The angular structure of ONC201, a TRAIL pathway-inducing compound,determines its potent anti-cancer activity

We previously identified TRAIL-inducing compound 10 (TIC10), also known as NSC350625 or ONC201, from a NCI chemical library screen as a small molecule that has potent anti-tumor efficacy and a benign safety profile in preclinical cancer models. The chemical structure that was originally published by Stahle, et. al. in the patent literature was described as an imidazo[1,2-a]pyrido[4,3-d]pyrimidine derivative. The NCI and others generally accepted this as the correct structure, which was consistent with the mass spectrometry analysis outlined in the publication by Allen et. al. that first reported the molecule''s anticancer properties. A recent publication demonstrated that the chemical structure of ONC201 material from the NCI is an angular [3,4-e] isomer of the originally disclosed, linear [4,3-d] structure. Here we confirm by NMR and X-ray structural analysis of the dihydrochloride salt form that the ONC201 material produced by Oncoceutics is the angular [3,4-e] structure and not the linear structure originally depicted in the patent literature and by the NCI. Similarly, in accordance with our biological evaluation, the previously disclosed anti-cancer activity is associated with the angular structure and not the linear isomer. Together these studies confirm that ONC201, produced by Oncoceutics or obtained from the NCI, possesses an angular [3,4-e] structure that represents the highly active anti-cancer compound utilized in prior preclinical studies and now entering clinical trials in advanced cancers.     

Targeting p53/TRAIL/caspase-8 signaling by adiponectin reverses thioacetamide-induced hepatocellular carcinoma in rats

Given the enormous impact of HCC on the patients' quality of life and healthcare economics, the current study was conducted to investigate the potential ability of adiponectin to reverse established HCC and to investigate the underlying mechanisms which control the chemotherapeutic and hepatoprotective effects. HCC was induced in Male Sprague Dawely rats by I.P. injection of thioacetamide(200 mg/kg) 3 times/week for 14 weeks.HCC development was confirmed by histopathological examination and assessment of serum levels of α-fetoprotein (AFP). Adiponectin was administered (5 μg/kg, I.P.) starting from week 13 of the experiment and for further 4 weeks. Adiponectinadministration revealed a significant antitumor activity with significant improvement in liver functions and oxidative status. Nevertheless, pathological features as cirrhosis, dysplastic changes, and tumoral nodules were significantly attenuated with significant enhancement in hepatic caspase-3 immunostaining. Mechanistically, adiponectin administration was associated with significant restoration of p53 activity; which increased by 133%, with a reduction in HCC-induced expression of-JNK which decreased by 53%as well as a significant enhancement of hepatic TRAIL and caspase-8 activities which increased by 27% and 20% respectively. In conclusion; Adiponectin can be proposed as a promising therapy for HCC. Adiponectin's tumoricidal activity can be partially mediated by blocking HCC-induced reduction in p53 expression as well as reactivation of TRAIL signaling and induction of apoptotic pathway providing more protection for the body against the tumor.     

Ixazomib promotes CHOP-dependent DR5 induction and apoptosis in colorectal cancer cells

Background: Ixazomib (Ninlaro), a novel proteasome inhibitor, has been developed for the treatment of many cancers and has demonstrated anti-tumor efficacy against various malignancies. However, the mechanism of the anti-tumor effect of ixazomib in colorectal cancer (CRC) cells remains unclear.

Methods: MTS and flow cytometry were performed to determine the effect of ixazomib on CRC cells. Western blotting and real-time RT-PCR were performed to detect ixazomib-induced DR5 upregulation. ChIP was performed to detect CHOP binding to DR5 promoter. Finally, xenograft experiments were carried out to measure the antitumor effect of ixazomib in vivo.

Results: In this study, we revealed the mechanism by which ixazomib inhibits the growth of CRC cells. Our findings indicated that ixazomib treatment induces CHOP-dependent DR5 induction, irrespective of p53 status. Furthermore, DR5 is necessary for ixazomib-mediated apoptosis. Ixazomib also synergized with TRAIL to induce marked apoptosis via DR5 in CRC cells.

Conclusions: Our findings further suggested that ixazomib sensitizes TRAIL/death receptor signaling pathway-targeted CRC and suggested that DR5 induction could be a valuable indicator of ixazomib sensitivity.     

TRAIL在肿瘤治疗中的研究进展

TRAIL是TNF家族的新成员,它通过与死亡受体结合,激活caspase通路传递和放大凋亡信号,从而诱导靶细胞发生快速、大量、高效的凋亡反应.TRAIL可以选择性地杀伤肿瘤细胞和病毒感染的细胞,而正常细胞则通过表达诱骗受体等方式逃逸其杀伤作用,这种特性使其具有在肿瘤治疗中应用的巨大潜力.     

TRAIL诱导白血病细胞凋亡的分子机制研究

目的　检测TRAIL引起的T淋巴白血病细胞凋亡及与之相关的蛋白和信号通路 ,为探索T淋巴细胞凋亡分子机制打下基础 ,为相关肿瘤的治疗提供依据。方法　用TRAIL( 1μg/ml)刺激Jurkat细胞 3 6h以诱导细胞发生凋亡 ,用流式细胞术和MTS比色法检测细胞存活率 ,计算细胞凋亡率 ;用免疫印迹 (Westernblot)检测NF κB的表达和胱天肽酶 (caspase) 3、cas pase 8的变化。 结果　TRAIL能诱导Jurkat细胞凋亡并伴随NF κB表达增加及caspase 3和caspase 8的活化。 结论　TRAIL诱导的T淋巴白血病细胞凋亡的分子机制涉及NF κB及caspase 3、caspase 8,为TRAIL用于治疗白血病提供实验基础。     

Aortic valvular interstitial cells apoptosis and calcification are mediated by TNF-related apoptosis-inducing ligand

Background/Objectives

Calcific aortic valvular disease (CAVD) is an actively regulated process characterized by the activation of specific osteogenic signaling pathways and apoptosis. We evaluated the involvement in CAVD of the TNF-related apoptosis-inducing ligand (TRAIL), an apoptotic molecule which induces apoptosis by interacting with the death receptor (DR)-4 and DR5, and whose activity is modulated by the decoy receptor (DcR)-1 and DcR2.

Methods

Sections of calcific and normal aortic valves, obtained at surgery time, were subjected to immunohistochemistry and confocal microscopy for TRAIL immunostaining. Valvular interstitial cells (VICs) isolated from calcific (C-VICs) and normal (N-VICs) aortic valves were investigated for the gene and protein expression of TRAIL receptors. Cell viability was assayed by MTT. Von Kossa staining was performed to verify C-VIC ability to produce mineralized nodules. TRAIL serum levels were detected by ELISA.

Results

Higher levels of TRAIL were detected in calcific aortic valves and in sera from the same patients respect to controls. C-VICs express significantly higher mRNA and protein levels of DR4, DR5, DcR1, DcR2 and Runx2 compared to N-VICs. C-VICs and N-VICs, cultured in osteogenic medium, express significantly higher mRNA levels of DR4, Runx2 and Osteocalcin compared to baseline. C-VICs and N-VICs were sensitive to TRAIL-apoptotic effect at baseline and after osteogenic differentiation, as demonstrated by MTT assay and caspase-3 activation. TRAIL enhanced mineralized matrix nodule synthesis by C-VICs cultured in osteogenic medium.

Conclusions

TRAIL is characteristically present within calcific aortic valves, and mediates the calcification of aortic valve interstitial cells in culture through mechanism involving apoptosis.     

Activation of conventional PKC isoforms increases expression of the pro-apoptotic protein Bad and TRAIL receptors

Background. Pancreatic cancer is a leading cause of cancer death worldwide; current treatment options have been ineffective in prolonging survival. Agents that target specific signaling pathways (e.g., protein kinase C [PKC]) may regulate apoptotic gene expression rendering resistant cancers sensitive to the effects of other chemotherapeutic drugs. The purpose of our study was to assess the effect of PKC stimulation on apoptotic gene expression in pancreatic cancer cells. Methods. The human pancreatic cancer cell line, PANC-1, was treated with PKC-stimulating agents, phorbol 12-myristate 13-acetate (PMA) or bryostatin-1, and analyzed for expression of apoptosis-related genes. Results. Both PMA and bryostatin-1 induced expression of the pro-apoptotic gene Bad in a dosedependent fashion. The expression of Bad was blocked by the PKC inhibitors GF109203x, G?6983, and Ro-31-8220, suggesting a role for the conventional isoforms of PKC. In addition, treatment with the MEK inhibitors PD98059 or UO126 reduced PMA-mediated induction of Bad gene expression. PMA also increased the expression of TRAIL receptors DR4 and DR5; this expression was inhibited by the PKC inhibitors GF109203x, G?6983, and Ro-31-8220 and the MEK inhibitor UO126, suggesting a role for conventional PKC isoforms and MEK in the regulation of TRAIL receptor expression. Conclusions. PKC stimulation in PANC-1 cells increases expression of the pro-apoptotic gene Bad and the TRAIL receptors, DR4 and DR5, through both conventional PKC- and MEK-dependent pathways. Agents that stimulate PKC may sensitize pancreatic cancer cells to apoptosis and provide a potential adjuvant therapy for the treatment of chemoresistant pancreatic cancers.     

TRAIL/Apo-2-ligand-induced apoptosis in human T cells

Members of the tumor necrosis factor (TNF) family such as CD95 (APO-1/Fas) ligand (L) trigger apoptosis in lymphoid cells. Recently, a new member of apoptosis-inducing ligands, TRAIL (TNF-related-apoptosis-inducing-ligand)/Apo-2 ligand, was identified that might act in a similar way. We compared TRAIL and CD95L-induced apoptosis in human lymphoid cells. Expression of TRAIL was found in CD4+ and CD8+ T cells following activation, suggesting that TRAIL participates in T cell-mediated induction of apoptosis. Similar to CD95L, TRAIL-induced apoptosis in target cells is mediated by activation of caspases (ICE/Ced-3 proteases). However, different human lymphoid cell lines and peripheral T cells differ in sensitivity towards induction of apoptosis by TRAIL and CD95L. In addition, T cells are highly sensitive towards CD95L-induced apoptosis after prolonged activation in vitro, but remain completely resistant to TRAIL-induced apoptosis. In contrast, T cells from HIV-1-infected patients previously shown to exhibit increased CD95 sensitivity are even more susceptible towards TRAIL-induced cell death. These data suggest that TRAIL might participate in CD95-independent apoptosis of lymphoid cells and might be involved in deregulated apoptosis in diseases such as leukemias and HIV-1 infection.     

TRAIL/APO-2L及其受体系统在脑肿瘤中的研究进展

TRAIL/APO - 2L及其受体是TNF家族中的新成员。该系统以其独特的凋亡诱导方式参与抗病毒免疫 ,免疫赦免以及肿瘤细胞的免疫逃逸等生理作用。TRAIL能诱导多种脑肿瘤细胞系发生凋亡而对神经细胞没有毒性 ,这为恶性脑肿瘤的治疗提供了新的途径     

Thapsigargin potentiates TRAIL-induced apoptosis in giant cell tumor of bone

TNF-related apoptosis-inducing ligand (TRAIL) is capable of causing apoptosis in tumor cells but not in normal cells; however, it has been shown that certain types of tumor cells are resistant to TRAIL-induced apoptosis. In this study, we examined the potentiation of TRAIL-induced apoptosis in the stromal-like tumor cells of giant cell tumor of bone (GCT). We show that both mRNA and protein of TRAIL receptors—death receptors (DR4, DR5) and decoy receptors (DcR1, DcR2) are present in GCT stromal tumor cells. However, the expression profiles in all GCT clones tested do not readily correlate with their differential sensitivity to TRAIL. To this end, we selected thapsigargin (TG), an agent known to cause perturbations in intracellular Ca²⁺ homeostasis to enhance the apoptotic action of TRAIL. When added alone, neither TRAIL nor TG induces a therapeutically important magnitude of cell death in GCT tumor cells. Interdependently, scheduled treatment of the cultures with TG followed by subsequent addition of TRAIL resulted in a significant synergistic apoptotic activity, while in contrast, no obvious augmentation was seen when TRAIL was added before TG. This effect was in accord with our observation that TG predominantly up-regulated both mRNA and protein expression of DR5, as well as DR4 mRNA while down-regulating DcR1 protein in GCT stromal-like tumor cells. Taken together, our findings suggest that TG is able to sensitize tumor cells of GCT to TRAIL-induced cell death, perhaps in part through up-regulating the death receptor DR5 and down-regulating the decoy receptor DcR1. These findings provide an additional insight into the design of new treatment modalities for patients suffering from GCT.