salinomycin and Carcinoma--Hepatocellular

Hepatocellular carcinoma (HCC) is a main cause of cancer-related mortality and its etiology is not fully understood. As prominent factors that regulate cellular homeostasis, both reactive oxygen species (ROS) and autophagy are considered to play an essential role in the liver carcinogenesis. However, the crosstalk between ROS and autophagy is not well characterized in the pathogenesis of HCC. This review summarizes the roles of autophagy in ROS-mediated hepatocarcinogenesis and discusses the role of ROS-induced autophagy in HCC cell fate decision following treatment with chemotherapeutic agents in preclinical settings, which may allow the identification of novel strategies for the treatment of HCC.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Bevacizumab; Carcinogenesis; Carcinoma, Hepatocellular; DNA Damage; Humans; Liver Neoplasms; Oxaliplatin; Oxidative Stress; Pyrans; Reactive Oxygen Species

There is currently no effective treatment for advanced hepatocellular carcinoma (HCC), and chemotherapy has little effect on long-term survival of HCC patients, largely due to the cancer stem cell (CSC) chemoresistance of HCC.. We constructed a small-molecule nanometer-sized prodrug (nanoprodrug) loaded with salinomycin (SAL) for the treatment of HCC. SAL was encapsulated by the prodrug LA-SN38 (linoleic acid modified 7-ethyl-10-hydroxycamptothecin) to construct a self-assembled nanoprodrug further PEGylated with DSPE-PEG. Delivery of the SAL- and LA-SN38-based nanoprodrugs effectively promoted apoptosis of HCC cells, exerted inhibition of HCC tumor-sphere formation as well as HCC cell motility and invasion, and reduced the proportion of CD133+ HCC-CSC cells. In nude mice, the nanoprodrug suppressed growth of tumor xenografts derived from human cell lines and patient.. Our results show that SAL-based nanoprodrugs are a promising platform for treating patients with HCC and a novel strategy for combination therapy of cancers.

Topics: Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Humans; Irinotecan; Linoleic Acid; Liver Neoplasms; Male; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Neoplastic Stem Cells; Phosphatidylethanolamines; Polyethylene Glycols; Prodrugs; Pyrans; Xenograft Model Antitumor Assays

Evidence indicated that GATA5 may suppress hepatocellular carcinoma (HCC) cell malignant transformation, but the mechanism of how GATA5 affects cancer cell reprogramming to inhibit HCC malignant behaviour is still unclear. In this study, we report that the expression of β-catenin and reprogramming genes p-Oct4, Nanog, Klf4, c-myc and EpCAM was significantly higher in HCC tissues compared to normal liver tissues. In contrast, the expression of GATA5 was significantly lower in HCC tissues compared to normal liver tissues. Transfection of CDH-GATA5 vectors into HCC cells (HLE, Bel 7402 and PLC/PRF/5 cells) increased the GATA5 expression and decreased the expression of β-catenin and reprogramming genes p-Oct4, Nanog, Klf4, c-myc and EpCAM. Increased GATA5 expression by transfection with its expression vectors was also able to inhibit the cell growth, colony formation and capability of migration, invasion, while promoting apoptosis in HCC cells. Results revealed that GATA5 co-localization with β-catenin in the cytoplasm, preventing β-catenin from entering the nucleus. Treatment with the specific Wnt/β-catenin pathway inhibitor salinomycin was able to reduce the expression of β-catenin and reprogramming genes. Salinomycin exerted a similar influence as GATA5, and siRNA-GATA5 restored β-catenin and reprogramming gene expression. This study demonstrates that an increase in the expression of GATA5 inhibits the expression of β-catenin and reprogramming genes and suppresses tumour growth, colony formation, metastasis and invasion, while promoting apoptosis in HCC cells. The mechanism of GATA5 inhibiting the malignant behaviours of HCC cells may involve in the disruption of the Wnt/β-catenin pathway and the reduction of reprogramming gene expression.

Topics: Adult; Aged; Apoptosis; beta Catenin; Carcinoma, Hepatocellular; Case-Control Studies; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Transformation, Neoplastic; Epithelial Cell Adhesion Molecule; Female; GATA5 Transcription Factor; Gene Expression Regulation, Neoplastic; Hepatocytes; Humans; Kruppel-Like Factor 4; Kruppel-Like Transcription Factors; Liver Neoplasms; Male; Middle Aged; Nanog Homeobox Protein; Octamer Transcription Factor-3; Proto-Oncogene Proteins c-myc; Pyrans; RNA, Small Interfering; Wnt Signaling Pathway

Salinomycin has recently been identified as an antitumour drug for several types of cancer stem cell (CSC) treatments. However, the effects of salinomycin on the migratory and invasive properties of liver cancer stem cells (LCSCs) are unclear. In present study, we investigated the effect of salinomycin on the migration and invasion of LCSCs, and examined the molecular mechanisms underlying the anticancer effects of salinomycin. Here we showed that the migration and invasion of LCSCs were significantly suppressed in a salinomycin dose-dependent manner. Moreover, western blot analysis showed that salinomycin repressed the phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK1/2). Taken together, these findings provide new evidence that salinomycin suppresses the migration and invasion of LCSCs by inhibiting the expression of the FAK-ERK1/2 signalling pathway. In addition, the analysis of the mechanical properties showed that salinomycin increased cell stiffness in LCSCs via the FAK, and ERK1/2 pathways, suggesting that the inhibition of LCSC migration might partially contribute to the increase in cell stiffness stimulated by salinomycin. To further examine the role of salinomycin on cell motility and stiffness, the actin cytoskeleton of LCSCs was detected. The increased F-actin filaments in LCSCs induced by salinomycin reflected the increase in cell stiffness and the decrease in cell migration. Overall, these results showed that salinomycin inhibits the migration and invasion of LCSCs through the dephosphorylated FAK and ERK1/2 pathways, reflecting the changes in cell stiffness resulting from the increased actin cytoskeleton.

Topics: Actins; Anti-Bacterial Agents; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Coccidiostats; Extracellular Signal-Regulated MAP Kinases; Focal Adhesion Kinase 1; Humans; Liver Neoplasms; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplastic Stem Cells; Pyrans

To develop salinomycin-loaded nanoliposomes (SLN), doxorubicin-loaded nanoliposomes (DLN) and nanoliposomes codelivering salinomycin and doxorubicin (SDLN) to target both liver cancer cells and cancer stem cells.. The characterization and antitumor activity of SLN, DLN and SDLN were evaluated.. The doxorubicin/salinomycin sodium mole ratio of 1:1 had the best synergistic combination index value, and was chosen as the drug ratio in SDLN. SDLN could maintain the drug ratio between 1:1 and 3:1 in 12 h in vivo. SDLN and SLN + DLN showed the best tumor inhibitory rate, and could significantly decrease the percentage of liver cancer stem cells in vivo. SDLN and SLN + DLN may serve as an effective approach to treat liver cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Hepatocellular; Cell Survival; Doxorubicin; Drug Liberation; Hep G2 Cells; Humans; Liposomes; Liver Neoplasms; Male; Mice, Nude; Nanocapsules; Neoplastic Stem Cells; Particle Size; Pyrans; Surface Properties

The antibiotic salinomycin (Salin) was recently identified as an antitumor drug for the treatment of several types of solid tumors. However, the effects of Salin on the migratory and invasive properties of hepatocellular carcinoma (HCC) cells are unclear. The present study aimed to determine the antitumor efficacy and mechanism of Salin in HCC cells. Human HCC cells (HCCLM3) treated with Salin showed a concentration-dependent reduction in cell migration and invasion, and this was associated with reduced MMP9 expression. The MMP9 promoter and enhancer in a luciferase reporter assay revealed that Salin can regulate MMP9 expression through an activator protein (AP-1) site within the MMP9 enhancer. JunD, one of the AP-1 components, was significantly decreased by Salin in a concentration- and time-dependent manner. Salin was able to induce c-Jun NH2-kinase (JNK) phosphorylation and to block both JunD and MMP9 expression. Our results showed that JNK phosphorylation and JunD may be involved in the Salin-regulated MMP9 signaling pathway in HCCLM3 cells and may mediate HCC cell biological characteristics. Our studies provide new insight into the antitumor effects of Salin.

Topics: Antineoplastic Agents; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Enzyme-Linked Immunosorbent Assay; Humans; Liver Neoplasms; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Proto-Oncogene Proteins c-jun; Pyrans

Doxorubicin is a conventional and effective chemotherapy drug against hepatocellular carcinoma (HCC). However, during long-term doxorubicin monotherapy, HCC cells may eventually develop acquired-resistance to doxorubicin which results in recurrence and a poor prognosis. Salinomycin, an ionophore antibiotic, was recently reported to selectively kill human cancer stem cells (CSCs) which were response for chemoresistance. In this study, salinomycin was found to exert synergistic cytotoxicity with doxorubicin in HCC cells and be capable of inhibiting doxorubicin-induced epithelial-mesenchymal transition (EMT), an important cellular process involved in the acquired chemoresistance of tumors. Further experiments revealed that FOXO3a, a multifunctional transcription factor that can be activated by salinomycin, was vital in mediating doxorubicin-induced EMT. In addition, activated FOXO3a disturbed the interaction between β-catenin and TCF and inhibited the expression of β-catenin/TCF target genes (ZEB1, c-Myc and CyclinD1), which played important roles in doxorubicin-induced EMT in HCC cells. Finally, the enhanced curative efficacy of combination treatment of doxorubicin and salinomycin for HCC was confirmed in established xenograft models. In summary, the present study identifies a new doxorubicin-based chemotherapy for advanced HCC and provides a potential anti-cancer strategy targeting FOXO3a and related cell pathway molecules.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; beta Catenin; Carcinoma, Hepatocellular; Cell Line, Tumor; Doxorubicin; Drug Resistance, Neoplasm; Drug Synergism; Forkhead Box Protein O3; Forkhead Transcription Factors; Humans; Liver Neoplasms; Male; Mice; Mice, Nude; Pyrans; Random Allocation; TCF Transcription Factors; Transfection; Xenograft Model Antitumor Assays

To develop salinomycin-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with both CD133 aptamers A15 and EGFR aptamers CL4 (CESN), to target hepatocellular carcinoma (HCC) cells simultaneously expressing EGFR and CD133.. The antitumor activity and mechanism of CESN were investigated.. The cytotoxicity of CESN in HCC cells and CD133(+) HCC cells was superior to that of A15 or CL4-conjugted or nontargeted salinomycin-loaded nanoparticles. The antitumor assay in mice bearing HCC xenograft tumors confirmed the superior antitumor activity of CESN over other controls. We speculated that the improved therapeutic effect of CESN may be attributed to both targeting a higher percentage of HCC cells and increased delivery of salinomycin to HCC cells.

Topics: AC133 Antigen; Antigens, CD; Antineoplastic Agents; Aptamers, Nucleotide; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Drug Synergism; ErbB Receptors; Glycoproteins; Humans; Lactic Acid; Liver Neoplasms; Molecular Targeted Therapy; Nanocapsules; Nanoconjugates; Particle Size; Peptides; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Pyrans; Treatment Outcome

Hepatocellular carcinoma (HCC) is one of the few cancers in which a continuous increase in incidence has been observed over several years. Drug resistance is a major problem in the treatment of HCC. In the present study, we used salinomycin (Sal) and 5-fluorouracil (5-FU) combination therapy on HCC cell lines Huh7, LM3 and SMMC-7721 and nude mice subcutaneously tumor model to study whether Sal could increase the sensitivity of hepatoma cells to the traditional chemotherapeutic agent such as 5-FU. The combination of Sal and 5-FU resulted in a synergistic antitumor effect against liver tumors both in vitro and in vivo. Sal reversed the 5-FU-induced increase in CD133(+) EPCAM(+) cells, epithelial-mesenchymal transition and activation of the Wnt/β-catenin signaling pathway. The combination of Sal and 5-FU may provide us with a new approach to reverse drug resistant for the treatment of patients with HCC.

Topics: Analysis of Variance; Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Line; Colony-Forming Units Assay; DNA Primers; Drug Synergism; Drug Therapy, Combination; Epithelial-Mesenchymal Transition; Flow Cytometry; Fluorescent Antibody Technique; Fluorouracil; Humans; Immunohistochemistry; Liver Neoplasms; Mice; Pyrans; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Wnt Signaling Pathway

Salinomycin raised hope to be effective in anti-cancer therapies due to its capability to overcome apoptosis-resistance in several types of cancer cells. Recently, its effectiveness against human hepatocellular carcinoma (HCC) cells both in vitro and in vivo was demonstrated. However, the mechanism of action remained unclear. Latest studies implicated interference with the degradation pathway of autophagy. This study aimed to determine the impact of Salinomycin on HCC-autophagy and whether primary human hepatocytes (PHH) likewise are affected. Following exposure of HCC cell lines HepG2 and Huh7 to varying concentrations of Salinomycin (0-10 µM), comprehensive analysis of autophagic activity using western-blotting and flow-cytometry was performed. Drug effects were analyzed in the settings of autophagy stimulation by starvation or PP242-treatment and correlated with cell viability, proliferation, apoptosis induction, mitochondrial mass accumulation and reactive oxygen species (ROS) formation. Impact on apoptosis induction and cell function of PHH was analyzed. Constitutive and stimulated autophagic activities both were effectively suppressed in HCC by Salinomycin. This inhibition was associated with dysfunctional mitochondria accumulation, increased apoptosis and decreased proliferation and cell viability. Effects of Salinomycin were dose and time dependent and could readily be replicated by pharmacological and genetic inhibition of HCC-autophagy alone. Salinomycin exposure to PHH resulted in transient impairment of synthesis function and cell viability without apoptosis induction. In conclusion, our data suggest that Salinomycin suppresses late stages of HCC-autophagy, leading to impaired recycling and accumulation of dysfunctional mitochondria with increased ROS-production all of which are associated with induction of apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Autophagy-Related Protein 7; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cells, Cultured; Coccidiostats; Dose-Response Relationship, Drug; Flow Cytometry; Green Fluorescent Proteins; Hep G2 Cells; Hepatocytes; Humans; Liver Neoplasms; Microtubule-Associated Proteins; Pyrans; Reactive Oxygen Species; RNA Interference; Ubiquitin-Activating Enzymes

The anti-tumor antibiotic salinomycin (Sal) was recently identified as a selective inhibitor of breast cancer stem cells; however, the effect of Sal on hepatocellular carcinoma (HCC) is not clear. This study aimed to determine the anti-tumor efficacy and mechanism of Sal on HCC. HCC cell lines (HepG2, SMMC-7721, and BEL-7402) were treated with Sal. Cell doubling time was determinated by drawing growth curve, cell viability was evaluated using the Cell Counting Kit 8. The fraction of CD133(+) cell subpopulations was assessed by flow cytometry. We found that Sal inhibits proliferation and decreases PCNA levels as well as the proportion of HCC CD133(+)cell subpopulations in HCC cells. Cell cycle was analyzed using flow cytometry and showed that Sal caused cell cycle arrest of the various HCC cell lines in different phases. Cell apoptosis was evaluated using flow cytometry and Hoechst 33342 staining. Sal induced apoptosis as characterized by an increase in the Bax/Bcl-2 ratio. Several signaling pathways were selected for further mechanistic analyses using real time-PCR and Western blot assays. Compared to control, β-catenin expression is significantly down-regulated upon Sal addition. The Ca(2+) concentration in HCC cells was examined by flow cytometry and higher Ca(2+) concentrations were observed in Sal treatment groups. The anti-tumor effect of Sal was further verified in vivo using the hepatoma orthotopic tumor model and the data obtained showed that the size of liver tumors in Sal-treated groups decreased compared to controls. Immunohistochemistry and TUNEL staining also demonstrated that Sal inhibits proliferation and induces apoptosis in vivo. Finally, the role of Sal on in vivo Wnt/β-catenin signaling was evaluated by Western blot and immunohistochemistry. This study demonstrates Sal inhibits proliferation and induces apoptosis of HCC cells in vitro and in vivo and one potential mechanism is inhibition of Wnt/β-catenin signaling via increased intracellular Ca(2+) levels.

Topics: AC133 Antigen; Animals; Antigens, CD; Antineoplastic Agents; Apoptosis; beta Catenin; Calcium; Carcinoma, Hepatocellular; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Glycoproteins; Humans; Intracellular Space; Liver Neoplasms; Male; Mice; Peptides; Pyrans; Signal Transduction; Wnt Proteins; Xenograft Model Antitumor Assays