• International Journal of Oral Biology
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• v.46 no.4
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• pp.176-183
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• 2021

Oral squamous cell carcinoma (OSCC) metastasis is characterized by distant metastasis and local recurrence. Combined chemotherapy with cisplatin and 5-fluorouracil is routinely used to treat patients with OSCC, and the combined use of gefitinib with cytotoxic drugs has been reported to enhance the sensitivity of cancer cells in vitro. However, the development of drug resistance because of prolonged chemotherapy is inevitable, leading to a poor prognosis. Therefore, understanding alterations in signaling pathways and gene expression is crucial for overcoming the development of drug resistance. However, the altered characterization of Ca²⁺ signaling in drug-resistant OSCC cells remains unclear. In this study, we investigated alterations in intracellular Ca²⁺ ([Ca²⁺]_i) mobilization upon the development of gefitinib resistance in human tongue squamous carcinoma cell line (HSC)-3 and HSC-4 using ratiometric analysis. This study demonstrated the presence of altered epidermal growth factor- and purinergic agonist-mediated [Ca²⁺]_i mobilization in gefitinib-resistant OSCC cells. Moreover, Ca²⁺ content in the endoplasmic reticulum, store-operated calcium entry, and lysosomal Ca²⁺ release through the transient receptor potential mucolipin 1, were confirmed to be significantly reduced upon the development of apoptosis resistance. Consistent with [Ca²⁺]_i mobilization, we identified modified expression levels of Ca²⁺ signaling-related genes in gefitinib-resistant cells. Taken together, we propose that the regulation of [Ca²⁺]_i mobilization and related gene expression can be a new strategy to overcome drug resistance in patients with cancer.

• International Journal of Oral Biology
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• v.41 no.3
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• pp.119-123
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• 2016

Acetylcholine receptors (AChR) including muscarinic and nicotinic AChR are widely expressed and mediate a variety of physiological cellular responses in neuronal and non-neuronal cells. Notably, a functional cholinergic system exists in oral epithelial cells, and nicotinic AChR (nAChR) mediates cholinergic anti-inflammatory responses. However, the pathophysiological roles of AChR in periodontitis are unclear. Here, we show that activation of AChR elicits increased cytosolic $Ca^{2+}([Ca^{2+}]_i)$, transient cytotoxicity, and induction of receptor activator of nuclear factor kappa-B ligand (RANKL) expression. Intracellular $Ca^{2+}$ mobilization in human gingival fibroblast-1 (hGF-1) cells was measured using the fluorescent $Ca^{2+}$ indicator, fura-2/AM. Cytotoxicity and induction of gene expression were evaluated by measuring the release of glucose-6-phosphate dehydrogenase and RT-PCR. Activation of AChR in hGF-1 cells by carbachol (Cch) induced $[Ca^{2+}]_i$ increase in a dose-dependent manner. Treatment with a high concentration of Cch on hGF-1 cells caused transient cytotoxicity. Notably, treatment of hGF-1 cells with Cch resulted in upregulated RANKL expression. The findings may indicate potential roles of AChR in gingival fibroblast cells in bone remodeling.

• Korean Journal of Pharmacognosy
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• v.51 no.2
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• pp.100-106
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• 2020

The extract of Cudrania tricuspidata is used in ethnomedicine throughout Eastern Asia in China, Korea and Japan. In Korean traditional medicine, Cudrania tricuspidata has been used to treat eczema, mumps, tuberculosis, contusions, insomnia and acute arthritis. In addition, it has been reported that root extract of Cudrania tricuspidata has anti-platelet effects. Therefore, we investigated which compound in Cudrania tricuspidata has inhibitory effect on platelet aggregation. In this study, we tried to explain the inhibitory mechanism of steppogenin from Cudrania tricuspidata on human platelet aggregation. Collagen-induced human platelet aggregation and [Ca²⁺]_i mobilization were dose-dependently inhibited by steppogenin and we determined the inhibition by steppogenin is due to the down regulation of extracellular-signal-regulated kinase(ERK) and inositol-1,4,5-triphosphate receptor type I(IP₃RI) phosphorylation. In addition, steppogenin inhibited collagen-induced fibronectin adhesion to αIIb/β3 and thromboxane A₂ generation. Thus, in the present study, steppogenin showed an inhibitory effect on human platelet aggregation, suggesting its potential use for preventing platelet-induced cardiovascular disease.

• International Journal of Oral Biology
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• v.45 no.2
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• pp.64-69
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• 2020

Short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate are secondary metabolites produced by anaerobic fermentation of dietary fibers in the intestine. Intestinal SCFAs exert various beneficial effects on intestinal homeostasis, including energy metabolism, autophagy, cell proliferation, immune reaction, and inflammation, whereas contradictory roles of SCFAs in the oral cavity have been reported. Herein, we found that low and high concentrations of SCFAs induce differential regulation of intracellular Ca²⁺ mobilization and expression of pro-inflammatory cytokines, such as interleukin (IL)-6 and IL-8, respectively, in gingival fibroblast cells. Additionally, cell viability was found to be differentially regulated in response to low and high concentrations of SCFAs. These findings demonstrate that the physiological functions of SCFAs in various cellular responses are more likely dependent on their local concentration.

• Journal of Applied Biological Chemistry
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• v.65 no.4
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• pp.463-469
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• 2022

Cudrania tricuspidata (C. tricuspidata), a medicinal plant widely employed throughout Asia in ethnomedicine, has various bioactive properties, including antidiabetic, antiobesity, antitumor, and anti-inflammatory activities. In addition, the C. tricuspidata root extract reportedly inhibits platelet aggregation. Therefore, we focused on the active substances present in the C. tricuspidata extract. Sanggenon N (SN) is a flavonoid found in the root bark of C. tricuspidata. In the present study, we examined the inhibitory effects of SN on platelet aggregation, phosphoproteins, thromboxane A₂ generation, and integrin αIIbβ3 activity. SN inhibited collagen-induced human platelet aggregation in a dose-dependent manner without cytotoxicity. Furthermore, SN suppressed Ca²⁺ mobilization and influx through associated signaling molecules, such as inositol 1, 4, 5-triphosphate receptor I (Ser¹⁷⁵⁶), and extracellular signal-regulated kinase. In addition, SN inhibited thromboxane A₂ generation and associated signaling molecules, including cytosolic phospholipase A₂ and mitogen-activated protein kinase p38. Finally, SN could inhibit integrin (αIIb/β₃) activity by regulating vasodilator-stimulated phosphoprotein and Akt. Collectively, SN possesses potent antiplatelet effects and is a potential therapeutic drug candidate to prevent platelet-related thrombosis and cardiovascular disease.

• Journal of Ginseng Research
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• v.39 no.4
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• pp.354-364
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• 2015

Background: Intracellular $Ca^{2+}$($[Ca^{2+}]_i$) is a platelet aggregation-inducing molecule. Therefore, understanding the inhibitory mechanism of $[Ca^{2+}]_i$mobilization is very important to evaluate the antiplatelet effect of a substance. This study was carried out to understand the $Ca^{2+}$-antagonistic effect of total saponin from Korean Red Ginseng (KRG-TS). Methods: We investigated the $Ca^{2+}$-antagonistic effect of KRG-TS on cyclic nucleotides-associated phosphorylation of inositol 1,4,5-trisphosphate receptor type I ($IP_3RI$) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) in thrombin (0.05 U/mL)-stimulated human platelet aggregation. Results: The inhibition of $[Ca^{2+}]_i$ mobilization by KRG-TS was increased by a PKA inhibitor (Rp-8-BrcAMPS), which was more stronger than the inhibition by a cyclic guanosine monophosphate (cGMP)- dependent protein kinase (PKG) inhibitor (Rp-8-Br-cGMPS). In addition, Rp-8-Br-cAMPS inhibited phosphorylation of PKA catalytic subunit (PKAc) ($Thr^{197}$) by KRG-TS. The phosphorylation of $IP_3RI$ ($Ser^{1756}$) by KRG-TS was very strongly inhibited by Rp-8-Br-cAMPS compared with that by Rp-8-BrcGMPS. These results suggest that the inhibitory effect of $[Ca^{2+}]_i$ mobilization by KRG-TS is more strongly dependent on a cAMP/PKA pathway than a cGMP/PKG pathway. KRG-TS also inhibited the release of adenosine triphosphate and serotonin. In addition, only G-Rg3 of protopanaxadiol in KRG-TS inhibited thrombin-induced platelet aggregation. Conclusion: These results strongly indicate that KRG-TS is a potent beneficial compound that inhibits $[Ca^{2+}]_i$ mobilization in thrombin-platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic disease.

• Biomedical Science Letters
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• v.25 no.2
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• pp.123-130
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• 2019

Platelet aggregation is essential for hemostatic process in case of blood vessels damages. However, excessive platelet aggregation can cause cardiovascular disorders including atherosclerosis, thrombosis and myocardial infarction. Scopoletin is usually found in the roots of genus Scopolia or Artemisia, and is known to have anticoagulant and anti-malarial effects. This study investigated the effect of scopoletin on human platelet aggregation induced by U46619, an analogue of thromboxane $A_2(TXA_2)$. Scopoletin had anti-platelet effects by down-regulating $TXA_2$ and intracellular $Ca^{2+}$ mobilization ($[Ca^{2+}]_i$), the aggregation-inducing molecules generated in activated platelets. On the other hand, scopoletin increased the levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are known to be intracellular $Ca^{2+}$ antagonists. This resulted in inhibition of fibrinogen binding to ${\alpha}IIb/{\beta}_3$ in U46619-induced human platelet aggregation. In addition, scopoletin inhibited the release of adenosine trisphosphate (ATP) in dose-dependent manner. This result means that the aggregation amplification activity through the granule secretion in platelets was suppressed by scopoletin. Therefore, we demonstrated that scopoletin has a potent antiplatelet effect and is highly likely to prevent platelet-derived vascular disease.

• Biomedical Science Letters
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• v.24 no.3
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• pp.253-262
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• 2018

Platelets are activated at sites of vascular injury via several molecules, such as adenosine diphosphate, collagen and thrombin. Full platelet aggregation is absolutely essential for normal hemostasis. Moreover, this physiological event can trigger circulatory disorders, such as thrombosis, atherosclerosis, and cardiovascular disease. Therefore, platelet function inhibition is a promising approach in preventing platelet-mediated circulatory disease. Many studies reported the involvement of mitogen-activated protein kinases (MAPKs) signaling pathways in platelet functions. However, these studies were limited. Thus, we examined MAPK signaling pathways in human platelets using specific MAPK inhibitors, such as PD98059, SB203580, and SP600125. We observed that these inhibitors were involved in calcium mobilization and influx in human platelets. They also suppressed thrombin-induced ${\alpha}$- and ${\delta}$-granule release. These results suggest that PD98059, SB203580, and SP600125 exhibit $Ca^{2+}$ antagonistic effects.

• Journal of Ginseng Research
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• v.47 no.1
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• pp.89-96
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• 2023

Background and aim: Panax ginseng, a key herbal medicine of replenishing Qi and tonifying Spleen, is widely used in the treatment of gastrointestinal diseases in East Asia. In this study, we aim to investigate the potential effects and mechanisms of polysaccharides from P. ginseng (PGP) on intestinal mucosal restitution which is one of the crucial repair modalities during the recovery of mucosal injury controlled by the Ca²⁺ signaling. Methods: Rat model of intestinal mucosal injury was induced by indomethacin. The fractional cell migration was carried out by immunohistochemistry staining with BrdU. The morphological observations on intestinal mucosal injury were also performed. Intestinal epithelial cell (IEC-6) migration in vitro was conducted by scratch method. Western-blot was adopted to determine the expressions of PLC-𝛾1, Rac1, TRPC1, RhoA and Cav-1. Immunoprecipitation was used to evaluate the levels of Rac1/PLC-𝛾1, RhoA/TRPC1 and Cav-1/TRPC1. Results: The results showed that PGP effectively reduced the assessment of intestinal mucosal injury, reversed the inhibition of epithelial cell migration induced by Indomethacin, and increased the level of Ca²⁺ in intestinal mucosa in vivo. Moreover, PGP dramatically promoted IEC-6 cell migration, the expression of Ca²⁺ regulators (PLC-𝛾1, Rac1, TRPC1, Cav-1 and RhoA) as well as protein complexes (Rac1/PLC-𝛾1, Cav-1/TRPC1 and RhoA/TRPC1) in vitro. Conclusion: PGP increases the Ca²⁺ content in intestinal mucosa partly through controlling the regulators of Ca²⁺ mobilization, subsequently promotes intestinal epithelial cell migration, and then prevents intestinal mucosal injury induced by indomethacin.

• Biomedical Science Letters
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• v.25 no.4
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• pp.302-312
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• 2019

The aim of this work was to investigate the effect of black tea extract (BTE) on collagen -induced platelet aggregation. In this study, BTE (10~500 ㎍/mL) was shown to inhibit platelet aggregation via thromboxane A₂ (TXA₂) down-regulation by blocking cyclooxygenase-1 (COX-1) activity. Also, BTE decreased intracellular Ca²⁺ mobilization ([Ca²⁺]_i). Additionally, BTE enhanced the levels of both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are aggregation-inhibiting molecules. BTE inhibited the phosphorylation of phospholipase C (PLC) γ2 and syk activated by collagen. BTE regulated platelet aggregation via cAMP-dependent phosphorylation of vasodilator-stimulated phosphoprotein (VASP) Ser¹⁵⁷. The anti-platelet effects of BTE in high fat diet (HFD)-induced obese rats were evaluated. After eight weeks of BTE treatment (300 and 600 mg/kg), the platelet aggregation rate in the treated groups was significantly less than that in the HFD-fed control group. Also, BTE exhibited a hepatoprotective effect and did not exert hepatotoxicity. Therefore, these data suggest that BTE has anti-platelet effects on collagen-stimulated platelet aggregation and may have therapeutic potential for the prevention of platelet-mediated thrombotic diseases.