• International Journal of Oral Biology
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• 제40권2호
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• pp.85-91
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• 2015

Quercetin is a natural flavonoid phytochemical that is extracted from various plants. Having an advantages due to its varied biological properties, such as anti-inflammatory, anti-viral, anti-oxidant, and anti-cancer effects, quercetin is used to treat many diseases. Recently, it has been reported that autophagy inhibition may play a key role in anti-cancer therapy. Therefore, in this study, we investigated the molecular mechanisms and anti-cancer effects of quercetin in human osteosarcoma cells via autophagy inhibition. We ascertained that quercetin inhibited cell proliferation and induced cell death, these process is demonstrated that apoptosis via the mitochondrial pathway and the caspase cascade. Quercetin also induced autophagy which was inhibited by 3-MA, autophagy inhibitor and the blockade of autophagy promoted the quercetin-induced apoptosis, confirming that autophagy is a pro-survival process. Thus, these findings demonstrate that quercetin is an effective anti-cancer agent, and the combination of quercetin and an autophagy inhibitor should enhance the effect of anti-cancer therapy.

• The Korean Journal of Physiology and Pharmacology
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• 제20권6호
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• pp.573-580
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• 2016

Caspases, a family of cysteine proteases, cleave substrates and play significant roles in apoptosis, autophagy, and development. Recently, our group identified 72 genes that interact with Death Caspase-1 (DCP-1) proteins in Drosophila by genetic screening of 15,000 EP lines. However, the cellular functions and molecular mechanisms of the screened genes, such as their involvement in apoptosis and autophagy, are poorly understood in mammalian cells. In order to study the functional characterizations of the genes in human cells, we investigated 16 full-length human genes in mammalian expression vectors and tested their effects on apoptosis and autophagy in human cell lines. Our studies revealed that ALFY, BIRC4, and TAK1 induced autophagy, while SEC61A2, N-PAC, BIRC4, WIPI1, and FALZ increased apoptotic cell death. BIRC4 was involved in both autophagy and apoptosis. Western blot analysis and luciferase reporter activity indicated that ALFY, BIRC4, PDGFA, and TAK1 act in a p53-dependent manner, whereas CPSF1, SEC61A2, N-PAC, and WIPI1 appear to be p53-independent. Overexpression of BIRC4 and TAK1 caused upregulation of p53 and accumulation of its target proteins as well as an increase in p53 mRNA levels, suggesting that these genes are involved in p53 transcription and expression of its target genes followed by p53 protein accumulation. In conclusion, apoptosis and/or autophagy mediated by BIRC4 and TAK1 may be regulated by p53 and caspase activity. These novel findings may provide valuable information that will aid in a better understanding of the roles of caspase-related genes in human cell lines and be useful for the process of drug discovery.

• 대한한의학방제학회지
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• 제28권1호
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• pp.15-27
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• 2020

Objectives : We selected three herb-combined remedies, Bigihwan (BGH), Daechilgitang (DCGT) and Mokwhyangbinranghwan (MHBRH), through Donguibogam text-mining analysis, and evaluated their anti-cancer effects on human hepatocellular carcinoma Hep3B cells. Methods : Cytotoxicity was assessed by an MTT assay. Apoptosis rate, autophagy and ROS level were detected by flow cytometry. The autophagy was also observed by Cyto-ID staining fluorescence microscopy. The expression of autophagy, mitophagy and pexophagy regulatory proteins was detected by Western blot analysis. Results : BGH showed the strongest effect among the three prescriptions in inhibiting Hep3B cell viability, which was associated with the induction of apoptosis and autophagy. Autophagy blockers improved cell viability and reduced apoptosis after BGH and DCGT treatments, indicating that autophagy by these prescriptions enhanced Hep3B cells against their cytotoxicity. However, MHBRH enhanced the reduction of cell viability and apoptosis by autophagy blockers. Induction of autophagy by BGH treatment was associated with mitophagy due to mitochondrial dysfunction than DCGT and MHBRH-treated cells. In addition, induction of apoptosis by BGH treatment was ROS-dependent and showed the possibility of pexophagy involvement. Conclusion : Although further studies need to be conducted to study the efficacy and mechanism of accurate anticancer activity, the present results will serve as important sources of understanding the mechanism of action of herbal remedies prescribed for liver disease as documented in Donguibogam.

• 대한한의학방제학회지
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• 제30권2호
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• pp.67-83
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• 2022

Objectives : In this study, the anticancer activity of water extracts of three herbal medicine formulas, Bigihwan (BGH), Daechilgitang (DCGT) and Mokwhyangbinranghwan (MHBRH) listed in Donguibogam, was evaluated in HepG2 cells, a human hepatocellular carcinoma cell line. Methods : We investigated whether the cell viability of HepG2 cells was inhibited by the treatment of water extracts of three prescriptions, and whether their viability inhibitory effect was related to the induction of apoptosis. In addition, the role of autophagy on the induction of apoptosis by the treatment of these extracts was investigated. Results : The anticancer activity of the three water extracts on HepG2 cells was due to induction of apoptosis, not necrosis. Among them, BGH activated the caspase-dependent intrinsic apoptosis pathway associated with mitochondrial dysfunction. However, autophagy was induced more than 2-fold in DCGT-treated HepG2 cells, and the anticancer activity of DCGT was enhanced 1.5-fold in the presence of an autophagy inhibitor, but was attenuated in BGH and MHBRH-treated cells. Conclusion : The results of this study indicate that DCGT-induced autophagy was involved in the inhibition of apoptosis, whereas autophagy by BGH and MHBRH was related to induction of apoptosis.

• International Journal of Oral Biology
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• 제42권1호
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• pp.17-23
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• 2017

Background: Periodontitis is generally a chronic disorder characterized by the breakdown of tooth-supporting tissues. P. gingivalis, a Gram-negative anaerobic rod, is one of the major pathogens associated with periodontitis. Frequently, P. gingivalis infection leads to cell death. However, the correlation between P. gingivalis-induced cell death and periodontal inflammation remains to be elucidated. Among cell deaths, the death of immune cells appears to play a significant role in inflammatory response. Thus, the aim of this study was to examine P. gingivalis-induced cell death, focusing on autophagy and apoptosis in THP-1 cells. Methods: Human acute monocytic leukemia cell line (THP-1) was used for all experiments. Autophagy induced by P. gingivalis in THP-1 cells was examined by Cyto ID staining. Intracellular autophagic vacuoles were observed by fluorescence microscopy using staining Acridine orange (AO); and 3-methyladenine (3-MA) was used to inhibit autophagy. Total cell death was measured by LDH assay. Cytokine production was measured by an ELISA method. Results: P. gingivalis induced autophagy in an MOI-dependent manner in THP-1 cells, but 3-MA treatment decreased autophagy and increased the apoptotic blebs. P. gingivalis infection did not increase apoptosis compared to the control cells, whereas inhibition of autophagy by 3-MA significantly increased apoptosis in P. gingivalis-infected THP-1 cells. Inhibition of autophagy by 3-MA also increased total cell deaths and inflammatory cytokine production, including $IL-1{\beta}$ and $TNF-{\alpha}$. Conclusion: P. gingivalis induced autophagy in THP-1 cells, but the inhibition of autophagy by 3-MA stimulated apoptosis, leading to increased cell deaths and pro-inflammatory cytokines production. Hence, the modulation of cell deaths may provide a mechanism to fight against invading microorganisms in host cells and could be a promising way to control inflammation.

• 대한한방내과학회지
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• 제35권3호
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• pp.317-331
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• 2014

Objectives: The root of Platycodon grandiflorum (PG) has been known to possess a range of pharmacological activities including anti-cancer, anti-inflammatory, and anti-oxidant effects. The present study was designed to investigate whether or not PG-induced cell death was connected with autophagy and apoptosis in NCI-H460 human lung cancer cells. Methods: Effects on the cell viability and apoptotic activity were quantified using MTT assays and flow cytometry analysis, respectively. Protein activation was measured by immunoblotting. Autophagy was measured by LC3 immunofluorescence and immunoblotting. ROS production and loss of mitochondria membrane potential (MMP) were checked with flow cytometry analysis. Results: Following exposure to PG, NCI-H460 cell proliferation decreased simultaneously inducing autophagic vacuoles and up-regulation of microtubule-associated protein 1 light chain 3 and beclin-1 protein expressions. Interestingly, pre-treated with autophagy inhibitors, 3-methyladenin or bafilomycin A1 further triggered reduction of cell viability. PG treatment also induced apoptosis that was related modulation of Bcl-2 family proteins, death receptors and activation of caspases. In addition, PG stimulation clearly enhanced loss of MMP and reactive oxygen species (ROS) generation. Conclusions: Our results suggest that PG elicited both autophagy and apoptosis by increasing loss of MMP and ROS production. PG induced-autophagy may play a cell protective role.

• Asian Pacific Journal of Cancer Prevention
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• 제15권19호
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• pp.8107-8113
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• 2014

The aim of this study was to investigate the effects of olanzapine on growth inhibition as well as autophagy in glioma cells in vitro and in vivo. The proliferation of both LN229 and T98 glioma cells, measured by MTT assay, was suppressed in a concentration-dependent and time-dependent manner. Moreover, apoptosis of both cells was significantly increased with the treatment of olanzapine as evidenced by increased Bcl-2 expression, Hoechst 33258 staining and annexinV-FITC/PI staining. Olanzapine treatment also enhanced activation of autophagy with increased expression of LC3-II, expression of protein p62, a substrate of autophagy, being decreased. The growth inhibition by olanzapine in both glioma cell lines could be blocked by co-treatment with 3-MA, an autophagy inhibitor. Furthermore, olanzapine effectively blocked the growth of subcutaneous xenografts of LN229 glioma cells in vivo. The increased level of protein LC3-II and decreased level of p62 followed by a decreased level of Bcl-2, suggesting that autophagy may contribute to apoptosis. In addition, reduced proliferation of glioma cells was shown by a decrease of Ki-67 staining and increased caspase-3 staining indicative of apoptosis in mouse xenografts. These results indicated that olanzapine inhibited the growth of glioma cells accompanied by induction of autophagy and apoptosis both in vitro and in vivo. Olanzapine-induced autophagy plays a tumor-suppressing role in glioma cells.

• Biomolecules & Therapeutics
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• 제20권4호
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• pp.393-398
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• 2012

Recently, we reported that defective autophagy may contribute to the inhibition of the growth in response to PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), a selective SFK inhibitor, in multidrug-resistant v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3/Mdr). In this study, we demonstrated that PP2 induces LC3 conversion via a mechanism that is uncoupled from autophagy and increases apoptosis in Ras-NIH 3T3/Mdr cells. PP2 preferentially induced autophagy in Ras-NIH 3T3 cells rather than in Ras-NIH 3T3/Mdr cells as determined by LC3-I to LC3-II conversion and GFP-LC3 fluorescence microscopy. Beclin 1 knockdown experiments showed that, regardless of drug resistance, PP2 induces autophagy via a Beclin 1-dependent mechanism. PP2 induced a conformational change in Beclin 1, resulting in the enhancement of the pro-autophagic activity of Beclin 1, in Ras-NIH 3T3 cells. Further, PI3K inhibition induced by wortmannin caused a significant increase in apoptosis in Ras-NIH 3T3 cells, as demonstrated by flow cytometric analysis of Annexin V staining, implying that autophagy inhibition through PI3K increases apoptosis in response to PP2 in Ras-NIH 3T3 cells. However, despite the fact that wortmannin abrogates PP2-induced GFP-LC3 punctae formation, some LC3 conversion remains in Ras-NIH 3T3/Mdr cells, suggesting that LC3 conversion may occur in an autophagy-independent manner. Taken together, these results suggest that PP2 induces LC3 conversion independent of PI3K, concomitant with the uncoupling of LC3 conversion from autophagy, in multidrug-resistant cells.

• Journal of Medicine and Life Science
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• 제17권2호
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• pp.41-46
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• 2020

Metformin, a predominantly prescribed anti-diabetic drug for decades, has gained new insights for its anti-tumor activity in a variety of cancer cells. Our previous studies also showed the obvious pro-apoptotic activity of metformin and the underlying action mechanisms in hepatocellular carcinoma cells. Together with apoptosis, autophagy is a crucial intracellular process to determine the survival or death of cells under some stressful environments. The present study aimed to determine the role of autophagy in metformin-induced death of H4IIE hepatocellular carcinoma cells. Metformin blocked the formation of autophagosome and the expression of LC3A, generally described as a biomarker of autophagy. Inhibition of AMPK reversed the metformin-induced blockade of autophagy. Antioxidant (NAC) suppressed the metformin-induced cell death but not affected LC3A. The inhibition of protein kinase C totally restored the metformin-suppressed expression of LC3A. In summary, our present study suggests that autophagy is an anti-apoptotic player in metformin-induced apoptosis in H4IIE cells.

• Asian Pacific Journal of Cancer Prevention
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• 제14권10호
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• pp.5849-5854
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• 2013

Background: Apoptosis may be induced after Bcl-2 expression is inhibited in proliferative cancer cells. This study focused on the effect of autophagy activation by ABT737 on anti-tumor effects of epirubicin. Methods: Cytotoxic effects of ABT737 on the HepG2 liver cancer cell line were assessed by MTT assay and cell apoptosis through flow cytometry. Mitochondrial membrane potential was measured by fluorescence microscopy. Monodansylcadaverin (MDC) staining was used to detect activation of autophagy. Expression of p53, p62, LC3, and Beclin1, apoptotic or autophagy related proteins, was detected by Western blotting. Results: ABT737 and epirubicin induced growth inhibition in HepG2 cells in a dose- and time-dependent manner. Both ABT737 and epirubicin alone could induce cell apoptosis with a reduction in mitochondrial membrane potential as well as increased apoptotic protein expression. Further increase of apoptosis was detected when HepG2 cells were co-treated with ABT373 and epirubicin. Furthermore, our results demonstrated that ABT373 or epirubicin ccould activate cell autophagy with elevated autophagosome formation, increased expression of autophagy related proteins and LC3 fluorescent puncta. Conclusions: ABT737 influences cancer cells through both apoptotic and autophagic mechanisms, and ABT737 may enhance the effects of epirubicin on HepG2 cells by activating autophagy and inducing apoptosis.