Quantitative proteomic analysis of the inhibitory effects of CIL-102 on viability and invasiveness in human glioma cells.

CIL-102 (1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone), the major active agent of the alkaloid derivative, has been demonstrated to exert anticancer effects. Herein, we present an investigation focused on the identification of the target(s) of CIL-102's action and the mechanism of its action in apoptotic and anti-invasive pathways. Proteomic approaches were used to purify and identify the protein substrates using 2D difference gel electrophoresis (2D SDS-PAGE) to assess changes in the expression of relevant protein treatment with CIL-102 that resulted in the inhibition of viability and invasion. Our results demonstrate that CIL-102 treatment of U87 cells decreased cell proliferation and invasiveness. CIL-102 dose-dependent induction of apoptosis and inhibitory invasiveness were accompanied by sustained phosphorylation of JNK1/2 and p70S6K as well as generation of the reactive oxygen species. In addition, differential proteins displayed between CIL-102-treated and untreated U87 were determined and validated. There were 11 differentially expressed proteins between the CIL-102-treated and untreated groups. Furthermore, we demonstrated that CIL-102 inhibited cancer cell proliferation and reduced anti-invasion properties by up-regulating the levels of FUMH (Fumarate hydratase). The investigation demonstrated that there was an increase in the cellular levels of FUMH in the CIL-102 reduction in viability and invasion via the activation of JNK1/2 and mTOR signaling modules. NAC administration and shRNA FUMH conferred resistance to CIL-102-inhibited HIF1α and MMP-2 levels via inhibition of JNK1/2 and mTOR activation. We concluded that CIL-102-induced an apoptosis cascade and decreased aggressiveness in astrocytoma cells by modulation of mitochondria function, providing a new mechanism for CIL-102 treatment.

Identification of Two Novel CIL-102 Upregulations of ERP29 and FUMH to Inhibit the Migration and Invasiveness of Colorectal Cancer Cells by Using the Proteomic Approach.

CIL-102 (1-[4-(furo[2,3-b]quinolin-4-ylamino) phenyl]ethanone) is a major active agent of Camptotheca acuminata's alkaloid derivative, and its anti-tumorigenic activity, a valuable biological property of the agent, has been reported in many types of cancer. In this study, we researched the novel CIL-102-induced protein for either the induction of cell apoptosis or the inhibition of cell migration/invasiveness in colorectal cancer cells (CRC) and their molecular mechanism. Firstly, our data showed that CIL-102 treatment not only increased the cytotoxicity of cells and the production of Reactive Oxygen Species (ROS), but it also decreased cell migration and invasiveness in DLD-1 cells. In addition, many cellular death-related proteins (cleavage caspase 9, cleavage caspase 3, Bcl-2, and TNFR1 and TRAIL) and JNK MAPK/p300 pathways were increased in a time-dependent manner. Using the proteomic approach with a MALDI-TOF-TOF analysis, CIL-102-regulated differentially expressed proteins were identified, including eight downregulated and 11 upregulated proteins. Among them, upregulated Endoplasmic Reticulum resident Protein 29 (ERP29) and Fumarate Hydratase (FUMH) by CIL-102 were blocked by the inhibition of ROS production, JNK activity, and p300/CBP (CREB binding protein) signaling pathways. Importantly, the knockdown of ERP29 and FUMH expression by shRNA abolished the inhibition of cell migration and invasion by CIL-102 in DLD-1 cells. Together, our findings demonstrate that ERP29 and FUMH were upregulated by CIL102 via ROS production, JNK activity, and p300/CBP pathways, and that they were involved in the inhibition of the aggressive status of colorectal cancer cells.

Novel regulator role of CIL-102 in the epigenetic modification of TNFR1/TRAIL to induce cell apoptosis in human gastric cancer.

CIL-102 (1-[4-(furo [2,3-b]quinolin-4-ylamino)phenyl]ethanone) is a major active agent and an alkaloid derivative of Camptotheca acuminata, which has valuable biological properties, including anti-tumorigenic activity. However, the molecular mechanisms of CIL-102 related to inductive apoptosis in human gastric cancer remain unclear. By using diphenyltetrazolium bromide (MTT), annexin-V-fluorescein-isothiocyanate (FITC)/propidium iodide staining and a 2',7' -dichlorofluorescin diacetate (DCFDA), a Fluo-3 fluorescence staining assay, the cell death and cell viability in gastric cancer cells and an in vivo xenograft mouse model, with or without the addition of CIL-102, were measured, respectively. Furthermore, signaling pathways and apoptotic molecules were also detected by western blots and an immunohistochemical assay. Our results demonstrated that CIL-102 treatment significantly induced the cell apoptosis of gastric cancer cells, along with increased ROS production and increased intracellular Ca2+ levels. In addition, through the inactivation of CDK1/cyclin B1, CIL-102 treatment induced the cell cycle G2/M arrest of gastric cancer cells. Moreover, our data revealed that multiple signaling pathways were involved in the H3K4 trimethylation of TNFR1 and TRAIL proteins by CIL-102, including ROS-derived and JNK/mTOR/p300 pathways in gastric cancer AGS cells. The CIL-102 treatment also consistently inhibited tumor growth and increased tumor apoptosis, as measured by TUNEL assay in an in vivo xenograft mouse model. An immunohistochemical analysis revealed that the upregulation of the TNFR1 and TRAIL proteins and the downregulation of PCNA and CDK1 proteins were found in the CIL-102-treated gastric cancer xenograft mouse model, compared to that of the saline control. Together, this study sheds light on the novel mechanism associated with CIL-102 for inducing gastric cancer apoptosis.

Novel CIL-102 derivatives as potential therapeutic agents for docetaxel-resistant prostate cancer.

The standard-of-care treatment for metastatic prostate cancer (PCa) is androgen deprivation therapy (ADT). Nevertheless, most tumors eventually relapse and develop into lethal castration-resistant prostate cancer (CRPC). Docetaxel is a FDA-approved agent for the treatment of CRPC; however, the tumor often quickly develops resistance to this drug. Thus, there is an immediate need for novel therapies to treat docetaxel-resistant PCa. In this study, we modified the structure of CIL-102 and investigated the efficacy of the derivatives against CRPC and docetaxel-resistant PCa. These novel CIL-102 derivatives inhibit CRPC tumorigenicity, including proliferation, migration and colony formation, and importantly, selectively inhibit CRPC cell proliferation over non-cancerous prostate epithelia. Computational modeling indicated the derivatives bind to ß-tubulin and immunocytochemistry revealed the depolymerization of microtubules upon treatment. Western blot analyses reveal that pro-apoptotic and anti-oxidant pathways are activated, and MitoSOX and DCF-DA analyses confirmed increased reactive oxygen species (ROS) production upon treatments. Furthermore, CIL-102 derivatives effectively reduce the proliferation of docetaxel-resistant CR PCa cell lines. Our data indicate the potential of these compounds as promising therapeutic agents for CRPC as well as docetaxel-resistant CRPC.