ABSTRACT
Regulatory factor X-1 (RFX-1) is a transcription factor that has been linked to negative regulation of tumor progression; however, its biological function and signaling cascades are unknown. Here, we performed several studies to elucidate the roles of RFX-1 in the regulation of SHP-1 in hepatocellular carcinoma (HCC) cells. Overexpression of RFX-1 resulted in the activation of SHP-1 and repressed colony formation of HCC cells. In addition, by a mouse xenograft model, we demonstrated that RFX-1 overexpression also inhibited the tumor growth of HCC cells in vivo, suggesting that RFX-1 is of potential interest for small-molecule-targeted therapy. We also found that SC-2001, a bipyrrole molecule, induced apoptosis in HCC cells through activating RFX-1 expression. SC-2001 induced RFX-1 translocation from the cytosol to nucleus, bound to the SHP-1 promoter, and activated SHP-1 transcription. In a xenograft model, knockdown of RFX-1 reversed the antitumor effect of SC-2001. Notably, SC-2001 is much more potent than sorafenib, a clinically approved drug for HCC, in in vitro and in vivo assays. Our study confirmed that RFX-1 acts as a tumor suppressor in HCC and might be a new target for HCC therapy. The findings of this study also provide a new lead compound for targeted therapy via the activation of the RFX-1/SHP-1 pathway.
Subject(s)
Carcinoma, Hepatocellular/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Liver Neoplasms/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , STAT3 Transcription Factor/antagonists & inhibitors , Transcription Factors/metabolism , Animals , Antineoplastic Agents/pharmacology , Blotting, Western , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Chromatin Immunoprecipitation , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Flow Cytometry , Humans , Immunoenzyme Techniques , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Luciferases/metabolism , Male , Mice , Mice, Nude , Niacinamide/analogs & derivatives , Niacinamide/pharmacology , Phenylurea Compounds/pharmacology , Promoter Regions, Genetic/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Pyrroles/pharmacology , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Regulatory Factor X Transcription Factors , Regulatory Factor X1 , Reverse Transcriptase Polymerase Chain Reaction , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , Sorafenib , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Obatoclax is a small molecule which targets the Bcl-2 family, and is to treat leukemia, lymphoma and lung carcinoma. Previously, an obatoclax analogue, SC-2001, was found to disrupt the protein-protein interactions of the Bcl-2 family and also repress Bcl-XL and Mcl-1 expression via STAT3 inactivation. Here, we report a novel mechanism of autophagy induction by SC-2001 in liver cancer cells. The findings indicate that SC-2001 induced the autophagy marker LC3-II in four hepatocellular carcinoma (HCC) cells. Autophagosomes induced by SC-2001-treated cells were confirmed by electron microscopy. SC-2001 activated SHP-1, dephosphorylated STAT3 and Mcl-1, and subsequently released free beclin 1. Overexpression of STAT3 and Mcl-1 in PLC5 cells attenuated the induction of SC-2001 on autophagy. Abolishment of SHP-1 by a specific inhibitor aboragated the autophagic effects induced by SC-2001. In addition, it was further revealed that RFX-1, a transcription factor of SHP-1, is a critical regulator in SC-2001-mediated autophagy. Downregulation of RFX-1 by si-RNA protected cells from SC-2001-induced autophagy. Importantly, Huh7 tumor-bearing nude mice treated with SC-2001 showed downregulation of Mcl-1 and p-STAT3 protein expression and upregulation of SHP-1, LC3II, and RFX-1 protein expression. In summary, our data suggest that SC-2001 induces autophagic cell death in a RFX1/SHP-1/STAT3/Mcl-1 signaling cascade.
Subject(s)
Autophagy/drug effects , DNA-Binding Proteins/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Pyrroles/pharmacology , Transcription Factors/metabolism , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptosis Regulatory Proteins/metabolism , Beclin-1 , Blotting, Western , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/prevention & control , Cell Line, Tumor , DNA-Binding Proteins/genetics , Hep G2 Cells , Humans , Liver Neoplasms/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/prevention & control , Male , Membrane Proteins/metabolism , Mice, Nude , Microscopy, Electron, Transmission , Microtubule-Associated Proteins/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Phagosomes/drug effects , Phagosomes/metabolism , Phagosomes/ultrastructure , Phosphorylation/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , RNA Interference , Regulatory Factor X Transcription Factors , Regulatory Factor X1 , STAT3 Transcription Factor/metabolism , Transcription Factors/genetics , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Detecting and concentrating cancer cells in peripheral blood is of great importance for cancer diagnosis and prognosis. Optically induced dielectrophoresis (ODEP) can achieve high resolution and low optical intensities, and the electrode pattern can be dynamically changed by varied light patterns. By changing the projected light pattern, it is demonstrated to separate high-purity gastric cancer cell lines. Traditionally, the purity of cancer cell isolation by negative selection is 0.9% to 10%; by positive selection it is 50% to 62%. An ODEP technology is proposed to enhance the purity of cancer cell isolation to about 77%.
Subject(s)
Cell Separation/methods , Electrophoresis/methods , Microfluidic Analytical Techniques/instrumentation , Optical Imaging/methods , Cell Line, Tumor , Cell Separation/instrumentation , Electrophoresis/instrumentation , Equipment Design , Humans , MCF-7 Cells , Neoplasms , Optical Imaging/instrumentationABSTRACT
The multiple kinase inhibitor dovitinib is currently under clinical investigation for hepatocellular carcinoma (HCC). Here, we investigated the mechanistic basis for the effects of dovitinib in HCCs. Dovitinib showed significant antitumor activity in HCC cell lines PLC5, Hep3B, Sk-Hep1, and Huh-7. Dovitinib downregulated phospho-STAT3 (p-STAT3) at tyrosine 705 and subsequently reduced the levels of expression of STAT3-related proteins Mcl-1, survivin, and cyclin D1 in a time-dependent manner. Ectopic expression of STAT3 abolished the apoptotic effect of dovitinib, indicating that STAT3 is indispensable in mediating the effect of dovitinib in HCC. SHP-1 inhibitor reversed downregulation of p-STAT3 and apoptosis induced by dovitinib, and silencing of SHP-1 by RNA interference abolished the effects of dovitinib on p-STAT3, indicating that SHP-1, a protein tyrosine phosphatase, mediates the effects of dovitinib. Notably, dovitinib increased SHP-1 activity in HCC cells. Incubation of dovitinib with pure SHP-1 protein enhanced its phosphatase activity, indicating that dovitinib upregulates the activity of SHP-1 via direct interactions. In addition, dovitinib induced apoptosis in two sorafenib-resistant cell lines through inhibition of STAT3, and sorafenib-resistant cells showed significant activation of STAT3, suggesting that targeting STAT3 may be a useful approach to overcome drug resistance in HCC. Finally, in vivo, dovitinib significantly suppressed growth of both Huh-7 and PLC5 xenograft tumors and downregulated p-STAT3 by increasing SHP-1 activity. In conclusion, dovitinib induces significant apoptosis in HCC cells and sorafenib-resistant cells via SHP-1-mediated inhibition of STAT3.
Subject(s)
Apoptosis/drug effects , Benzimidazoles/pharmacology , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Quinolones/pharmacology , STAT3 Transcription Factor/metabolism , Animals , Benzenesulfonates/pharmacology , Benzimidazoles/chemistry , Blotting, Western , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/drug effects , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Male , Mice , Mice, Nude , Molecular Structure , Niacinamide/analogs & derivatives , Phenylurea Compounds , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , Pyridines/pharmacology , Quinolones/chemistry , RNA Interference , STAT3 Transcription Factor/genetics , Sorafenib , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
A series of azulene-based derivatives were synthesized as potent inhibitors for receptor tyrosine kinases such as FMS-like tyrosine kinase 3 (FLT-3). Systematic side chain modification of prototype 1a was carried out through SAR studies. Analogue 22 was identified from this series and found to be one of the most potent FLT-3 inhibitors, with good pharmaceutical properties, superior efficacy, and tolerability in a tumor xenograft model.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Azulenes/chemistry , Azulenes/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/pharmacology , Azulenes/blood , Azulenes/pharmacology , Cell Line, Tumor , Cell Proliferation , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Rats , Receptor Protein-Tyrosine Kinases/metabolism , fms-Like Tyrosine Kinase 3/antagonists & inhibitorsABSTRACT
A series of new ureidoindolin-2-one derivatives were synthesized and evaluated as inhibitors of receptor tyrosine kinases. Investigation of structure-activity relationships at positions 5, 6, and 7 of the oxindole skeleton led to the identification of 6-ureido-substituted 3-pyrrolemethylidene-2-oxindole derivatives that potently inhibited both the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) families of receptor tyrosine kinases. Several derivatives showed potency against the PDGFR inhibiting both its enzymatic and cellular functions in the single-digit nanomolar range. Among them, compound 35 was a potent inhibitor against tyrosine kinases, including VEGFR and PDGFR families, as well as Aurora kinases. Inhibitor 36 (non-substituted on the pyrrole or phenyl ring) had a moderate pharmacokinetic profile and completely inhibited tumor growth initiated with the myeloid leukemia cell line, MV4-11, in a subcutaneous xenograft model in BALB/c nude mice.
