背景:NSCLC是死亡的主要原因之一,通常在晚期诊断,没有替代治疗方法。DC是专业的抗原呈递细胞,基于DC的免疫疗法因其抗癌特性而备受关注。表观遗传修饰包括DNA甲基化和组蛋白修饰在调节其功能如成熟和激活中起着至关重要的作用。先天免疫反应,T细胞启动,抗原呈递,和细胞因子的产生。在目前的研究中,我们研究了阿霉素在非细胞毒性浓度下的抗癌特性,该浓度可以外推作为DC成熟的表观遗传调节因子,从而引发抗肿瘤活性.
方法:分离来自正常和NSCLC血液样品的PBMC并用生长因子处理。用低剂量阿霉素使DC成熟,并且通过使用流式细胞术检查DC成熟标记。Further,进行ELISA,并将低剂量阿霉素诱导的DC用LCA(肺癌抗原)脉冲并用CD4+T辅助(Th)细胞进行细胞毒性评估。Further,T:DC缀合的表观遗传标记在显微镜下免疫荧光可视化。ChIP-qPCR和Invitro测定,如组蛋白甲基化,DNA甲基化,进行m6A甲基化以研究低剂量Dox治疗下的表观遗传变化。进行IL-12中和测定以检查DC的IL-12依赖性及其在低剂量治疗下在Dox下的作用。随后进一步进行组蛋白和非组蛋白蛋白的Western印迹分析。
结果:低剂量阿霉素诱导DC的表观遗传变化,通过产生CTL并伴随抗炎细胞因子的细胞外分泌增加,在NSCLC中引发抗肿瘤反应。我们还发现,低剂量的阿霉素在用LCA脉冲并用CD4+T辅助细胞引发时成熟的DC,分泌IFN-γ,其在通过激活CD8+细胞毒性T淋巴细胞协调适应性免疫中是重要的。此外,IL-12的分泌帮助我们推断保护性免疫也是通过Th1反应诱导的,Th1反应选择性地触发PKCθ向DC和Th之间的免疫突触易位。Further,甲基化和乙酰化标志物H3K4me3和H3K14Ac分别上调,而STAT5,NFkB,NOTCH1和DNAPKcs下调。然后DNA和RNA甲基化测定导致关于由低剂量Dox治疗引起的表观遗传变化的确认。DNA甲基化减少,导致抑癌基因p53和Th1相关转录因子TBX21的激活。另一方面,在低剂量的Dox存在下,绝对和相对RNA甲基化定量均增加。
结论:从这项研究来看,我们了解到,非细胞毒性浓度的阿霉素通过IL-12依赖性机制增加DCs的Ag递呈能力,并在NSCLC中引起表观遗传修饰.
BACKGROUND: NSCLC is one of the leading causes of death and is often diagnosed at late stages with no alternative therapeutic approach. DCs are professional antigen-presenting cells and DC-based immunotherapy has been under the spotlight for its anti-cancer properties. Epigenetic modifications including DNA methylation and histone modification in DCs play a crucial role in regulating their functions such as maturation and activation,innate immune responses, T cell priming, antigen presentation, and cytokine production. In the current study, we investigated the anti-cancer properties of Doxorubicin at a noncytotoxic concentration that could be extrapolated as an epigenetic regulator for DC maturation to elicit anti-tumor activity.
METHODS: PBMCs from normal and NSCLC blood samples were isolated and treated with growth factors. DCs were matured with low dose Doxorubicin and the DC maturation markers were checked by using flow-cytometry. Further, ELISA was performed and low dose Doxorubicin-induced DCs were pulsed with LCA (Lung Cancer Antigen) and primed with CD4 +T helper (Th) cells for cytotoxicity assessment. Further, epigenetic markers of T: DC conjugation were immunofluorescently visualized under a microscope. ChIP-qPCR and Invitro assays such as histone methylation, DNA methylation, and m6A methylation were performed to study the epigenetic changes under low dose Dox treatment. IL-12 neutralization assay was performed to check for the IL-12 dependency of DCs and their effect under Dox at low dose treatment. This was further followed by a Western Blotting analysis for histone and non-histone proteins.
RESULTS: Low dose Doxorubicin induces epigenetic changes in DCs to elicit an anti-tumor response in NSCLC through the generation of CTLs with a concomitant increase in the extracellular secretions of anti-inflammatory cytokines. We also found that low dosage of Doxorubicin matured DCs when pulsed with LCA and primed with CD4 +T helper cells, secrete IFN-γ which is important in orchestrating adaptive immunity by activating CD8 + cytotoxic T-lymphocytes. Also, the secretions of IL-12 help us infer that protective immunity is also induced via Th1 response which triggered selectively the translocation of PKCθ to immunological synapse in between DC and Th. Further, methylation and acetylation markers H3K4me3 and H3K14Ac respectively upregulated whereas levels of STAT5, NFkB, NOTCH1, and DNAPKcs were downregulated. DNA and RNA methylation assays then lead to confirmations about the epigenetic changes caused by low dose Dox treatment. DNA methylation was reduced which resulted in the activation of tumor suppressor gene p53 and Th1-associated transcription factor TBX21. On the other hand, both absolute and relative RNA methylation quantification increased in the presence of Dox at a low dose.
CONCLUSIONS: From this study, we understand that non-cytotoxic concentration of Doxorubicin increases the Ag-presenting ability of DCs via an IL-12-dependent mechanism and causes epigenetic modifications in NSCLC.