2019_Hepcidin TH1-5 Induced Apoptosis in HEPG2 Hepatocarcinoma and Hela Cervical Carcinoma Cell and Its Structure-Activity Relationship
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| copyright | Copyright©PWB2025 |
| country | Malaysia |
| date | 2019-06-30 |
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| id | 15344 |
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| internalnotes | Sila masukkan subject wajib Dissertations, Academic. Terima kasih... |
| originalfilename | HEPCIDIN TH1-5 INDUCED APOPTOSIS IN HEPG2 HEPATOCARCINOMA AND HELA CERVICAL CARCINOMA CELL AND ITS STRUCTURE-ACTIVITY RELATIONSHIP (PHD_2019).pdf |
| person | Wan Siti Nur Atirah Binti Wan Mohd Azemin |
| recordtype | oai_dc |
| resourceurl | https://intelek.unisza.edu.my/intelek/pages/view.php?ref=15344 |
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| spelling | 15344 https://intelek.unisza.edu.my/intelek/pages/view.php?ref=15344 https://intelek.unisza.edu.my/intelek/pages/search.php?search=!collection3 General Document Malaysia Library Staff (Top Management) Library Staff (Management) Library Staff (Support) Terengganu Faculty of Bio-resources & Food Industry English application/pdf 28 1.7 Server storage Scanned document Universiti Sultan Zainal Abidin UniSZA Private Access Universiti Sultan Zainal Abidin PDFium HEPCIDIN TH1-5 INDUCED APOPTOSIS IN HEPG2 HEPATOCARCINOMA AND HELA CERVICAL CARCINOMA CELL AND ITS STRUCTURE-ACTIVITY RELATIONSHIP (PHD_2019).pdf 2019-06-30 2019_Hepcidin TH1-5 Induced Apoptosis in HEPG2 Hepatocarcinoma and Hela Cervical Carcinoma Cell and Its Structure-Activity Relationship Wan Siti Nur Atirah Binti Wan Mohd Azemin Hepcidin Apoptosis HepG2 Hepatocarcinoma Copyright©PWB2025 Hepcidin is a principal regulator of systemic iron homeostasis in human. Dysregulation of hepcidin has been shown to be a causative factor in cancer and iron disorders. Highly conserved fish hepcidin protein, HepTH1-5 was used to study its mechanism, interaction and potential as an antitumour agent by testing its cytotoxicity effects, apoptosis inducing mechanism and antimitotic effect on several cancer cell lines. Additional in silico studies were conducted to understand the structure-activity relationship of the synthesized HepTH1-5. Structure-activity relationship of HepTH1-5 was investigated using bioinformatics tools; molecular docking analysis to determine their binding mechanism and protein-protein interaction analysis to determine biological functional distributions. Cytotoxicity of HepTH1-5 against HepG2, HeLa, HL-60 and normal mouse fibroblast NIH/3T3 cell lines were determined by MTT assay with methotrexate as a positive control. Cells in apoptosis and necrosis were quantified using AO/PI staining. Proportion of cell death in early and late apoptosis and necrotic cells were analysed with flow cytometer after Annexin V-FITC/PI staining. Systematic analysis of cell cycle effects of HepTH1-5 was analysed using flow cytometry technique. Apoptotic pathways were elucidated based on caspase-8, -9, -3/7, anti- and pro-apoptotic genes and proteins expression. Antimitotic effects were identified by DAPI staining and phospho HH3-Ser10 (pHH3) labelling. Modelled structure of HepTH1-5 showed the presence of iron-sulfur binding sites as catalytic domain, ligands (copper, zinc) act as cofactors and distorted β-sheets stabilised by vicinal disulfide bonds. Cysteine residues of HepTH1-5 were shown to bind with ligands and interact with protein that is associated with apoptosis signalling pathway that includes p53, PRKCD, Bcl-2, Bcl-xL and mitochondrial DNA, suggesting strong antitumour potential. HepTH1-5 has selective toxicity in HepG2 and HeLa cells with CD50 values of 2µg/mL and 11.5µg/mL, respectively. No cytotoxic effect was observed in HL-60 cells and very weak cytotoxicity against NIH/3T3 normal cells. In contrast, methotrexate was cytotoxic in all cells being tested. The CD50 values were 2µg/mL, 4.7µg/mL, 0.7µg/mL and 14.6µg/mL towards HepG2, HeLa, HL-60 and NIH/3T3 cells, respectively. Results of AO/PI staining revealed majority of both cells treated with HepTH1-5 died via apoptosis (91.7%, 88.8%) and necrosis (4%, 7.8%) in HepG2 cells and HeLa cells, respectively after 72 hours. Analysis of Annexin V/FITC-PI showed early apoptotic cells (4.1%, 5.1%) and late apoptotic cells (43.1%, 45.5%) in HepG2 and HeLa cells, respectively. Necrotic cells showed at 0.3% and 1.2% while viable cells were seen at 52.4% and 48.2% in HepG2 and HeLa cells, respectively after 72 hours. HepTH1-5 arrested at G2/M phase which significantly (p < 0.05) accumulated at 30.9% in HepG2 cells and 27.7% in HeLa cells at 48 hours before inducing a marked apoptosis at 32.7%, 33.1% in HepG2 and HeLa cells, respectively at sub G0/G1 phase after 72 hours. This trend corresponded to the antimicrotubule drug, paclitaxel. Antimitotic effect of HepTH1-5 blocked at anaphase (27.3%) and metaphase (23.7%) in HepG2 and HeLa cells, respectively. This blockage corroborated by peak level of mitotic delay via cyclin A, cyclin B1 and pHH3 proteins detection. The induction of mitotic cell cycle arrest and apoptosis occurred due to p53 activation through phosphorylation residues at Arg283, Thr118 and His115 in the nucleus. It was then simultaneously decreased the PCNA levels and upregulated the TGFβ1 and PRKCD expressions. This led to the translocation of p53 to mitochondria and interacted with associated proteins including superoxidase dismutase 2, mitochondrial DNA, frataxin and ferredoxin as shown in PPI network analysis. HepTH1-5 also increased the expression of caspase-2 protein, Bax gene and abrogated the function of Bcl-2 and Bcl-xL proteins in mitochondria. The downregulation of these proteins could be seen via Bcl-2 phosphorylation at Thr7, Thr21, Thr41, Ser49 and Bcl-xL phosphorylation at Ser106, Tyr195 which was analysed by molecular docking approach. This led to cytochrome c release, caspase cascade (caspase-9, -3/7) activation, downregulation of XIAP gene and cleavage of PARP protein at 89-kDa. This study demonstrated that HepTH1-5 caused apoptotic induction and arrest mitotically at G2/M phase in HepG2 and HeLa cells. Cysteine residues and ligands of HepTH1-5 are predicted to be involved in microtubule disruption and are linked to mitochondrial dysfunction. Thus, HepTH1-5 can potentially be developed as an antitumour agent for liver and cervical cancers. Hepcidin – Structure, function, biological activity Dissertations, Academic Sila masukkan subject wajib Dissertations, Academic. Terima kasih... Thesis |
| spellingShingle | 2019_Hepcidin TH1-5 Induced Apoptosis in HEPG2 Hepatocarcinoma and Hela Cervical Carcinoma Cell and Its Structure-Activity Relationship |
| state | Terengganu |
| subject | Hepcidin – Structure, function, biological activity Dissertations, Academic |
| summary | Hepcidin is a principal regulator of systemic iron homeostasis in human. Dysregulation of hepcidin has been shown to be a causative factor in cancer and iron disorders. Highly conserved fish hepcidin protein, HepTH1-5 was used to study its mechanism, interaction and potential as an antitumour agent by testing its cytotoxicity effects, apoptosis inducing mechanism and antimitotic effect on several cancer cell lines. Additional in silico studies were conducted to understand the structure-activity relationship of the synthesized HepTH1-5. Structure-activity relationship of HepTH1-5 was investigated using bioinformatics tools; molecular docking analysis to determine their binding mechanism and protein-protein interaction analysis to determine biological functional distributions. Cytotoxicity of HepTH1-5 against HepG2, HeLa, HL-60 and normal mouse fibroblast NIH/3T3 cell lines were determined by MTT assay with methotrexate as a positive control. Cells in apoptosis and necrosis were quantified using AO/PI staining. Proportion of cell death in early and late apoptosis and necrotic cells were analysed with flow cytometer after Annexin V-FITC/PI staining. Systematic analysis of cell cycle effects of HepTH1-5 was analysed using flow cytometry technique. Apoptotic pathways were elucidated based on caspase-8, -9, -3/7, anti- and pro-apoptotic genes and proteins expression. Antimitotic effects were identified by DAPI staining and phospho HH3-Ser10 (pHH3) labelling. Modelled structure of HepTH1-5 showed the presence of iron-sulfur binding sites as catalytic domain, ligands (copper, zinc) act as cofactors and distorted β-sheets stabilised by vicinal disulfide bonds. Cysteine residues of HepTH1-5 were shown to bind with ligands and interact with protein that is associated with apoptosis signalling pathway that includes p53, PRKCD, Bcl-2, Bcl-xL and mitochondrial DNA, suggesting strong antitumour potential. HepTH1-5 has selective toxicity in HepG2 and HeLa cells with CD50 values of 2µg/mL and 11.5µg/mL, respectively. No cytotoxic effect was observed in HL-60 cells and very weak cytotoxicity against NIH/3T3 normal cells. In contrast, methotrexate was cytotoxic in all cells being tested. The CD50 values were 2µg/mL, 4.7µg/mL, 0.7µg/mL and 14.6µg/mL towards HepG2, HeLa, HL-60 and NIH/3T3 cells, respectively. Results of AO/PI staining revealed majority of both cells treated with HepTH1-5 died via apoptosis (91.7%, 88.8%) and necrosis (4%, 7.8%) in HepG2 cells and HeLa cells, respectively after 72 hours. Analysis of Annexin V/FITC-PI showed early apoptotic cells (4.1%, 5.1%) and late apoptotic cells (43.1%, 45.5%) in HepG2 and HeLa cells, respectively. Necrotic cells showed at 0.3% and 1.2% while viable cells were seen at 52.4% and 48.2% in HepG2 and HeLa cells, respectively after 72 hours. HepTH1-5 arrested at G2/M phase which significantly (p < 0.05) accumulated at 30.9% in HepG2 cells and 27.7% in HeLa cells at 48 hours before inducing a marked apoptosis at 32.7%, 33.1% in HepG2 and HeLa cells, respectively at sub G0/G1 phase after 72 hours. This trend corresponded to the antimicrotubule drug, paclitaxel. Antimitotic effect of HepTH1-5 blocked at anaphase (27.3%) and metaphase (23.7%) in HepG2 and HeLa cells, respectively. This blockage corroborated by peak level of mitotic delay via cyclin A, cyclin B1 and pHH3 proteins detection. The induction of mitotic cell cycle arrest and apoptosis occurred due to p53 activation through phosphorylation residues at Arg283, Thr118 and His115 in the nucleus. It was then simultaneously decreased the PCNA levels and upregulated the TGFβ1 and PRKCD expressions. This led to the translocation of p53 to mitochondria and interacted with associated proteins including superoxidase dismutase 2, mitochondrial DNA, frataxin and ferredoxin as shown in PPI network analysis. HepTH1-5 also increased the expression of caspase-2 protein, Bax gene and abrogated the function of Bcl-2 and Bcl-xL proteins in mitochondria. The downregulation of these proteins could be seen via Bcl-2 phosphorylation at Thr7, Thr21, Thr41, Ser49 and Bcl-xL phosphorylation at Ser106, Tyr195 which was analysed by molecular docking approach. This led to cytochrome c release, caspase cascade (caspase-9, -3/7) activation, downregulation of XIAP gene and cleavage of PARP protein at 89-kDa. This study demonstrated that HepTH1-5 caused apoptotic induction and arrest mitotically at G2/M phase in HepG2 and HeLa cells. Cysteine residues and ligands of HepTH1-5 are predicted to be involved in microtubule disruption and are linked to mitochondrial dysfunction. Thus, HepTH1-5 can potentially be developed as an antitumour agent for liver and cervical cancers. |
| title | 2019_Hepcidin TH1-5 Induced Apoptosis in HEPG2 Hepatocarcinoma and Hela Cervical Carcinoma Cell and Its Structure-Activity Relationship |
| title_full | 2019_Hepcidin TH1-5 Induced Apoptosis in HEPG2 Hepatocarcinoma and Hela Cervical Carcinoma Cell and Its Structure-Activity Relationship |
| title_fullStr | 2019_Hepcidin TH1-5 Induced Apoptosis in HEPG2 Hepatocarcinoma and Hela Cervical Carcinoma Cell and Its Structure-Activity Relationship |
| title_full_unstemmed | 2019_Hepcidin TH1-5 Induced Apoptosis in HEPG2 Hepatocarcinoma and Hela Cervical Carcinoma Cell and Its Structure-Activity Relationship |
| title_short | 2019_Hepcidin TH1-5 Induced Apoptosis in HEPG2 Hepatocarcinoma and Hela Cervical Carcinoma Cell and Its Structure-Activity Relationship |
| title_sort | 2019_hepcidin th1-5 induced apoptosis in hepg2 hepatocarcinoma and hela cervical carcinoma cell and its structure-activity relationship |