• IMMUNE NETWORK
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• v.3 no.4
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• pp.268-275
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• 2003

Background: Hemopoietic cells require the constant presence of growth factors for survival in vitro and in vivo. Caspases have been known as central executors of apoptotic cell death. We have, therefore, investigated the pathways that regulate caspase activity and apoptosis using the $CD34^+$ cell line, TF-1 which requires GM-CSF for survival. Methods: Apoptosis was measured by annexin V staining and mitochondrial membrane potential was measured by DiOC6 labelling. Intracellular pH was measured using pH sensitive fluorochrome, BCECF or SNARF-1, followed by flow cytometry analysis. Caspase activation was analyzed by PARP cleavage using anti-PARP antibody. Results: Removal of GM-CSF induceed PARP cleavage, a hallmark of caspase activity, concomitant with pHi acidification and a drop in mitochondrial potential. Treatment with ZVAD, a competitive inhibitor of caspases, partially rescued cell death without affecting pHi acidification and the reduction of mitochondrial potential, suggesting that both these events act upstream of caspases. Overexpression of Bcl-2 prevented cell death induced by GM-CSF deprivation as well as pHi acidification and the reduction in mitochondrial membrane potential. In parental cells maintained with GM-CSF, EIPA, a competitive inhibitor of $Na^+/H^+$ antiporter induced apoptosis, accompanied by a drastic reduction in mitochondrial potential. In contrast, EIPA induced apoptosis in Bcl-2 transfectants without causing mitochondrial membrane depolarization. Conclusion: Taken together, our results suggest that the regulation of $H^+$fluxes, either through a mitochondriondependent or independent pathway, is central to caspase activation and apoptosis.

• Journal of Life Science
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• v.26 no.4
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• pp.398-405
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• 2016

Statins, the inhibitors of 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase, are widely used in treatments of hypercholesterolemia and newly known as anti-cancer effect of various cancer cells. Recently, several studies suggested that reactive oxygen species (ROS) play a critical role on cell death signaling. However, mechanism of ROS by rosuvastatin is currently unclear. This study aimed to explore the molecular mechanism of apoptosis by rosuvastatin in human prostate cancer PC-3 cells. Cell viability and apoptosis-related protein expression were measured by MTT assay and western blotting, respectively. In addition, the levels of apoptosis and ROS were analyzed. The results showed that rosuvastatin dramatically reduced cell viability in a dose- and time-dependent manner. We confirmed that rosuvastatin induced apoptosis through reduction of procaspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP) in PC-3 cells. In addition, rosuvastatin stimulated ROS production in a dose-dependent manner and pre-treatment with N-acetylcysteine (NAC), a ROS scavenger, significantly recovered rosuvastatin-induced ROS and apoptosis. Thus, we concluded that rosuvastain induces apoptosis through generation of ROS in human prostate cancer PC-3 cells and provides a promising approach to improve the efficacy of cancer therapy.

• Journal of Life Science
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• v.25 no.12
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• pp.1384-1392
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• 2015

Sagantang (SGT), a Korean multiherb formula comprising six medicinal herbs, Paeonia lactiflora Pall., Belamcanda chinensis (L.) DC, Gardenia jasminoides Ellis, Poria cocos Wolf, Cimicifuga heracleifolia Komarov, and Artractylodes japonica Koidzumi, was recorded in “Dongeuibogam.” The present study investigated the anticancer potential of SGT in AGS human gastric carcinoma cells. The results indicated that SGT treatment significantly inhibited the growth and viability of AGS cells in a dose-dependent manner, which was associated with the induction of apoptotic cell death, as evidenced by the formation of apoptotic bodies, in addition to chromatin condensation and DNA fragmentation, and the accumulation of annexin-V positive cells. The induction of apoptotic cell death by the SGT treatment was associated with up-regulation of Fas protein expression, truncation of Bid, and down-regulation of the anti-apoptotic Bcl-2 protein. The SGT treatment also effectively induced the loss of mitochondrial membrane potential, which was associated with the activation of caspases (caspase-3, -8, and -9) and degradation of poly (ADP-ribose) polymerase. However, a pan-caspase inhibitor significantly blocked the SGT-induced apoptosis and growth suppression in AGS cells. This study suggests that SGT induces caspase-dependent apoptosis through an extrinsic pathway by upregulating Fas, as well as through an intrinsic pathway by modulating Bcl-2 family members in AGS cells. The results suggest that SGT may be a potential chemotherapeutic agent for the control of human gastric cancer cells. However, further studies will be needed to confirm the potential of SGT in cancer prevention and therapy in an in vivo model and to identify biological active compounds of SGT.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.9
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• pp.3901-3906
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• 2014

Aim: The purpose of this study was to investigate anti-tumor effects and safety of DH332, a new ${\beta}$-carboline alkaloids derivatives in vitro and in vivo. Materials and Methods: The effects of DH332 on human (RAMOS RA.1) and mouse (J558) B lymphoma cell lines were detected using a CCK-8 kit (Cell Counting Kit-8), and apoptosis was detected by flow cytometry with PI/annexinV staining. Western blotting was used to detected caspase-3 and caspase-8. Neurotoxic and anti-tumor effects were evaluated in animal experiments. Results: DH332 exerts a lower neurotoxicity compared with harmine. It also possesses strong antitumor effects against two B cell lymphoma cell lines with low $IC_{50s}$. Moreover, DH332 could inhibit the proliferation and induce the apoptosis of RAMOS RA.1 and J558 cell lines in a dose-dependent manner. Our results suggest that DH332 triggers apoptosis by mainly activating the caspase signaling pathway. In vivo studies of tumor-bearing BALB/c mice showed that DH332 significantly inhibited growth of J558 xenograft tumors. Conclusions: DH332 exerts effective antitumor activity in vitro and in vivo, and has the potential to be a promising drug candidate for lymphoma therapy.

• The Journal of Internal Korean Medicine
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• v.36 no.1
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• pp.1-12
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• 2015

Objectives : This study was performed to investigate the anti-cancer effects of Rhus verniciflua Stokes (RVS) extracted with sterile distilled water on cholangiocarcinoma cell lines. Materials and Methods : Two cholangiocarcinoma cell lines, SNU-1079 and SNU-1196, were used in this study. Cells were treated with different concentrations of RVS for 24, 48, and 72 hours. Cell count, viability, apoptosis, and mRNA expression of Bax, Bcl-2, Mcl-1, survivin, caspase-3, and cyclin D1 and P21 were determined with an automatic cell counter (ADAM-MC), MTT assay, apoptosis assay (Annexin-V/PI staining), and RT-PCR. Results : All cells treated with RVS showed decreased cell counts in a dose-dependent manner. RVS inhibited proliferation of SNU-1196 in a dose-dependent manner, but SNU-1079 proliferation was inhibited in the long-time culture group in a dose-dependent manner. The proportion of early and late-stage apoptotic cells was increased by RVS in a dose-dependent manner in SNU-1196. In contrast, it was increased significantly in SNU-1079 treated with high-dose RVS. After treatment with RVS, the mRNA expression of Bcl-2 was decreased while Bax was increased in SNU-1079. Cyclin D1 mRNA levels were decreased in SNU-1196 in a dose-dependent manner. P21 expression was increased in all cells after the treatment with RVS. Conclusions : RVS appears to have potential as a therapeutic agent for cholangiocarcinoma.

• Journal of Life Science
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• v.31 no.2
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• pp.199-208
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• 2021

Euonymus porphyreus, a species of plant in the Celastraceae family, is widely distributed in East Asia, especially in Southern China. The botanical characteristics of E. porphyreus have been reported, but its antioxidative and anticancer activities remain unclear. In this study, we evaluated the antioxidative and anticancer effects of ethanol extracts of E. porphyreus (EEEP) and the molecular mechanism of its anticancer activity in human lung adenocarcinoma A549 cells. The total polyphenol and flavonoid compound contents from EEEP were 115.42 mg/g and 23.07 mg/g, respectively. EEEP showed significant antioxidative effects with a concentration at 50% of the inhibition (IC50) value of 11.09 ㎍/ml, as measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. EEEP showed cytotoxic activity by increasing the SubG1 cell population of A549 cells in a dose-dependent manner. Apoptosis in A549 cells treated with EEEP was evident due to increased apoptotic cells and apoptotic bodies, as detected by Annexin V and 4,6-diamidino-2-phenylindole (DAPI) staining, respectively. EEEP-induced apoptosis resulted in increased expression of the First apoptosis signal (Fas), p53, and Bax, with decreased expression of Bcl-2 and subsequent activation of caspase-8, -9, and caspase-3, leading to cleavage of poly (ADP-ribose) polymerase (PARP). Collectively, these results suggest that EEEP may exert an anticancer effect by inducing apoptosis in A549 cells through both intrinsic and extrinsic pathways.

• BMB Reports
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• v.34 no.3
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• pp.250-258
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• 2001

For a long time Rhus verniciflua Stokes (RVS) has traditionally been used as a herbal plant. It is known to contain various biological activities. Previously, a crude ethanol extract from RVS was reported to have antioxidant effects, and antiproliferative activities, on human cancer cell lines. In this report, we prepared a highly purified ethanol extract from RVS, which did not contain the urushiol derivatives, named REEE-1 ($\underline{R}$hus $\underline{e}$thanol $\underline{e}$luted $\underline{e}$xtract-1), to investigate the mechanisms of the scavenging activity of hydroxyl radicals using mouse thymocytes. The results from the deoxyribose, DNA nicking, and glucose/glucose oxidase enzyme assays showed that REEE-1 contained a strong scavenging activity of oxygen free radicals, especially of hydroxyl radicals. However, interestingly, REEE-1 also showed cytotoxicity against the thymocytes, although the effect was variable, depending on the concentrations and times of treatment. The REEE-1-mediated cytotoxicity against thymocytes, which has been used as one of the well-characterized models for apoptosis studies, was verified to be apoptotic. This was proven by the following: the appearance of DNA laddering, increases in DNA fragmentation, low fluorescence intensity in the nuclei after propidium iodide staining, and positive Annexin V staining of the cells. These results suggested that REEE-1 had both antioxidative activity and cytotoxicity against the thymocytes, although the effect of the cytotoxicity was variable, depending on the dose and time of the treatment.

• BMB Reports
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• v.48 no.9
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• pp.531-536
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• 2015

Gecko proteins have long been used as anti-tumor agents in oriental medicine, without any scientific background. Although anti-tumor effects of Gecko proteins on several cancers were recently reported, their effect on bladder cancer has not been investigated. Thus, we explored the anti-tumor effect of Gecko proteins and its cellular mechanisms in human bladder cancer 5637 cells. Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells. These proteins increased the Annexin-V staining and the amount of condensed chromatin, demonstrating that the Gecko proteinsinduced cell death was caused by apoptosis. Gecko proteins suppressed Akt activation, and the overexpression of constitutively active form of myristoylated Akt prevented Gecko proteins-induced death of 5637 cells. Furthermore, Gecko proteins activated caspase 9 and caspase 3/7. Taken together, our data demonstrated that Gecko proteins suppressed the Akt pathway and activated the intrinsic caspase pathway, leading to the apoptosis of bladder cancer cells. [BMB Reports 2015; 48(9): 531-536]

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.4
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• pp.1395-1399
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• 2012

Background: Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation of tumor cells. The present study aimed to examine the effects of trichostatin A (TSA), one such inhibitor, on the cell cycle, apoptosis and invasiveness of osteosarcoma cells. Methods: MG-63 cells were treated with TSA at various concentrations. Then, cell growth and apoptosis were determined by 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively; cell cycling was assessed by flow cytometry; invasion assays were performed with the transwell Boyden Chamber system. Results: MTT assays revealed that TSA significantly inhibited the growth of MG-63 cells in a concentration and time dependent manner. TSA treated cells demonstrated morphological changes indicative of apoptosis and TUNEL assays revealed increased apoptosis of MG-63 cells after TSA treatment. Flow cytometry showed that TSA arrested the cell cycle in G1/G2 phase and annexin V positive apoptotic cells increased markedly. In addition, the invasiveness of MG-63 cells was inhibited by TSA in a concentration dependent manner. Conclusion: Our findings demonstrate that TSA inhibits the proliferation, induces apoptosis and inhibits invasiveness of osteosarcoma cells in vitro. HDAC inhibitors may thus have promise to become new therapeutic agents against osteosarcoma.

• Molecules and Cells
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• v.38 no.5
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• pp.416-425
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• 2015

NF-E2-related factor 2 (Nrf2), a basic leucine zipper transcription factor, has recently received a great deal of attention as an important molecule that enhances antioxidative defenses and induces resistance to chemotherapy or radiotherapy. In this study, we investigated the apoptosis-inducing and Nrf2- upregulating effects of quercetin on malignant mesothelioma (MM) MSTO-211H and H2452 cells. Quercetin treatment inhibited cell growth and led to upregulation of Nrf2 at both the mRNA and protein levels without altering the ubiquitination and extending the half-life of the Nrf2 protein. Following treatment with quercetin, analyses of the nuclear level of Nrf2, Nrf2 antioxidant response element-binding assay, Nrf2 promoter-luc assay, and RT-PCR toward the Nrf2-regulated gene, heme oxygenase-1, demonstrated that the induced Nrf2 is transcriptionally active. Knockdown of Nrf2 expression with siRNA enhanced cytotoxicity due to the induction of apoptosis, as evidenced by an increase in the level of proapoptotic Bax, a decrease in the level of antiapoptotic Bcl-2 with enhanced cleavage of caspase-3 and PARP proteins, the appearance of a sub-$G_0/G_1$ peak in the flow cytometric assay, and increased percentage of apoptotic propensities in the annexin V binding assay. Effective reversal of apoptosis was observed following pretreatment with the pan-caspase inhibitor Z-VAD. Moreover, Nrf2 knockdown exhibited increased sensitivity to the anticancer drug, cisplatin, presumably by potentiating the oxidative stress induced by cisplatin. Collectively, our data demonstrate the importance of Nrf2 in cytoprotection, survival, and drug resistance with implications for the potential significance of targeting Nrf2 as a promising strategy for overcoming resistance to chemotherapeutics in MM.