Novel engineered TRAIL-based chimeric protein strongly inhibits tumor growth and bypasses TRAIL resistance

Targeting of the TRAIL-DR4/5 pathway was proposed as a promising approach for specific induction of apoptosis in cancer cells. Clinical trials, however, showed inadequate efficiency of TRAIL as a monotherapy. It is a widely held view that the application of multifunctional molecules or combination therapy may lead to substantial improvement. Here, we demonstrate the effectiveness and safety of a novel chimeric protein, AD-O51.4, which is a TRAIL equipped with positively charged VEGFA-derived effector peptides. The study was performed in multiple cancer cell line- and patient-derived xenografts. A pharmacokinetic profile was established in monkeys. AD-O51.4 strongly inhibits tumor growth, even leading to complete long-term tumor remission. Neither mice nor monkeys treated with AD-O51.4 demonstrate symptoms of drug toxicity. AD-O51.4 exhibits a satisfactory half-life in plasma and accumulates preferentially in tumors. The cellular mechanism of AD-O51.4 activity involves both cytotoxic effects in tumor cells and antiangiogenic effects on the endothelium. The presence of DRs in cancer cells is crucial for AD-O51.4-driven apoptosis execution. The TRAIL component of the fusion molecule serves as an apoptosis inducer and a cellular anchor for the effector peptides in TRAIL-sensitive and TRAIL-resistant cancer cells, respectively. The FADD-dependent pathway, however, seems to be not indispensable in death signal transduction; thus, AD-O51.4 is capable of bypassing the refractoriness of TRAIL. AD-O51.4-driven cell death, which exceeds TRAIL activity, is achieved due to the N-terminally fused polypeptide, containing VEGFA-derived effector peptides. The high anticancer efficiency of AD-O51.4 combined with its safety has led to the entry of AD-O51.4 into toxicological studies.     

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目的探讨不同时期婴幼儿口腔颌面部血管瘤TRAIL受体DR4、DR5 m RNA的表达及意义。方法本研究于2013年3—10月在中国医科大学中心实验室完成。收集婴幼儿口腔颌面部血管瘤组织标本40例(增殖期血管瘤26例、退化期血管瘤14例),以及正常皮肤组织标本12例,应用RT-PCR检测TRAIL的2个受体DR4和DR5m RNA在其中的分布情况。结果增殖期血管瘤组织中DR4和DR5 m RNA的表达强度显著低于退化期血管瘤和正常皮肤组织,差异有统计学意义(P<0.01)。退化期血管瘤和正常皮肤组织中DR4、DR5 m RNA的表达强度较高,但二者之间差异无统计学意义。结论退化期血管瘤组织DR4和DR5 m RNA表达程度高,与增殖期血管瘤相比,退化期血管瘤可以更多的结合TRAIL,细胞凋亡增多,引发血管瘤消退。     

TRAIL联合化疗药物对人宫颈癌Hela细胞增殖与凋亡的影响

目的探讨肿瘤坏死因子相关诱导凋亡配体 (TRAIL)对人宫颈癌Hela细胞的作用 ,以及与化疗药物的协同作用。方法将Hela细胞接种至 96孔培养板后分别加入浓度为l.0、10 .0、10 0 .0 μl/L的TRAIL、0 .1、1.0、10 .0mg/L的阿霉素 (ADM )和丝裂霉素 (MMC) ,及不同匹配的TRAIL MMC和TRAIL ADM ,采用四甲基偶氮唑盐 (MTT)比色法分别检测肿瘤细胞的生存率 ;将Hela细胞接种至 12孔板 ,培育 2 4h后加入不同浓度的TRAIL、ADM、MMC、TRAIL ADM、TRAL MMC ,用流式细胞术检测不同处理组肿瘤细胞的凋亡率和死亡率。结果 10 0 μg/LTRAIL引起细胞的凋亡率为 2 0 .1% ,与无药物组 1.1%的凋亡率有非常显著性差异 (P <0 .0 1) ;单独采用 10mg/LMMC、ADM对细胞的抑制率为 3 6.0 %和 44 .1% ,而 10 0 μg/LTRAIL与 10mg/LMMC、ADM联合后对细胞的抑制率分别达到 5 8.4%和 73 .7% ,两者有协同作用 (P <0 .0 5 )。结论在体外实验中 ,TRAIL可通过诱导宫颈癌Hela细胞的凋亡而产生抗肿瘤作用 ;TRAIL与化疗药物ADM、MMC有协同抗肿瘤作用。     

TNF相关凋亡诱导配体联合阿糖胞苷诱导HL-60细胞凋亡及其机制的研究

本研究探讨人重组可溶性肿瘤坏死因子相关凋亡诱导配体（hrsTRAIL）单用或联合阿糖胞苷（Ara—C）诱导HL-60细胞凋亡作用及其机制。在体外以人类急性白血病细胞株HL-60为研究对象，分5组进行细胞培养：对照组、Ara—C组、rsTRAIL组、同时给药组（Ara—C＋rsTRAIL）、先后给药组（Ara—C→rsTRAIL）。采用MTT比色法测定细胞生长抑制率；应用Annexin V／PI染色和流式细胞术检测细胞凋亡率；使用流式细胞术检测不同浓度的Ara—C对细胞表面DR5表达水平的影响及rsTRAIL单药组、先后给药组细胞表面DR5表达水平和细胞内caspase-8活性。结果表明：rsTRAIL能抑制HL-60细胞生长并诱导HL-60细胞凋亡。先后给药组诱导HL-60细胞凋亡率大于同时给药组。先后给药组细胞表面DR5表达水平和细胞内caspase-8的活性高于rsTRAIL组。5mg／L、10mg／LAra—C作用于HL-60细胞24小时，其细胞表面DR5表达水平升高，大于对照组。结论：rsTRAIL抑制HL-60细胞生长．诱导HL-60细胞凋亡。Ara—C上调HL-60细胞表面DR5表达水平。增强rsTRAIL诱导HL-60细胞凋亡的作用。Ara—C和rsTRAIL联合用药时，在先加Ara—C、再加rsTRAIL组诱导HL-60细胞凋亡的效果比同时给药组效果好，其机制可能与Ara—C上调细胞表面DIL5表达水平和（或）增强细胞内caspase-8的活性有关。     

Mechanisms and biomarkers of immune quiescence in kidney transplantation

This review discusses the current understanding of biomarkers of immune quiescence based on reviews of published literature in kidney transplant operational tolerance and mechanistic studies based on a better characterization of the stable, well-functioning renal allograft.     

Smac mimetics and TRAIL cooperate to induce MLKL-dependent necroptosis in Burkitt's lymphoma cell lines

Burkitt''s lymphoma (BL) is a highly aggressive form of B-cell non-Hodgkin''s lymphoma. The clinical outcome in children with BL has improved over the last years but the prognosis for adults is still poor, highlighting the need for novel treatment strategies. Here, we report that the combinational treatment with the Smac mimetic BV6 and TRAIL triggers necroptosis in BL when caspases are blocked by zVAD.fmk (TBZ treatment). The sensitivity of BL cells to TBZ correlates with MLKL expression. We demonstrate that necroptotic signaling critically depends on MLKL, since siRNA-induced knockdown and CRISPR/Cas9-mediated knockout of MLKL profoundly protect BL cells from TBZ-induced necroptosis. Conversely, MLKL overexpression in cell lines expressing low levels of MLKL leads to necroptosis induction, which can be rescued by pharmacological inhibitors, highlighting the important role of MLKL for necroptosis execution. Importantly, the methylation status analysis of the MLKL promoter reveals a correlation between methylation and MLKL expression. Thus, MLKL is epigenetically regulated in BL and might serve as a prognostic marker for treatment success of necroptosis-based therapies. These findings have crucial implications for the development of new treatment options for BL.     

Thymoquinone Crosstalks with DR5 to Sensitize TRAIL Resistance and Stimulate ROS-Mediated Cancer Apoptosis

Objective: Cancer treatment using a targeted inducer of apoptosis like tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) faced the obstacle of resistance, thus providing a plus drug like Thymoquinone (TQ) could be of great interest to tackle breast cancer cells. The aim of the present work is to examine the genetic modulation impacts of the TRAIL receptors and apoptotic markers upon the combinatorial remedy of TRAIL plus TQ on human breast cancer cell lines. Methods: To achieve this rationale, the protein content-based cytotoxicity using SRB assay, as well as the genetic expressions of the TRAIL receptors (DR4 and DR5) and apoptotic markers (Bcl-2, Cas-8, and FADD) using real time qRT-PCR technique were preceded against breast cancer MCF-7 and MDA-MB-231 cancerous cell lines. Results: The current study showed that the combination therapy of TQ+TRAIL significantly inhibited the protein content-based proliferation of MDA-MB-231 cells more than MCF-7 cells. The synergistic effect of them significantly up-regulated the genetic expressions of DR4, DR5, Cas-8, and FADD genes and inhibited the genetic expression of the Bcl-2 gene in the proposed cell lines treated for 24 h. The induction of the apoptotic genes using the combined therapy was stimulated by the elevation of the reactive oxygen species (ROS); nitric oxide (NO) and malondialdehyde (MDA) levels. Conclusions: The synergistic influence between TQ which induced the DR5 and TRAIL, facilitating the connection between TRAIL and its receptors on the cancerous cell membrane. Hence, the proposed combination therapy induced the ROS-mediated apoptotic stimulus.     

Caspase-8和Bcl-2在脑肿瘤干细胞肿瘤坏死因子相关凋亡诱导配体耐药中的作用研究

 目的 探讨肿瘤坏死因子相关凋亡诱导配体(TRAIL)对脑肿瘤干细胞生长的影响,研究Caspase-8和Bcl-2在肿瘤坏死因子相关凋亡诱导配体耐药中的作用。方法 应用CD133免疫磁珠分选法获得脑肿瘤干细胞,流式细胞术检测分选阳性率;亚神经球形成实验分析脑肿瘤干细胞的自我更新能力和增殖能力;四甲基偶氮唑盐法检测肿瘤坏死因子相关凋亡诱导配体对脑肿瘤干细胞生长的影响;Western blot法检测DR5、FADD、Caspase-8和Bcl-2蛋白的表达。结果 分选后CD133⁺细胞即脑肿瘤干细胞可达71%;脑肿瘤干细胞以神经球的方式生长;肿瘤坏死因子相关凋亡诱导配体作用前后,脑肿瘤干细胞都有极强的形成神经球的能力;肿瘤坏死因子相关凋亡诱导配体可以引起脑肿瘤干细胞死亡,加入Caspase-8或Caspases抑制剂,可明显阻断肿瘤坏死因子相关凋亡诱导配体所引起的细胞死亡(P<0.05);Western blot结果显示,脑肿瘤干细胞表达Caspase-8 蛋白水平降低,表达Bcl-2蛋白水平增加(P<0.05)。结论 脑肿瘤干细胞是脑肿瘤对肿瘤坏死因子相关凋亡诱导配体耐药的根源,Caspase-8蛋白表达减少及Bcl-2蛋白表达增强使Caspase-8不能活化,导致了脑肿瘤干细胞发生肿瘤坏死因子相关凋亡诱导配体耐药。     

Engineered adenoviruses combine enhanced oncolysis with improved virus production by mesenchymal stromal carrier cells

Oncolytic viruses have demonstrated in pre‐clinical and clinical studies safety and a unique pleiotropic activity profile of tumor destruction. Yet, their delivery suffers from virus inactivation by blood components and sequestration to healthy tissues. Therefore, mesenchymal stromal cells (MSCs) have been applied as carrier cells for shielded virus delivery to tumors after ex vivo infection with oncolytic viruses. However, infection and particle production by MSCs have remained unsatisfying. Here, we report engineered oncolytic adenoviruses (OAds) for improved virus production and delivery by MSCs. OAds are uniquely amenable to molecular engineering, which has facilitated improved tumor cell destruction. But for MSC‐mediated regimens, OAd engineering needs to achieve efficient infection and replication in both MSCs and tumor cells. We show that an Ad5/3 chimeric OAd capsid, containing the adenovirus serotype 3 cell‐binding domain, strongly increases the entry into human bone marrow‐derived MSCs and into established and primary pancreatic cancer cells. Further, we reveal that OAd with engineered post‐entry functions—by deletion of the anti‐apoptotic viral gene E1B19K or expression of the death ligand TRAIL—markedly increased virus titers released from MSCs, while MSC migration was not hampered. Finally, these virus modifications, or viral expression of FCU1 for local 5‐FC prodrug activation, improved tumor cell killing implementing complementary cytotoxicity profiles in a panel of pancreatic cancer cell cultures. Together, our study establishes post‐entry modification of OAd replication for improving virus delivery by carrier cells and suggests a panel of optimized OAds for future clinical development in personalized treatment of pancreatic cancer.