• The Journal of Korean Obstetrics and Gynecology
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• v.19 no.2
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• pp.77-91
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• 2006

Purpose : To address the ability of Dohaekseungkitang (DST: a commonly used herb formulation in Korea, Japan and China to have anti-cancer effect on cervical carcinoma), we investigated the effects of DST on programmed cell death (apoptosis) in HeLa human cervical carcinoma cells. Methods : We cultured HeLa cell which is human metrocarcinoma cell in D-MEM included 10% fetal bovine serum(Hyclone Laboratories) below $37^{\circ}C$, 5% CO2. Then we observed apoptosis of log phage cell which is changed cultivation liquid 24 Hours periodically. Results : After the treatment of DST for 48 hours, apoptosis occurred in a dose-dependent manner. In this study, we have shown that DST induces calpain and the associated caspase-8 and -9 activations. Apoptosis was prevented by pre-incubation of the cells with the calcium cHeLator-BAPTA-AM, calcium channel blocker-Nif edipine or Ryonidine agonist-Ryonidine peptide, implicating calcium in the apoptotic process. Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, especially in calcium-related apoptosis. However this study showed 1hat either calpain inhibitor-calpastin or caspase-3 inhibitor-DEVD- did not blocked the herb formulation-induced apoptosis in HeLa human cervical carcinoma cells. D ST initiates a cell death pathway that is partially dependent of caspases. DST-induced apoptosis requires caspase-independent mechanism. Conclusion : We conclude that DST-induced calpain activation triggers the intrinsic apoptotic pathway in which caspase-independent mechanism is also involved.

• Molecules and Cells
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• v.19 no.1
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• pp.60-66
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• 2005

Low density lipoproteins (LDL) play important roles in the pathogenesis of atherosclerosis. Diabetes is associated with accelerated atherosclerosis leading to cardiovascular disease in diabetic patients. Although LDL stimulates the proliferation of arterial smooth muscle cells (SMC), the mechanisms are not fully understood. We examined the effects of native LDL and glycated LDL on the extracellular signal-regulated kinase (ERK) pathway. Addition of native and glycated LDL to rat aorta SMCs (RASMCs) stimulated ERK phosphorylation. ERK phosphorylation was not affected by exposure to the $Ca^{2+}$ chelator BAPTA-AM but inhibition of protein kinase C (PKC) with GF109203X, inhibition of Src kinase with PP1 ($5{\mu}M$) and inhibition of phospholipase C (PLC) with U73122/U73343 ($5{\mu}M$) all reduced ERK phosphorylation in response to glycated LDL. In addition, pretreatment of the RASMCs with a cell-permeable mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059, $5{\mu}M$) markedly decreased ERK phosphorylation in response to native and glycated LDL. These findings indicate that ERK phosphorylation in response to glycated LDL involves the activation of PKC, PLC, and MEK, but is independent of intracellular $Ca^{2+}$.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.1
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• pp.19-27
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• 1999

Apoptosis has been implicated in the pathophysiological mechanisms of various neurodegenerative diseases. In a variety of cell types, oxidative stress has been demonstrated to play an important role in the apoptotic cell death. However, the exact mechanism of oxidative stress-induced apoptosis in neuronal cells is not known. In this study, we induced oxidative stress in IMR-32 human neuroblastoma cells with tert- butylhydroperoxide (TBHP), which was confirmed by significantly reduced glutathione content and glutathione reductase activity, and increased glutathione peroxidase activity. TBHP induced decrease in cell viability and increase in DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. TBHP also induced a sustained increase in intracellular $Ca^{2+}$ concentration, which was completely prevented either by EGTA, an extracellular $Ca^{2+}$ chelator or by flufenamic acid (FA), a non-selective cation channel (NSCC) blocker. These results indicate that the TBHP-induced intracellular $Ca^{2+}$ increase may be due to $Ca^{2+}$ influx through the activation of NSCCs. In addition, treatment with either an intracellular $Ca^{2+}$ chelator (BAPTA/AM) or FA significantly suppressed the TBHP-induced apoptosis. Moreover, TBHP increased the expression of p53 gene but decreased c-myc gene expression. Taken together, these results suggest that the oxidative stress-induced apoptosis in neuronal cells may be mediated through the activation of intracellular $Ca^{2+}$ signals and altered expression of p53 and c-myc.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.46-46
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• 2003

Salviae Miltiorrhizae Radix has been used for treatment of cardiovascular diseases in oriental medicine. To investigate the possible involvement of cardiac ion channel in this effect, we examined electrophysiological effects of the extract of Salviae Miltiorrhizae Radix on action potentials and ionic currents in rat ventricular myocytes. The extracts of Salviae Miltiorrhizae Radix were fractionated into nine fractions, and the effect of each fraction on action potential was tested. The fraction containing monomethyl lithospermic acid-A (LSA-A) induced a significant prolongation of action potential duration (APD). LSA-B which is a major component of Salviae Miltiorrhizae Radix, however, did not cause a significant effect. In voltage clamp experiments, the effects of LSA-A on K currents, Ca currents and Na currents were tested. Neither K currents nor L-type Ca currents were affected by LSA-A. On the contrary, LSA-A significantly slowed down the inactivation kinetics of the Na current with no effect on the fast component of the inactivation process. The amplitude of the peak current and the voltage-dependence of activation were not changed by LSA-A. The effect of LSA-A on Na current was abolished when high concentration of $Ca^{2+}$ buffer (10 mM BAPTA) was included in the pipette solution or when Ca2+ current was blocked by nicardipine (1 $\mu$M) in the bath solution.n.

• Archives of Pharmacal Research
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• v.22 no.4
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• pp.404-409
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• 1999

The inhibitory effects of the constituents of Gastrodia elata Bl. (GE) on glutamate-induced apoptosis in human neuronal cells were investigated using IMR32 human neuroblastoma cells. Glutamate (GLU) induced DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. GLU also induced a slow and sustained increase in intracellular $Ca^{2+}$ concentration. Treatment with EGTA, an extracellular $Ca^{2+}$ chelator, in a nominal $Ca^{2+}$ -free buffer solution abolished the GLU-induced intracellular $Ca^{2+}$ increase, indicating that GLU stimulated Ca2+ influx pathway in the IMR32 cells. BAPTA, an intracellualr $Ca^{2+}$ chelator, significantly inhibited the GLU-induced apoptosis assessed by the flow cytometry measuring hypodiploid DNA content indicative of apoptosis, implying that intracellular $Ca^{2+}$ rise may mediate the apoptotic action of GLU. Vanillin (VAN) and p-hydroxybenzaldehyde(p-HB), known constituents of GE, significantly inhibited both intracellular $Ca^{2+}$ rise and apoptosis induced by GLU. These results suggest that the apoptosis-inhibitory actions of the constituents of GE may account, at least in part, for the basis of their antiepileptic activities. These results further suggest that intracelluarl $Ca^{2+}$ signaling pathway may be a molecular target of the constituents of GE.

• YAKHAK HOEJI
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• v.57 no.1
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• pp.24-31
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• 2013

Although statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, have been shown to increase melanin synthesis, the exact mechanism of this action is not fully understood. In this study we investigated the possible involvement of intracellular $Ca^{2+}$ signal in the mechanism of stimulation of melanin synthesis induced by lovastatin in B16 cells. Lovastatin stimulated the production of melanin in a dose-dependent manner in the cells. Treatment with mevalonate, FPP and GGPP, precursors of cholesterol, did not significantly suppress the lovastatin-induced melanin production, suggesting that inhibition of cholesterol synthesis may not be involved in the mechanism of the action of lovastatin. In addition, lovastatin did not significantly alter the cAMP concentration and the stimulated production of melanin by lovastatin was not significantly changed by treatment with H89, a potent inhibitor of protein kinase A, which demonstrates that cAMP pathway may not be involved. However, lovastatin increased intracellular $Ca^{2+}$ concentration in a dose-related fashion. Treatment with EGTA, an extracellular $Ca^{2+}$ chelator did not significantly alter the lovastatin-induced intracellular $Ca^{2+}$ increase and melanin synthesis, whereas intracellular $Ca^{2+}$ reduction with BAPTA/AM and intracellular $Ca^{2+}$ release blockers (dantrolene and TMB-8) completely blunted these actions of lovastatin. Taken together, these results suggest that the intracellular $Ca^{2+}$ release may play an important role in the lovastatin-induced stimulation of melanin synthesis in B16 cells. These results further suggest that lovastatin may be useful for the treatment of hypopigmentation disorders, such as vitiligo.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.5
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• pp.345-356
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• 2019

Docosahexaenoic acid (DHA), an omega-3-fatty acid, modulates multiple cellular functions. In this study, we addressed the effects of DHA on human umbilical vein endothelial cell calcium transient and endothelial nitric oxide synthase (eNOS) phosphorylation under control and adenosine triphosphate (ATP, $100{\mu}M$) stimulated conditions. Cells were treated for 48 h with DHA concentrations from 3 to $50{\mu}M$. Calcium transient was measured using the fluorescent dye Fura-2-AM and eNOS phosphorylation was addressed by western blot. DHA dose-dependently reduced the ATP stimulated $Ca^{2+}$-transient. This effect was preserved in the presence of BAPTA (10 and $20{\mu}M$) which chelated the intracellular calcium, but eliminated after withdrawal of extracellular calcium, application of 2-aminoethoxy-diphenylborane ($75{\mu}M$) to inhibit store-operated calcium channel or thapsigargin ($2{\mu}M$) to delete calcium store. In addition, DHA ($12{\mu}M$) increased ser1177/thr495 phosphorylation of eNOS under baseline conditions but had no significant effect on this ratio under conditions of ATP stimulation. In conclusion, DHA dose-dependently inhibited the ATP-induced calcium transient, probably via store-operated calcium channels. Furthermore, DHA changed eNOS phosphorylation suggesting activation of the enzyme. Hence, DHA may shift the regulation of eNOS away from a $Ca^{2+}$ activated mode to a preferentially controlled phosphorylation mode.

• Progress in Medical Physics
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• v.14 no.1
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• pp.54-67
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• 2003

The voltage-dependence of N-type calcium current inactivation is U-shaped with the degree of inactivation roughly mirroring inward current. This voltage-dependence has been reported to result from a purely voltage-dependent mechanism. However, $Ca^{2＋}$-dependent inactivation of N-channels has also been reported. We have investigated the role of $Ca^{2＋}$ in N-channel inactivation by comparing the effects of $Ba^{2＋}$and $Ca^{2＋}$ on whole-cell N-current in rat superior cervical ganglion neurons. For individual cells in-activation was always larger in $Ca^{2＋}$ than in $Ba^{2＋}$ even when internal EGTA (11 mM) was replaced with BAPTA (20 mM). The inactivation vs. voltage relationship was U-shaped in both divalent cations. The enhancement of inactivation by $Ca^{2＋}$ was inversely related with the magnitude of inactivation in $Ba^{2＋}$ as if the mechanisms of inactivation were the same in both $Ba^{2＋}$ and $Ca^{2＋}$. In support of this idea we could separate fast ( ${\gamma}$ ～150 ms) and slow ( ${\gamma}$ ～ 2500 ms) components of inactivation in both $Ba^{2＋}$and $Ca^{2＋}$ using 5 sec voltage steps. Differential effects were observed on each component with $Ca^{2＋}$ enhancing the magnitude of the fast component and the speed of the slow component. The larger amplitude of fast component indicates that the more channels inactivate via this pathway with $Ca^{2＋}$ than with $Ba^{2＋}$, but the stable time constants support the idea the fast inactivation mechanism is identical in $Ba^{2＋}$and $Ca^{2＋}$. The results do not support a $Ca^{2＋}$-dependent mechanism for fast inactivation. However, the $Ca^{2＋}$-induced acceleration of the slowly inactivating component could result from a $Ca^{2＋}$-dependent process.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.45 no.1
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• pp.57-67
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• 2019

Particulate matters with a diameter of < $10{\mu}m$ (PM10) exert oxidative stress and inflammatory events in various organs. The purpose of this study was to examine the molecular mechanism of reactive oxygen species (ROS) production induced by PM10 in the human epidermal keratinocytes (HEKs). When cultured HEKs were exposed to PM10, ROS production was induced and it was inhibited by apocynin, an antioxidant. The mRNA expression of NADPH oxidase (NOX) family was analyzed in order to examine their role in PM10-induced ROS production. PM10 increased the mRNA expression of NOX1, NOX2, dual oxidase (DUOX) 1 and DUOX2. HEKs expressed DUOX1 and DUOX2 at higher levels compared to other NOXs. The mRNA expression of dual oxidase maturation factors, DUOXA1 and DUOXA2, was also increased by PM10. We examined whether these calcium-dependent enzymes, DUOX1 and DUOX2, mediate the PM10-induced ROS production. A selective intracellular calcium chelator, BAPTA-AM, attenuated ROS production induced by PM10 or calcium ionophore A23187. The small intereference RNA (siRNA)-mediated down-regulation of DUOX2, but not DUOX1, attenuated the ROS production induced by PM10. PM10 increased the expression of inflammatory cytokines such as interleukin $(IL)-1{\beta}$, IL-6, IL-8 and interferon $(IFN)-{\gamma}$. SiRNA-mediated down-regulation of DUOX2 suppressed the PM10-induced expression of $IFN-{\gamma}$ but not other cytokines. This study suggests that DUOX2 plays a crucial role in ROS production and inflammatory response in PM10-exposed keratinocytes.

• Biomolecules & Therapeutics
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• v.13 no.2
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• pp.78-83
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• 2005

2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) has previously shown to induce neurotoxicity through intracellular $Ca^{2+}$ increase in rat neurons. In this study we investigated the role and signaling pathway of intracellular $Ca^{2+}$ in TCDD-induced inhibition of neuronal cell proliferation in SK-N-SH human neuronal cells. We found that TCDD(10nM) rapidly increased the level of intracellular $Ca^{2+}$, which was completely blocked by the extracellular $Ca^{2+}$ chelation with EGTA (1 mM) or by pretreatment of the cells with the non-selective cation channel blocker. flufenamic acid (200 ${\mu}M$). However, pretreatment of the cells with dantrolene (25 ${\mu}M$) and TMB-8(10 ${\mu}M$), intracellular $Ca^{2+}$-release blockers, or a voltage-sensitive $Ca^{2+}$ channel blocker, varapamil (100 ${\mu}M$), failed to block the TCDD-induced $Ca^{2+}$ increase in the cells. In addition, TCDD induced a rapid and transient activation of phatidvlinositol 3-kinase (PI3K) and extracellular signal-regulated kinase 1/2(ERK1/2), which was ingnificantly blocked by the pretreatment with BAPTA, an intracellular $Ca^{2+}$ chelator, and LY294002, a PI3K inhibitor. Furthermore, inhibitors of PI3K, ERK, or an intracellular $Ca^{2+}$ chelator further potentiated the anti-proliferative effect of TCDD in the cells. Collectively, the results suggest that intracellular $Ca^{2+}$ and PI3K-dependent activation of ERK 1/2 may be involved in the TCDD-induced inhibition of cell proliferation in SK-N-SH human neuronal cells.