• Archives of Pharmacal Research
• /
• v.28 no.12
• /
• pp.1358-1364
• /
• 2005

In this study, we investigated whether a novel anti-ischemic $K_{ATP}$ opener KR-31378 [(2S,3S,4R)­N'-cyano-N-(6-amino-3,4-dihydro-3-hydroxy-2 -methly-2-dimethoxymethly-2H-benzopyran-4-yl)­N'-benzylguanidine] has protective effect against oxidative stress-induced death in heart-derived H9c2 cells. Cell death was induced by BSO, butionine sulfoximine, which inhibits GSH synthesis and subsequently increases reactive oxygen species (ROS) level. Cell death was quantitatively determined by measuring lactate dehydrogenase (LDH) activity and stained by Hoechst 33258. BSO-induced ROS production and mitochondrial membrane potential (MMP) were measured using 2',7'-dichlorofluorescein diacetate oxidation and rhodamine 123, respectively. Both the LDH release and the ROS elevation induced by treatment of H9c2 cells with 10 mM BSO, were significantly decreased by KR-31378. These protective effect and antioxidant effect of KR-31378 appeared to be independent on $K_{ATP}$ channel opening. Cells exposed to BSO showed an early reduction in MMP, and this reduction in MMP was significantly reversed by treatment with KR-31378. Caspase-3 activity in BSO treated H9c2 cells was remarkably increased, and this increased caspase-3 activity was significantly reversed by KR-31378. In conclusion, our results suggest that KR-31378 can produce cardioprotective effect against oxidative stress-induced cell death through antioxidant mechanism.

• The Journal of Korean Obstetrics and Gynecology
• /
• v.20 no.3
• /
• pp.1-12
• /
• 2007

Purpose: Uterine leiomyomas (fibroids) are common estrogen-dependent uterine tumors. Houttuynia cordata thunberg has cancer-preventing properties and often used in Chinese medicine. In the present study we used Houttuynia cordata thunberg to determine its effect on cell proliferation and apoptosis in human uterine leiomyoma cells. Methods: Primary cultured human uterine leiomyoma cells were treated with Houttuynia cordata thunberg. Cell viability analysis was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTS) assay and Flow cytometry was performed to ascertain the effects Houttuynia cordata thunberg. Expression of cell cycle related proteins and apoptosis related proteins were evaluated by Western blot analysis. Results: Cell viability was significantly influenced by Houttuynia cordata thunberg treatment in a dose-dependent manner in leiomyoma cells compare to normal myometrial cells. Flow cytometric analysis showed that Houttuynia cordata thunberg induced Sub G1 arrest. DNA fragmentation assay was carried out and apoptosis was detected. Activation of caspase-3, down-regulation of Bcl-2, with concomitant increase in p21 was observed. Houttuynia cordata thunberg treatment of uterine leiomyoma cellsresulted in a concentration-dependent cell death induced via the caspase dependent mechanism. Conclusion: These results suggest that Houttuynia cordata thunberg treatment in uterine leiomyoma cells leads to growth inhibition and induced apoptosis. These results suggest that Houttuynia cordata thunberg will be a promising agent for use in therapeutics agents against human uterine endometrial cancer.

• Toxicological Research
• /
• v.35 no.1
• /
• pp.83-91
• /
• 2019

Nanoparticles (NPs) have been recognized as both useful tools and potentially toxic materials in various industrial and medicinal fields. Previously, we found that zinc oxide (ZnO) NPs that are neurotoxic to human dopaminergic neuroblastoma SH-SY5Y cells are mediated by lipoxygenase (LOX), not cyclooxygenase-2 (COX-2). Here, we examined whether human bone marrow-derived mesenchymal stem cells (MSCs), which are different from neuroblastoma cells, might exhibit COX-2- and/or LOX-dependent cytotoxicity of ZnO NPs. Additionally, changes in annexin V expression, caspase-3/7 activity, and mitochondrial membrane potential (MMP) induced by ZnO NPs and ZnO were compared at 12 hr and 24 hr after exposure using flow cytometry. Cytotoxicity was measured based on lactate dehydrogenase activity and confirmed by trypan blue staining. Rescue studies were executed using zinc or iron chelators. ZnO NPs and ZnO showed similar dose-dependent and significant cytotoxic effects at concentrations ${\geq}15{\mu}g/mL$, in accordance with annexin V expression, caspase-3/7 activity, and MMP results. Human MSCs exhibited both COX-2 and LOX-mediated cytotoxicity after exposure to ZnO NPs, which was different from human neuroblastoma cells. Zinc and iron chelators significantly attenuated ZnO NPs-induced toxicity. Conclusively, these results suggest that ZnO NPs exhibit both COX-2- and LOX-mediated apoptosis by the participation of mitochondrial dysfunction in human MSC cultures.

• Journal of Microbiology and Biotechnology
• /
• v.31 no.12
• /
• pp.1615-1623
• /
• 2021

Picropodophyllotoxin (PPT), an epimer of podophyllotoxin, is derived from the roots of Podophyllum hexandrum and exerts various biological effects, including anti-proliferation activity. However, the effect of PPT on colorectal cancer cells and the associated cellular mechanisms have not been studied. In the present study, we explored the anticancer activity of PPT and its underlying mechanisms in HCT116 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to monitor cell viability. Flow cytometry was used to evaluate cell cycle distribution, the induction of apoptosis, the level of reactive oxygen species (ROS), assess the mitochondrial membrane potential (Δψm), and multi-caspase activity. Western blot assays were performed to detect the expression of cell cycle regulatory proteins, apoptosis-related proteins, and p38 MAPK (mitogen-activated protein kinase). We found that PPT induced apoptosis, cell cycle arrest at the G1 phase, and ROS in the HCT116 cell line. In addition, PPT enhanced the phosphorylation of p38 MAPK, which regulates apoptosis and PPT-induced apoptosis. The phosphorylation of p38 MAPK was inhibited by an antioxidant agent (N-acetyl-L-cysteine, NAC) and a p38 inhibitor (SB203580). PPT induced depolarization of the mitochondrial inner membrane and caspase-dependent apoptosis, which was attenuated by exposure to Z-VAD-FMK. Overall, these data indicate that PPT induced G1 arrest and apoptosis via ROS generation and activation of the p38 MAPK signaling pathway.

• Biomolecules & Therapeutics
• /
• v.31 no.6
• /
• pp.682-691
• /
• 2023

Cell transformation induced by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) is a critical event in cancer initiation and progression, and understanding the underlying mechanisms is essential for the development of new therapeutic strategies. Licorice extract contains various bioactive compounds, which have been reported to have anticancer and anti-inflammatory effects. This study investigated the cancer preventive efficacy of licochalcone D (LicoD), a chalcone derivative in licorice extract, in EGF and TPA-induced transformed skin keratinocyte cells. LicoD effectively suppressed EGF-induced cell proliferation and anchorage-independent colony growth. EGF and TPA promoted the S phase of cell cycle, while LicoD treatment caused G1 phase arrest and down-regulated cyclin D1 and up-regulated p21 expression associated with the G1 phase. LicoD also induced apoptosis and increased apoptosis-related proteins such as cleaved-caspase-3, cleaved-caspase-7, and Bax (Bcl2-associated X protein). We further investigated the effect of LicoD on the AKT signaling pathway involved in various cellular processes and found decreased p-AKT, p-GSK3β, and p-NFκB expression. Treatment with MK-2206, an AKT pharmacological inhibitor, suppressed EGF-induced cell proliferation and transformed colony growth. In conclusion, this study demonstrated the potential of LicoD as a preventive agent for skin carcinogenesis.

• Journal of Veterinary Science
• /
• v.24 no.5
• /
• pp.55.1-55.12
• /
• 2023

Background: Peste des petits ruminants (PPR), caused by the PPR virus (PPRV), is an acute and fatal contagious disease that mainly infects goats, sheep, and other artiodactyls. Peripheral blood mononuclear cells (PBMCs) are considered the primary innate immune cells. Objectives: PBMCs derived from goats were infected with PPRV and analyzed to detect the relationship between PPRV replication and apoptosis or the inflammatory response. Methods: Quantitative real-time polymerase chain reaction was used to identify PPRV replication and cytokines expression. Flow cytometry was conducted to detect apoptosis and the differentiation of CD4⁺ and CD8⁺ T cells after PPRV infection. Results: PPRV stimulated the differentiation of CD4⁺ and CD8⁺ T cells. In addition, PPRV induced apoptosis in goat PBMCs. Furthermore, apoptosis and the inflammatory response induced by PPRV could be suppressed by Z-VAD-FMK and Z-YVAD-FMK, respectively. Moreover, the virus titer of PPRV was attenuated by inhibiting caspase-1-dependent apoptosis and inflammation. Conclusions: This study showed that apoptosis and the inflammatory response play an essential role in PPR viral replication in vitro, providing a new mechanism related to the cell host response.

• Asian Pacific Journal of Cancer Prevention
• /
• v.14 no.4
• /
• pp.2325-2331
• /
• 2013

Objective: To detect effects of plumbagin on proliferation and apoptosis in non-small cell lung cancer cell lines, and investigate the underlying mechanisms. Materials and Methods: Human non-small cell lung cancer cell lines A549, H292 and H460 were treated with various concentrations of plumbagin. Cell proliferation rates was determined using both cell counting kit-8 (CCK-8) and clonogenic assays. Apoptosis was detected by annexin V/propidium iodide double-labeled flow cytometry and TUNEL assay. The levels of reactive oxygen species (ROS) were detected by flow cytometry. Activity of NF-${\kappa}B$ was examined by electrophoretic mobility shift assay (EMSA) and luciferase reporter assay. Western blotting was used to assess the expression of both NF-${\kappa}B$ regulated apoptotic-related gene and activation of p65 and $I{\kappa}B{\kappa}$. Results: Plumbagin dose-dependently inhibited proliferation of the lung cancer cells. The IC50 values of plumbagin in A549, H292, and H460 cells were 10.3 ${\mu}mol/L$, 7.3 ${\mu}mol/L$, and 6.1 ${\mu}mol/L$ for 12 hours, respectively. The compound concentration-dependently induced apoptosis of the three cell lines. Treatment with plumbagin increased the intracellular level of ROS, and inhibited the activation of NK-${\kappa}B$. In addition to inhibition of NF-${\kappa}B$/p65 nuclear translocation, the compound also suppressed the degradation of $I{\kappa}B{\kappa}$. ROS scavenger NAC highly reversed the effect of plumbagin on apoptosis and inactivation of NK-${\kappa}B$ in H460 cell line. Treatment with plumbagin also increased the activity of caspase-9 and caspase-3, downregulated the expression of Bcl-2, upregulated the expression of Bax, Bak, and CytC. Conclusions: Plumbagin inhibits cell growth and induces apoptosis in human lung cancer cells through an NF-${\kappa}B$-regulated mitochondrial-mediated pathway, involving activation of ROS.

• Toxicological Research
• /
• v.35 no.2
• /
• pp.167-179
• /
• 2019

Ovarian cancer is the fifth main cause of pre-senescent death in women. Although chemotherapy is generally an efficient treatment, its side effects and the occurrence of chemotherapeutic resistance have prompted the need for alternative treatments. In this study, ${\alpha}$-mangostin and apigenin were evaluated as possible anticancer alternatives to the chemotherapeutic drug doxorubicin, used herein as a positive control. The ovarian adenocarcinoma cell line SKOV-3 (ATCC No. HTB77) was used as model ovarian cancer cells, whereas the skin fibroblast line CCD-986Sk (ATCC No. CRL-1947) and lung fibroblast line WI-38 (ATCC No. CCL-75) were used as model untransformed cells. Apigenin and doxorubicin inhibited the growth of SKOV-3 cells in a dose- and time-dependent manner. After 72 hr exposure, doxorubicin was mostly toxic to SKOV-3 cells, whereas apigenin was toxic to SKOV-3 cells but not CCD-986Sk and WI-38 cells. ${\alpha}$-Mangostin was more toxic to SKOV-3 cells than to CCD-986Sk cells. A lower cell density, cell shrinkage, and more unattached (floating round) cells were observed in all treated SKOV-3 cells, but the greatest effects were observed with ${\alpha}$-mangostin. With regard to programmed cell death, apigenin caused early apoptosis within 24 hr, whereas ${\alpha}$-mangostin and doxorubicin caused late apoptosis and necrosis after 72 hr of exposure. Caspase-3 activity was significantly increased in ${\alpha}$-mangostin-treated SKOV-3 cells after 12 hr of exposure, whereas only caspase-9 activity was significantly increased in apigenin-treated SKOV-3 cells at 24 hr. Both ${\alpha}$-mangostin and apigenin arrested the cell cycle at the $G_2/M$ phase, but after 24 and 48 hr, respectively. Significant upregulation of BCL2 (apoptosis-associated gene) and COX2 (inflammation-associated gene) transcripts was observed in apigenin- and ${\alpha}$-mangostin-treated SKOV-3 cells, respectively. ${\alpha}$-Mangostin and apigenin are therefore alternative options for SKOV-3 cell inhibition, with apigenin causing rapid early apoptosis related to the intrinsic apoptotic pathway, and ${\alpha}$-mangostin likely being involved with inflammation.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.20 no.6
• /
• pp.1459-1466
• /
• 2006

The water extract of Hwansodan has been traditionally used for treatment of ischemic brain damage in oriental medicine. However, little is known about the mechanism by which the water extract of Hwansodan rescues cells from neurodegenerative disease. PC12 pheochromocytoma cells have been used extensively as a model for studying the cellular and molecular mechanisms of neuronal cell damages. Under deprivation of growth factor and ischemic injury, PC12 cells spontaneously undergoes apoptotic cell death. Serum and glucose deprivation markedly decreased the viability of PC12 cells, which was characterized with apparent apoptotic features such as membrane blebbing as well as fragmentation of genomic DNA and nuclei. However, the aqueous extract of Hwansodan significantly reduced serum and glucose deprivation-induced cell death and apoptotic characteristics through reduction of intracellular peroxide generation. Pretreatment of Hwansodan also ingibited the activation of caspase-3, in turn, degradation of ICAD/DFF45 was completely abolished in serum and glucose deprivated cells. Furthermore, pretreatment of Hwansodan obviously increased heme oxygenase 1 (HO-1) expression in PC12 cells. Taken together, the data suggest that the protective effects of Hwansodan against serum and glucose deprivation induced oxidative injuries may be achieved through the scavenging of reactive oxygene species accompanying with HO-1 induction.

• THE JOURNAL OF KOREAN ORIENTAL ONCOLOGY
• /
• v.6 no.1
• /
• pp.81-97
• /
• 2000

Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to $0.4{\mu}g$) and periods (6 to 30 hr) of $H_2O$ and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with $200{\mu}g/ml$ of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ of methanol extract for various periods. Lysate from the cells used to measure the activity of Caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively. Cells were treated with $200{\mu}g/ml$ of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with $32^p-ATP$ and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at $CO_2$ incubator for 6 days. The number of colony was counted under light microscopy ($\time100$). Results: The death of HL-60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL-60 cells. In addition, it was shown nucleus chromatin condensation and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, $NF-{\kappa}B$ was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator $NF-{\kappa}B$, In addition, our results also suggest that methanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming effciency through cleavage of Bc12 as well as induction of Bax.