• The Journal of Internal Korean Medicine
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• v.21 no.2
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• pp.259-266
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• 2000

Objectives : The effects of aqueous extracts of Cortex ulmi pumilae (a traditional medicine for cancer treatment in oriental medicine) on the induction of apoptotic cell death were investigated in human liver origm hepatoma cell lines, HepG2. Methods : The death of HepG2 cells was markedly induced by the addition of extracts of Cortex ulmi pumilae in a dose-dependent manner. The apoptotic characteristic ladder pattern of DNA strand break was not observed in cell death of HepG2. In addition, it was not shown nucleus chromatin condensation and fragmentation under hoechst staining. However, by the using annexin V staining assay, externalizations of phosphatidylserine in HepG2 cell which were treated with Cortex ulmi pumilae extracts were detected in the early time (at 9 hr after extract treatment). Furthermore, LDH release was not detected in this early stage. Therefore, Cortex ulmi pumilae extracts-induced cell death of HepG2 cells is mediated by apoptotic death signal processes. Result : The activity of caspase 3-like proteases remained in a basal level in HepG2 cells which treated with the extract of Cordyceps sinensis. However, it was markedly increased in HepG2 cells which treated with two extracts of Cortex ulmi pumilae (C.U.P.-C, C.U.P.-K) which were differently extracted (respectively, 2.3 and 3.3 fold). On a while, the phosphotransferase activities of JNK1 was markedly induced in HepG2 cells which were treated with two extracts of Cortex ulmi pumilae. On the contrary, the activation of transcriptional activator, activating protein1(AP-1) and NF-kB were severely decreased by these two extracts of Cortex ulmi pumilae (C.U.P.-C, C.U.P.-K). In addition, antioxidants (GSH and NAC) and intracellular $Ca2^+$ level regulator (Bapta/AM and Thapsigargin) did not affect Cortex ulmi pumilae extracts-induced apoptotic death of HepG2 cells. Conclusions : In conclusion, our results suggest that two extracts of Cortex ulmi pumilae (C.U.P.-C, C.U.P.-K) induces the apoptotic death of human liver origin hepatoma HepG2 cells via activation of caspase 3-like proteases as well as JNK1, and inhibition of transcriptional activators, AP-1 and $NK-{\kappa}B$.

• Journal of Ginseng Research
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• v.42 no.2
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• pp.165-174
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• 2018

Background: Extended endoplasmic reticulum (ER) stress may initiate apoptotic pathways in cancer cells, and ER stress has been reported to possibly increase tumor death in cancer therapy. We previously reported that caspase-8 played an important role in compound K-induced apoptosis via activation of caspase-3 directly or indirectly through Bid cleavage, cytochrome c release, and caspase-9 activation in HL-60 human leukemia cells. The mechanisms leading to apoptosis in A549 and SK-MES-1 human lung cancer cells and the role of ER stress have not yet been understood. Methods: The apoptotic effects of compound K were analyzed using flow cytometry, and the changes in protein levels were determined using Western blot analysis. The intracellular calcium levels were monitored by staining with Fura-2/AM and Fluo-3/AM. Results: Compound K-induced ER stress was confirmed through increased phosphorylation of $eIF2{\alpha}$ and protein levels of GRP78/BiP, XBP-1S, and $IRE1{\alpha}$ in human lung cancer cells. Moreover, compound-K led to the accumulation of intracellular calcium and an increase in m-calpain activities that were both significantly inhibited by pretreatment either with BAPTA-AM (an intracellular $Ca^{2+}$ chelator) or dantrolene (an RyR channel antagonist). These results were correlated with the outcome that compound K induced ER stress-related apoptosis through caspase-12, as z-ATAD-fmk (a specific inhibitor of caspase-12) partially ameliorated this effect. Interestingly, 4-PBA (ER stress inhibitor) dramatically improved the compound K-induced apoptosis. Conclusion: Cell survival and intracellular $Ca^{2+}$ homeostasis during ER stress in human lung cancer cells are important factors in the induction of the compound K-induced apoptotic pathway.

• Journal of Life Science
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• v.18 no.9
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• pp.1177-1185
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• 2008

In the present work, we show that spermine (spm)-induced cytotoxicity is due to the mitochondrial-dependent pathway triggered by the intracellular $Ca^{2+}$ increase in MCF-7 human breast cancer cells. Spm induced the intracellular $Ca^{2+}$ increase in a dose-dependent manner in the medium containing 1.5 mM $Ca^{2+}$. Even in the $Ca^{2+}$-free medium, spm could induce a minor $Ca^{2+}$ increase in a dose-dependent fashion, suggesting a probable leak from the internal storage. The cytotoxic effect of $Ca^{2+}$ could be further proved by using either BAPTA or ionophore. Spm-induced $Ca^{2+}$ increase led to the release of cytochrome c from mitochondria into the cytosol and the change of mitochondrial membrane potential. In MCF-7 cells, caspase-7 plays a key role in the downstream of apoptosis because caspase-3 is absent. In the cells treated with spm, the cleavage of caspase-7 and -12 was increased almost two-fold. The level of anti-apoptotic Bcl-2 protein decreased to 35% of the control; however, the cells showed increased expression of pro-apoptotic Bax protein about two-fold in response to spm. These results imply that the apoptotic signaling pathway activated by spm is likely to be mediated via the mitochondrial-dependent pathway.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.1
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• pp.101-109
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• 2016

Reducing $[Mg^{2+}]_o$ to 0.1 mM can evoke repetitive $[Ca^{2+}]_i$ spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM $[Mg^{2+}]_o$ are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether $Ca^{2+}$ spikes are involved in this process. Preconditioning by an exposure to 0.1 mM $[Mg^{2+}]_o$ for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type $Ca^{2+}$ channel antagonist nimodipine, which blocked 0.1 mM $[Mg^{2+}]_o$-induced $[Ca^{2+}]_i$ spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the $[Ca^{2+}]_i$ spikes. The intracellular $Ca^{2+}$ chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the $[Ca^{2+}]_i$ spikes. While $G{\ddot{o}}6976$, a specific inhibitor of $PKC{\alpha}$ had no effect on the tolerance, both the $PKC{\varepsilon}$ translocation inhibitor and the $PKC{\zeta}$ pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the $[Ca^{2+}]_i$ spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low $[Mg^{2+}]_o$ preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the $[Ca^{2+}]_i$ spike-induced activation of $PKC{\varepsilon}$ and $PKC{\xi}$, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.

• Molecules and Cells
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• v.37 no.9
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• pp.656-663
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• 2014

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits $[Ca^{2+}]_i$ transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier $K^+$ ($I_{Ks}$) channel is a cardiac $K^+$ channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating $I_{Ks}$ channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human $I_{Ks}$ channel activity by expressing human $I_{Ks}$ channels in Xenopus oocytes. We found that gintonin enhances $I_{Ks}$ channel currents in concentration- and voltage-dependent manners. The $EC_{50}$ for the $I_{Ks}$ channel was $0.05{\pm}0.01{\mu}g/ml$. Gintonin-mediated activation 1 of the $I_{Ks}$ channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an $IP_3$ receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the $I_{Ks}$ channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 $[Ca^{2+}]_i$/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on $I_{Ks}$ channel. However, gintonin had no effect on hERG $K^+$ channel activity. These results show that gintonin-mediated enhancement of $I_{Ks}$ channel currents is achieved through binding of the $[Ca^{2+}]_i$/CaM complex to the C terminus of KCNQ1 subunit.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.3
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• pp.249-255
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• 2015

Wnk kinase maintains cell volume, regulating various transporters such as sodium-chloride cotransporter, potassium-chloride cotransporter, and sodium-potassium-chloride cotransporter 1 (NKCC1) through the phosphorylation of oxidative stress responsive kinase 1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). However, the activating mechanism of Wnk kinase in specific tissues and specific conditions is broadly unclear. In the present study, we used a human salivary gland (HSG) cell line as a model and showed that $Ca^{2+}$ may have a role in regulating Wnk kinase in the HSG cell line. Through this study, we found that the HSG cell line expressed molecules participating in the WNK-OSR1-NKCC pathway, such as Wnk1, Wnk4, OSR1, SPAK, and NKCC1. The HSG cell line showed an intracellular $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) increase in response to hypotonic stimulation, and the response was synchronized with the phosphorylation of OSR1. Interestingly, when we inhibited the hypotonically induced $[Ca^{2+}]_i$ increase with nonspecific $Ca^{2+}$ channel blockers such as 2-aminoethoxydiphenyl borate, gadolinium, and lanthanum, the phosphorylated OSR1 level was also diminished. Moreover, a cyclopiazonic acid-induced passive $[Ca^{2+}]_i$ elevation was evoked by the phosphorylation of OSR1, and the amount of phosphorylated OSR1 decreased when the cells were treated with BAPTA, a $Ca^{2+}$ chelator. Finally, through that process, NKCC1 activity also decreased to maintain the cell volume in the HSG cell line. These results indicate that $Ca^{2+}$ may regulate the WNK-OSR1 pathway and NKCC1 activity in the HSG cell line. This is the first demonstration that indicates upstream $Ca^{2+}$ regulation of the WNK-OSR1 pathway in intact cells.

• The Korean Journal of Physiology and Pharmacology
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• v.17 no.3
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• pp.223-228
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• 2013

The calcium-activated $K^+$ ($BK_{Ca}$) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. $Ca^{2+}$ is the main regulator of $BK_{Ca}$ channel activation. The $BK_{Ca}$ channel contains two high affinity $Ca^{2+}$ binding sites, namely, regulators of $K^+$ conductance, RCK1 and the $Ca^{2+}$ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein-coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular $Ca^{2+}$ levels through diverse G proteins such as $G{\alpha}_{q/11}$, $G{\alpha}_i$, $G{\alpha}_{12/13}$, and $G{\alpha}s$ and the related signal transduction pathway. In the present study, we examined LPA effects on $BK_{Ca}$ channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated $BK_{Ca}$ channel activation was also attenuated by the PLC inhibitor U-73122, $IP_3$ inhibitor 2-APB, $Ca^{2+}$ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated $BK_{Ca}$ channel activation. The present study indicates that LPA-mediated activation of the $BK_{Ca}$ channel is achieved through the PLC, $IP_3$, $Ca^{2+}$, and PKC pathway and that LPA-mediated activation of the $BK_{Ca}$ channel could be one of the biological effects of LPA in the nervous and vascular systems.

• Proceedings of the Ginseng society Conference
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• 2002.10a
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• pp.66-83
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• 2002

Recently, we have provided evidence that ginsenosides, the active components of Panax ginseng, utilize pertussis toxin (PTX)-insensitive $G{\alpha}_{q/11}-phospholipase\;C-{\beta}3(PLC-{\beta}3)$ signal transduction pathway for the enhancement of $Ca^{2+}-activated\;Cl^{-}$ current in the Xenopus oocyte (British J. Pharmacol. 132, 641-647, 2001; JBC 276, 48797-48802, 2001). Other investigators have shown that stimulation of receptors linked to $G{\alpha}-PLC$ pathway inhibits the activity of G proteincoupled inwardly rectifying $K^+$ (GIRK) channel. In the present study, we sought to determine whether ginsenosides influenced the activity of GIRK 1 and GIRK 4 (GIRK 1/4) channels expressed in the Xenopus oocyte, and if so, the underlying signal transduction mechanism. In oocyte injected with GIRK 1/4 channel cRNAs, bath-applied ginsenosides inhibited high potassium (HK) solution-elicited GIRK current $(EC_{50}:4.9{\pm}4.3\;{\mu}g/ml).$ Pretreatment of the oocyte with PTX reduced the HK solution-elicited GIRK current by $49\%,$ but it did not alter the inhibitory ginsenoside effect on GIRK current. Prior intraoocyte injection of cRNA(s) coding $G{\alpha}_q,\;G{\alpha}_{11}\;or\;G{\alpha}_q/G{\alpha}_{11},\;but\;not\;G{\alpha}_{i2}\;or\;G{\alpha}_{oA}$ attenuated the inhibitory ginsenoside effect. Injection of cRNAs coding $G{\beta}_{1{\gamma}2}$ also attenuated the ginsenoside effect. Similarly, injection of the cRNAs coding regulators of G protein signaling 1, 2 and 4 (RGS1, RGS2 and RGS4), which interact with $G{\alpha}_i\;and/or\;G{\alpha}_{q/11}$ and stimulates the hydrolysis of GTP to GDP in active GTP-bound $G{\alpha}$ subunit, resulted in a significant reduction of ginsenoside effect on GIRK current. Preincubation of GIRK channel-expressing oocyte in PLC inhibitor (U73122) or protein kinase C (PKC) inhibitor (staurosporine or chelerythrine) blocked the inhibitory ginsenoside effect on GIRK current. On the other hand, intraoocyte injection of BAPTA, a free $Ca^{2+}$ chelator, had no significant effect on the ginsenoside action. Taken together, these results suggest that ginsenosides inhibit the activity of GIRK 1/4 channel expressed in the Xenopus oocyte through a PTX-insensitive and $G{\alpha}_{q/11}$-,PLC-and PKC-mediated signal transduction pathway.

• Journal of Ginseng Research
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• v.43 no.2
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• pp.305-311
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• 2019

Background: Gintonin is a ginseng-derived exogenous ligand of the G protein-coupled lysophosphatidic acid (LPA) receptor. We previously reported that gintonin stimulates gliotransmitter release in primary cortical astrocytes. Astrocytes play key roles in the functions of neurovascular systems. Although vascular endothelial growth factor (VEGF) is known to influence the normal growth and maintenance of cranial blood vessels and the nervous system, there is little information about the effect of gintonin on VEGF regulation in primary astrocytes, under normal and hypoxic conditions. Methods: Using primary cortical astrocytes of mice, the effects of gintonin on the release, expression, and distribution of VEGF were examined. We further investigated whether the gintonin-mediated VEGF release protects astrocytes from hypoxia. Results: Gintonin administration stimulated the release and expression of VEGF from astrocytes in a concentration- and time-dependent manner. The gintonin-mediated increase in the release of VEGF was inhibited by the LPA1/3 receptor antagonist, Ki16425; phospholipase C inhibitor, U73122; inositol 1,4,5- triphosphate receptor antagonist, 2-APB; and intracellular $Ca^{2+}$ chelator, BAPTA. Hypoxia further stimulated astrocytic VEGF release. Gintonin treatment stimulated additional VEGF release and restored cell viability that had decreased due to hypoxia, via the VEGF receptor pathway. Altogether, the regulation of VEGF release and expression and astrocytic protection mediated by gintonin under hypoxia are achieved via the LPA receptor-VEGF signaling pathways. Conclusion: The present study shows that the gintonin-mediated regulation of VEGF in cortical astrocytes might be neuroprotective against hypoxic insults and could explain the molecular basis of the beneficial effects of ginseng on the central nervous system.