Supernatant of bacterial fermented soybean induces apoptosis of human hepatocellular carcinoma Hep 3B cells via activation of caspase 8 and mitochondria. |
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| Authors: | Chun-Li Su Chao-Jung Wu Fang-Nan Chen Be-Jen Wang Shane-Rong Sheu Shen-Jeu Won |
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| Affiliation: | Department of Nursing, Chang Jung Christian University, Tainan 711, Taiwan, ROC. |
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| Abstract: | SC-1, the aqueous phase of soybean fermentation products by bacteria (Bacillus subtilis and Bacillus brevis), significantly inhibited the growth and clonogenesity of human hepatocellular (Hep 3B), mouse hepatocellular (ML-1), and human colorectal (HCT 116 and HT-29) carcinoma cells. Cytotoxicity of SC-1 in Hep 3B cells was through the process of apoptosis characterizing by increase in cell population of sub-G(1) phase, fragmentation of DNA, and change of nuclear morphology. Treatment of Hep 3B cells with SC-1 activated caspase 8 and caspase 3. Elevation of nuclear DNA fragmentation factor 40 (DFF40) and cleavage form of poly(ADP-ribose) polymerase (PARP) were also observed. SC-1 also activated intrinsic pathway via increase of pro-apoptotic (tBid, Bak and Bax) and decrease of anti-apoptotic (Bcl-2 and Bcl-x(L)) proteins on mitochondria, disruption of mitochondrial membrane potential, release of cytochrome c and Smac (second mitochondria-derived activator of caspase/direct IAP binding protein with low PI) from mitochondria, and activation of caspase 9. Inhibition on protein expression of Ku70 in cytosol and cyclooxygenase (COX)-2, but not COX-1, in whole cell lystes were revealed in SC-1-treated Hep 3B cells. These results suggest caspase 8, Ku70 and mitochondria are involved in the antitumor mechanism of SC-1 in Hep 3B cells. |
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| Keywords: | Apaf-1, Apoptotic protease activating factor-1 ATCC, American type culture collection B. brevis, Bacillus brevis B. subtilis, Bacillus subtilis COX, Cyclooxygenase DFF, DNA fragmentation factor DMEM, Dulbecco’s modified Eagle medium DTT, Dithiothreitol EDTA, Ethylenediamine-tetraacetic acid EGTA, Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid FBS, Fetal bovine serum HBS, HEPES buffer solution HBV, Hepatitis B virus HCC, Hepatocellular carcinoma HEPES, N-2-hydroxyethylpiperazine-N′-2-ethane sulfonic acid HRP, Horseradish peroxidase MTT, 3-[4,5]-dimethylthiazol-2-yl-2,5-diphenyltetra-zolium bromide PARP, Poly(ADP-ribose)polymerase PBS, Phosphate buffered saline PI, Propidium iodide PMSF, Henylmethysufonyl fluoride SC-1, The aqueous phase of soybean fermentation products by bacteria B. subtilis and B. brevis SDS, Sodium dodecylsulphate SDS-PAGE, SDS-polyacrylamide-gel electrophoresis Smac, Second mitochondria-derived activator of caspase/direct IAP binding protein with low PI tBid, Truncated Bid TBST, Tris buffer solution with tween XIAP, X-linked apoptosis-inhibiting protein Δψm, Mitochondrial membrane potential |
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