• Nutrition Research and Practice
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• v.8 no.2
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• pp.132-137
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• 2014

BACKGROUND/OBJECTIVES: In this study, the apoptogenic activity and mechanisms of cell death induced by hexane extract of aged black garlic (HEABG) were investigated in human leukemic U937 cells. MATERIALS/METHODS: Cytotoxicity was evaluated by MTT (3-(4, 5-dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide) assay. Apoptosis was detected using 4,6-diamidino-2-phenyllindile (DAPI) staining, agarose gel electrophoresis and flow cytometry. The protein levels were determined by Western blot analysis. Caspase activity was measured using a colorimetric assay. RESULTS: Exposure to HEABG was found to result in a concentration- and time-dependent growth inhibition by induction of apoptosis, which was associated with an up-regulation of death receptor 4 and Fas legend, and an increase in the ratio of Bax/Bcl-2 protein expression. Apoptosis-inducing concentrations of HEABG induced the activation of caspase-9, an initiator caspase of the mitochodrial mediated intrinsic pathway, and caspase-3, accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase. HEABG also induced apoptosis via a death receptor mediated extrinsic pathway by caspase-8 activation, resulting in the truncation of Bid, and suggesting the existence of cross-talk between the extrinsic and intrinsic pathways. However, pre-treatment of U937 cells with the caspase-3 inhibitor, z-DEVD-fmk, significantly blocked the HEABG-induced apoptosis of these cells, and increased the survival rate of HEABG-treated cells, confirming that HEABG-induced apoptosis is mediated through activation of caspase cascade. CONCLUSIONS: Based on the overall results, we suggest that HEABG reduces leukemic cell growth by inducing caspase-dependent apoptosis through both intrinsic and extrinsic pathways, implying its potential therapeutic value in the treatment of leukemia.

• BMB Reports
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• v.40 no.6
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• pp.899-910
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• 2007

Extracellular Regulated Kinases (ERK) and Protein Kinase B (Akt) are intermediaries in relaying extracellular growth signals to intracellular targets. Each pathway can become activated upon stimulation of G protein-coupled receptors mediated by $G_q$ and $G_{i/o}$ proteins subjected to regulation by RGS proteins. The goal of the study was to delineate the specificity in which cardiac RGS proteins modulate $G_{q^-}$ and $G_{i/o}$-induced ERK and Akt phosphorylation. To isolate $G_{q^-}$ and $G_{i/o}$-mediated effects, we exclusively expressed muscarinic $M_2$ or $M_3$ receptors in COS-7 cells. Western blot analyses demonstrated increase of phosphorylation of ERK 1.7-/3.3-fold and Akt 2.4-/6-fold in $M_{2^-}/M_{3^-}$ expressing cells through carbachol stimulation. In co-expressions, $M_3/G_q$-induced activation of Akt was exclusively blunted through RGS3s/RGS3, whereas activation of ERK was inhibited additionally through RGS2/RGS5. $M_2/G_{i/o}$ induced Akt activation was inhibited by all RGS proteins tested. RGS2 had no effect on $M_2/G_{i/o}$-induced ERK activation. The high degree of specificity in RGS proteins-depending modulation of $G_{q^-}$ and $G_{i/o}$-mediated ERK and Akt activation in the muscarinic network cannot merely be attributed exclusively to RGS protein selectivity towards $G_q$ or $G_{i/o}$ proteins. Counter-regulatory mechanisms and inter-signaling cross-talk may alter the sensitivity of GPCR-induced ERK and Akt activation to RGS protein regulation.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.14
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• pp.5835-5842
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• 2015

Background: Melatonin, which is mainly produced by the pineal gland, has a good inhibitory effect on cell growth of multiple cancer types. However, the underlying molecular mechanisms of anti-tumor activity for colon cancer have not been fully elucidated. In this study, we investigated the effects of melatonin on migration in human colon cancer RKO cells and the potential molecular mechanisms. Materials and Methods: The viability of RKO cells was investigated by MTT assay after treatment with melatonin, SB203580 (p38 inhibitor) and phorbol 12-myristate 13-acetate (PMA, MAPK activator) alone or in combination for 48h. The effects of melatonin, and ML-7, a selective inhibitor of myosin light chain kinase (MLCK), and SB203580, and PMA on the migration of RKO cells were analyzed by in vitro scratch-wound assay. The relative mRNA levels of MLCK was assessed by real-time quantitative RT-PCR. Western blotting analysis was performed to examine the expression of MLCK, phosphorylation of myosin light chain (pMLC) and p38 (pp38). Results: The proliferation and migration of human colon cancer RKO cells were inhibited significantly after treatment with melatonin. The expression levels of MLCK and phosphorylation of MLC of RKO cells were reduced, and real-time quantitative RT-PCR showed that melatonin had significant effects on suppressing the expression of MLCK. Furthermore, the phosphorylation level of p38, which showed the same trend, was also reduced when cells were treated by melatonin. In addition, ML-7 (25umol/l) could down-regulate the phosphorylation of p38. Conclusions: Melatonin could inhibit the proliferation and migration of RKO cells, and further experiments confirmed that p38 MAPK plays an important role in regulating melatonin-induced migration inhibition through down-regulating the expression and activity of MLCK.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.04a
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• pp.111-111
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• 2019

Glycyrrhizae radix is one of the most frequently prescribed ingredients in Oriental medicine, and G. radix extract has been shown to exert anti-cancer effects. However, the cellular and molecular mechanisms of apoptosis by G. radix are poorly defined. In the present study, it was examined the biochemical mechanisms of apoptosis by water extract of G. radix (WEGR) in human bladder T24 cancer cells. It was found that WEGR could inhibit the cell growth of T24 cells in a dose-dependent manner, which was associated with the induction of apoptotic cell death, as evidenced by the formation of apoptotic bodies, DNA fragmentation and increased populations of annexin-V positive cells. The induction of apoptotic cell death by WEGR was connected with an up-regulation of pro-apoptotic Bax protein expression and down-regulation of anti-apoptotic Bcl-2 and Bcl-xL proteins, and inhibition of apoptosis family proteins (XIAP, cIAP-1 and cIAP-2). In addition, apoptosis-inducing concentrations of WEGR induced the activation of caspase-9, an initiator caspase of the mitochondrial-mediated intrinsic pathway, and caspase-3, accompanied by proteolytic degradation of poly (ADP-ribose)-polymerase. WEGR also induced apoptosis via a death receptor-mediated extrinsic pathway by caspase-8 activation, resulting in the down-regulation of total Bid and suggesting the existence of cross-talk between the extrinsic and intrinsic pathways. Taken together, the present results suggest that WEGR may be a potential chemotherapeutic agent for the control of human bladder cancer cells.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2019.04a
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• pp.113-113
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• 2019

Diallyl trisulfide (DATS) is an organic polysulfide compound found in garlic. Although certain studies have demonstrated that DATS possesses strong anti-inflammatory activity, the underlying molecular mechanisms remain largely unresolved. In this study, we examined whether DATS exerts anti-inflammatory activity and investigated the possible mechanisms. Our results indicated that DATS significantly suppressed the lipopolysaccharide (LPS)-induced release of nitric oxide (NO) and prostaglandin E2 by inhibiting inducible NO synthase and cyclooxygenase-2 expression at the transcriptional and post-transcriptional levels in RAW 264.7 macrophages. DATS also down-regulated Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 expression, and inhibited nuclear translocation of nuclear transcription factor-kappa B (NF-${\kappa}B$) in LPS-stimulated 264.7 macrophages. Furthermore, we found that these inhibitory effects of DATS were associated with the inhibition of chemokine receptor (CXCR4) and ligand (CXCL12) expression, and reactive oxygen species generation. Overall, the present data indicated that DATS had anti-inflammatory effects on LPS-activated macrophages, possibly via inhibiting the TLR4/NF-kB and/or chemokine signaling pathways, and DATS could be a potential drug therapy for inflammation and its associated diseases.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.14
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• pp.5715-5719
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• 2014

Autophagy is crucial in the maintenance of homeostasis and regenerated energy of mammalian cells. Macroautophagy and chaperone-mediated autophagy(CMA) are the two best-identified pathways. Recent research has found that in normal cells, decline of macroautophagy is appropriately parallel with activation of CMA. However, whether it is also true in cancer cells has been poorly studied. Here we focused on cross-talk and conversion between macroautophagy and CMA in cultured Burkitt lymphoma Raji cells when facing serum deprivation and exposure to a toxic compound, arsenic trioxide. The results showed that both macroautophagy and CMA were activated sequentially instead of simultaneously in starvation-induced Raji cells, and macroautophagy was quickly activated and peaked during the first hours of nutrition deprivation, and then gradually decreased to near baseline. With nutrient deprivation persisted, CMA progressively increased along with the decline of macroautophagy. On the other hand, in arsenic trioxide-treated Raji cells, macroautophagy activity was also significantly increased, but CMA activity was not rapidly enhanced until macroautophagy was inhibited by 3-methyladenine, an inhibitor. Together, we conclude that cancer cells exhibit differential responses to diverse stressor-induced damage by autophagy. The sequential switch of the first-aider macroautophagy to the homeostasis-stabilizer CMA, whether active or passive, might be conducive to the adaption of cancer cells to miscellaneous intracellular or extracellular stressors. These findings must be helpful to understand the characteristics, compensatory mechanisms and answer modes of different autophagic pathways in cancer cells, which might be very important and promising to the development of potential targeting interventions for cancer therapies via regulation of autophagic pathways.

• Journal of Life Science
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• v.25 no.1
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• pp.93-100
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• 2015

Pachymic acid (PA) is a lanostane-type triterpenoid derived from the Poria cocos mushroom. Several beneficial biological features of PA provide medicine with a wide variety of valuable effects, such as anticancer and anti-inflammatory activity; it also has antioxidant effects against oxidative stress. Nonetheless, the biological properties and mechanisms that produce this anti-cancer action of PA remain largely undetermined. In this study, we investigated the pro-apoptotic effects of PA in T24 human bladder cancer cells. It was found that PA could inhibit the cell growth of T24 cells in a dose-dependent manner, which was associated with the induction of apoptotic cell death, as evidenced by the formation of apoptotic bodies and chromatin condensation and accumulation of cells in the sub-G1 phase. The induction of apoptotic cell death by PA was connected with an up-regulation of pro-apoptotic Bax and Bad protein expression and down-regulation of anti-apoptotic Bcl-2 and Bcl-xL proteins, and inhibition of apoptosis family proteins. In addition, apoptosis-inducing concentrations of PA induced the activation of caspase-9, an initiator caspase of the mitochondrial-mediated intrinsic pathway, and caspase-3, accompanied by proteolytic degradation of poly (ADP-ribose)-polymerase. PA also induced apoptosis via a death receptor-mediated extrinsic pathway by caspase-8 activation, resulting in the truncation of Bid and suggesting the existence of cross-talk between the extrinsic and intrinsic pathways. Taken together, the present results suggest that PA may be a potential chemotherapeutic agent for the control of human bladder cancer cells.

• Korean Journal of Plant Resources
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• v.32 no.4
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• pp.255-263
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• 2019

Glycyrrhizae radix is one of the most frequently prescribed ingredients in Oriental medicine, and Glycyrrhizae radix extract has been shown to exert anti-cancer effects. However, the cellular and molecular mechanisms of programed cell death (apoptosis) by Glycyrrhizae radix are poorly defined. In the present study, it was examined the molecular mechanisms of apoptosis by water extracts of Glycyrrhizae radix (GRW) in human bladder T24 cancer cells. It was found that GRW could inhibit the cell growth of T24 cells in a concentration-dependent manner, which was associated with the induction of apoptotic cell death, as evidenced by the formation of apoptotic bodies, DNA fragmentation and increased populations of annexin-V positive cells. The induction of apoptotic cell death by GRW was connected with an up-regulation of pro-apoptotic Bax protein expression and down-regulation of anti-apoptotic proteins (Bcl-2 and Bcl-xL), and inhibition of apoptosis family proteins (XIAP, cIAP-1 and cIAP-2). In addition, apoptosis-inducing concentrations of GRW induced the activation of caspase-9, an initiator caspase of the mitochondrial-mediated intrinsic pathway, and caspase-3, accompanied by proteolytic degradation of PARP. GRW also induced apoptosis via a death receptor-mediated extrinsic pathway by caspase-8 activation, resulting in the down-regulation of total Bid and suggesting the existence of cross-talk between the extrinsic and intrinsic pathways. Taken together, the present results suggest that GRW may be a potential chemotherapeutic agent for the control of human bladder cancer cells.

• Tuberculosis and Respiratory Diseases
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• v.47 no.3
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• pp.347-355
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• 1999

Background: Phospholipase C(PLC) plays a central role in cellular signal transduction and is important in cellular growth, differentiation and transformation. There are currently ten known mammalian isozymes of PLC reported to this date. Hydrolysis of phosphatidylinositol 4,5-bisphosphate($PIP_2$) by PLC produces two important second messengers, inositol 1,4,5-trisphosphate($IP_3$) and diacylglycerol. PLC-${\gamma}1$, previously, was known to be activated mainly through growth factor receptor tyrosine kinase. Other mechanisms of activating PLC-yl have been reported such as activation through tau protein in the presence of arachidonic acid in bovine brain and activation by $IP_3$, phosphatidic acid, etc. Very recently, another PLC-${\gamma}1$ activator protein such as tau has been found in bovine lung tissue, which now is considered to be AHNAK protein. But there has been no report concerning AHNAK and its associated disease to this date. In this study, we examined the expression of the PLC-${\gamma}1$ activator, AHNAK, in lung cancer specimens and their paired normal. Methods: From surgically resected human lung cancer tissues taken from twenty-eight patients and their paired normal counterparts, we evaluated expression level of AHNAK protein using immunoblot analysis of total tissue extract Immunohistochemical stain was performed with primary antibody against AHNAK protein. Results: Twenty-two among twenty-eight lung cancer tissues showed overexpression of AHNAK protein (eight of fourteen squamous cell lung cancers, all of fourteen adenocarcinomas). The resulting bands were multiple ranging from 70 to 200 kDa in molecular weight and each band was indistinct and formed a smear, reflecting mobility shift mainly due to proteolysis during extraction process. On immunohistochemistry, lung cancer tissues showed a very heavy, dense staining with anti-AHNAK protein antibody as compared to the surrounding normal lung tissue, coresponding well with the results of the western blot Conclusion: The overexpression of PLC-${\gamma}1$ activator protein, AHNAK in lung cancer may provide evidence that the AHNAK protein and PLC-${\gamma}1$ act in concerted manner in carcinogenesis.