p53/p73 downstream target genes, such as p21, Bax, survivin and cdc2. Bexarotene‐mediated ataxia telangiectasia mutated protein (ATM) activation in all studied lines suggests that ATM is likely to be the p53/p73 upstream activator. Conclusions Our data indicate for the first time that bexarotene exerts its effect in CTCL mainly by triggering the p53/p73‐dependent cell cycle inhibition pathway, probably by upstream ATM activation. Therefore, bexarotene‐modulated genes represent potential biomarkers to assess the response to treatment of patients with CTCL.     

The retinoid derivant ECPIRM selectively exhibited anti-proliferation effects in cutaneous T-Cell lymphoma via ITK-mediated signaling pathway

BackgroundThe treatment of Cutaneous T-cell lymphoma (CTCL) met huge challenges because of the heterogeneity and the scarcity of targeted drugs. ECPIRM derived from isotretinoin exhibited strong anti-proliferation effects in Hut78 and MJ cells rather than Myla cells. However, there was no data regarding the potential target of ECPIRM for its selective activity.ObjectivesTo investigate the potential target of ECPIRM for its selective anti-proliferation activity.MethodsWe evaluated the cell viability of CTCL cells after ECPIRM treatment, and detected the effects of ECPIRM on the biomarker genes of CTCL. Subsequently, the mRNA and protein level of Interleukin-2-inducible T-cell kinase (ITK) was determined. Then the induction of apoptosis triggered by ITK inhibitor BMS-509744 and ITK siRNAs were detected, and the docking of ECPIRM interacted with ITK and the effects of ECPIRM on ITK-mediated signaling pathway were analyzed. Finally, we evaluated the anti-growth activity of ECPIRM in Hut78-xenografted nude mice, and the relative expression of cleaved caspase-3, ITK, p-ERK and p-Akt were determined.ResultsITK was highly expressed in Hut78 and MJ cells rather than Myla cells, and targeted inhibition of ITK triggered cell apoptosis. ECPIRM efficiently bound the hydrophobic active pocket of ITK, and significantly inhibited ITK-mediated signaling pathway. In addition, ECPIRM suppressed tumor growth in Hut78-xenografted model, and upregulated the expression of cleaved caspase 3 and inhibited the expression of ITK, p-ERK and p-Akt in tumor tissues, which was consistent with in vitro study.ConclusionECPIRM might provide a novel strategy for CTCL by inhibiting ITK-mediated signaling pathway.     

Apoptosis Induction by SAHA in Cutaneous T-Cell Lymphoma Cells Is Related to Downregulation of c-FLIP and Enhanced TRAIL Signaling

Suberoylanilide hydroxamic acid (SAHA) has been approved for the treatment of cutaneous T-cell lymphoma (CTCL), but its mode of action remained largely elusive. As shown here in four CTCL cell lines, loss of cell viability correlated with significant time- and dose-dependent induction of apoptosis, whereas cytotoxicity was less pronounced. Both extrinsic and intrinsic apoptosis pathways were activated, as seen by processing of initiator caspases 8 and 9, loss of mitochondrial membrane potential, and cytochrome c release. Characteristically, antiapoptotic mediators such as Mcl-1, XIAP, survivin, and c-FLIP were downregulated. Consistent with its critical function, c-FLIP overexpression resulted in a significant decrease of SAHA-mediated apoptosis. Enhanced sensitivity to TRAIL (TNF-related apoptosis-inducing ligand) and enhanced TRAIL signaling was seen in CTCL cell lines with high sensitivity, whereas cell lines with moderate response were characterized by downregulation of TRAIL-R2 and weaker TRAIL expression. Comparable proapoptotic responses to SAHA and to the combination with TRAIL were seen in ex vivo tumor T cells of CTCL patients. Thus, activation of extrinsic apoptosis pathways, related to c-FLIP downregulation and enhanced TRAIL signaling, appeared as characteristic for CTCL cell responsiveness to SAHA. An improved understanding of the pathways may facilitate its targeted use and the selection of suitable combinations.     

Blockade of death receptor-mediated pathways early in the signaling cascade coincides with distinct apoptosis resistance in cutaneous T-cell lymphoma cells

Control of apoptosis via death ligands plays a basic role for lymphocyte homeostasis and lymphoma development. In this study, cutaneous T-cell lymphoma (CTCL) cell lines revealed pronounced resistance to death ligands as compared to cell lines of T-cell acute lymphoblastic leukemia (T-ALL). The proapoptotic activity of tumor necrosis factor (TNF)-alpha was blocked, sensitivity to TNF-related apoptosis-inducing ligand was significantly reduced, and 1/4 CTCL cell lines was resistant to CD95 activation. In parallel, there was no activation of effector caspase-3 and initiator caspase-8 in nonresponsive CTCL cells, whereas caspase-10 was cleaved selectively in sensitive CTCL cells. No indication for a responsibility of typical downstream regulators of apoptosis was obtained, but loss of CD95 was found in 1/4, loss of TNF-R1 in 3/4, loss of caspase-10 in 2/4, loss of Bid in 1/4, and overexpression of cellular flice inhibitory protein was found in 4/4 CTCL cell lines. This clearly indicates an inhibition of apoptosis early in the extrinsic cascade, namely at the formation of the death-inducing signaling complex. Parallels with regard to expression of apoptosis regulators were seen in peripheral blood mononuclear cells and biopsies of CTCL patients. This study may indicate defects in apoptosis in CTCL and may help to guide CTCL therapy.     

hTERT反义寡核苷酸抑制Hut78细胞端粒酶活性及诱导细胞凋亡的研究

目的 探讨人端粒酶逆转录酶(hTERT)基因反义寡核苷酸对皮肤T细胞淋巴瘤(CTCL)细胞株端粒酶活性及细胞生长的影响,为CTCL基因治疗提供新的基因靶点。方法 采用脂质体介导的基因转染方法,将不同浓度(10μmol/L、20μmol/L、30μmol/L)的寡核苷酸分别导入CTCL细胞株Hut78中;分别于不同的时间,应用端粒酶重复序列扩增及酶联免疫吸附方法(PCR-ELISA)、逆转录-聚合酶链反应(RT-PCR)、流式细胞仪技术等动态观察转染细胞中端粒酶活性、hTERT mRNA表达以及细胞凋亡的变化。结果 经hTERT反义寡核苷酸(AODN)作用72h后,细胞端粒酶的活性明显下降,hTERT mRNA的表达减弱,并可观察到细胞凋亡,凋亡细胞发生率为13.05%。细胞生长抑制作用有明显的时效性,以30μmol/L、72h时下降最明显。而有义寡核苷酸(SODN)组和对照组以上指标均无明显变化(P>0.05)。结论 hTERT反义寡核苷酸可显著抑制CTCL细胞的端粒酶活性,抑制细胞生长增殖,诱导细胞凋亡。     

Antitumour effects in mycosis fungoides of the immunomodulatory, tellurium-based compound, AS101

BACKGROUND: The immunomodulator AS101 [ammonium trichloro (dioxoethylene-O,O') tellurate], a nontoxic tellurium (IV) compound, has antitumoral effects which were demonstrated in several preclinical and clinical studies. OBJECTIVES: To investigate the antitumour activity of AS101 on cutaneous T-cell lymphoma (CTCL), of which mycosis fungoides (MF) is the most frequent disease variant. METHODS: We used a newly established mouse xenograft model for MF to test the effect of AS101 in vivo and analysed apoptosis induction in vitro. RESULTS: When injected intratumorally, AS101 delayed tumour growth in a dose-dependent manner. In vitro, AS101 induced a dose-dependent G2/M arrest in the CTCL cell lines Hut78 and MyLa. Moreover, higher concentrations of AS101 induced apoptosis in MyLa cells. Programmed cell death was associated with the loss of mitochondrial transmembrane potential and activation of caspase 9 and caspase 3. AS101 also elevated intracellular reactive oxygen species (ROS) production; the antioxidant, Mn superoxide dismutase, significantly reduced the degree of apoptosis, suggesting that ROS play a key role in apoptosis induction. CONCLUSIONS: These findings indicate that AS101 may be a promising antitumour drug for CTCL.     

IFN-α对人皮肤淋巴瘤细胞系Hut78的影响

目的研究IFN-α对人皮肤T细胞淋巴瘤(CTCL)细胞的增殖抑制和凋亡诱导作用,探讨IFN-α治疗CTCL的机制。方法分别用3000,5000,10000,20000U/mL的IFN-α作用Hut78细胞24,48,72h后,利用MTS法检测Hut78细胞的生存率;并用流式细胞术检测不同浓度IFN-α作用细胞24h后,Hut78细胞的凋亡情况。运用Real-time PCR检测不同时间点IFN-α作用后,BCL11B mRNA的表达情况,Western blot检测不同时间点IFN-α作用后BCL11B蛋白的表达情况,免疫荧光检测IFN-α不同时间点作用后,BCL11B蛋白在Hut78细胞表达程度和分布情况。结果 IFN-α能明显抑制Hut78细胞的增殖,且这种作用呈剂量依赖性和时间依赖性。其抑制效应在48h达到峰值。IFN-α还能显著诱导Hut78细胞的凋亡,其作用亦呈浓度依赖性。10000U/mL IFN-α作用细胞3,6,9,12h后,BCL11B mR-NA表达均显著降低,其中在6h时间点BCL11B的mRNA表达下降到最低点,10000U/mL IFN-α作用细胞6,12,24h后BCL11B的蛋白也明显降低,在12h时间点,BCL11B的蛋白表达量下降至最低。结论 IFN-α通过抑制细胞增殖和促进细胞凋亡对CTCL起到治疗作用,并可通过降低BCL11B的表达,促进细胞凋亡,并提高CTCL细胞对常规化疗的敏感性。     

Nonsteroidal anti-inflammatory drugs induce apoptosis in cutaneous T-cell lymphoma cells and enhance their sensitivity for TNF-related apoptosis-inducing ligand

Cutaneous T-cell lymphomas (CTCL) form a heterogeneous group of non-Hodgkin's lymphomas of the skin. In previous studies, we had characterized CTCL cells as resistant to the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which correlated to pronounced expression of the caspase-8/-10 inhibitor c-FLIP. For identification of proapoptotic strategies in CTCL cells and for overcoming their death ligand resistance, we investigated the effects of nonsteroidal anti-inflammatory drugs (NSAIDs) such as acetylsalicylic acid, sodium salicylate, and diclofenac (DF). These drugs strongly enhanced apoptosis, as well as decreased CTCL cell proliferation and vitality, and DF furthermore sensitized for TRAIL-induced apoptosis. Full activation of the caspase cascade (caspase-3, -8, -9) and decreased mitochondrial membrane potential were characteristic for NSAID treatment, whereas cytochrome c release was seen only for DF. Downregulation of Mcl-1 and enhanced surface expression of TRAIL were seen in response to NSAIDs. Most characteristic for apoptosis induction was the downregulation of c-FLIP. In agreement with the critical role of c-FLIP for apoptosis deficiency of CTCL cells, its overexpression decreased NSAID-mediated apoptosis and its downregulation by small hairpin RNA-enhanced apoptosis. The study provides a rationale for the use of NSAIDs as a new therapeutic option for CTCL patients. Supporting this concept, ex vivo lymphoma cells of CTCL patients also revealed significant sensitivity for NSAID treatment.     

西达本胺联合姜黄素对人皮肤T细胞淋巴瘤细胞系Hut78的影响及其分子机制

目的 研究西达本胺联合姜黄素对皮肤T细胞淋巴瘤（CTCL）细胞的增殖抑制和凋亡诱导作用,探讨西达本胺联合姜黄素治疗CTCL的机制。 方法 分别用0.3、0.6、1.2、2.4 μmol/L西达本胺,10 μmol/L姜黄素,1.2 μmol/L西达本胺联合10 μmol/L姜黄素处理Hut78细胞24、48、72 h后,采用MTS法检测Hut78细胞的生存率。用0.6、1.2 μmol/L西达本胺,10 μmol/L姜黄素,1.2 μmol/L西达本胺联合10 μmol/L姜黄素分别处理Hut78细胞24 h,用流式细胞仪检测细胞凋亡情况及细胞周期,用实时PCR和Western印迹法检测凋亡相关基因Fas、caspase 8、NF-κB p65及细胞周期相关基因P21、CDK2、细胞周期蛋白E（cyclin E） mRNA和蛋白的表达。统计分析采用重复测量方差分析、单因素方差分析和LSD-t检验。 结果 西达本胺能明显抑制Hut78细胞的增殖,且呈剂量依赖性（F = 266.558,P < 0.001）和时间依赖性（F = 564.966,P < 0.001）。培养48 h和72 h时,1.2 μmol/L西达本胺联合10 μmol/L姜黄素对细胞增殖的抑制作用显著强于1.2 μmol/L西达本胺及10 μmol/L姜黄素（均P < 0.001）。流式细胞仪结果显示,联合组的凋亡细胞比例均显著高于0.6、1.2 μmol/L西达本胺组和10 μmol/L姜黄素组（均P < 0.001）。此外,联合组和1.2 μmol/L西达本胺组G0/G1期细胞比例明显高于0.6 μmol/L西达本胺组和10 μmol/L姜黄素组,而其S期细胞比例及G2/M期细胞比例均明显低于0.6 μmol/L西达本胺组和10 μmol/L姜黄素组（均P < 0.05）,联合组与1.2 μmol/L西达本胺组各细胞周期比例差异均无统计学意义（均P > 0.05）。实时PCR结果显示,联合组和1.2 μmol/L西达本胺组Fas、caspase 8、P21 mRNA的表达均明显高于0.6 μmol/L西达本胺组和10 μmol/L姜黄素组（均P < 0.001）,而其NF-κB p65、CDK2、cyclin E mRNA的表达均明显低于0.6 μmol/L西达本胺组和10 μmol/L姜黄素组（均P < 0.001）。联合组Fas mRNA的表达明显高于1.2 μmol/L西达本胺组（P < 0.001）,而caspase 8、P21、NF-κB p65、CDK2、cyclin E mRNA的表达与1.2 μmol/L西达本胺组差异无统计学意义（均P > 0.05）。Western印迹结果显示,联合组与0.6、1.2 μmol/L西达本胺、不加药物的对照组比较,Fas、caspase 8、P21蛋白的表达明显增加,而NF-κB p65、CDK2、CyclinE蛋白的表达明显降低,与以上基因mRNA的表达一致。 结论 西达本胺通过抑制细胞增殖和促进细胞凋亡来抑制CTCL细胞系Hut78生长,联合姜黄素能明显提高抑制CTCL细胞系Hut78的生长率。     

Proteasome inhibition as a novel mechanism of the proapoptotic activity of γ‐secretase inhibitor I in cutaneous T‐cell lymphoma

Background We have previously discovered that Notch1 is expressed on malignant T cells in cutaneous T‐cell lymphoma (CTCL), and is required for survival of CTCL cell lines. Notch can be inhibited by γ‐secretase inhibitors (GSIs), which differ widely in their ability to induce apoptosis in CTCL. Objectives To investigate whether GSI‐I, in addition to inhibiting Notch, induces apoptosis in CTCL by proteasome inhibition, as GSI‐I is very potent and has structural similarity to the proteasome inhibitor MG‐132. Methods Cell lines derived from CTCL (MyLa, SeAx, JK, Mac1 and Mac2a) were treated with GSI‐I and two other proteasome inhibitors (MG‐132 and bortezomib). The effects on cell viability, apoptosis and proteasome activity were measured, as was the impact on the prosurvival, nuclear factor κB (NF‐κB) pathway. Results In CTCL, GSI‐I had proteasome‐blocking activity with a potency comparable to the proteasome inhibitors MG‐132 and bortezomib. Proteasome inhibition was the main mechanism responsible for GSI‐I‐induced cell death, as tiron, a compound known to reverse the effect of MG‐132, restored proteasome activity and largely abrogated the cytotoxic effect of GSI‐I. Although inactivation of NF‐κB is an important mechanism of action for proteasome inhibitors, we demonstrated an apparent activation of NF‐κB. Furthermore, we showed that while the tumour suppressor protein p53 was induced during proteasome inhibition, it was dispensable for CTCL apoptosis, as both SeAx cells, which harbour p53 mutations that attenuate the apoptotic capacity, and HuT‐78 cells, which have a deleted p53 gene, demonstrated potent apoptotic response. Conclusions GSI‐I represents an interesting drug with a dual mechanism of action comprising inhibition of both Notch and the proteasome.     

溶血磷脂酸受体6在蕈样肉芽肿向大细胞转变中的意义及其对皮肤T细胞淋巴瘤细胞增殖和凋亡的影响

【摘要】 目的 探讨皮肤T细胞淋巴瘤（CTCL）蕈样肉芽肿（MF）患者中溶血磷脂酸受体6（LPAR6）的表达水平及其对CTCL疾病发展及预后的影响与可能机制。方法 收集北京大学第一医院皮肤性病科2011—2020年间确诊的110例MF患者,包括49例发现队列的24例大细胞转变（LCT）及25例非大细胞转变（NLCT）,61例验证队列的24例LCT及37例NLCT。提取患者组织总RNA分别采用RNA测序及RT-PCR检测LPAR6的表达,比较LPAR6在LCT与NLCT患者间的表达差异及对生存预后的影响。构建2种CTCL细胞系MyLa、Sz4过表达LPAR6细胞模型,以正常表达细胞为对照,检测过表达LPAR6对MyLa、Sz4细胞增殖活力的影响;通过加入LPAR6相关配体溶血磷脂酸（LPA）、2S-OMPT、三磷酸腺苷（ATP）、腺苷（ADO）,采用细胞增殖实验检测试剂盒及流式细胞仪检测激活LPAR6对过表达LPAR6 MyLa、Sz4细胞增殖活力与凋亡的影响。采用log-rank检验进行预后分析,两组间数据的比较采用t检验或Mann-Whitney U检验。结果 RNA测序结果显示,发现队列中LPAR6是LCT中显著性低表达的基因之一;验证队列中,LCT组的LPAR6表达水平［M（Q1,Q3）为204.90（81.90,512.70）］低于NLCT组［809.40（417.50,1 829.20）,U = 242.00,P = 0.002］;两队列中,低表达LPAR6均与不良预后的发生风险显著相关（P < 0.01）。细胞增殖实验结果显示,在实验的0、24、48、72 h,过表达组MyLa、Sz4细胞与对照组细胞的细胞增殖活力差异均无统计学意义（均P > 0.05）;分别采用LPA、2S-OMPT、ATP、ADO激活MyLa细胞LPAR6后48 h,过表达组MyLa细胞的增殖活力（1.38 ± 0.01、1.04 ± 0.01、1.09 ± 0.03、1.23 ± 0.01）均低于对照组（1.73 ± 0.04、1.23 ± 0.01、1.24 ± 0.01、1.42 ± 0.03,t值分别为30.33、18.38、4.78、5.75,均P < 0.05）,细胞凋亡率（17.93% ± 0.88%、17.75% ± 0.35%、23.97% ± 0.57%、31.44% ± 0.34%）均高于对照组（3.98% ± 0.03%、7.81% ± 0.59%、11.95% ± 0.85%、12.02% ± 0.48%,t值分别为15.93、14.49、11.74、33.01,均P < 0.05）;分别采用2S-OMPT、ADO激活Sz4细胞LPAR6后48 h,Sz4细胞增殖活力2S-OMPT对照组为1.48 ± 0.01、过表达组为1.29 ± 0.04;ADO对照组为1.51 ± 0.02、过表达组为1.27 ± 0.01,过表达组均低于对照组（P < 0.05）,细胞凋亡率均高于对照组（2S-OMPT对照组29.35% ± 0.55%、过表达组41.70% ± 0.70%;ADO对照组24.60% ± 1.00%、过表达组37.05% ± 0.15%,均P < 0.05）。结论 LPAR6在LCT患者中低表达并与不良预后的发生风险显著相关,体外激活LPAR6能够抑制CTCL细胞的增殖,促进细胞凋亡,LCT患者LPAR6表达水平降低可能是疾病进展的重要机制之一。     

鬼臼毒素纳米脂质载体通过线粒体途径诱导人阴道上皮细胞凋亡的机制研究

目的:研究鬼臼毒素纳米脂质载体(POD-NLC)对体外培养的永生化人阴道上皮细胞(VK2/E6E7细胞)线粒体途径相关凋亡基因表达的影响,探索POD-NLC诱导VK2/E6E7细胞凋亡的作用机制。方法:自制0.5%POD-NLC,用不同浓度的POD-NLC(0.25,1μg/mL)和空白NLC作用VK2/E6E7细胞24 h后,采用实时荧光定量核酸扩增法(Real-time Quantitative PCR,qPCR)检测bcl-2、bax、caspace-3、caspase-9等mRNA表达水平,Western Bolt检测bcl-2、bax蛋白的表达水平。结果:POD-NLC呈剂量依赖性的上调bax mRNA的表达水平(P值均<0.01),同时也增加caspase-3、caspase-9 mRNA的表达(P值均<0.05),并降低bcl-2 mRNA表达量(P值均<0.01);空白-NLC组对上述mRNA的影响均无统计学意义(P值均>0.05)。POD-NLC可剂量依赖性地上调bax蛋白表达量(P值均<0.01),同时下调bcl-2蛋白表达量(P值均<0.01),但各药物处理组之间差异无统计学意义(P值均>0.05)。结论:POD-NLC能通过线粒体途径诱导VK2/E6E7细胞发生凋亡。