• Biomolecules & Therapeutics
• /
• 제28권2호
• /
• pp.202-210
• /
• 2020

Fluoxetine is used widely as an antidepressant for the treatment of cancer-related depression, but has been reported to also have anti-cancer activity. In this study, we investigated the cytotoxicity of fluoxetine to human gastric adenocarcinoma cells; as shown by the MTT assay, fluoxetine induced cell death. Subsequently, cells were treated with 10 or 20 µM fluoxetine for 24 h and analyzed. Apoptosis was confirmed by the increased number of early apoptotic cells, shown by Annexin V- propidium iodide staining. Nuclear condensation was visualized by DAPI staining. A significant increase in the expression of cleaved PARP was observed by western blotting. The pan-caspase inhibitor Z-VAD-FMK was used to detect the extent of caspase-dependent cell death. The induction of autophagy was determined by the formation of acidic vesicular organelles (AVOs), which was visualized by acridine orange staining, and the increased expression of autophagy markers, such as LC3B, Beclin 1, and p62/SQSTM 1, observed by western blotting. The expression of upstream proteins, such as p-Akt and p-mTOR, were decreased. Autophagic degradation was evaluated by using bafilomycin, an inhibitor of late-stage autophagy. Bafilomycin did not significantly enhance LC3B expression induced by fluoxetine, which suggested autophagic degradation was impaired. In addition, the co-administration of the autophagy inhibitor 3-methyladenine and fluoxetine significantly increased fluoxetine-induced apoptosis, with decreased p-Akt and markedly increased death receptor 4 and 5 expression. Our results suggested that fluoxetine simultaneously induced both protective autophagy and apoptosis and that the inhibition of autophagy enhanced fluoxetine-induced apoptosis through increased death receptor expression.

• Biomolecules & Therapeutics
• /
• 제26권6호
• /
• pp.608-615
• /
• 2018

Benzalkonium chloride, diazolidinyl urea, and imidazolidinyl urea are commonly used preservatives in cosmetics. Recent reports suggested that these compounds may have cellular and systemic toxicity in high concentration. In addition, diazolidinyl urea and imidazolidinyl urea are known formaldehyde (FA) releasers, raising concerns for these cosmetic preservatives. In this study, we investigated the effects of benzalkonium chloride, diazolidinyl urea, and imidazolidinyl urea on ROS-dependent apoptosis of rat neural progenitor cells (NPCs) in vitro. Cells were isolated and cultured from embryonic day 14 rat cortices. Cultured cells were treated with 1-1,000 nM benzalkonium chloride, and $1-50{\mu}M$ diazolidinyl urea or imidazolidinyl urea at various time points to measure the reactive oxygen species (ROS). PI staining, MTT assay, and live-cell imaging were used for cell viability measurements. Western blot was carried out for cleaved caspase-3 and cleaved caspase-8 as apoptotic protein markers. In rat NPCs, ROS production and cleaved caspase-8 expression were increased while the cell viability was decreased in high concentrations of these substances. These results suggest that several cosmetic preservatives at high concentrations can induce neural toxicity in rat brains through ROS induction and apoptosis.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• 제30권6호
• /
• pp.474-481
• /
• 2004

Squamous cell carcinoma is the most prevalent oral cancer, which is characterized by its low survival rate, high malignancy, mortality with facial defects, and poor prognosis. Exact cause and pathogenesis of the squamous cell carcinoma is still unknown. Various routes including smoking, radiation, and viral infections predispose its genesis, and recent studies revealed that genetic defects which fail to prevent cancer proliferation play a role. Generally, a cancer develops from the decreased rate of apoptosis which is an active and voluntary cell death, and from the altered cell cycles. Anticancer effect can be obtained by recovering the apoptotic process, and by suppressing the cell cycles. Among the apoptosis related factors, bcl-2, caspase-9, and VDAC (voltage-dependent anion channel)are produced in mitochondria of the cell. Cyclosporin-A is known to induce apoptosis through its activation with VDAC. This study was to reveal the anticancer effect of Cyclosporin A to the oral squamous cell carcinoma. The inverted microscope was used to find alterations in the tissue, and sensitivity test to the anticancer cells was performed with MTT (Tetrazolium-based colorimetric) assay. Following cell line culture of primary and metastastic oral squamous cell carcinoma, electrophoresis was performed with extracted total RNA. Finally, semi-quantitative study was carried out through RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The results of this study are as follows: 1. The inverted microscopic observation revealed a poorly defined cytoplasm at $2000ng{\sim}3000ng/ml$, indistinct nucleus, and apoptosis. 2. The Growth of cancer cells was decreased at 1000ng/ml of cyclosporin-A. No cancer cell growth was observed at over 2000ng/ml concentration of cyclosporin-A, and at one week, growth of cancer cells was ceased. 3. The MTT assays were decreased as cyclosporin-A concentration was increased. This means that the activation of succinyl dehydrogenase in mitochondria was decreased following administration of cyclosporin A. 4. A result of RT-PCR showed that amount of mRNA of VDAC-2 was decreased half times at a cyclosporine-A concentration of 2000ng/ml. In bcl-2, amount of mRNA was significantly decreased 1/5 times at 2000ng/ml. caspase-9, however, showed slight increase compared to the control group. From the results obtained in this study, administration of cyclosporin-A to the cell lines of oral squamous cell carcinoma induced alterations in morphology and growth of the cells as its concentration increased. Since apoptosis related factors such as VDAS-2, bcl-2, and caspase-9 also showed distinct alterations on their mRNAs, further research on cyclosporin A as an anti-cancer agent will be feasible.

• The Korean Journal of Physiology and Pharmacology
• /
• 제13권4호
• /
• pp.273-279
• /
• 2009

The accumulation of ${\beta}$-amyloid (A${\beta}$) aggregates is a characteristic of Alzheimer's disease (AD). Furthermore, these aggregates have neurotoxic effects on cells, and thus, molecules that inhibit A${\beta}$ aggregate formation could be valuable therapeutics for AD. It is well known that aggregation of A${\beta}$ depends on its hydrophobicity, and thus, in order to increase the hydrophilicity of A${\beta}$, we considered using citrate, an anionic surfactant with three carboxylic acid groups. We hypothesized that citrate could reduce hydrophobicity and increase hydrophilicity of A${\beta}_{1-40}$ molecules via hydrophilic/electrostatic interactions. We found that citrate significantly inhibited A${\beta}_{1-40}$ aggregation and significantly protected SH-SY5Y cell line against A${\beta}_{1-40}$ aggregates-induced neurotoxicity. In details, we examined the effects of citrate on A${\beta}_{1-40}$ aggregation and on A${\beta}_{1-40}$ aggregates-induced cytotoxicity, cell viability, and apoptosis. Th-T assays showed that citrate significantly inhibited A${\beta}_{1-40}$ aggregation in a concentration-dependent manner (Th-T intensity: from 91.3% in 0.01 mM citrate to 82.1% in 1.0 mM citrate vs. 100.0% in A${\beta}_{1-40}$ alone). In cytotoxicity and viability assays, citrate reduced the toxicity of A${\beta}_{1-40}$ in a concentration-dependent manner, in which the cytotoxicity decreased from 107.5 to 102.3% as compared with A${\beta}_{1-40}$ aggregates alone treated cells (127.3%) and the cell viability increased from 84.6 to 93.8% as compared with the A${\beta}_{1-40}$ aggregates alone treated cells (65.3%). Furthermore, Hoechst 33342 staining showed that citrate (1.0 mM) suppressed A${\beta}_{1-40}$ aggregates-induced apoptosis in the cells. This study suggests that citrate can inhibit A${\beta}_{1-40}$ aggregation and protect neurons from the apoptotic effects of A${\beta}_{1-40}$ aggregates. Accordingly, our findings suggest that citrate administration should be viewed as a novel neuroprotective strategy for AD.

• 한국식품과학회지
• /
• 제41권5호
• /
• pp.597-601
• /
• 2009

간성상세포(HSC)는 간섬유화와 간경변에 중요한 역할을 한다고 알려져 있다. 간손상에 의해 둥근 모양의 간성상세포는 활성화되어 세포외기질(ECM)을 생산하는 myofibroblast와 같은 모양으로 활성화 된다. 활성화된 간성상세포의 특징은 빠른 증식 속도와 collagen과 같은 세포외 기질의 생산이다. 활성화된 간성상 세포의 제거방법은 apoptosis를 유도하는 것이다. 가자 추출물은 정상 간세포(rat primary hepatocyte), 간세포주(HepG2) 및 활성화된 간성상세포주인 T-HSC/Cl-6에 $1,000{\mu}g/mL$의 농도까지 처리하여 세포독성을 확인하였다. 그 결과 hepatocyte나 HepG2에서는 최고 농도에서도 독성이 없었으나 T-HSC/Cl-6는 U-shape 모양으로 사멸하는 것을 확인 하였다. T-HSC/Cl-6의 사멸이 apoptosis에 의한 것인지를 Annexin-V/PI double staining을 통하여 확인한 결과 apoptosis에 의해 T-HSC/Cl-6의 사멸이 일어나는 것을 확인하였다.

• 한국미생물·생명공학회지
• /
• 제32권1호
• /
• pp.67-71
• /
• 2004

남미 아마존 유역의 열대 우림 자생식물인 Pata de Vaca(Bauhinia forficata)의 추출물로부터 활성분획을 분리하여, 세포성장 억제활성 및 세포사멸 유도에 관해 조사하였다. MTT assay를 통해 그 저해 활성을 비교 분석해 가면서 Pata de Vaca의 70% ethanol 추출물을 HP-20 Diaion column chromatograph 및 C-18 column chromatography에 순차적으로 적용시켰다. 그 결과, 저해 활성이 가장 강하며 수율이 높은 Pata-50을 분리하여, 6종의 암세포주에 대한 MTT assay를 실시하였다. Pata-50에 의한 세포성장 억제는, MCF-7을 제외한 나머지 세포주인 HepG2, HT-29, AGS, A549 및 HeLa세포에 대해서 농도 의존적인 저해활성을 보였으며, $IC_{50}$ 는 40.4-77.5$\mu\textrm{g}$/$m\ell$의 농도 범위에서 나타났다. Pata-50의 세포성장 억제를 확인하였으므로, HepG2 세포에.If Pata-50에 의한 세포주기의 변화와 세포사별로 유도되는 세포의 분포를 측정하기 위하여 flow cytometry analysis를 실시하였다. 그 결과, Pata-50은 HepG2에 대하여 농도 의존적으로 세포사멸을 유도하지만, 세포주기 조절에는 아무런 영향을 미치지 많고 있음을 알 수 있었다. 특히, Pata-50을 50$\mu\textrm{g}$/$m\ell$ 이하로 처리할 경우에는 apoptosis가 유도된 세포기 비율이 10%미만이지만, 80$\mu\textrm{g}$/$m\ell$ 및 110$\mu\textrm{g}$/$m\ell$을 처리하면 28.6%및 56.2%로_급격히 증가하였다. 다음으로, 세사멸에 결정적인 단백질의 발현 양상을 관찰하기 위해, HeLa세포주에 Pata-50을 농도별로 처리하여 Western blot으로 분석하였다. Fig. 4에 나타난 바와 같이, HeLa 세포주에 Pata-50을 80$\mu\textrm{g}$/$m\ell$의 농도로 처리하였을 때, caspase-3.가 활성화되었으며, 이로 인해, PARP가 116 kDa에서 85kDa로 cleavag되어, 불활성화 되는 것을 확인할 수 있었다.

• Journal of Dental Anesthesia and Pain Medicine
• /
• 제17권1호
• /
• pp.21-28
• /
• 2017

Background: The skin consists of tightly connected keratinocytes, and prevents extensive water loss while simultaneously protecting against the entry of microbial pathogens. Excessive cellular levels of reactive oxygen species can induce cell apoptosis and also damage skin integrity. Propofol (2,6-diisopropylphenol) has antioxidant properties. In this study, we investigated how propofol influences intracellular autophagy and apoptotic cell death induced by oxidative stress in human keratinocytes. Method: The following groups were used for experimentation: control, cells were incubated under normoxia (5% $CO_2$, 21% $O_2$, and 74% $N_2$) without propofol; hydrogen peroxide ($H_2O_2$), cells were exposed to $H_2O_2$ ($300{\mu}M$) for 2 h; propofol preconditioning (PPC)/$H_2O_2$, cells pretreated with propofol ($100{\mu}M$) for 2 h were exposed to $H_2O_2$; and 3-methyladenine $(3-MA)/PPC/H_2O_2$, cells pretreated with 3-MA (1 mM) for 1 h and propofol were exposed to $H_2O_2$. Cell viability, apoptosis, and migration capability were evaluated. Relation to autophagy was detected by western blot analysis. Results: Cell viability decreased significantly in the $H_2O_2$ group compared to that in the control group and was improved by propofol preconditioning. Propofol preconditioning effectively decreased $H_2O_2$-induced cell apoptosis and increased cell migration. However, pretreatment with 3-MA inhibited the protective effect of propofol on cell apoptosis. Autophagy was activated in the $PPC/H_2O_2$ group compared to that in the $H_2O_2$ group as demonstrated by western blot analysis and autophagosome staining. Conclusion: The results suggest that propofol preconditioning induces an endogenous cellular protective effect in human keratinocytes against oxidative stress through the activation of signaling pathways related to autophagy.

• 한국식품영양과학회지
• /
• 제38권12호
• /
• pp.1679-1684
• /
• 2009

본 연구에서는 천년초선인장 열매의 총 폴리페놀과 플라보노이드 함량을 분석하고, 열매의 추출물이 인간의 유방상피조직에서 유래한 암세포인 MCF-7의 증식과 세포사멸에 미치는 영향을 조사하였다. 천년초선인장 열매의 총 폴리페놀 함량은 약 4.49 g/100 g, 플라보노이드 함량은 약 1.31g/100 g로 나타났다. 열매의 물 추출물 100과 500 ${\mu}$g/mL 농도에서 정상세포인 BJ에 대한 세포독성을 나타내지 않으면서 MCF-7 세포의 살아있는 세포수를 농도 의존적으로 감소시키는 효과를 보였다. 또한 세포배양액에 열매의 물 추출물을 첨가한 경우, G1 arrest가 일어나 세포주기 진행이 지연되었으나, apoptosis에는 영향을 주지 않음이 확인되어 천년초선인장 열매 물 추출물이 apoptosis보다는 G1 arrest를 유도하여 유방암세포를 억제한다는 것을 규명하였다. 이러한 결과들은 천년초선인장 열매의 물 추출물이 세포독성에는 영향을 주지 않으면서 유방암세포의 증식과 세포주기 진행을 억제하는 효과적인 항암물질이 될 수 있는 기능성 소재로 활용 가능하다는 것을 암시한다.

• Asian Pacific Journal of Cancer Prevention
• /
• 제16권15호
• /
• pp.6463-6468
• /
• 2015

Background: Chemotherapy is one of the common approaches in treatment of cancers, especially leukemia. However, drug resistance phenomena reduce the likelihood of treatment success. Resveratrol is a herbal compound which through complicated processes makes some selected cells sensitive to treatment and induction of apoptosis. In the present study, the effects of resveratrol on the expression of miR 15a and miR16-1 and apoptosis in the CCRF-CEM cell line were investigated. Materials and Methods: The CCRF-CEM cell line was cultured under standard conditions and changes in miR 15a and miR 16-1 expression were analyzed by real time-PCR technique, with attention to reveratrol dose and time dependence. Also, apoptosis is evaluated by flow cytometry using annexin V and PI. Results: CCRF-CEM cells underwent dose-dependent apoptotic cell death in response to resveratrol. MiR 15a and miR 16-1 expression was up-regulated after 24 and 48 hours resveratrol treatment (p<0.05). Conclusions: The results of our study indicate that resveratrol induces apoptosis in a time and dose-dependent manner in CCRF-CEM cells. Also, increased expression level of miR 16-1 and miR 15a by means of resveratrol in CCRF-CEM cells might have a role in apoptosis induction and predisposition. According to our results resveratrol can be regarded as a dietary supplement to improve efficacy of anti-leukemia therapies.

• Asian Pacific Journal of Cancer Prevention
• /
• 제15권18호
• /
• pp.7825-7829
• /
• 2014

Objective: To investigate the effect of high expression of XAF1 in vivo or in vitro on lung cancer cell growth and apoptosis. Methods: 1. The A549 human lung cancer cell line was transfected with Ad5/F35 - XAF1, or Ad5/F35 - Null at the same multiplicity of infection (MOI); (hereinafter referred to as transient transfected cell strain); XAF1 gene mRNA and protein expression was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively. 2. Methyl thiazolyl tetrazolium (MTT) and annexin V-FITC/PI double staining were used to detect cell proliferation and apoptosis before and after infection of Ad5/F35 - XAF1 with Western blotting for apoptosis related proteins, caspase 3, caspase - 8 and PARP. 3. After the XAF1 gene was transfected into lung cancer A549 cells by lentiviral vectors, and selected by screening with Blasticidin, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were applied to detect mRNA and protein expression, to establish a line with a stable high expression of XAF1 (hereinafter referred to as stable expression cell strain). Twenty nude mice were randomly divided into groups A and B, 10 in each group: A549/XAF1 stable expression cell strain was subcutaneously injected in group A, and A549/Ctrl stable cell line stable expression cell strain in group B (control group), to observe transplanted tumor growth in nude mice. Results: The mRNA and protein expression of XAF1 in A549 cells transfected by Ad5/F35 - XAF1 was significantly higher than in the control group. XAF1 mediated by adenovirus vector demonstrated a dose dependent inhibition of lung cancer cell proliferation and induction of apoptosis. This was accompanied by cleavage of caspase -3, -8, -9 and PARP, suggesting activation of intrinsic or extrinsic apoptotic pathways. A cell strain of lung cancer highly expressing XAF1 was established, and this demonstrated delayed tumor growth after transplantation in vivo. Conclusion: Adenovirus mediated XAF1 gene expression could inhibit proliferation and induce apoptosis in lung cancer cells in vitro; highly stable expression of XAF1 could also significantly inhibit the growth of transplanted tumors in nude mouse, with no obvious adverse reactions observed. Therefore, the XAF1 gene could become a new target for lung cancer treatment.