Curcumin Induces p53-Null Hepatoma Cell Line Hep3B Apoptosis through the AKT-PTEN-FOXO4 Pathway.
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ABSTRACT:
SUBMITTER: Liou AT
PROVIDER: S-EPMC5523542 | biostudies-other | 2017
REPOSITORIES: biostudies-other
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Project description:Curcumin is an anticancer agent, but adverse effects and low bioavailability are its main drawbacks, which drives efforts in chemical modifications of curcumin. This study evaluated antiproliferative activity and cancer cell selectivity of a curcumin derivative, curcumin nicotinate (CN), in which two niacin molecules were introduced. Our data showed that CN effectively inhibited proliferation and clonogenic growth of colon (HCT116), breast (MCF-7) and nasopharyngeal (CNE2, 5-8F and 6-10B) cancer cells with IC50 at 27.7 ?M, 73.4 ?M, 64.7 ?M, 46.3 ?M, and 31.2 ?M, respectively. In cancer cells, CN induced apoptosis and cell cycle arrest at G2/M phase through a p53-mediated mechanism, where p53 was activated, p21 and pro-apoptotic proteins Bid and Bak were upregulated, and PARP was cleaved. In non-transformed human mammary epithelial cells MCF10A, CN at 50 µM had no cytotoxicity and p53 was not activated, but curcumin at 12.5 µM activated p53 and p21 and inhibited MCF10A cell growth. These data suggest that CN inhibits cell growth and proliferation through p53-mediated apoptosis and cell cycle arrest with cancer cell selectivity.
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Project description:Hyperactivated AKT kinase due to loss of its negative regulator PTEN influences many aspects of cancer biology, including chromatin. AKT primarily regulates acetyl-CoA production and phosphorylates many histone-modulating enzymes, resulting in their activation or inhibition. Therefore, understanding the therapeutic impact of AKT inhibition on chromatin-related events is essential. Here, we report that AKT inhibition in prostate-specific PTEN knockout mice significantly induces di- and trimethylation of H3K4 with concomitant reduction in H3K9 acetylation. Mechanistically, we observed that AKT inhibition reduces expression of the H3K4 methylation-specific histone demethylases KDM5 family, especially KDM5B expression at transcriptional levels. Furthermore, we observed that AKT negatively regulates miR-137 levels, which transcriptionally represses KDM5B expression. Overexpression of miR-137 significantly reduced KDM5B and increased H3K4 methylation levels but failed to change AKT phosphorylation. Overall, we observed that AKT transcriptionally regulates KDM5B mainly via repression of miR-137. Our data identify a mechanism by which AKT kinase modulates the prostate cancer epigenome through regulating H3K4 methylation. Additional studies on AKT inhibition-mediated induction of H3K4 methylation will help in designing strategies to enhance the therapeutic efficacy of PI3K/AKT inhibitors.
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Project description:SOX14 is a member of the SOX family of transcription factors mainly involved in the regulation of neural development. Recently, it became evident that SOX14 is one of four hypermethylated genes in cervical carcinoma, considered as a tumor suppressor candidate in this type of malignancy. In this paper we elucidated the role of SOX14 in the regulation of malignant properties of cervical carcinoma cells in vitro. Functional analysis performed in HeLa cells revealed that SOX14 overexpression decreased viability and promoted apoptosis through altering the expression of apoptosis related genes. Our results demonstrated that overexpression of SOX14 initiated accumulation of p53, demonstrating potential cross-talk between SOX14 and the p53 signaling pathway. Further analysis unambiguously showed that SOX14 triggered posttranslational modification of p53 protein, as detected by the significantly increased level of phospho-p53 (Ser-15) in SOX14-overexpressing HeLa cells. Moreover, the obtained results revealed that SOX14 activated p53 protein, which was confirmed by elevated p21Waf1/Cip1, a well known target gene of p53. This study advances our understanding about the role of SOX14 and might explain the molecular mechanism by which this transcription factor could exert tumor suppressor properties in cervical carcinoma.
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Project description:BackgroundIn recent years, copper complexes have gradually become the focus of potential anticancer drugs due to their available redox properties and low toxicity. In this study, a novel mitochondrion-targeting copper (II) complex, [Cu (ttpy-tpp)Br2] Br (simplified as CTB), is first synthesized by our group. CTB with tri-phenyl-phosphine (TPP), a targeting and lipophilic group, can cross the cytoplasmic and mitochondrial membranes of tumor cells. The present study aims to investigate how CTB affects mitochondrial functions and exerts its anti-tumor activity in hepatoma cells.MethodsMultiple molecular experiments including Flow cytometry, Western blot, Immunofluorescence, Tracker staining, Transmission Electron Microscopy and Molecular docking simulation were used to elucidate the underlying mechanisms. Human hepatoma cells were subcutaneously injected into right armpit of male nude mice for evaluating the effects of CTB in vivo.ResultsCTB induced apoptosis via collapse of mitochondrial membrane potential (MMP), ROS production, Bax mitochondrial aggregation as well as cytochrome c release, indicating that CTB-induced apoptosis was associated with mitochondrial pathway in human hepatoma cells. Mechanistic study revealed that ROS-related mitochondrial translocation of p53 was involved in CTB-mediated apoptosis. Simultaneously, elevated mitochondrial Drp1 levels were also observed, and interruption of Drp1 activation played critical role in p53-dependent apoptosis. CTB also strongly suppressed the growth of liver cancer xenografts in vivo.ConclusionIn human hepatoma cells, CTB primarily induces mitochondrial dysfunction and promotes accumulation of ROS, leading to activation of Drp1. These stimulation signals accelerate mitochondrial accumulation of p53 and lead to the eventual apoptosis. Our research shows that CTB merits further evaluation as a chemotherapeutic agent for the treatment of Hepatocellular carcinoma (HCC).
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Project description:Hepatocellular carcinoma (HCC) is the most common liver cancer and a major cause of adult death. The current treatments for HCC suffer from drug resistance and poor prognosis; therefore, novel therapeutic agents are urgently needed. Phytochemicals have been proposed to treat a range of cancers. Among them, ?-lipoic acid (?-LA), a naturally synthesized antioxidant found in various dietary animal and plant sources, prevents oxidant-mediated cell death in normal cells while inducing apoptosis in several cancer cell lines. Previously, we demonstrated that the treatment of hepatoma cells with ?-LA induced apoptosis, which was preceded by the generation of reactive oxygen species (ROS) and activation of the p53 protein, a known inducer of mitochondria-mediated apoptosis. Several studies have shown that ROS-induced apoptosis is associated with endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) activation. Herein, we investigated if ?-LA-induced apoptosis in hepatoma cell lines was ER stress- and UPR-mediated by gene expression profiling analyses. UPR and ER stress pathways were the most up-regulated after treatment with ?-LA. This finding, which has been confirmed by expression analyses of ER- and UPR-associated proteins, provides a better understanding of the molecular mechanisms behind the anti-tumoral action of ?-LA on hepatoma cells.
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Project description:Aims: Hilar cholangiocarcinoma (HCCA) is a tumour with high malignancy, low surgical resection potential, and a poor prognosis. Ecotropic Viral Integration site 1 (EVI1) is a transcriptional regulator that has been proven to be associated with tumourigenesis and progression in many human solid tumours. However, the expression of EVI1 and its role in HCCA progression remain unclear. The aim of this study was to clarify the association between EVI1 expression and clinical outcomes in patients with HCCA. Methods: The expression of EVI1 in HCCA tissue samples and cell lines was examined by quantitative real-time PCR (qRT-PCR), Western blotting, and immunohistochemistry (IHC). Kaplan-Meier analysis was used for survival analysis. A log-rank test was performed for univariate analysis of survival, and a Cox regression model was utilized for multivariate analysis of survival. Cell proliferation was measured by cell counting kit-8 (CCK-8), colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) assays. The cell cycle was evaluated by flow cytometry. Cell apoptosis was detected by flow cytometry and a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) assay. In vivo tumour growth was observed for xenografts in nude mice. Results: EVI1 expression was upregulated in HCCA tissue samples and correlated with a poor prognosis. In clinical specimens, the expression of EVI1 correlated with tumour histological grade and tumour size. Knocking down EVI1 expression reduced HCCA cell proliferation, blocked cell cycle progression, and promoted apoptosis in vitro and in vivo. Furthermore, we found that EVI1 could regulate the AKT signalling pathway by regulating PTEN levels in HCCA. Conclusion: Our data revealed that EVI1 played important roles in HCCA tumourigenesis and development. Our findings suggest that EVI1 may be a potentially useful therapeutic target in HCCA.
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Project description:BackgroundOesophageal cancer incidence is increasing and survival rates remain extremely poor. Natural agents with potential for chemoprevention include the phytochemical curcumin (diferuloylmethane). We have examined the effects of curcumin on a panel of oesophageal cancer cell lines.MethodsMTT (3-(4,5-dimethyldiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assays and propidium iodide staining were used to assess viability and DNA content, respectively. Mitotic catastrophe (MC), apoptosis and autophagy were defined by both morphological criteria and markers such as MPM-2, caspase 3 cleavage and monodansylcadaverine (MDC) staining. Cyclin B and poly-ubiquitinated proteins were assessed by western blotting.ResultsCurcumin treatment reduces viability of all cell lines within 24 h of treatment in a 5-50 muM range. Cytotoxicity is associated with accumulation in G2/M cell-cycle phases and distinct chromatin morphology, consistent with MC. Caspase-3 activation was detected in two out of four cell lines, but was a minor event. The addition of a caspase inhibitor zVAD had a marginal or no effect on cell viability, indicating predominance of a non-apoptotic form of cell death. In two cell lines, features of both MC and autophagy were apparent. Curcumin-responsive cells were found to accumulate poly-ubiquitinated proteins and cyclin B, consistent with a disturbance of the ubiquitin-proteasome system. This effect on a key cell-cycle checkpoint regulator may be responsible for the mitotic disturbances and consequent cytotoxicity of this drug.ConclusionCurcumin can induce cell death by a mechanism that is not reliant on apoptosis induction, and thus represents a promising anticancer agent for prevention and treatment of oesophageal cancer.
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Project description:The thioredoxin (Trx) system plays critical roles in regulating intracellular redox levels and defending organisms against oxidative stress. Recent studies indicated that Trx reductase (TrxR) was overexpressed in various types of human cancer cells indicating that the Trx-TrxR system may be a potential target for anti-cancer drug development. This study investigated the synergistic effect of auranofin, a TrxR-specific inhibitor, on sulforaphane-mediated apoptotic cell death using Hep3B cells. The results showed that sulforaphane significantly enhanced auranofin-induced apoptosis by inhibiting TrxR activity and cell proliferation compared to either single treatment. The synergistic effect of sulforaphane and auranofin on apoptosis was evidenced by an increased annexin-V-positive cells and Sub-G1 cells. The induction of apoptosis by the combined treatment caused the loss of mitochondrial membrane potential (??m) and upregulation of Bax. In addition, the proteolytic activities of caspases (-3, -8, and -9) and the degradation of poly (ADP-ribose) polymerase, a substrate protein of activated caspase-3, were also higher in the combined treatment. Moreover, combined treatment induced excessive generation of reactive oxygen species (ROS). However, treatment with N-acetyl-L-cysteine, a ROS scavenger, reduced combined treatment-induced ROS production and apoptosis. Thereby, these results deduce that ROS played a pivotal role in apoptosis induced by auranofin and sulforaphane. Furthermore, apoptosis induced by auranofin and sulforaphane was significantly increased through inhibition of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Taken together, the present study demonstrated that down-regulation of TrxR activity contributed to the synergistic effect of auranofin and sulforaphane on apoptosis through ROS production and inhibition of PI3K/Akt signaling pathway.
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