Hyung-Rho Kim

Induction of apoptosis by diallyl disulfide through activation of caspase-3 in human leukemia HL-60 cells

Diallyl disulfide (DADS), a component of garlic (Allium sativum), has been known to exert potent chemopreventative activity against colon, lung, and skin cancers. However, its molecular mechanism of action is still obscure. The present study demonstrated that DADS induces apoptosis of human leukemia HL-60 cells in a concentration- and time-dependent manner with an IC50 for cell viability of less than 25 microM. DADS activated caspase-3 as evidenced by both the proteolytic cleavage of the proenzyme and increased protease activity. Activation of caspase-3 was maximal at 3 hr and led to the cleavage of 116 kDa poly(ADP-ribose) polymerase (PARP), resulting in the accumulation of an 85 kDa cleavage product. Both activation of caspase-3 and cleavage of PARP were blocked by pretreatment with either antioxidants or a caspase-3 inhibitor, but not a caspase-1 inhibitor. DADS increased the production of intracellular hydrogen peroxide, which was blocked by preincubation with catalase. These results indicate that DADS-induced apoptosis is triggered by the generation of hydrogen peroxide, activation of caspase-3, degradation of PARP, and fragmentation of DNA. The induction of apoptosis by DADS may be the pivotal mechanism by which its chemopreventative action against cancer is based.

Hypoxia decreases Runx2/Cbfa1 expression in human osteoblast-like cells.

To elucidate the molecular mechanism in relation to vascular supply and osteoporosis, we investigated the effect of hypoxia on Runx2 expression in MG63 cells. Also investigated was expression of type I collagen and osteocalcin, which are regulated by Runx2, alkaline phosphatase (ALPase) to see if they are affected by hypoxia. Quiescent cultures of MG63 cells were exposed to hypoxia (2% O(2)) and normoxia (18% O(2)) for 24, 48, 72 and 96 h. In cells exposed to hypoxia, reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that mRNA expression of Runx2, type I collagen, osteocalcin, and ALPase were decreased in a time dependent manner to 96 h. Activity of ALPase was also reduced in the same manner. Western blotting showed a marked decrease in Runx2 protein at 96 h in cells under hypoxia compared to normoxia. These data indicate that Runx2 expression in osteoblasts is reduced by hypoxia, and may be a mechanism of osteoporosis by decreased vascular supply.

Hemolytic mechanism of cytolysin produced from V. vulnificus

The characteristics of hemolytic action of cytolysin produced from V. vulnificus were investigated in mouse erythrocytes. The cytolysin bound erythrocyte membranes in temperature-independent manner and then lysed cells temperature-dependently. Hemoglobin release by the cytolysin was completely inhibited by the presence of raffinose or melezitose, but K+ release was not affected. The cytolysin-induced hemolysis was always accompanied with the conversion of membrane-bound cytolysin into an oligomer of 210 kDa, corresponding to a tetramer of native cytolysins. Nonesterified cholesterol inactivated the cytolysin by converting active monomeric cytolysin into inactive oligomer. The results suggest that the cytolysin lyses erythrocytes due to the formation of small pores on erythrocyte membrane by cholesterol-mediated oligomerization of the cytolysin.

Role of Ca2+ in alloxan-induced pancreatic β-cell damage

Abstract Pretreatment of rats with verapamil, a Ca 2+ -antagonist, completely prevented alloxan-induced hyperglycemia. Verapamil also abolished the inhibition of insulin secretion by alloxan and H 2 O 2 in isolated rat pancreatic islets. H 2 O 2 generation from alloxan was not affected by verapamil, but alloxan- and H 2 O 2 -induced DNA strand breaks were completely prevented. Treatment of β-cells with alloxan and H 2 O 2 caused elevation of cytosolic free Ca 2+ , and this increase of Ca 2+ was also abolished by verapamil. These results suggest that alloxan-derived oxygen radicals may disturb intracellular Ca 2+ homeostasis by increasing Ca 2+ influx, which results in secondary reactions ultimately leading to DNA strand breaks and cytotoxicity of β-cells.

Membrane-associated NAD+ glycohydrolase from rabbit erythrocytes is solubilized by phosphatidylinositol-specific phospholipase C

NAD+ glycohydrolase (NADase) present on the surface of rabbit erythrocytes is a membrane-bound ectoenzyme that can be solubilized by phosphatidylinositol-specific phospholipase C (PIPLC). As much as 70% of the cell-associated NADase was made soluble by treatment with PIPLC. The portion of NADase that remained cell-associated after an initial PIPLC treatment proved to be resistant to subsequent solubilization attempts. Further analysis showed that release of NADase from erythrocytes could not be attributed to the action of proteinases or phospholipase C. Erythrocytes obtained from other mammals were analyzed and found to have variable amounts of PIPLC-susceptible NADase. Practically, this finding can be used to easily solubilize membrane-bound NADase as a first step in its purification.

Protective mechanism of glucose against alloxan-induced beta-cell damage: pivotal role of ATP.

Glucose prevents the development of diabetes induced by alloxan. In the present study, the protective mechanism of glucose against alloxan-induced β-cell damage was investigated using HIT-T 15 cell, a Syrian hamster transformed β-cell line. Alloxan caused β-cell damages with DNA fragmentation, inhibition of glucose-stimulated insulin release, and decrease of cellular ATP level, but all of these β-cell damages by alloxan were prevented by the presence of 20 mM glucose. Oligomycin, a specific inhibitor of ATP synthase, completely abolished the protective effects of glucose against alloxan-induced cell damage. Furthermore, treatment of nuclei isolated from HIT-T15 cells with ATP significantly prevented the DNA fragmentation induced by Ca2+. The results indicate that ATP produced during glucose metabolism plays a pivotal role in the protection of glucose against alloxan-induced β-cell damage.

Protective Effect of C-Reactive Protein against the Lethality Induced by Vibrio vulnificus Lipopolysaccharide

Vibrio vulnificus infection has attracted special interest because of its high mortality. A strong clinical association exists between hepatic dysfunction and increased morbidity and mortality from V. vulnificus infection. In this study, the effect of C‐reactive protein (CRP), a typical hepatogenic acute phase protein, on the lethality induced by V. vulnificus lipopolysaccharide (LPS) was investigated in galactosamine‐sensitized mice. The pretreatment of CRP, in a dose of at least 2 mg/kg, 2 hr before the challenge of LPS completely protected mice against the lethality by V. vulnificus LPS. The elevation of serum tumor necrosis factor‐α (TNF‐α) induced by LPS administration was not affected by CRP pretreatment. However, the LPS‐ or TNF‐α‐induced hepatotoxicity was completely prevented by CRP. These results indicate that CRP does not prevent the synthesis, but prevents the hepatotoxic action of TNF‐α. The possibility that impaired production of acute phase proteins in patients with pre‐existing hepatic dysfunction may predispose the higher risk of V. vulnificus infection needs to be evaluated further.

Cytotoxic mechanism of Vibrio vulnificus cytolysin in CPAE cells.

Vibrio vulnificus is an estuarian bacterium that causes septicemia and serious wound infection. The cytolysin, one of the important virulence determinants in V. vulnificus infection, has been reported to have lethal activity primarily by increasing pulmonary vascular permeability. In the present study, we investigated the cytotoxic mechanism of V. vulnificus cytolysin in cultured pulmonary artery endothelial (CPAE) cells, which are possible target cells of cytolysin in vivo. V. vulnificus cytolysin caused the CPAE cell damages with elevation of the cytosolic free Ca2+, DNA fragmentation, and decrease of the cellular NAD+ and ATP level. These cytotoxic effects of V. vulnificus cytolysin were prevented by EGTA and aminobenzamide, but were not affected by verapamil or catalase. These results indicate that the elevation of cytosolic free Ca2+ induced by V. vulnificus cytolysin causes the increase of DNA fragmentation and the damaged DNA activates nuclear poly(ADP-ribose) synthetase, which depletes the cellular NAD+ and ATP, resulting in cell death.

Effect of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on the dimerization of lipoprotein lipase.

Lipoprotein lipase (LPL), an enzyme playing the central role in triglyceride metabolism, is a glycoprotein and a homodimer of identical subunits. Dimerization and proper processing of oligosaccharide chains are important maturation steps in post-translational regulation of enzyme activity. Indirect evidences suggest that dimerization of LPL occurs in endoplasmic reticulum (ER) or Golgi. In this study, we investigated the dimerization status of LPL in 3T3-L1 adipocytes, using sucrose density gradient ultracentrifugation and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of ER-Golgi protein transport. In the presence of CCCP, no increase of cellular LPL activity was detected during 2 b of recovery period after the depletion of LPL, with heparin and cycloheximide. Only endoglycosidase H (endo H)-sensitive subunits were found in CCCP-treated cells after endo H digestion, suggesting that inactive LPL was retained in ER. In the presence of castanospermine, an inhibitor of ER glucosidase I, LPL subunits of both control and CCCP-treated cells had same molecular weight, indicating that complete oligosaccharides were transferred to LPL subunits in the presence of CCCP. In sucrose density gradient ultracentrifugation, all the LPL protein synthesized in the presence of CCCP was found at the dimeric fractions as in control cells. Most of LPL protein in control cells showed high affinity for heparin, and there was no difference between the control and CCCP-treated cells. These results suggest that dimerization and acquisition of high affinity for heparin of LPL can occur in ER of CCCP-treated cells without acquisition of catalytic activity.

Glycosylation, dimerization, and heparin affinity of lipoprotein lipase in 3T3-L1 adipocytes.

The relationship between glycosylation, dimerization, and heparin affinity of lipoprotein lipase (LPL) was studied in 3T3-L1 adipocytes. Three forms of LPL subunits were found in normal cells; totally endo H-resistant (57 kDa), partially sensitive (54 kDa), and totally sensitive (51 kDa) forms. LPL in normal cells was active, dimeric, and showed high affinity for heparin. LPL in cells treated with tunicamycin, preventing the transfer of N-linked oligosaccharide chain, was unglycosylated (51 kDa) and inactive. LPL proteins were found as an aggregate, and had low affinity for heparin. After treatment with castanospermine, an inhibitor of ER glucosidase I, 80% of LPL activity was inhibited. Most of LPL proteins were totally endo H-sensitive, present as an aggregate, and had low affinity for heparin. LPL in cells treated with deoxymannojirimycin, an inhibitor of Golgi mannosidase I, was active, dimeric, and had high affinity for heparin as in normal cells. But LPL subunits were all endo H-sensitive. These results suggest that core glycosylation and subsequent removal of glucose residue is required, but processing after Golgi mannosidase I is not necessary for dimerization and acquisition of high heparin affinity of LPL.