1. Molecular mechanisms of apoptosis induced by Scorpio water extract in human hepatoma HepG2 cells
Eun-Kyung Kim, Young-Se Jeon, Eun-Sil Jeong, Jung-Gook Lim, Eun-A Seo, Kang-Beom Kwon, Do-Gon Ryu, Kang-San Kim, Byung-Cheul Shin World J Gastroenterol . 2005 Feb 21;11(7):943-7. doi: 10.3748/wjg.v11.i7.943.
Aim:To clarify the mechanism underlying the anti-mutagenic and anti-cancer activities of Scorpio water extract (SWE).Methods:Human hepatoma HepG2 cells were incubated with various concentrations of SWE. After 24-h incubation, cytotoxicity and apoptosis evaluations were determined by MTT and DNA fragmentation assay, respectively. After treatment with SWE, mitochondrial membrane potential (MMP) was determined by measuring the retention of the dye 3,3'-dihexyloxacarbocyanine (DiOC(6)(3)) and the protein expression including cytochrome C and poly-(ADP-ribose) polymerase (PARP) were measured by Western blotting. Caspase-3 and -9 enzyme activities were measured using specific fluorescence dyes such as Ac-DEVD-AFC and Ac-LEHD-AFC.Results:We found that treatment with SWE induced apoptosis as confirmed by discontinuous DNA fragmentation in cultured human hepatoma HepG2 cells. Our investigation also showed that SWE-induced apoptosis of HepG2 cells were associated with intracellular events including disruption of MMP, increased translocation of cytochrome C from mitochondria to cytosol, activation of caspase-3, and PARP. Pre-treatment of N-acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO), a caspase-3 specific inhibitor, or cyclosporin A (CsA), an inhibitor of MMP disruption, completely abolished SWE-induced DNA fragmentation.Conclusion:These results suggest that SWE possibly causes mitochondrial damage, leading to cytochrome C release into cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic cell death in HepG2 cells. These results further suggest that Scorpio may be a valuable agent of therapeutic intervention of human hepatomas.
2. Cysteine protease attribute of eukaryotic ribosomal protein S4
Abani K Bhuyan, Dasari Ramakrishna, Babu Sudhamalla, Madasu Yadaiah Biochim Biophys Acta . 2012 Oct;1820(10):1535-42. doi: 10.1016/j.bbagen.2012.05.001.
Background:Ribosomal proteins often carry out extraribosomal functions. The protein S4 from the smaller subunit of Escherichia coli, for instance, regulates self synthesis and acts as a transcription factor. In humans, S4 might be involved in Turner syndrome. Recent studies also associate many ribosomal proteins with malignancy, and cell death and survival. The list of extraribosomal functions of ribosomal proteins thus continues to grow.Methods:Enzymatic action of recombinant wheat S4 on fluorogenic peptide substrates Ac-XEXD↓-AFC (N-acetyl-residue-Glu-residue-Asp-7-amino-4-trifluoromethylcoumarin) and Z-FR↓-AMC (N-CBZ-Phe-Arg-aminomethylcoumarin) as well as proteins has been examined under a variety of solution conditions.Results:Eukaryotic ribosomal protein S4 is an endoprotease exhibiting all characteristics of cysteine proteases. The K(m) value for the cleavage of Z-FR↓-AMC by a cysteine mutant (C41F) is about 70-fold higher relative to that for the wild-type protein under identical conditions, implying that S4 is indeed a cysteine protease. Interestingly, activity responses of the S4 protein and caspases toward environmental parameters, including pH, temperature, ionic strength, and Mg(2+) and Zn(2+) concentrations, are quite similar. Respective kinetic constants for their cleavage action on Ac-LEHD↓-AFC are also similar. However, S4 cannot be a caspase, because unlike the latter it also hydrolyzes the cathepsin substrate Z-FR↓-AMC.General significance:The eukaryotic S4 is a generic cysteine protease capable of hydrolyzing a broad spectrum of synthetic substrates and proteins. The enzyme attribute of eukaryotic ribosomal protein S4 is a new phenomenon. Its possible involvement in cell growth and proliferations are presented in the light of known extraribosomal roles of ribosomal proteins.
3. Je-chun-jun induced apoptosis of human cervical carcinoma HeLa cells
Shim-Keun Yoo, Gi-Seup Jeong, Han-Jung Chae, Soo-Wan Chae, Hyung-Min Kim, Hyung-Ryong Kim, Kyung-Mi Park, Geun-Youn Lee, Hyung-Rae Park Acta Pharmacol Sin . 2004 Oct;25(10):1372-9.
Aim:To study the mechanism of je-chun-jun (JCJ)-inducing the apoptosis of the human cervical carcinoma, HeLa cells.Methods:The cell viability was assessed using MTT assay. The optical density was measured at 570 nm. The caspase activity was measured using 50 mmol/L of fluorogenic substrate, AC-DEVD-AMC (caspase-3), AC-VEID-AMC (caspase-8) or AC-LEHD-AFC (caspase-9). To confirm the expression of proteins, Western blotting was performed. To detect the characteristic of apoptosis chromatin condensation, HeLa cells were stained with Hoechst 33258 in the presence of JCJ. For the cell cycle analysis, HeLa cells were incubated with Propidium iodide (PI) solution. Fluorescence intensity of cell cycle was measured using flow cytometry system.Results:The loss of viability occurred following the exposure of 10 g/L JCJ. Cells treated with 10 g/L JCJ for 3 d exhibited the apoptotic morphology (brightly blue-fluorescent condensed nuclei by Hoechst 33258-staining) and the reduction of cell volume. Cells incubated with JCJ for 48 h were arrested at the G1 phase of cell cycle and their G1 checkpoint related gene products such as cyclin D1 were transiently decreased. We showed that JCJ induced the p38 MAPK activation in HeLa cells. The p38 MAPK inhibitor, SB203580 protected Hela cells from the JCJ-induced death as well as intervened the JCJ-induced accumulation of cells at the G1 phase. In contrast, MEK1 (-ERK upstream) inhibitor, PD98059 had no effect on HeLa cells.Conclusion:JCJ induced cell cycle arrest and apoptosis of HeLa cells through p38 MAPK pathway.