Expression Changes and Impact of the Extracellular Matrix on Etoposide Resistant Human Retinoblastoma Cell Lines
- 作者列表："Jacqueline Reinhard","Natalie Wagner","Miriam M. Krämer","Marvin Jarocki","Stephanie C. Joachim","H. Burkhard Dick","Andreas Faissner","Vinodh Kakkassery
Retinoblastoma (RB) represents the most common malignant childhood eye tumor worldwide. Several studies indicate that the extracellular matrix (ECM) plays a crucial role in tumor growth and metastasis. Moreover, recent studies indicate that the ECM composition might influence the development of resistance to chemotherapy drugs. The objective of this study was to evaluate possible expression differences in the ECM compartment of the parental human cell lines WERI-RB1 (retinoblastoma 1) and Y79 and their Etoposide resistant subclones via polymerase chain reaction (PCR). Western blot analyses were performed to analyze protein levels. To explore the influence of ECM molecules on RB cell proliferation, death, and cluster formation, WERI-RB1 and resistant WERI-ETOR cells were cultivated on Fibronectin, Laminin, Tenascin-C, and Collagen IV and analyzed via time-lapse video microscopy as well as immunocytochemistry. We revealed a significantly reduced mRNA expression of the proteoglycans <i>Brevican</i>, <i>Neurocan,</i> and <i>Versican</i> in resistant WERI-ETOR compared to sensitive WERI-RB1 cells. Also, for the glycoproteins <i>α1-Laminin</i>, <i>Fibronectin</i>, <i>Tenascin-C,</i> and <i>Tenascin-R</i> as well as <i>Collagen IV</i>, reduced expression levels were observed in WERI-ETOR. Furthermore, a downregulation was detected for the matrix metalloproteinases <i>MMP2</i>, <i>MMP7</i>, <i>MMP9</i>, the tissue-inhibitor of metalloproteinase <i>TIMP2</i>, the Integrin receptor subunits <i>ITGA4</i>,<i> ITGA5 </i>and <i>ITGB1, </i>and all <i>receptor protein tyrosine phosphatase β/ζ</i> isoforms. Downregulation of<i> Brevican</i>, <i>Collagen IV</i>, <i>Tenascin-R</i>, <i>MMP2</i>, <i>TIMP2,</i> and <i>ITGA5</i> was also verified in Etoposide resistant Y79 cells compared to sensitive ones. Protein levels of Tenascin-C and MMP-2 were comparable in both WERI cell lines. Interestingly, Fibronectin displayed an apoptosis-inducing effect on WERI-RB1 cells, whereas an anti-apoptotic influence was observed for Tenascin-C. Conversely, proliferation of WERI-ETOR cells was enhanced on Tenascin-C, while an anti-proliferative effect was observed on Fibronectin. In WERI-ETOR, cluster formation was decreased on the substrates Collagen IV, Fibronectin, and Tenascin-C. Collectively, we noted a different ECM mRNA expression and behavior of Etoposide resistant compared to sensitive RB cells. These findings may indicate a key role of ECM components in chemotherapy resistance formation of RB.
视网膜母细胞瘤 (Retinoblastoma，RB) 代表了全球最常见的恶性儿童眼部肿瘤。一些研究表明，细胞外基质 (ECM) 在肿瘤的生长和转移中起着至关重要的作用。此外，最近的研究表明，ECM 成分可能会影响化疗药物耐药性的发展。本研究的目的是评估亲代人类细胞系 WERI-RB1 (视网膜母细胞瘤 1) 的 ECM 室中可能的表达差异。和 Y79 及其依托泊苷抗性亚克隆通过聚合酶链反应 (PCR)。进行 Western blot 分析以分析蛋白水平。为探讨细胞外基质 (ECM) 分子对 RB 细胞增殖、死亡及成簇形成的影响，以纤维连接蛋白 (fn) 、层粘连蛋白 (Laminin) 、 Tenascin-C (Tenascin-C) 为材料，对 RB 细胞进行抗 WERI-RB1 (WERI-ETOR) 培养。和 IV 型胶原，并通过延时视频显微镜以及免疫细胞化学进行分析。我们发现蛋白多糖 <i>Brevican</i> 、 <i>Neurocan 的 mRNA 表达显著降低,</i> 和 <i>Versican</i> 在耐药 WERI-ETOR 与敏感的 WERI-RB1 细胞相比。此外，对于糖蛋白 <i>α 1-层粘连蛋白 </i>，<i> 纤维连接蛋白 </i>，<i>Tenascin-C,</i> 和 <i>Tenascin-R</i> 以及 <i> 胶原 IV</i>，在 WERI-ETOR 中观察到表达水平降低。此外，检测到基质金属蛋白酶 <i>MMP2</i> 、 <i>MMP7</i> 、 <i>MMP9</i> 下调,金属蛋白酶组织抑制剂 <i>TIMP2</i>，整合素受体亚基 <i>ITGA4</i>，<i> ITGA5 </i> 和 <i>ITGB1, </i> 和所有 <i> 受体蛋白酪氨酸磷酸酶 β/ζ</i> 亚型。下调 <i> Brevican</i> 、 <i>Collagen IV</i> 、 <i>Tenascin-R</i> 、与敏感细胞相比，在依托泊苷耐药的 Y79 细胞中也验证了 <i>MMP2</i> 、 <i>TIMP2 </i> 和 <i>ITGA5</i>。Tenascin-C 和 MMP-2 的蛋白水平在两种 WERI 细胞系中相当。有趣的是，纤维连接蛋白对 WERI-RB1 细胞显示凋亡诱导作用，而 Tenascin-C 则观察到抗凋亡作用。相反，WERI-ETOR 细胞在 Tenascin-C 上的增殖增强，而在纤维连接蛋白上观察到抗增殖作用。在 WERI-ETOR 中，基质胶原 IV 、纤维连接蛋白和 Tenascin-C 上的簇形成减少。总的来说，我们注意到与敏感的 RB 细胞相比，依托泊苷耐药的 ECM mRNA 表达和行为不同。这些发现可能表明 ECM 成分在 RB 化疗耐药形成中的关键作用。
METHODS::Chimeric antigen receptor (CAR) T cells are a novel class of anti-cancer therapy in which autologous or allogeneic T cells are engineered to express a CAR targeting a membrane antigen. In Europe, tisagenlecleucel (Kymriah™) is approved for the treatment of refractory/relapsed acute lymphoblastic leukemia in children and young adults as well as relapsed/refractory diffuse large B-cell lymphoma, while axicabtagene ciloleucel (Yescarta™) is approved for the treatment of relapsed/refractory high-grade B-cell lymphoma and primary mediastinal B-cell lymphoma. Both agents are genetically engineered autologous T cells targeting CD19. These practical recommendations, prepared under the auspices of the European Society of Blood and Marrow Transplantation, relate to patient care and supply chain management under the following headings: patient eligibility, screening laboratory tests and imaging and work-up prior to leukapheresis, how to perform leukapheresis, bridging therapy, lymphodepleting conditioning, product receipt and thawing, infusion of CAR T cells, short-term complications including cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, antibiotic prophylaxis, medium-term complications including cytopenias and B-cell aplasia, nursing and psychological support for patients, long-term follow-up, post-authorization safety surveillance, and regulatory issues. These recommendations are not prescriptive and are intended as guidance in the use of this novel therapeutic class.
METHODS:The aim of the present study was to explore the possible mechanisms of phosphatase and tensin homolog (PTEN) in the pathogenesis of Burkitt's lymphoma, and provide novel information that can be used in the targeted treatment of this disease. PTEN lentiviral overexpression vector and short‑hairpin PTEN silencing vectors were constructed. The effect of PTEN on the growth and proliferation of CA46 and RAJI cells was analyzed using a Cell Counting Kit‑8 assay. Apoptosis was detected by Hoechst 33342 and propidium iodide double staining. Flow cytometry was used to analyze the cell cycle. A Transwell chamber was used to detect cell migration and invasion abilities. Western blot analysis was used to detect related protein changes. The mechanism of the effect of PTEN on the biological characteristics of Burkitt's lymphoma cells was subsequently analyzed. The results revealed that PTEN inhibited the proliferation of CA46 and RAJI cells by downregulating the expression of p‑AKT, It was indicated that the upregulation of proapoptotic proteins (including Bad and Bax) induced apoptosis, regulated cyclin (including P53, P21, CDK4, CDK6, cyclin D3 and cyclin H) to inhibit cell cycle progression, and mediated epithelial‑mesenchymal transition‑like cell markers (including E‑cadherin, N‑cadherin, β‑catenin, TCF‑8, vimentin, Slug and Snail) to inhibit cell migration and invasion. In conclusion, the tumor‑suppressor gene PTEN inhibited the phosphoinositide 3‑kinase/protein kinase B (PI3K/AKT) signaling pathway and inhibited the proliferation and migration of Burkitt's lymphoma cells, induced apoptosis and cell cycle arrest, thus playing a crucial role in the pathogenesis of Burkitt's lymphoma.
METHODS:Melatonin (Mel) has been shown to involve in many essential cell functions via modulating many signaling pathways. We for the first time investigated that Mel exerted anti-tumor activities in Hodgkin lymphoma (HL) via inhibiting cell proliferation and promoting cell apoptosis. Further study revealed that Mel treatment increased expression of LC3-II and decreased p62 proteins with the enhanced production of autolysosome, indicating it induced activation of autophagy. Nevertheless, Mel treatment together with autophagy inhibitors 3-MA or CQ exacerbated the damage effect of Mel in HL cells, which means autophagy plays a protective role in this process. Furthermore, we found Mel treatment increased the expression of G protein-coupled receptors MT2 and retinoic acid-related orphan receptors (RORs), eg. RORA, RORB and RORC. While RORC has the highest increase in Mel treated HL cells. In addition, RORC overexpression induced autophagy activation. Therefore, Mel showed tumor-suppressive role due to an increased level of RORC induced autophagy in HL.