Chemotherapy and immunotherapy of malignant glioma: molecular mechanisms and clinical perspectives

Despite the considerable progress in modern tumor therapy, the prognosis for patients with glioblastoma, the most frequent malignant brain tumor, has not been substantially improved. Although cytoreductive surgery and radiotherapy are the mainstays of treatment for malignant glioma at present, novel cytotoxic drugs and immunotherapeutic approaches hold great promise as effective weapons against these malignancies. Thus, great efforts are being made to enhance antitumoral efficacy by combining various cytotoxic agents, by novel routes of drug administration, or by combining anticancer drugs and immune modulators. Immunotherapeutic approaches include cytotoxic cytokines, targeted antibodies, and vaccination strategies. However, the success of most of these experimental therapies is prevented by the marked molecular resistance of glioma cells to diverse cytotoxic agents or by glioma-associated immunosuppression. One promising experimental strategy to target glioma is the employment of death ligands such as CD95 (Fas/Apo1) ligand or Apo2 ligand (TRAIL). Specific proapoptotic approaches may overcome many of the obvious obstacles to a satisfactory management of malignant brain tumors. Received 8 March 1999; received after revision 27 May 1999; accepted 14 June 1999     

Ubiquitin-proteasome pathway as a primary defender against TRAIL-mediated cell death

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptotic cell death as well as expression of proinflammatory genes such as CXCL8 in malignant human astrocytoma cells. However, the molecular mechanisms that determine the fate of cells are not yet understood. The ubiquitin (Ub)-proteasome pathway regulates a wide range of cellular functions through degradation of various regulatory proteins; given this, we hypothesized that this pathway may play a central role in TRAIL-mediated signaling. We demonstrate here that inhibition of the Ub-proteasome pathway enhanced TRAIL-mediated cell death of human astrocytoma CRT-MG cells within hours by blocking degradation of active caspase-8 and -3. Proteasome inhibitors suppressed TRAIL-mediated activation of NF-B; however, inhibition of the NF-B pathway alone was not sufficient to enhance TRAIL-mediated cell death. Collectively, these results suggest that the Ub-proteasome pathway may play an important role as an antiapoptotic surveillance system by eliminating activated caspases as well as mediating NF-B-dependent signals.Received 30 December 2003; received after revision 9 February 2004; accepted 13 February 2004     

XIAP在TRAIL抗性细胞HCT116 bax-/-中的作用机制研究

为了探索结直肠癌细胞HCT116抗TRAIL诱导凋亡的分子机制,本课题以其抗性细胞HCT116 bax~(-/-)为实验对象进行了研究.通过利用目前最热的基因定点编辑技术Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas)9系统将HCT116bax~(-/-)的XIAP(X-linked inhibitor of apoptosis protein)基因彻底敲除后,用TRAIL处理,发现其恢复了对TRAIL的敏感,形态学发生了明显凋亡,而且western blot检测显示PARP蛋白发生了完全剪切.由此证明敲除XIAP基因能克服HCT116 bax~(-/-)对TRAIL的抗性.这些发现对肿瘤细胞抗TRAIL的分子机理研究以及肿瘤的个性化治疗有非常重要的意义.     

可溶性人TRAIL蛋白诱导肝癌细胞的凋亡

为研究可溶性人TRAIL蛋白（sTRAIL）对肝癌细胞株SMMC7721的生长抑制效应及凋亡诱导作用．采用显微镜、台盼蓝排斥试验、MTT比色试验、TUNEL法和DNA断裂实验等方法检测细胞增殖和细胞凋亡．通过显微镜观察到核染色质凝集等凋亡的形态学变化,台盼蓝排斥试验、MTT比色试验结果显示,sTRAIL蛋白可显著抑制SMMC7721细胞的生长和增殖,并且TUNEL法检测到经sTRAIL处理后的细胞凋亡指数与对照比较有显著差异,DNA断裂实验亦观察到典型的DNA梯形条带,这些结果提示sTRAIL可诱导肝癌细胞株SMMC7721发生凋亡,具有抗肝癌的作用．     

Recombinant soluble TRAIL induces apoptosis of cancer cells

TRAIL is a tumor necrosis factor family member that selectively induces apoptosis of cancer cells but not of normal cells. To develop TRAIL into a potential cancer drug, three different sizes of soluble TRAIL fragments, including sTRAIL(74—281), sTRAIL(95—281) and sTRAIL(101—281), were expressed in E. coli and purified to homogeneity. Apoptosis assays indicated that sTRAIL(95—281) and sTRAIL(101—281), but not sTRAIL(74—281), can potently induce apoptosis of various cancer cell lines in 6 h, suggesting that the N-terminal fragment of aa101 has inhibitory effect on TRAIL-induced apoptosis. Moreover, we found that some cancer cells were resistant to TRAIL and the resistant cells could be converted into sensitive cells by treatment with the protein synthesis inhibitor cycloheximide, suggesting that one or more short-lived proteins are responsible for cells’ resistance to TRAIL.     

TRAIL induces proliferation of human glioma cells by c-FLIPL-mediated activation of ERK1/2

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in TRAIL-sensitive human malignant glioma cells. We show for the first time that TRAIL stimulates cell growth in TRAIL-resistant glioma cells. TRAIL-induced cell growth in resistant cells occurred through increased cell cycle progression as determined by flow cytometry and Western blot analysis of retinoblastoma protein phosphorylation. Western blot analysis of TRAIL-treated resistant cells revealed phosphorylation of ERK1/2 proteins and in vitro kinase analysis confirmed the activation of the ERK1/2 kinases. Inhibition of MEK1 eliminated both TRAIL-induced ERK1/2 activation and cell proliferation. In addition, siRNA inhibition of c-FLIP expression eliminates TRAIL-induced ERK1/2 activation and proliferation. Furthermore, overexpression of c-FLIP_L potentiates TRAIL-induced ERK1/2 activation and proliferation of resistant glioma cells. Our results have shown for the first time that TRAIL-induced ERK1/2 activation and proliferation of TRAIL-resistant human glioma cells is dependent upon the expression of the long form of the caspase-8 inhibitor c-FLIP_L. Received 2 November 2007; received after revision 14 December 2007; accepted 21 December 2007     

Modulation by polyamines of apoptotic pathways triggered by procyanidins in human metastatic SW620 cells

We showed previously that inhibition of polyamine catabolism with the polyamine oxidase inhibitor MDL 72527 (MDL) potentiates the apoptotic effects of apple procyanidins (Pcy) in SW620 cells. Here we report that Pcy caused an activation of the intrinsic apoptotic pathway through enhanced polyamine catabolism and mitochondrial membrane depolarization. MDL in the presence of Pcy caused a profound intracellular depletion of polyamines and exerted a protective effect on mitochondrial functions. MDL potentiation of Pcy-triggered apoptosis was reversed by addition of exogenous polyamines. In addition, MDL in combination with Pcy activated the extrinsic apoptotic pathway through enhanced TRAIL-death receptor (DR4/DR5) expression. Potentiation of Pcy-triggered apoptosis by MDL was inhibited when cells were exposed to specific inhibitors of DR4/DR5. These data indicate that the depletion of intracellular polyamines by MDL in the presence of Pcy caused a switch from intrinsic to extrinsic apoptotic pathways in human colon cancer-derived metastatic cells. Received 15 January 2008; received after revision 19 February 2008; accepted 7 March 2008     

Expression of human soluble TRAIL in Chlamydomonas reinhardtii chloroplast

The use of plants as bioreactors for the expression oftherapeutic proteins has developed into a new field in biotechnology research[1]. Plant systems provide simple genetic manipulation, low production costs, and low risk of contamination by animal viruse…     

Sorafenib联合重组人可溶性TRAIL蛋白对K562细胞增殖抑制和凋亡诱导的研究

目的观察比较联合应用重组人可溶性TRAIL蛋白及Sorafenib于人慢性髓系白血病细胞株K562与单独应用时细胞增殖抑制及诱导凋亡的差异。方法通过CCK-8法检测两者对K562细胞的增殖抑制作用;Western-Blot分析TRAIL或与Sorafenib联用对K562细胞的凋亡诱导作用。结果 Sorafenib与TRAIL联合作用于K562细胞时,其细胞的增殖抑制率及凋亡率均显著高于二者单独应用。结论 Sorafenib与TRAIL对K562细胞的增殖抑制及凋亡诱导有增敏及协同作用。     

PRMT5 suppresses DR4-mediated CCL20 release via NF-κB pathway

Construct expression vectors of pCMV-DR4-HA and pCMV-PRMT5-Flag,and transfect them into HEK293 cells to identify the interaction between TRAIL-R1 and PRMT5 and the molecular mechanism underlying DR4-mediated inhibition of chemokine CCL20 release via TRAIL receptor 1(DR4).Inflammatory cytokine was detected by RT-PCR and ELISA after TRAIL-R1 and/or PRMT5 transfection,respectively.NF-κB activity was detected by Dual Luciferase Reporter Gene Assay.ERK1/2 phosphorylation was analyzed by Western blot.PRMT5 could inhibit DR4-activated NF-κB activity and ERK1/2 phosphorylation.PRMT5 could inhibit NF-κB activition,ERK1/2 phosphorylation as well as CCL20 secretion via binding with DR4 in HEK293 cell,suggesting that PRMT5 may involve in DR4 dependent immune regulation.