ABSTRACT
The cancer stem cell (CSC) hypothesis postulates that heterogeneous human cancers harbor a population of stem-like cells which are resistant to cytotoxic therapies, thus providing a reservoir of relapse following conventional therapies like chemotherapy and radiation (RT). CSCs have been observed in multiple human cancers, and their presence has been correlated with worse clinical outcomes. Here, we sought to evaluate the impact of drug dosing of the multi-tyrosine kinase inhibitor, sorafenib, on CSC and non-CSCs in soft tissue sarcoma (STS) models, hypothesizing differential effects of sorafenib based on dose and target cell population. In vitro, human cancer cell lines and primary STS from surgical specimens were exposed to escalating doses of sorafenib to determine cell viability and expression of CSC marker aldehyde dehydrogenase (ALDH). In vivo, ALDHbright CSCs were isolated, exposed to sorafenib, and xenograft growth and survival analyses were performed. We observed that sarcoma CSCs appear to paradoxically respond to the tyrosine kinase inhibitor sorafenib at low doses with increased proliferation and stem-like function of CSCs, whereas anti-viability effects dominated at higher doses. Importantly, STS patients receiving neoadjuvant sorafenib and RT on a clinical trial (NCT00864032) showed increased CSCs post therapy, and higher ALDH scores post therapy were associated with worse metastasis-free survival. These data suggest that low-dose sorafenib may promote the CSC phenotype in STS with clinically significant effects, including increased tumor growth and higher rates of metastasis formation in sarcoma patients.
Subject(s)
Sarcoma , Humans , Sorafenib/pharmacology , Sorafenib/therapeutic use , Sorafenib/metabolism , Aldehyde Dehydrogenase/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Kinase Inhibitors/metabolism , Sarcoma/pathology , Neoplastic Stem Cells/metabolism , Cell Line, TumorABSTRACT
BACKGROUND: SHP1 has been documented as a tumor suppressor and it was thought to play an antagonistic role in the pathogenesis of Helicobacter pylori infection. In this study, the exact mechanism of this antagonistic action was studied. MATERIALS AND METHODS: AGS, MGC803, and GES-1 cells were infected with H. pylori, intracellular distribution changes of SHP1 were first detected by immunofluorescence. SHP1 overexpression and knockdown were then constructed in these cells to investigate its antagonistic roles in H. pylori infection. Migration and invasion of infected cells were detected by transwell assay, secretion of IL-8 was examined via ELISA, the cells with hummingbird-like alteration were determined by microexamination, and activation of JAK2/STAT3, PI3K/Akt, and ERK pathways were detected by immunoblotting. Mice infection model was established and gastric pathological changes were evaluated. Finally, the SHP1 activator sorafenib was used to analyze the attenuating effect of SHP1 activation on H. pylori pathogenesis in vitro and in vivo. RESULTS: The sub-localization of SHP1 changed after H. pylori infection, specifically that the majority of the cytoplasmic SHP1 was transferred to the cell membrane. SHP1 inhibited H. pylori-induced activation of JAK2/STAT3 pathway, PI3K/Akt pathway, nuclear translocation of NF-κB, and then reduced EMT, migration, invasion, and IL-8 secretion. In addition, SHP1 inhibited the formation of CagA-SHP2 complex by dephosphorylating phosphorylated CagA, reduced ERK phosphorylation and the formation of CagA-dependent hummingbird-like cells. In the mice infection model, gastric pathological changes were observed and increased IL-8 secretion, indicators of cell proliferation and EMT progression were also detected. By activating SHP1 with sorafenib, a significant curative effect against H. pylori infection was obtained in vitro and in vivo. CONCLUSIONS: SHP1 plays an antagonistic role in H. pylori pathogenesis by inhibiting JAK2/STAT3 and PI3K/Akt pathways, NF-κB nuclear translocation, and CagA phosphorylation, thereby reducing cell EMT, migration, invasion, IL-8 secretion, and hummingbird-like changes.
Subject(s)
Helicobacter Infections , Helicobacter pylori , Animals , Mice , Bacterial Proteins/metabolism , Antigens, Bacterial/metabolism , Helicobacter pylori/physiology , NF-kappa B/metabolism , Interleukin-8/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Helicobacter Infections/pathology , Sorafenib/metabolism , Epithelial Cells/metabolismABSTRACT
Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that poses a significant threat to the global livestock industry. However, specific antiviral treatments against FMDV are currently unavailable. This study aimed to evaluate the antiviral activity of anticancer drugs, including kinase and non-kinase inhibitors against FMDV replication in BHK-21 cells. Sorafenib, a multi-kinase inhibitor, demonstrated a significant dose-dependent reduction in FMDV replication. It exhibited a half maximal effective concentration (EC50) value of 2.46 µM at the pre-viral entry stage and 2.03 µM at the post-viral entry stage. Further intracellular assays revealed that sorafenib effectively decreased 3Dpol activity with a half maximal inhibitory concentration (IC50) of 155 nM, while not affecting 3Cpro function. The study indicates that sorafenib influences host protein pathways during FMDV infection, primarily by potentiating the c-RAF canonical pathway and AKT/PI3K pathway. Molecular docking analysis demonstrated specific binding of sorafenib to the active site of FMDV 3Dpol, interacting with crucial catalytic residues, including D245, D338, S298, and N307. Additionally, sorafenib exhibited significant binding affinity to the active site motifs of cellular kinases, namely c-RAF, AKT, and PI3K, which play critical roles in the viral life cycle. The findings suggest that sorafenib holds promise as a therapeutic agent against FMDV infection. Its mechanism of action may involve inhibiting FMDV replication by reducing 3Dpol activity and regulating cellular kinases. This study provides insights for the development of novel therapeutic strategies to combat FMDV infections.
Subject(s)
Foot-and-Mouth Disease Virus , Foot-and-Mouth Disease , Animals , Sorafenib/pharmacology , Sorafenib/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 3-Kinases/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/pharmacology , Molecular Docking Simulation , Cell Line , Antiviral Agents/pharmacology , Virus ReplicationABSTRACT
Hepatic stellate cell (HSC) activation is the key process in hepatic fibrosis (HF) development. Targeted death of HSCs could be effective in the prevention and treatment of HF. Phosphatidylethanolamine-binding protein (PEBP)1 can trigger ferroptosis by mediating peroxide production, but how it modulates HSC ferroptosis is not known. We screened natural small molecules that could bind with PEBP1, and investigated the mechanism by which it promotes HSC ferroptosis. The maximum binding energy of berberine with PEBP1 was - 8.51 kcal/mol, indicating that berberine could bind strongly with PEBP1. Berberine binding to PEBP1 could promote HSC ferroptosis via synergy of its actions with those of sorafenib, but it could not induce ferroptosis alone. Combined administration of berberine enhanced the ferroptotic effects of low-dose sorafenib upon HSCs. Herein, we revealed that PEBP1 might be a target that could enhance the effects of sorafenib, which could provide a new therapeutic approach for HF treatment.
Subject(s)
Berberine , Ferroptosis , Humans , Sorafenib/pharmacology , Sorafenib/metabolism , Sorafenib/therapeutic use , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/pathology , Berberine/pharmacology , Berberine/metabolism , Berberine/therapeutic use , Liver Cirrhosis/drug therapy , Liver Cirrhosis/metabolism , Phosphatidylethanolamine Binding Protein/genetics , Phosphatidylethanolamine Binding Protein/metabolismABSTRACT
SCOPE: Cachexia, which is often marked by skeletal muscular atrophy, is one of the leading causes of death in cancer patients. Astaxanthin, a carotenoid obtained from marine organisms that can aid in the prevention and treatment of a variety of disorders. In this study, to assess whether astaxanthin ameliorates weight loss and skeletal muscle atrophy in sorafenib-treated hepatocellular carcinoma mice is aimed. METHODS AND RESULTS: H22 mice are treated with 30 mg kg-1 day-1 of sorafenib and 60 mg kg-1 day-1 of astaxanthin by gavage lasted for 18 days. Sorafenib does not delay skeletal muscle atrophy and weight loss, although it does not reduce tumor burden. Astaxanthin dramatically delays weight loss and skeletal muscle atrophy in sorafenib-treating mice, without affecting the food intake. Astaxanthin inhibits the tumor glycolysis, slows down gluconeogenesis, and improves insulin resistance in tumor-bearing mice. Astaxanthin increases glucose competition in skeletal muscle by targeting the PI3K/Akt/GLUT4 signaling pathway, and enhances glucose utilization efficiency in skeletal muscle, thereby slowing skeletal muscle atrophy. CONCLUSION: The findings show the significant potential of astaxanthin as nutritional supplements for cancer patients, as well as the notion that nutritional interventions should be implemented at the initiation of cancer treatment, as instead of waiting until cachexia sets in.
Subject(s)
Cachexia , Glucose , Mice , Animals , Cachexia/drug therapy , Cachexia/etiology , Sorafenib/pharmacology , Sorafenib/metabolism , Glucose/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Muscular Atrophy/drug therapy , Muscular Atrophy/etiology , Muscular Atrophy/metabolism , Muscle, Skeletal/metabolism , Weight Loss , Dietary SupplementsABSTRACT
BACKGROUND: Tyrosine kinase inhibitors (TKIs) are anti-cancer therapeutics often prescribed for long-term treatment. Many of these treatments cause cardiotoxicity with limited cure. We aim to clarify molecular mechanisms of TKI-induced cardiotoxicity so as to find potential targets for treating the adverse cardiac complications. METHODS: Eight TKIs with different levels of cardiotoxicity reported are selected. Phenotypic and transcriptomic responses of human cardiomyocytes to TKIs at varying doses and times are profiled and analyzed. Stress responses and signaling pathways that modulate cardiotoxicity induced by three TKIs are validated in cardiomyocytes and rat hearts. RESULTS: Toxicity rank of the eight TKIs determined by measuring their effects on cell viability, contractility, and respiration is largely consistent with that derived from database or literature, indicating that human cardiomyocytes are a good cellular model for studying cardiotoxicity. When transcriptomes are measured for selected TKI treatments with different levels of toxicity in human cardiomyocytes, the data are classified into 7 clusters with mainly single-drug clusters. Drug-specific effects on the transcriptome dominate over dose-, time- or toxicity-dependent effects. Two clusters with three TKIs (afatinib, ponatinib, and sorafenib) have the top enriched pathway as the endoplasmic reticulum stress (ERS). All three TKIs induce ERS in rat primary cardiomyocytes and ponatinib activates the IRE1α-XBP1s axis downstream of ERS in the hearts of rats underwent a 7-day course of drug treatment. To look for potential triggers of ERS, we find that the three TKIs induce transient reactive oxygen species followed by lipid peroxidation. Inhibiting either PERK or IRE1α downstream of ERS blocks TKI-induced cardiac damages, represented by the induction of cardiac fetal and pro-inflammatory genes without causing more cell death. CONCLUSIONS: Our data contain rich information about phenotypic and transcriptional responses of human cardiomyocytes to eight TKIs, uncovering potential molecular mechanisms in modulating cardiotoxicity. ER stress is activated by multiple TKIs and leads to cardiotoxicity through promoting expression of pro-inflammatory factors and cardiac fetal genes. ER stress-induced inflammation is a promising therapeutic target to mitigate ponatinib- and sorafenib-induced cardiotoxicity.
Subject(s)
Myocytes, Cardiac , Protein Serine-Threonine Kinases , Humans , Rats , Animals , Myocytes, Cardiac/metabolism , Protein Serine-Threonine Kinases/metabolism , Cardiotoxicity/etiology , Sorafenib/metabolism , Sorafenib/pharmacology , Endoribonucleases/metabolism , Endoribonucleases/pharmacology , Apoptosis , Endoplasmic Reticulum Stress/physiologyABSTRACT
Rab22a-NeoF fusion protein has recently been reported as a promising target for osteosarcoma lung metastasis. However, how this fusion protein is regulated in cells remains unknown. Here, using multiple screenings, it is reported that Rab22a-NeoF1 fusion protein is degraded by an E3 ligase STUB1 via the autophagy receptor NDP52-mediated lysosome pathway, which is facilitated by PINK1 kinase. Mechanistically, STUB1 catalyzes the K63-linked ubiquitin chains on lysine112 of Rab22a-NeoF1, which is responsible for the binding of Rab22a-NeoF1 to NDP52, resulting in lysosomal degradation of Rab22a-NeoF1. PINK1 is able to phosphorylate Rab22a-NeoF1 at serine120, which promotes ubiquitination and degradation of Rab22a-NeoF1. Consistently, by upregulating PINK1, Sorafenib and Regorafenib can inhibit osteosarcoma lung metastasis induced by Rab22a-NeoF1. These findings reveal that the lysosomal degradation of Rab22a-NeoF1 fusion protein is targetable for osteosarcoma lung metastasis, proposing that Sorafenib and Regorafenib may benefit cancer patients who are positive for the RAB22A-NeoF1 fusion gene.
Subject(s)
Lung Neoplasms , Oncogene Proteins, Fusion , Osteosarcoma , Humans , Lung Neoplasms/secondary , Lysosomes/metabolism , Osteosarcoma/drug therapy , Osteosarcoma/genetics , Protein Kinases/metabolism , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism , Sorafenib/metabolism , Ubiquitin-Protein Ligases/metabolism , Oncogene Proteins, Fusion/metabolism , Oncogene Proteins, Fusion/therapeutic useABSTRACT
BACKGROUND: Sorafenib is a multikinase inhibitor currently used in the treatment of hepatocellular carcinoma, renal cell carcinoma and thyroid cancer. OBJECTIVES: The literature on this agent is scarce. This study aimed to evaluate the effects of sorafenib when administered to both healthy and cisplatin-induced rats. MATERIAL AND METHODS: The animals were divided into 4 groups: 1) control group that received 0.9% saline intraperitoneally (C); 2) group administered a single dose (7 mg/kg) of cisplatin (Cis); 3) a group administered 20 mg/kg of sorafenib for 7 days (Sor); 4) group administered 20 mg/kg of sorafenib followed by 7 mg/kg of cisplatin for 7 days (Cis+Sor). All animals were sacrificed 7 days after the completion of their treatment arm, and serum and tissue samples were taken. RESULTS: Alanine aminotransferase (ALT), aspartate aminotransferase (AST) and interleukin 38 (IL-38) levels were increased in the Sor and Cis+Sor groups compared to the control group. When compared with the control group, serum urea, creatinine, kidney IL-1ß, and tumor necrosis factor alpha (TNF-α) levels did not change in the Sor group. When compared to the Cis group, the levels of these parameters decreased in the Cis+Sor group. CONCLUSIONS: According to the data obtained, sorafenib caused liver toxicity when given to both healthy and cisplatin-induced rats. While sorafenib did not cause any significant changes in the kidneys when given to healthy rats, it had a healing effect in kidneys after stress induced by cisplatin.
Subject(s)
Cisplatin , Liver Neoplasms , Rats , Animals , Cisplatin/pharmacology , Sorafenib/metabolism , Sorafenib/pharmacology , Kidney/metabolism , Antioxidants/pharmacology , Liver Neoplasms/pathology , Oxidative StressABSTRACT
Edible plants are gaining importance as an integrative therapy for many chronic diseases, including cancer. We first reported that the edible wild plant Crithmum maritimum L. inhibits the growth of hepatocellular carcinoma (HCC) cells by exerting a multitarget action on cellular metabolism and bioenergetic profile. Here, we show that Crithmum maritimum ethyl acetate extract significantly increases the responsiveness of HCC cells to the chemotherapeutic drug sorafenib by reducing lactic acid fermentation and inducing a pro-hepatocyte biomarker profile. Our findings strengthen the role of Crithmum maritimum L. as a valuable nutraceutical tool to support pharmacological therapeutic interventions in HCC.
Subject(s)
Apiaceae , Carcinoma, Hepatocellular , Liver Neoplasms , Sorafenib/metabolism , Fermentation , Apiaceae/metabolism , HepatocytesABSTRACT
Sorafenib (Sor), a novel multi-target anticancer drug also induces severe toxicity in heart, while the mechanism of its cardiotoxicity remains to be fully elucidated. Dysregulation of autophagy and mitochondrial dynamics imbalance have been implicated in cardiomyocyte death. The aim of this study is to test the hypothesis that Sor disrupts autophagy and mitochondrial dynamics, thereby aggravating Sor-induced oxidative stress damage to cardiomyocytes. Our results revealed that Sor (≥ 5 µM) concentration- and time-dependently reduced cell viability and induced apoptosis in H9c2 myoblasts. Sor treatment promoted intracellular reactive oxygen species (ROS) generation, and subsequent Ca2+ overload as well as apoptosis, which were abolished by the ROS scavenger MPG. Sor inhibited the basal autophagy activity of cells, as supported by the fact that ERK1/2 inhibition-dependent decreases of autophagosomes and autolysosomes, and p62 accumulation in a concentration- and time-dependent manner. Improving autophagy with rapamycin abrogated Sor-induced ROS and Ca2+ overloads, and cell apoptosis. Furthermore, Sor compromised mitochondrial morphology and caused excessive mitochondrial fragmentation in cells. The imbalance of mitochondrial dynamics was attributed to ROS-mediated CaMKII overactivity, and increased phosphorylation of dynamin-related protein 1 (phosph-Drp1). Suppression of CaMKII with KN-93 or mitochondrial fission with mitochondrial division inhibitor-1 (Mdivi-1) attenuated Sor-induced ROS and Ca2+ overloads as well as apoptosis. In conclusion, these results provide the first evidence that impairments in autophagy and mitochondrial dynamics are involved in Sor-induced cardiomyocyte apoptosis. The present study may provide a potential strategy for preventing or reducing cardiotoxicity of Sor.
Subject(s)
Mitochondrial Dynamics , Myocytes, Cardiac , Humans , Sorafenib/toxicity , Sorafenib/metabolism , Reactive Oxygen Species/metabolism , Myocytes, Cardiac/metabolism , Cardiotoxicity/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Dynamins/metabolism , Apoptosis , AutophagyABSTRACT
Hepatocellular carcinoma (HCC) is one of the most malignant human cancers, with a high mortality rate worldwide. Within an HCC tumor, cancer stem cells (CSCs) are responsible for tumor maintenance and progression and may contribute to resistance to standard HCC treatments. Previously, we characterized CD133+ cells as CSCs in primary HCC and identified chromenopyrimidinone (CPO) as a novel therapeutic for the effective treatment of CD133+ HCC. However, the biological function and molecular mechanism of CD133 remain unclear. Epigenetic alterations of CSCs have impacts on tumor initiation, progression, and therapeutic response. Here, we found that pharmacological and genetic depletion of CD133 in HCC attenuated the activity of DNA methyltransferases via control of DNMT3B stabilization. Genes were ranked by degree of promoter hypo/hyper methylation and significantly differential expression to create an "epigenetically activated by CPO" ranked genes list. Through this epigenetic analysis, we found that CPO treatment altered DNA methylation-mediated oncogenic signaling in HCCs. Specifically, CPO treatment inhibited Adenylyl cyclase-associated protein 1 (CAP1) expression, thereby reducing FAK/ERK activity and EMT-related proteins in HCC. Moreover, CPO improved the efficacy of sorafenib by inhibiting CAP1 expression and FAK/ERK activation in sorafenib-resistant HCC. These novel mechanistic insights may ultimately open up avenues for strategies targeting DNA methylation in liver cancer stem cells and provides novel therapeutic function of CPO for the effective treatment of sorafenib-resistant HCC.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular , Liver Neoplasms , Pyrimidinones/pharmacology , Adenylyl Cyclases/metabolism , Adenylyl Cyclases/pharmacology , Adenylyl Cyclases/therapeutic use , Carcinoma, Hepatocellular/metabolism , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Cytoskeletal Proteins/metabolism , Humans , Liver Neoplasms/metabolism , Neoplastic Stem Cells/metabolism , Oligopeptides , Sorafenib/metabolism , Sorafenib/pharmacology , Sorafenib/therapeutic useABSTRACT
BACKGROUND: Hepatocellular carcinoma (HCC) is the fifth most diagnosed cancer and the second leading cause of cancer-related deaths worldwide. Sorafenib is the standard treatment used in the advanced stages of HCC. Cell therapy with mesenchymal stem cells (MSCs)-based cell therapy has proven effective in immune regulation and tumour growth inhibition. OBJECTIVES: In this study, we investigated the anti-inflammatory effect of MSCs on HCC xenografts. METHODS: Human HepG2 cell lines were subcutaneously implanted into the flank of 12 nude mice, divided into three groups: the control group, the IV group (intravenous MSCs injection) and the local group (local MSCs injection). Mice were sacrificed 6 weeks after tumour implantation, and tumours were resected entirety. Quantitative real-time polymerase chain reaction (qRT-PCR) measured the gene expression of inflammatory markers, including tumour necrosis factor-α (TNF-α), interleukin (IL)-1α and IL-10. Aspartate transaminase (AST), alanine transaminase (ALT) and urea levels were measured using spectrophotometry to ensure the safety of MSC therapy. RESULTS: Gene expressions for all three inflammatory markers were reduced in both MSCs groups compared to the control group. AST, ALT and urea levels remained in normal ranges. CONCLUSIONS: MSC therapy can reduce inflammation in HCC xenograft mouse models.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Rodent Diseases , Alanine Transaminase/metabolism , Alanine Transaminase/pharmacology , Alanine Transaminase/therapeutic use , Animals , Anti-Inflammatory Agents/pharmacology , Aspartate Aminotransferases/metabolism , Aspartate Aminotransferases/pharmacology , Aspartate Aminotransferases/therapeutic use , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/veterinary , Disease Models, Animal , Heterografts , Humans , Interleukin-10/metabolism , Interleukin-10/pharmacology , Interleukin-10/therapeutic use , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Liver Neoplasms/veterinary , Mesenchymal Stem Cell Transplantation/veterinary , Mesenchymal Stem Cells/metabolism , Mice , Mice, Nude , Sorafenib/metabolism , Sorafenib/pharmacology , Sorafenib/therapeutic use , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Cancer stem cells (CSCs) are reported to play essential roles in chemoresistance and metastasis. Pathways regulating CSC self-renewal and proliferation, such as Hedgehog, Notch, Wnt/ß-catenin, TGF-ß, and Myc, may be potential therapeutic targets. Here, a functional screening from the focused library with 365 compounds is performed by a step-by-step strategy. Among these candidate molecules, phenyl-2-pyrimidinyl ketone 4-allyl-3-amino selenourea (CU27) is chosen for further identification because it proves to be the most effective compound over others on CSC inhibition. Through ingenuity pathway analysis, it is shown CU27 may inhibit CSC through a well-known stemness-related transcription factor c-Myc. Gene set enrichment analysis, dual-luciferase reporter assays, expression levels of typical c-Myc targets, molecular docking, surface plasmon resonance, immunoprecipitation, and chromatin immunoprecipitation are conducted. These results together suggest CU27 binds c-Myc bHLH/LZ domains, inhibits c-Myc-Max complex formation, and prevents its occupancy on target gene promoters. In mouse models, CU27 significantly sensitizes sorafenib-resistant tumor to sorafenib, reduces the primary tumor size, and inhibits CSC generation, showing a dramatic anti-metastasis potential. Taken together, CU27 exerts inhibitory effects on CSC and CSC-associated traits in hepatocellular carcinoma (HCC) via c-Myc transcription activity inhibition. CU27 may be a promising therapeutic to treat sorafenib-resistant HCC.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Selenium Compounds , Selenium , Animals , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Early Detection of Cancer , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Mice , Molecular Docking Simulation , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Selenium/metabolism , Selenium/pharmacology , Selenium Compounds/metabolism , Selenium Compounds/pharmacology , Sorafenib/metabolism , Sorafenib/pharmacologyABSTRACT
BACKGROUND: Distinct subsets of cancer stem cells (CSCs) drive the initiation and progression of malignant tumors via enhanced self-renewal and development of treatment/apoptosis resistance. Endometrial CSC-selective drugs have not been successfully developed because most endometrial cell lines do not contain a sufficient proportion of stable CSCs. Here, we aimed to identify endometrial CSC-containing cell lines and to search for endometrial CSC-selective drugs. METHODS: We first assessed the presence of CSCs by identifying side populations (SPs) in several endometrial cancer cell lines. We then characterized cell viability, colony-formation, transwell invasion and xenotransplantion capability using the isolated SP cells. We also conducted real-time RT-PCR, immunoblot and immunofluorescence analyses of the cells' expression of CSC-associated markers. Focusing on 14 putative CSC-selective drugs, we characterized their effects on the proliferation and apoptosis of endometrial cancer cell lines, examining cell viability and annexin V staining. We further examined the inhibitory effects of the selected drugs, focusing on proliferation, invasion, expression of CSC-associated markers and tumor formation. RESULTS: We focused on HHUA cells, an endometrial cancer cell line derived from a well-differentiated endometrial adenocarcinoma. HHUA cells contained a sufficient proportion of stable CSCs with an SP phenotype (HHUA-SP). HHUA-SP showed greater proliferation, colony-formation, and invasive capabilities compared with the main population of HHUA cells (HHUA-MP). HHUA-SP generated larger tumors with higher expression of proliferation-related markers, Ki67, c-MYC and phosphorylated ERK compared with HHUA-MP when transplanted into immunodeficient mice. Among the 14 candidate drugs, sorafenib, an inhibitor of RAF pathways and multiple kinase receptors, inhibited cell proliferation and invasion in both HHUA-SP and -MP, but more profoundly in HHUA-SP. In vivo treatment with sorafenib for 4 weeks reduced the weights of HHUA-SP-derived tumors and decreased the expression of Ki67, ZEB1, and RAF1. CONCLUSIONS: Our results suggest that HHUA is a useful cell line for discovery and identification of endometrial CSC-selective drugs, and that sorafenib may be an effective anti-endometrial cancer drug targeting endometrial CSCs.
Subject(s)
Endometrial Neoplasms , MAP Kinase Signaling System , Animals , Carcinogenesis/pathology , Cell Line, Tumor , Cell Proliferation , Endometrial Neoplasms/drug therapy , Endometrial Neoplasms/genetics , Endometrial Neoplasms/metabolism , Female , Humans , Ki-67 Antigen/metabolism , Mice , Neoplastic Stem Cells/metabolism , Sorafenib/metabolism , Sorafenib/pharmacologyABSTRACT
Hypoxia promotes drug resistance and induces the expression of hypoxia inducible factor (HIF)1α in liver cancer cells. However, to date, no selective HIF1α inhibitor has been clinically approved. The aim of this study is to investigate a drugtargetable molecule that can regulate HIF1α under hypoxia. The present study demonstrated that hyperactivation of dualspecificity tyrosinephosphorylationregulated kinase 1A (DYRK1A)/HIF1α signaling was associated with an increased risk of liver cancer. In addition, DYRK1A knockdown using small interfering RNA transfection or treatment with harmine, a natural alkaloid, significantly reduced the protein expression levels of HIF1α in liver cancer cells under hypoxic conditions in vitro. Conversely, DYRK1A overexpressionvector transfection in liver cancer cell lines notably induced HIF1α expression under the same conditions. Furthermore, DYRK1A was shown to interact and activate STAT3 under hypoxia to regulate HIF1α expression. These findings indicated that DYRK1A may be a potential upstream activator of HIF1α and positively regulate HIF1α via the STAT3 signaling pathway in liver cancer cells. Additionally, treatment with harmine attenuated the proliferative ability of liver cancer cells under hypoxic conditions using sulforhodamine B and colony formation assay. Furthermore, DYRK1A knockdown could significantly enhance the antiliver cancer effects of regorafenib and sorafenib under hypoxia. Cotreatment with harmine and either regorafenib or sorafenib also promoted cell death via the STAT3/HIF1α/AKT signaling pathway under hypoxia using PI staining and western blotting. Overall, the results from the present study suggested that DYRK1A/HIF1α signaling may be considered a novel pathway involved in chemoresistance, thus providing a potentially effective therapeutic regimen for treating liver cancer.
Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia/metabolism , Liver Neoplasms/drug therapy , Phenylurea Compounds/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyridines/pharmacology , Sorafenib/pharmacokinetics , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Liver Neoplasms/physiopathology , Phenylurea Compounds/metabolism , Protective Factors , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Pyridines/metabolism , Sorafenib/metabolism , Dyrk KinasesABSTRACT
OBJECTIVE: Cytochrome P450 3A5 (CYP3A5) is a highly polymorphic gene and the encoded protein variants differ in catalytic activity, leading to inter-individual variation in metabolic ability. The aim of the current study was to investigate the effects of seven allelic variants on the ability of CYP3A5 to metabolize sorafenib in vitro and further explore the impacts of CYP3A5 polymorphism on the proliferation and apoptosis of hepatocellular carcinoma cell line (HepG2) induced by sorafenib. METHODS: Wild-type and variant CYP3A5 enzymes were expressed in Spodoptera frugiperda insect cells using a baculovirus dual-expression system, and protein expression was checked by western blot. The enzymes were incubated with sorafenib at 37°C for 30 min, and formation of the major metabolite sorafenib N-oxide was assayed using ultra-performance liquid chromatography and tandem mass spectrometry. Intrinsic clearance values (Vmax/Km) were calculated for each enzyme. Additionally, recombinant HepG2 cells transfecting with CYP3A5 variants were used to investigate the effects of sorafenib on the proliferation of HepG2 cells. RESULTS: Intrinsic clearance of the six variants CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 was 26.41-71.04% of the wild-type (CYP3A5*1) value. In contrast, the clearance value of the variant CYP3A5*6 was significantly higher (174.74%). Additionally, the decreased ATP levels and cell viability and the increased cell apoptosis in HepG2 cells transfected with CYP3A5*2, CYP3A5*3A, CYP3A5*3C, CYP3A5*4, CYP3A5*5, and CYP3A5*7 were observed, whereas, the increased ATP levels and cell viability and the reduced cell apoptosis in HepG2 cells transfected with CYP3A5*6 were also investigated when compared to CYP3A5*1. CONCLUSION: Our results suggest that CYP3A5 polymorphism influences sorafenib metabolism and pharmacotherapeutic effect in hepatic carcinomas. These data may help explain differential response to drug therapy for hepatocellular carcinoma, and they support the need for individualized treatment.
Subject(s)
Antineoplastic Agents/toxicity , Antineoplastic Agents/therapeutic use , Carcinoma, Hepatocellular/drug therapy , Cytochrome P-450 CYP3A/genetics , Liver Neoplasms/drug therapy , Sorafenib/toxicity , Sorafenib/therapeutic use , Antineoplastic Agents/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Humans , Polymorphism, Genetic , Sorafenib/metabolism , Tumor Cells, CulturedABSTRACT
We conducted large-scale screening test on drugs that were already approved for other diseases to find pigmentation-modulating agents. Among drugs with potential for pigmentation control, we selected sorafenib and further investigated the effect on pigmentation using HM3KO melanoma cells. As a result of treating melanoma cells with sorafenib, pigmentation was promoted in terms of melanin content and tyrosinase activity. Sorafenib increased mRNA and protein levels of pigmentation-related genes such as MITF, tyrosinase and TRP1. To uncover the action mechanism, we investigated the effect of sorafenib on the intracellular signalling pathways. Sorafenib reduced phosphorylation of AKT and ERK, suggesting that sorafenib induces pigmentation through inhibition of the AKT and ERK pathways. In addition, sorafenib significantly increased the level of active ß-catenin, together with activation of ß-catenin signalling. Mechanistic study revealed that sorafenib decreased phosphorylation of serine 9 (S9) of GSK3ß, while it increased phosphorylation of tyrosine 216 (Y216) of GSK3ß. These results suggest that sorafenib activates the ß-catenin signalling through the regulation of GSK3ß phosphorylation, thereby affecting the pigmentation process.
Subject(s)
Antineoplastic Agents/pharmacology , Melanoma/pathology , Pigmentation/drug effects , Skin Neoplasms/pathology , Sorafenib/pharmacology , beta Catenin/metabolism , Antineoplastic Agents/metabolism , Cell Line, Tumor , Humans , Signal Transduction/drug effects , Sorafenib/metabolismABSTRACT
In this study, we examined the regulatory role of CCDC34 in the sorafenib sensitivity of hepatocellular carcinoma (HCC) and its functional partners. Wide-type Huh7 and Hep3B and induced sorafenib-resistant (SR) Huh7/SR and Hep3B/SR cells were used as in vitro cell models. Immunofluorescent staining and coimmunoprecipitation were performed to check protein-protein interaction. Cell Counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL), PI/Annexin V staining, and western blot analysis were performed to assess cell response to sorafenib. The results showed that CCDC34 upregulation in HCC was associated with poor survival. Huh7/SR and Hep3B/SR cells had significantly higher CCDC34 expression than the parental cell lines. RABL2A expression was significantly upregulated in SR HCC cells and interacted with CCDC34 in its GTP-bound state in Huh7/SR and Hep3B/SR cells. RABL2A depletion sensitized Huh7/SR and Hep3B/SR cells to sorafenib. RABL2A Q80L mutant (GTP-bound state locked), but not S35N mutant (GDP-bound state locked) overexpression increased sorafenib IC50 of Huh7 and Hep3B cells. CCDC34 depletion nearly abrogated the protective effects of RABL2A Q80L overexpression both in vitro and in vivo. RABL2A Q80L overexpression significantly increased the expression of p-p38 and p-JNK, the effects of which were significantly attenuated by CCDC34 depletion. In summary, we infer that the RABL2A-CCDC34 axis plays an important role in mediating p38/MAPK and JNK/MAPK signaling, thereby contributing to acquired sorafenib resistance in HCC.
Subject(s)
Antigens, Neoplasm/metabolism , Carcinoma, Hepatocellular/metabolism , Neoplasm Proteins/metabolism , Sorafenib/metabolism , Antigens, Neoplasm/physiology , Antineoplastic Agents/pharmacology , Carcinoma, Hepatocellular/drug therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , China , Drug Resistance, Neoplasm/physiology , Gene Expression/drug effects , Gene Expression/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Liver Neoplasms/drug therapy , Liver Neoplasms/metabolism , Neoplasm Proteins/physiology , Signal Transduction/drug effects , Sorafenib/pharmacology , rab GTP-Binding Proteins/metabolism , rab GTP-Binding Proteins/physiologyABSTRACT
Sorafenib was suggested to cause drug-drug interaction (DDI) with the common anticoagulant, warfarin based on published studies. The inhibition on CYP2C9 enzyme was thought to be the mechanism, but further studies are warranted. Thus, a mechanistic PBPK/PD model for warfarin enantiomers was developed to predict DDI potential with sorafenib, aiming at providing reference for the rational use of both drugs. PBPK models of warfarin enantiomers were constructed by Simcyp software. A mechanistic PK/PD model was built in NONMEM software. PBPK model of sorafenib was fitted via a top-down method. The final PBPK/PD model of warfarin enantiomers was verified and validated by different dosing regimens, ethnicities and genetic polymorphisms, and used to perform DDI simulations between warfarin racemate and sorafenib among general populations and sub-populations with various CYP2C9 and VKORC1 genotypes. Results suggested low DDI risk between warfarin and sorafenib for general populations. Potentially serious consequence was seen for those carrying both CYP2C9 ∗2 and ∗3 and VKORC1 A/A genotypes. This PBPK/PD modeling approach for warfarin enantiomers enabled DDI evaluation with sorafenib. Close monitoring and warfarin dosage adjustment were recommended for patients carrying mutant genotypes. The novel model could be applied to investigate other drugs that may interact with warfarin.
Subject(s)
Blood Coagulation , Cytochrome P-450 CYP2C9/genetics , Drug Interactions/physiology , Hemorrhage , Sorafenib , Vitamin K Epoxide Reductases/genetics , Warfarin , Anticoagulants/metabolism , Anticoagulants/pharmacokinetics , Blood Coagulation/drug effects , Blood Coagulation/physiology , Computer Simulation , Dose-Response Relationship, Drug , Hemorrhage/chemically induced , Hemorrhage/prevention & control , Humans , International Normalized Ratio , Models, Biological , Models, Theoretical , Pharmacogenomic Testing/methods , Risk Assessment/methods , Sorafenib/metabolism , Sorafenib/pharmacokinetics , Warfarin/metabolism , Warfarin/pharmacokineticsABSTRACT
Despite liver cancer being the second-leading cause of cancer-related death worldwide, few systemic drugs have been approved. Sorafenib, the first FDA-approved systemic drug for unresectable hepatocellular carcinoma (HCC), is limited by resistance. However, the precise mechanisms underlying this phenomenon are unknown. Since fibrinogen-like 1 (FGL1) is involved in HCC progression and upregulated after anticancer therapy, we investigated its role in regulating sorafenib resistance in HCC. FGL1 expression was assessed in six HCC cell lines (HepG2, Huh7, Hep3B, SNU387, SNU449, and SNU475) using western blotting. Correlations between FGL1 expression and sorafenib resistance were examined by cell viability, colony formation, and flow cytometry assays. FGL1 was knocked-down to confirm its effects on sorafenib resistance. FGL1 expression was higher in HepG2, Huh7, and Hep3B cells than in SNU387, SNU449, and SNU475 cells; high FGL1-expressing HCC cells showed a lower IC50 and higher sensitivity to sorafenib. In Huh7 and Hep3B cells, FGL1 knockdown significantly increased colony formation by 61% (p = 0.0013) and 99% (p = 0.0002), respectively, compared to that in controls and abolished sorafenib-induced suppression of colony formation, possibly by modulating ERK and autophagy signals. Our findings demonstrate that sorafenib resistance mediated by FGL1 in HCC cells, suggesting FGL1 as a potential sorafenib-resistance biomarker and target for HCC therapy.