• Archives of Pharmacal Research
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• v.24 no.2
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• pp.126-135
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• 2001

Quinacrine (QU), a phospholipase-A2 (PLA-2) inhibitor has been used clinically as a chemotherapeutic adjuvant. To understand the mechanisms leading to its chemotherapeutic effect, we have investigated QU-induced apoptotic signaling pathways in human cervical squamous carcinoma HeLa cells. In this study, we found that QU induced cytochrome c-dependent apoptotic signaling. The release of pro-apoptotic cytochrome c was QU concentration- and time-dependent, and preceded activation of caspase-9 and -3. Flow cytometric FACScan analysis using fluorescence intensities of $DiOC_6$/ demonstrated that QU-induced cytochrome c release was independent of mitochondrial permeability transition (MPT), since the concentrations of QU that induced cytochrome c release did not alter mitochondrial membrane potential (${\blacktriangle}{\Psi}_m$). Moreover, kinetic analysis of caspase activities showed that cytochrome c release led to the activation of caspase-9 and downstream death effector caspase-3, Caspase-3 inhibitor (Ac-DEVD-CHO) partially blocked QU-induced apoptosis, suggesting the importance of caspase-3 in this apoptotic signaling mechanism. Supplementation with arachidonic acid (AA) sustained caspase-3 activation induced by QU. Using inhibitors against cellular arachidonate metabolism of lipooxygenase (Nordihydroxyguaiaretic Acid, NDGA) and cyclooxygenase (5,8,11,14-Eicosatetraynoic Acid, ETYA) demonstrated that QU-induced apoptotic signaling may be dependent on its role as a PLA-2 inhibitor. Interestingly, NDCA attenuated QU-induced cytochrome c release, caspase activity as well as apoptotic cell death. The blockade of cytochrome c release by NDCA was much more effective than that attained with cyclosporin A (CsA), a MPT inhibitor. ETYA was not effective in blocking cytochrome c release, except under very high concentrations. Caspase inhibitor z-VAD blocked the release of cytochrome c suggesting that this signaling event is caspase dependent, and caspase-8 activation may be upstream of the mitochondrial events. In summary, we report that QU induced cytochrome c-dependent apoptotic signaling cascade, which may be dependent on its role as a PLA-2 inhibitor. This apoptotic mechanism induced by QU may contribute to its known chemotherapeutic effects.

• BMB Reports
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• v.49 no.5
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• pp.247-248
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• 2016

Necroptosis is a well-known form of caspase-independent cell death. Necroptosis can be triggered by various extrinsic stimuli, including death ligands in the presence of receptorinteracting protein kinase 3 (RIPK3), a key mediator of necroptosis induction. Our recent studies have revealed that C-terminus HSC-70 interacting protein (CHIP), an E3 ligase, can function as an inhibitor of necroptosis. CHIP^−/− mouse embryonic fibroblast showed higher sensitivity to necrotic stimuli than wild-type mouse embryonic fibroblast cells. Deleterious effects of CHIP knockout MEFs were retrieved by RIPK3 depletion. We found that CHIP negatively regulated RIPK3 and RIPK1 by ubiquitylation- and lysosome- dependent degradation. In addition, CHIP^−/− mice showed postnatal lethality with intestinal defects that could be rescued by crossing with RIPK3^−/− mice. These results suggest that CHIP is a negative regulator of RIPK1 and RIPK3, thus inhibiting necroptosis.

• Tuberculosis and Respiratory Diseases
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• v.58 no.1
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• pp.43-53
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• 2005

Emphysema is characterized by air space enlargement and alveolar destruction. The mechanism responsible for the development of emphysema was thought to be protease/antiprotease imbalance and oxidative stress. A very recent study shows that alveolar cell apoptosis causes lung destruction and emphysematous changes. Thus, this study was performed to support the evidence for the role of apoptosis in the development of emphysema by characterizing cigarette smoke extract (CSE)-induced apoptosis in A549 (type II pneumocyte) lung epithelial cells. CSE induced apoptosis at low concentration (10% or less) and both apoptosis and necrosis at high concentration (20%). Apoptosis was demonstrated by DNA fragmentation using FACScan for subG1 fraction. Discrimination between apoptosis and necrosis was done by morphologic analysis using fluorescent microscopy with Hoecst 33342/propium iodide double staing and electron microscopy. Cytochrome c release was confirmed by using immunofluorescence with monoclonal anti-cytochrome c antibody. However, CSE-induced cell death did not show the activation of caspase 3 and was not blocked by caspase inhibitors. This suggests that CSE-induced apoptosis might be caspase-independent apoptosis. CSE-induced cell death was near completely blocked by N-acetylcystein and bcl-2 overexpression protected CSE-induced cell death. This results suggests that CSE might induce apoptosis through intracellular oxidative stress. CSE also activated p53 and functional knock-out of p53 using stable overexpression of HPV-E6 protein inhibited CSE-induced cell death. The characterization of CSE-induced cell death in lung epithelial cells could support the role of lung cell apoptosis in the pathogenesis of emphysema.

• IMMUNE NETWORK
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• v.12 no.6
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• pp.284-290
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• 2012

Innate immune cells sense and respond to the cytoplasmic infection of bacterial pathogens through NLRP3, NLRC4 or AIM2 inflammasome depending on the unique molecular pattern of invading pathogens. The infection of flagellin- or type III secretion system (T3SS)-containing Gram-negative bacteria such as Salmonella enterica serovar Typhimurium (S. typhimurium) or Pseudomonas aeruginosa (P. aeruginosa) triggers NLRC4-dependent caspase-1 activation leading to the secretion of proinflammatory cytokines such as interleukin-1-beta (IL-$1{\beta}$) and IL-18. Previous studies have shown that apoptosis-associated speck-like protein containing a CARD (ASC) is also required for Salmonella-induced caspase-1 activation, but it is still unclear how ASC contributes to the activation of NLRC4 inflammasome in response to S. typhimurium infection. In this study, we demonstrate that S. typhimurium triggers the formation of ASC oligomer in a potassium depletion-independent manner as determined by in vitro crosslinking and in situ fluorescence imaging. Remarkably, inhibition of potassium efflux failed to block Salmonella-promoted caspase-1 activation and macrophage cell death. These results collectively suggest that ASC is substantially oligomerized to facilitate the activation of caspase-1 in response to S. typhimurium infection. Contrary to NLRP3 inflammasome, intracellular potassium depletion is not critical for NLRC4 inflammasome signaling by S. typhimurium.

• Journal of Life Science
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• v.18 no.8
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• pp.1042-1048
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• 2008

Prostate cancer is the most predominant cancer in men and related death rate increases every year. Till date, there is no effective therapy for androgen independent prostate cancer. To investigate the mechanism for cell growth inhibition or apoptosis in human androgen independent prostate cancer PC-3 cells after gamma irradiation. The aim of this study was to examine the potential of gamma irradiation to induce apoptosis in PC-3 cells and to assess the mechanism of gamma irradiation-induced apoptosis. Five different assays were employed in this study: cell proliferation assay, morphological assessments of apoptotic cells, DNA fragmentation analysis, quantification of apoptosis by annexin V (AV) and propidium iodide (PI) staning, and western blot analysis. Cell viability was inversely related to radiation dose. DAPI-positive cells were detected 48 hr after 40 Gy radiation exposure. And nuclear morphological changes of cells were observed by gamma irradiation. DNA ladder patterns in the cells exposed to gamma-radiation were appeared at 24 hr. Also, gamma irradiation induces apoptosis of PC-3 cells via Caspase3, Bax and PARP-dependent fashion.

• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.6
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• pp.1661-1665
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• 2004

Herba Patriniae(HP) has been known to exert anti-tumoral activity in Korea. However, its molecular mechanism, of action is not understood. In this study, we found that HP induced apoptosis in androgen-dependent prostate cancer DU145 cells as evidenced by DNA fragmentation and chromatine condensation in hoechst dye staining. Our data demonstrated that HP-induced apoptotic cell death was accompanied by activation of caspase-3 and cleavages of its substrates, poly(ADP-ribose) polymerase(PARP) in a time- and concentration-dependent manner. Taken together, these results suggest that HP induces the activation of caspase-3, degradation of PARP, and eventually leads to apoptotic cell death.

• Biomolecules & Therapeutics
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• v.28 no.2
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• pp.195-201
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• 2020

Prostate cancer is one of the most common cancers in men. Choline PET or PET/CT has been used to visualize prostate cancer, and high levels of choline accumulation have been observed in tumors. However, the uptake system for choline and the functional expression of choline transporters in prostate cancer are not completely understood. In this study, the molecular and functional aspects of choline uptake were investigated in the LNCaP prostate cancer cell line along with the correlations between choline uptake and cell viability in drug-treated cells. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA were highly expressed in LNCaP cells. CTL1 and CTL2 were located in the plasma membrane and mitochondria, respectively. [³H]Choline uptake was mediated by a single Na⁺-independent, intermediate-affinity transport system in the LNCaP cells. The anticancer drugs, flutamide and bicalutamide, inhibited cell viability and [³H]choline uptake in a concentration-dependent manner. The correlations between the effects of these drugs on cell viability and [³H]choline uptake were significant. Caspase-3/7 activity was significantly increased by both flutamide and bicalutamide. Furthermore, these drugs decreased CTL1 expression in the prostate cancer cell line. These results suggest that CTL1 is functionally expressed in prostate cancer cells and are also involved in abnormal proliferation. Identification of this CTL1-mediated choline transport system in prostate cancer cells provides a potential new therapeutic target for the treatment of this disease.

• Animal cells and systems
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• v.5 no.4
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• pp.267-274
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• 2001

Calpains are a family of cysteine proteases existing primarily in two forms designated by the $Ca^{2+}$ concentration needed for activation in vitro, $\mu$-calpain (calpain-I) and m-calpain (calpain-II). The physiologica1 roles of calpains remain unclear. Many groups have proposed a role for calpains In apoptosis, but their patterns of activation are not well characterized. Calpains have been implicated in neutrophil apoptosis, glucocorticoid-induced thymocyte apoptosis, as well as many other apoptotic pathways. Calpain activation in apoptosis is usually linked upstream or downstream to caspase activation, or in a parallel pathway alongside caspase activation. Calpains have been suggested to be involved in DNA fragmentation (via endonuclease activation), but also as effector proteases that cleave cellular proteins involved in DNA repair, membrane associated proteins and other homeostatic regulatory proteins. Recently, our laboratory demonstrated $\mu$-calpain activation in NAD(P)H: quinone oxidoreducatse 1 (NQO1)-expressing cells after exposure to $\beta$-lapachone, a novel quinone and potential chemo- and radio-therapeutic agent. Increased cytosolic $Ca^{2+}$ in NQO1-expressing cells after $\beta$-lapachone exposures were shown to lead to $\mu$-calpain activation. In turn, $\mu$-calpain activation was important for substrate proteolysis and DNA fragmentation associated with apoptosis. Upon activation, $\mu$-calpain translocated to the nucleus where it could proteolytically cleave PARP and p53. We provided evidence that $\beta$-lapachone-induced, $\mu$-calpain stimulated, apoptosis did not involve any of the known caspases; known apoptotic caspases were not activated after $\beta$-lapachone treatment of NQO1-expressing cells, nor did caspase inhibitors have any effect on $\beta$-1apachone-induced cell death. Elucidation of processes by which $\beta$-1apachone-stimulated $\mu$-calpain activation and calpains ability to activate endonucleases and induce apoptosis independent of caspase activity will be needed to further develop/modulate $\beta$-lapachone for treatment of human cancers that over-express NQO1.

• Journal of Gastric Cancer
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• v.8 no.3
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• pp.120-128
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• 2008

Purpose: Mistletoe extract was widely used for cancer treatment as complementary or alternative therapy in European area from early twenty century. It is currently used as alternative anti-cancer remedy by piecemeal in domestic medical group, however, the anti-cancer mechanism of mistletoe extract was not known precisely until now. In this study the effect of mistletoe extract on gastric cancer was studied vis cell line experiments. Materials and Methods: The SNU719 gastric cancer cell line was used, and ABNOBAviscum-Q and ABNOBAviscum-F were treated to cells as mistletoe extract, or 5-FU and cisplatin were used with mistletoe extract. The cell viability and cell death rate were estimated by CCK-8 assay kit and lactate dehydrogenase (LDH) assay kit in each. Caspase 3 assay kit was used to measure caspase 3 activity. The protein expression amounts of Bcl2, p53, and PTEN were estimated through Western blot analysis. Results: The co-treatments of mistletoe extract Q/F and 5-FU/cisplatin decreased lesser cell viability than only mistletoe treat. Caspase 3 activity was increased 4~6 times in co-treatment of mistletoe extracts and 5-FU than control. Bcl2 protein expression was reduced by mistletoe extracts or anti-cancer drugs, further more, the co-treatment of mistletoe extracts and 5-FU/cisplatin diminished more the expression than only mistletoe treatment. Mistletoe extracts did not affect the protein expressions of p53 and PTEN. Conclusion: It was concluded that the anti-cancer mechanism of mistletoe extracts was made by caspase 3 activation and lowered Bcl2 expression, and this apoptosis inducing mechanism was independent to p53.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.17
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• pp.7943-7957
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• 2015

Background and Aims: Colorectal cancer is one of the leading causes of death in the world. The aim of this study was to investigate the growth-suppression potentiality of a crude saponin extract (CSENS) prepared from medicinal herb, Nigella sativa, on human colon cancer cells, HCT116. Materials and Methods: HCT116 cells were subjected to increasing doses of CSENS for 24, 48 and 72 h, and then harvested and assayed for cell viability by WST-1. Flow cytometry analyses, cell death detection ELISA, fluorescent stains (Hoechst 33342 and acridine orange/ethidium bromide), DNA laddering and comet assays were carried out to confirm the apoptogenic effects of CSENS. Luciferase reporter gene assays, quantitative reverse transcription-polymerase chain reaction and Western blot analyses were performed to assess the impact of CAERS and CFEZO on the expression levels of key regulatory proteins in HCT116 cells. Results: The results demonstrated that CSENS inhibited proliferation and induced apoptosis. Apoptosis was confirmed by flow cytometry analyses, while CSENS-treated cells exhibited morphological hallmarks of apoptosis including cell shrinkage, irregularity in cellular shape, cellular detachment and chromatin condensation. Biochemical signs of apoptosis, such as DNA degradation, were observed by comet assay and gel electrophoresis. The pro-apoptotic effect of CSENS was caspase-3-independent and associated with increase of the Bax/Bcl-2 ratio. CSENS treatment down-regulated transcriptional and DNA-binding activities of NF-${\kappa}B$ and AP-1 proteins, associated with down-regulation of their target oncogenes, c-Myc, cyclin D1 and survivin. On the other hand, CSENS up-regulated transcriptional and DNA-binding activities of Nrf2 and expression of cytoprotective genes. In addition, CSENS modulated the expression levels of ERK1/2 MAPK, p53 and p21. Conclusions: These findings suggest that CSENS may be a valuable agent for treatment of colon cancer.