7-ethyl-7-hydroxy-10h-1-3-dioxolo(4-5-g)pyrano(3--4--6-7)indolizino(1-2-b)quinoline-8-11(7h-12h)-dione and Colorectal-Neoplasms

Pancreatic ductal adenocarcinoma (PDAC), a difficult-to-treat cancer, is expected to become the second-largest cause of cancer-related deaths by 2030, while colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer deaths. Currently, there is no effective treatment for PDAC patients. The development of novel agents to effectively treat these cancers remains an unmet clinical need. FL118, a novel anticancer small molecule, exhibits high efficacy against cancers; however, the direct biochemical target of FL118 is unknown.. FL118 affinity purification, mass spectrometry, Nanosep centrifugal device and isothermal titration calorimetry were used for identifying and confirming FL118 binding to DDX5/p68 and its binding affinity. Immunoprecipitation (IP), western blots, real-time reverse transcription PCR, gene silencing, overexpression (OE) and knockout (KO) were used for analysing gene/protein function and expression. Chromatin IP was used for analysing protein-DNA interactions. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromid assay and human PDAC/CRC cell/tumour models were used for determining PDAC/CRC cell/tumour in vitro and in vivo growth.. We discovered that FL118 strongly binds to dephosphorylates and degrades the DDX5 oncoprotein via the proteasome degradation pathway without decreasing DDX5 mRNA. Silencing and OE of DDX5 indicated that DDX5 is a master regulator for controlling the expression of multiple oncogenic proteins, including survivin, Mcl-1, XIAP, cIAP2, c-Myc and mutant Kras. Genetic manipulation of DDX5 in PDAC cells affects tumour growth. PDAC cells with DDX5 KO are resistant to FL118 treatment. Our human tumour animal model studies further indicated that FL118 exhibits high efficacy to eliminate human PDAC and CRC tumours that have a high expression of DDX5, while FL118 exhibits less effectiveness in PDAC and CRC tumours with low DDX5 expression.. DDX5 is a bona fide FL118 direct target and can act as a biomarker for predicting PDAC and CRC tumour sensitivity to FL118. This would greatly impact FL118 precision medicine for patients with advanced PDAC or advanced CRC in the clinic. FL118 may act as a 'molecular glue degrader' to directly glue DDX5 and ubiquitination regulators together to degrade DDX5.

Topics: Animals; Benzodioxoles; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Colorectal Neoplasms; DEAD-box RNA Helicases; Humans; Indolizines; Oncogene Proteins; Pancreatic Neoplasms; Proto-Oncogene Proteins p21(ras); Survivin

Pyroptosis is a newly discovered inflammatory programmed cell death. This study was to investigate whether pyroptosis is involved in the anti-colorectal cancer process of FL118.. The relationship between NLRP3 and caspase-1 and colorectal cancer was analyzed by bioinformatics. MTT was used to detect the cell viability. Cell membrane integrity was examined by LDH release. Wound healing assay and Transwell were used to detect the cell migration and invasion respectively. TUNEL was to check the cell death. The expression of pyroptosis-related factors was detected using qRT-PCR, Western blotting, Immunofluorescence and Elisa. And H&E staining was used to detect the toxicity of FL118 in colorectal cancer.. In vitro, FL118 significantly inhibited the proliferation, migration and invasion of colorectal cancer, and the morphological characteristics of pyroptosis were observed under the microscope. With the change of FL118 concentration, the release rate of LDH in the supernatant and the expression of pyroptosis-related factors emerged an increase. However, pyroptosis induced by FL118 was reversed with the participation of MCC950 and VX-765, which suppressed the antitumor effect of FL118. In vivo, the result in the xenograft animal model and lung metastasis model experimental showed that FL118 could activate pyroptosis and thus inhibit the metastasis of colorectal cancer.. FL118 restrains the growth and metastasis of colorectal cancer by inducing NLRP3-ASC-Caspase-1 mediated pyroptosis, which provides important evidence in the study on the role of pyroptosis and different tumors.

Topics: Animals; Antineoplastic Agents; Benzodioxoles; Caspase 1; Cell Movement; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Humans; Indolizines; Male; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; NLR Family, Pyrin Domain-Containing 3 Protein; Pyroptosis; Xenograft Model Antitumor Assays

FL118, a novel camptothecin analogue, has been extensively studied for its superior antitumor potency. The aim of this research study is to explore its potential mechanism of action in anti- colorectal cancer (CRC).. The effect of FL118 on CRC cell proliferation was assessed using CCK-8 assay, while apoptosis was detected using Hoechst staining and Flow cytometry assays. The expression levels of CIP2A were analyzed using qRT-PCR. The expression of CIP2A, PP2A-C, Bax, cleaved caspase-3 and PARP were analyzed using western blotting analysis. The expressions of related proteins in CRC tissues were detected using immunohistochemical staining. TUNEL assay was used to detect apoptosis of tissue. Toxicity of FL118 in primary organs were examined using H&E staining.. The results show that FL118 can inhibit the proliferation and clonogenic potential of CRC cells and increase the expression of pro-apoptosis proteins, Bax, cleaved caspase-3 and PARP. Microarray analyses found that FL118 treatment significantly decreases cancerous inhibition of protein phosphatase 2A (CIP2A). Further validation found that CIP2A is aberrantly upregulated in CRC tissues, and is positively correlated with the progression of CRC. In vitro findings confirm that FL118 mediates the downregulation of CIP2A, at both protein and mRNA levels. Co-treatment with Okadaic acid (OA) (a PP2A inhibitor) partially abolishes the anti-proliferative and pro-apoptotic effect of FL118. Consistently, in vivo experiment demonstrates that FL118 can effectively suppress tumorigenesis without any obvious toxic effects.. Collectively, these findings exhibit the anti-neoplastic effects of FL118 against CRC through the down regulation of CIP2A, which subsequently enhances the activity of PP2A.

Topics: Animals; Apoptosis; Autoantigens; Benzodioxoles; Camptothecin; Cell Line, Tumor; Cell Proliferation; Cell Survival; China; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Indolizines; Intracellular Signaling Peptides and Proteins; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Protein Phosphatase 2; Proto-Oncogene Proteins c-akt; Signal Transduction; Xenograft Model Antitumor Assays

Irinotecan is a camptothecin analogue currently used in clinical practice to treat advanced colorectal cancer. However, acquired resistance mediated by the drug efflux pump ABCG2 is a recognized problem. We reported on a novel camptothecin analogue, FL118, which shows anticancer activity superior to irinotecan. In this study, we sought to investigate the potency of FL118 versus irinotecan or its active metabolite, SN-38, in both in vitro and in vivo models of human cancer with high ABCG2 activity. We also sought to assess the potency and ABCG2 affinity of several FL118 analogues with B-ring substitutions.. Colon and lung cancer cells with and without ABCG2 overexpression were treated with FL118 in the presence and absence of Ko143, an ABCG2-selective inhibitor, or alternatively by genetically modulating ABCG2 expression. Using two distinct in vivo human tumor animal models, we further assessed whether FL118 could extend time to progression in comparison with irinotecan. Lastly, we investigated a series of FL118 analogues with B-ring substitutions for ABCG2 sensitivity.. Both pharmacological inhibition and genetic modulation of ABCG2 demonstrated that, in contrast to SN-38, FL118 was able to bypass ABCG2-mediated drug resistance. FL118 also extended time to progression in both in vivo models by more than 50% compared with irinotecan. Lastly, we observed that FL118 analogues with polar substitutions had higher affinity for ABCG2, suggesting that the nonpolar nature of FL118 plays a role in bypassing ABCG2-mediated resistance.. Our results suggest that in contrast to SN-38 and topotecan, FL118 is a poor substrate for ABCG2 and can effectively overcome ABCG2-mediated drug resistance. Our findings expand the uniqueness of FL118 and support continued development of FL118 as an attractive therapeutic option for patients with drug-refractory cancers resulting from high expression of ABCG2.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzodioxoles; Camptothecin; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Colorectal Neoplasms; Disease Progression; Drug Resistance, Neoplasm; Female; Gene Silencing; Humans; Indolizines; Irinotecan; Lung Neoplasms; Mice, SCID; Neoplasm Proteins; Topoisomerase Inhibitors; Treatment Outcome; Xenograft Model Antitumor Assays

Anticancer agent FL118 was recently identified in screening of small-molecule inhibitors of human survivin expression. Although FL118 is a camptothecin analogue, its antitumor potency is much superior to other FDA-approved camptothecin analogues (irinotecan and topotecan). The mechanism of action (MOA) underlying the antitumor effects of FL118 remains to be fully elucidated. Here, we report that FL118 activates tumor suppressor p53 as a novel MOA in p53 wild-type cancer cells. Our studies show that this MOA involves an induction of proteasomal degradation of MdmX, a critical negative regulator of p53, in a manner largely independent of ATM-dependent DNA damage signaling pathway but dependent on E3-competent Mdm2. FL118 inhibits p53 polyubiquitination and monoubiquitination by Mdm2-MdmX E3 complex in cells and in cell-free systems. In contrast, FL118 stimulates Mdm2-mediated MdmX ubiquitination. Coimmunoprecipitation revealed that FL118 slightly decreases Mdm2-p53 interactions and moderately increases Mdm2-MdmX interactions, suggesting a change of targeting specificity of Mdm2-MdmX E3 complex from p53 to MdmX, resulting in accelerated MdmX degradation. As a result, p53 ubiquitination by Mdm2-MdmX E3 complex is reduced, which in turn activates p53 signaling. Activation of the p53 pathway by FL118 induces p53-dependent senescence in colorectal cancer cells. However, in the absence of p53 or in the presence of MdmX overexpression, FL118 promotes p53-independent apoptosis. These two distinct cellular consequences collectively contribute to the potent effects of FL118 to inhibit clonogenic potential of colon cancer cells. This study identifies a potential application of FL118 as an MdmX inhibitor for targeted therapies.

Topics: Apoptosis; Benzodioxoles; Cell Cycle Proteins; Cell Line, Tumor; Cellular Senescence; Colorectal Neoplasms; DNA Damage; Gene Expression Regulation, Neoplastic; Humans; Indolizines; Inhibitor of Apoptosis Proteins; Nuclear Proteins; Proteolysis; Proto-Oncogene Proteins; Signal Transduction; Survivin; Tumor Suppressor Protein p53; Ubiquitination