Sung Zoo Kim

An ROS generator, antimycin A, inhibits the growth of HeLa cells via apoptosis

Antimycin A (AMA), an inhibitor of electron transport in mitochondria, has been used as a reactive oxygen species (ROS) generator in biological systems. Here, we investigated the in vitro effect of AMA on apoptosis in HeLa cells. AMA inhibited the growth of HeLa cells with an IC50 of about 50 µM. AMA efficiently induced apoptosis, as evidenced by flow cytometric detection of sub‐G1 DNA content, annexin V binding assay, and DAPI staining. This apoptotic process was accompanied by the loss of mitochondrial membrane potential (ΔΨm), Bcl‐2 down‐regulation, Bax up‐regulation, and PARP degradation. All caspase inhibitors used in this experiment, especially pan‐caspase inhibitor (Z‐VAD), could rescue some HeLa cells from AMA‐induced cell death. When we examined the changes of the ROS, H2O2 or O u20092·− , in AMA‐treated cells, H2O2 and O u20092·− were markedly increased. In addition, we detected the depletion of GSH content in AMA‐treated cells. Pan‐caspase inhibitor showing the efficient anti‐apoptotic effect significantly reduced GSH depletion by AMA. Superoxide dismutase (SOD) and catalase did not reduce intracellular ROS, but these could strongly rescue the cells from apoptosis. However, these anti‐apoptotic effects were not accompanied by the recovery of GSH depletion. Interestingly, catalase significantly decreased the CMF negative (GSH depletion) and propidium iodide (PI) positive cells, indicating that catalase strongly maintained the integrity of the cell membrane in CMF negative cells. Taken together, these results demonstrate that AMA potently generates ROS, induces the depletion of GSH content in HeLa cells, and strongly inhibits the growth of HeLa cells throughout apoptosis. J. Cell. Biochem. 102: 98–109, 2007.

Renin Angiotensin System in Rabbit Corpus Cavernosum: Functional Characterization of Angiotensin II Receptors

PURPOSEnThe regulation of the corporal smooth muscle tone is important in the process of penile erection. Although specific angiotensin (ANG) II binding to and effects of ANG II on some reproductive structures have been studied, the presence of the renin-angiotensin system has not yet been defined in the corpus cavernosum. ANG II is formed from ANG I by angiotensin I-converting enzyme (ACE). ANG II and ANG I produce contractions in vascular smooth muscles. Two subtypes of ANG II receptors (AT1 and AT2) have been characterized. The purpose of the present experiments was to determine whether the renin-angiotensin system regulates rabbit corpus cavernosum smooth muscle tone.nnnMATERIALS AND METHODSnA strip of rabbit corpus cavernosum was mounted in an organ chamber to measure the isometric tension. The specific binding for 125I-ANG II was characterized by in vitro autoradiography.nnnRESULTSnANG II and ANG I, precursor of ANG II, contracted corpus cavernosum smooth muscle dose-dependently, but the response of smooth muscle to ANG I was 10-fold less than that to ANG II. Contractile responses of smooth muscle to ANG II and ANG I were blocked by Dup 753, a specific inhibitor of ANG II type 1 receptor, but not by PD 123,319, a specific inhibitor of ANG II type 2 receptor. The effect of ANG I was attenuated by captopril, an inhibitor of ACE. Specific binding sites for 125I-ANG II were found in the corpus cavernosum. The dissociation constant (Kd) was 5.32 +/- 1.65 nM. and maximum binding capacity (Bmax) was 305.72 +/- 85.24 amol/mm. Specific binding of 125I-ANG II was displaced by Dup 753 (10(-6) M) but not by PD 123,319 (10(-5) M). The inhibitory constant (Ki) for Dup 753 was 8.09 +/- 2.51 nM.nnnCONCLUSIONnThe present results suggest that the renin-angiotensin system is involved in the regulation of corpus cavernosum smooth muscle tone of rabbit and the ANG II receptor subtype AT1 is important in the regulation of penile erection.

Arsenic trioxide inhibits the growth of Calu-6 cells via inducing a G2 arrest of the cell cycle and apoptosis accompanied with the depletion of GSH

Arsenic trioxide (ATO) can regulate many biological functions such as apoptosis and differentiation in various cells. We evaluated the effects of ATO on the viability, cell cycle and apoptosis of human pulmonary adenocarcinoma, Calu-6 and A549 cells. ATO reduced the viability of Calu-6 cells with an IC50 of approximately 3 or 4 microM. However, A549 cells were very resistant to ATO. Calu-6 cells treated with 1, 3 or 5 microM ATO showed a G2 phase arrest of the cell cycle at 72 h. The G2 phase arrest was accompanied with the down-regulation of cdc2 protein. Treatment with ATO-induced apoptosis in Calu-6 cells. The apoptotic process was accompanied by the down-regulation of Bcl-2 protein, the activation of caspase-3, and the loss of the mitochondrial membrane potential (Delta Psi m). All of the caspase inhibitors, especially pan-caspase inhibitor (Z-VAD), markedly rescued Calu-6 cells from ATO-induced cell death. Caspase inhibitors also prevented the loss of mitochondrial membrane potential (Delta Psi m). The inhibitors significantly increased the number of G2 phase cells in 10 microM ATO-treated cells. In addition, the levels of O2- were significantly increased in 10 microM ATO-treated cells. However, the changes of ROS by 10 microM ATO are not correlated with apoptosis in Calu-6 cells. Treatment with 10 microM ATO depleted GSH content in Calu-6 cells and caspase inhibitors significantly prevented the GSH depletion in these cells. In conclusion, we have demonstrated that ATO inhibits the growth of Calu-6 cells by inducing a G2 arrest of the cell cycle and by triggering apoptosis accompanied with the depletion of GSH.

2,4-dinitrophenol induces G1 phase arrest and apoptosis in human pulmonary adenocarcinoma Calu-6 cells.

2,4-dinitrophenol (DNP) is an uncoupler of oxidative phosphorylation in the mitochondria. Here, we investigated the effect of DNP on the growth of Calu-6 lung cancer cells in view of cell cycle, apoptosis, ROS production and GSH content. DNP dose-dependently decreased cell viability at 72 h (EC50 of about 200 microM) as measured by a MTT assay. The lower doses of DNP induced a G1 arrest of the cell cycle in Calu-6 cells. Analysis of the cell cycle regulatory proteins demonstrated that DNP decreased the steady-state levels of cyclin proteins and cyclin dependent kinase (CDK), but increased the protein levels of cyclin dependent kinase inhibitor (CDKI) p27. DNP also caused a marked increase in apoptosis, as evidenced by DNA fragmentation (sub-G1 DNA content), DAPI staining, the loss of mitochondrial membrane potential (DeltaPsim), externalization of phosphatidylserine (PS). In addition, DNP-treated cells significantly increased the intracellular H2O2 and O2.- levels. All of caspase inhibitors could markedly rescue Calu-6 cells from DNP-induced cell death and only pan-caspase inhibitor, Z-VAD-FMK, could slightly prevent the loss of mitochondrial membrane potential (DeltaPsim). However, none of the caspase inhibitors reduced the increased H2O2 levels, but the increased O2.- levels was slightly attenuated by pan-caspase inhibitor. In addition, the depletion of GSH content in DNP-treated cells was prevented by all of caspase inhibitors. In conclusion, DNP, which induced ROS and reduced GSH content, inhibited the growth of Calu-6 cells via cell cycle arrest at G1 phase and apoptosis.

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) as an O2(*-) generator induces apoptosis via the depletion of intracellular GSH contents in Calu-6 cells.

Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) is an uncoupler of mitochondrial oxidative phosphorylation in eukaryotic cells. Here, we investigated an involvement of O(2)(*-) and GSH in FCCP-induced Calu-6 cell death and examined whether ROS scavengers rescue cells from FCCP-induced cell death. Levels of intracellular O(2)(*-) were markedly increased depending on the concentrations (5-100 microM) of FCCP. A depletion of intracellular GSH content was also observed after exposing cells to FCCP. Stable SOD mimetics, Tempol and Tiron did not change the levels of intracellular O(2)(*-), apoptosis and the loss of mitochondrial membrane potential (DeltaPsi(m)). Treatment with thiol antioxidants, NAC and DTT, showed the recovery of GSH depletion and the reduction of O(2)(*-) levels in FCCP-treated cells, which were accompanied by the inhibition of apoptosis. In contrast, BSO, a well-known inhibitor of GSH synthesis, aggravated GSH depletion, oxidative stress of O(2)(*-) and cell death in FCCP-treated cells. Taken together, our data suggested that FCCP as an O(2)(*-) generator, induces apoptosis via the depletion of intracellular GSH contents in Calu-6 cells.

A superoxide anion generator, pyrogallol, inhibits the growth of HeLa cells via cell cycle arrest and apoptosis

We investigated the in vitro effects of pyrogallol on cell growth, cell cycle regulation, and apoptosis in HeLa cells. Pyrogallol inhibited the growth of HeLa cells with an IC50 of approximately 45 µM. Pyrogallol induced arrest during all phases of the cell cycle and also very efficiently resulted in apoptosis in HeLa cells, as evidenced by flow cytometric detection of sub‐G1 DNA content, annexin V binding assay, and DAPI staining. This apoptotic process was accompanied by the loss of mitochondrial transmembrane potential (ΔΨm), Bcl‐2 decrease, caspase‐3 activation, and PARP cleavage. Pan‐caspase inhibitor (Z‐VAD) could rescue some HeLa cells from pyrogallol‐induced cell death, while caspase‐8 and ‐9 inhibitors unexpectedly enhanced the apoptosis. When we examined the changes of the ROS, H2O2 or O u20092.− in pyrogallol‐treated cells, H2O2 was slightly increased and O u20092.− significantly was increased. In addition, we detected a decreased GSH content in pyrogallol‐treated cells. Only pan‐caspase inhibitor showing recovery of GSH depletion and reduced intracellular O u20092.− level decreased PI staining in pyrogallol‐treated HeLa cells, which indicates dead cells. In summary, we have demonstrated that pyrogallol as a generator of ROS, especially O u20092.− , potently inhibited the growth of HeLa cells through arrests during all phases of the cell cycle and apoptosis.

Decreases in ANP Secretion by Lysophosphatidylcholine Through Protein Kinase C

Abstract— Lysophosphatidylcholine (LPC) is an endogenous phospholipid released from the cell membrane during ischemia, and it has potent, local effects on cardiac tissues. LPC has been implicated in arrhythmogenesis during ischemia by increasing intracellular Ca2+. However, it is not known whether LPC influences atrial release of atrial natriuretic peptide (ANP). The aim of this study was to investigate the effect of LPC on ANP secretion from isolated, perfused, beating rat atria. LPC (10 and 30 &mgr;mol/L) caused decreases in ANP secretion in a dose‐dependent manner, with slight increases in intra‐atrial pressure and extracellular fluid (ECF) translocation. Therefore, the ANP secretion in terms of ECF translocation was markedly decreased by LPC. The order of the suppressive effect of ANP release was stearoyl‐LPC>LPC>myristoyl‐LPC=lauroyl‐LPC. Staurosporine and wortmannin significantly attenuated suppression of the ANP release and an increase in intra‐atrial pressure by LPC. High extracellular Mg2+ also attenuated the LPC‐induced suppression of ANP release. However, other protein kinase C inhibitors such as chelerythrine, GF 109203X, and tamoxifen citrate did not affect LPC‐induced suppression of ANP release. In single atrial myocytes, LPC caused increases in intracellular Ca2+ in a dose‐dependent manner. The order of an increase in intracellular Ca2+ by LPC was stearoyl‐LPC>LPC>myristoyl‐LPC=lauroyl‐LPC. An increase in intracellular Ca2+ by LPC was attenuated by staurosporine. These results suggest that LPC‐induced suppression of ANP release through protein kinase C/Ca2+ and phosphoinositol‐3‐kinase might in part play an important role in the development of hypertension.

Apoptosis in arsenic trioxide‐treated Calu‐6 lung cells is correlated with the depletion of GSH levels rather than the changes of ROS levels

Arsenic trioxide (ATO) can regulate many biological functions such as apoptosis and differentiation in various cells. We investigated an involvement of ROS such as H2O2 and O u20092.− , and GSH in ATO‐treated Calu‐6 cell death. The levels of intracellular H2O2 were decreased in ATO‐treated Calu‐6 cells at 72 h. However, the levels of O u20092.− were significantly increased. ATO reduced the intracellular GSH content. Many of the cells having depleted GSH contents were dead, as evidenced by the propidium iodine staining. The activity of CuZn‐SOD was strongly down‐regulated by ATO at 72 h while the activity of Mn‐SOD was weakly up‐regulated. The activity of catalase was decreased by ATO. ROS scavengers, Tiron and Trimetazidine did not reduce levels of apoptosis and intracellular O u20092.− in ATO‐treated Calu‐6 cells. Tempol showing a decrease in intracellular O u20092.− levels reduced the loss of mitochondrial transmembrane potential (ΔΨm). Treatment with NAC showing the recovery of GSH depletion and the decreased effect on O u20092.− levels in ATO‐treated cells significantly inhibited apoptosis. In addition, BSO significantly increased the depletion of GSH content and apoptosis in ATO‐treated cells. Treatment with SOD and catalase significantly reduced the levels of O u20092.− levels in ATO‐treated cells, but did not inhibit apoptosis along with non‐effect on the recovery of GSH depletion. Taken together, our results suggest that ATO induces apoptosis in Calu‐6 cells via the depletion of the intracellular GSH contents rather than the changes of ROS levels. J. Cell. Biochem. 104: 862–878, 2008.

Antimycin A as a mitochondria damage agent induces an S phase arrest of the cell cycle in HeLa cells

Antimycin A (AMA), an electron transport chain inhibitor in mitochondria can produce reactive oxygen species (ROS) in cells. It has been reported that ROS may have roles in cell cycle progression via regulating cell cycle-related proteins. In the present study, we investigated the changes of the cell cycle distribution in AMA-treated HeLa cells in relation to cell cycle-related proteins. DNA flow cytometric analysis indicated that treatment with AMA significantly induced an S phase arrest of the cell cycle at 72 h. AMA decreased the expression of cyclin-dependent kinase inhibitor (CDKI), p21 and p27, CDK4, and cdc2 proteins. The expression of CDK6, cyclin D1, cyclin E, cyclin A, and cyclin B proteins was increased by 0.5 microM AMA, but was decreased by 2 and 10 microM AMA. The phosphorylation of Rb on the Ser (780) residue was increased by 0.5 microM AMA. Furthermore, treatment with AMA caused the accumulation of cells expressing cyclin A, B, and D1 proteins at the S phase of the cell cycle. However, treatment with 100 microM AMA nonspecifically extended all phases of the cell cycle. In conclusion, treatment with AMA (2, 10 and 50 microM) induced an S phase arrest of the cell cycle. An S phase arrest was accompanied by the alteration of other cell cycle-regulated proteins as well as S phase-related proteins.

Pyrogallol inhibits the growth of lung cancer Calu-6 cells via caspase-dependent apoptosis

Pyrogallol (PG) is a polyphenol compound and a known O2- generator. We evaluated the effects of PG on the growth and apoptosis of human pulmonary adenocarcinoma Calu-6 cells. PG decreased the viability of Calu-6 cells in a dose- and time-dependent manner. The induction of apoptosis by PG was accompanied by the loss of mitochondrial membrane potential (DeltaPsi(m)), cytochrome c release from mitochondria and activation of caspase-3 and caspase-8. All tested caspase inhibitors, especially the pan-caspase inhibitor (Z-VAD), markedly rescued Calu-6 cells from PG-induced cell death. Rescue was accompanied by inhibition of caspase-3 activation and PARP cleavage. Treatment with Z-VAD also prevented the loss of mitochondrial membrane potential (DeltaPsi(m)). In conclusion, PG inhibits the growth of Calu-6 cells via caspase-dependent apoptosis.