• Asian Pacific Journal of Cancer Prevention
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• 제15권8호
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• pp.3731-3736
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• 2014

Phytic acid (PA) has been reported to have positive nutritional benefits and prevent cancer formation. This study investigated the anticancer activity of rice bran PA against hepatocellular carcinoma (HepG2) cells. Cytotoxicty of PA (0.5 to 4mM) was examined by MTT and LDH assays after 24 and 48h treatment. Apoptotic activity was evaluated by expression analysis of apoptosis-regulatory genes [i.e. p53, Bcl-2, Bax, Caspase-3 and -9] by reverse transcriptase-PCR and DNA fragmentation assay. The results showed antioxidant activity of PA in Fe3+ reducing power assay ($p{\leq}0.03$). PA inhibited the growth of HepG2 cells in a concentration dependent manner ($p{\leq}0.04$). After 48h treatment, cell viability was recorded 84.7, 74.4, 65.6, 49.6, 36.0 and 23.8% in MTT assay and 92.6, 77.0%, 66.8%, 51.2, 40.3 and 32.3% in LDH assay at concentrations of 1, 1.5, 2.0, 2.5, 3.0, and 3.5mM, respectively. Hence, treatment of PA for 24h, recorded viability of cells 93.5, 88.6, 55.5, 34.6 and 24.4% in MTT assay and 94.2, 86.1%, 59.7%, 42.3 and 31.6%, in LDH assay at concentrations of 1, 2.2, 3.0, 3.6 and 4.0mM, respectively. PA treated HepG2 cells showed up-regulation of p53, Bax, Caspase-3 and -9, and down-regulation of Bcl-2 gene ($p{\leq}0.01$). At the $IC_{50}$ (2.49mM) of PA, the p53, Bax, Caspase-3 and-9 genes were up-regulated by 6.03, 7.37, 19.7 and 14.5 fold respectively. Also, the fragmented genomic DNA in PA treated cells provided evidence of apoptosis. Our study confirmed the biological activity of PA and demonstrated growth inhibition and induction of apoptosis in HepG2 cells with modulation of the expression of apoptosis-regulatory genes.

• Journal of Microbiology and Biotechnology
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• 제22권11호
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• pp.1591-1595
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• 2012

We previously isolated a novel compound, HY251, with the molecular structure of 3-propyl-2-vinyl-1,2,3,3a,3b,6,7,7a,8,8a-decahydrocyclopenta[a]indene-3,3a,7a,8a-tetraol from the roots of Aralia continentalis. The current study was designed to evaluate the detailed molecular mechanisms underlying the apoptotic induction by HY251 in human ovarian cancer PA-1 cells. TUNEL assay and Western blot analyses revealed an appreciable apoptotic induction in PA-1 cells treated with $60{\mu}M$ of HY251 for 24 h. This apoptotic induction was associated with caspase-8-dependent Bid cleavage, which in turn resulted in the formation of pro-apoptotic truncated Bid (tBid), and activation of caspase-9 and -3, as well as the cleavage of poly(ADP-ribose) polymerase (PARP). Moreover, we found that this death event was also associated with the significant up-regulation and activation of the p53 tumor-suppressor protein through phosphorylation at Ser15. Therefore, we suggest that HY251 may be a potent cancer chemotherapeutic candidate for the treatment of ovarian cancer.

• Journal of Acupuncture Research
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• 제22권3호
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• pp.169-183
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• 2005

Cobrotoxin의 항암효과를 알아보고자 암세포인 PC-3 cell에 cobrotoxin을 처리한 후 cell viability, cell death, apoptosis, cell cycle 및 관련단백질, Adk, 및 MAP kinase 관련 단백질의 변화를 관찰하여 다음과 같은 결론을 얻었다. 1. PC-3 세포에 각각 cobrotoxin을 각각 0-16 nM까지 투여시 대조군에 비하여 모두 농도 의존적으로 세포들의 모양이 길죽한 나선형 모양에서 둥글게 응축되는 모습으로 변하였으며 고농도로 갈수록 세포들의 성장이 억제를 보였다. 2. MTT assay를 이용하여 세포 생존력을 측정 한 결과, 0.1, 1 및 4nM의 cobrotoxin 처리군은 정상군에 비하여 세포활성의 감소를 나타내었고, 8, 16nM의 cobrotoxin 처리군은 정상군에 비하여 세포활성의 유의한 감소를 보였다. 3. PC3-cell에 cobrotoxin을 처리한후 FACS analysis를 통하여 세포주기를 측정한 결과 세포주기 중 S phase에서 0.0lnM cobrotoxin 처리군은 변함이 없지만, 1, 2, 4, 8 및 16 nM 의 cobrotoxin 처리군에서는 정상군에 비해 감소를 보였다. G2-M phase에서 0.1, 1, 2, 4, 8 및 16M의 cobrotoxin 처리군에서 정상군 에 비하여 증가를 보였다. 4. Cobrotoxin 처리후 Cox-2의 발현을 48시간 관찰한 결과 12시간에서 최대치를 이루었고, 6, 12 및 24시간후에서 대조군에 비하여 유의한 감소를 나타내었다. 5. G1 phase에서 활성을 이루는 Cdk4, cyclin Dl의 발현을 살펴본 결과 Cdk4는 cobrotoxin 1, 2, 4 및 8nM 처리군에서 정상군에 비하여 농도 의존적으로 감소하였고, cobrotoxin 4, 8nM 처리군은 정상군에 비하여 유의한 감소를 나타내었다. Cyclin Dl은 cobrotoxin 1, 2, 4 및 8nM 처리 군에서 정상군에 비하여 농도 의존적으로 감소하였다. Cycline E는 cobrotoxin 1, 2, 4 및 8nM 처리 군에서 정상군에 비해 큰 변화가 없었다. G2/M phase에 관여하는 단백질인 Cyclin Bl 은 cobrotoxin 1, 2, 4 및 8nM 처리군에서 정상군에 비하여 농도 의존적으로 감소하였고, cobrotoxin 2, 4, 8M 처리군은 정상군에 비하여 유의한 감소를 나타내었다. 6. 세포성장 단백질인 Akt의 발현을 살펴본 결과 1, 2, 4 및 8nM의 cobrotoxin 처리군에서 정상군에 비하여 농도 의존적으로 감소하였고, 4, 8nM친 cobrotoxln 처리군은 정상에 비하여 유의한 감소를 나타내었다. 7. MAP kinase에 관여하는 단백질인 ERK, p-ERK, JNK, p-JNK p38, p-p38에 미치는 영향을 살펴본 결과, ERK은 1, 2nM의 cobrotoxin 처리군에서 정상군에 비하여 증가를, 4, 8nM의 cobrotokin 처리군에서 정상군에 비하여 감소를 나타내었다. p-ERK은 1, 2, 4nM의 cobrotoxin 처리군에서 정상군에 비하여 증가를, 8nM의 cobrotoxin 처리군에서 정상군에 비하여 감소를 나타내었다. JNK와 p-JNK는 1, 2, 4 및 8nM의 cobrotoxin 처리군에서 정상군에 비하여 증가를 나타내었다. p38는 1, 4, 8nM의 cobrotoxin 처리군에서 정상군에 비하여 감소를, 2nM의 cobrotoxin 처리군에서는 정상군에 비하여 감소를 나타내었다. p-p38는 1nM의 cobrotoxin 처리군에서 정상군에 비하여 증가를, 2, 4, 8nM의 cobrotoxin 처리군에서는 정상군에 비하여 유의한 감소를 나타내었다. 8. PC 3-cells 성장의 억제 역시 세포사멸을 유도한 세포성장억제 인지를 알아보기 위해 DAPI staining을 통한 세포의 핵을 염색하여 그 모양을 관찰한 결과, 정상세포가 동글동글 하고 균일한데 비하여 세포사멸이 일어난 세 포는 핵이 응축되고, 여러 조각으로 나뉜 모습을 관찰할 수 있었다. 또한, 세포사멸을 살펴본 결과 1, 2, 4, 8 및 16nM cobrotoxin 처 8nM의 cobrotoxin 처리군에서 정상군에 비하여 유의한 변동을 나타내지 않았다. Bcl-2 는 1, 2, 4 및 8nM의 cobrotoxin 처리군에서 정상군에 비해 농도 의존적으로 유의한 감소를 나타내었다. 9. 세포사멸 관련 유전자인 caspase family (caspase 3, 9)와 Bcl-2 family (Bcl-2, Bax), p53의 발현을 살펴본 결과, Bax는 1, 2, 4 및 Caspase 3과 9는 1, 2, 4nM의 cobrotoxin 처리군에서 정상군에 비하여 유의한 변동을 나타내지 않았으나, 8nM cobrotoxin 처리군에서는 유의한 감소를 나타내었다. 이상의 결과를 종합해보면, 일정수준(pico 또는 namo molar 수준)의 cobrotoxin이 prostate cancer cell의 성장을 억제하고, 세포사멸을 유도 하여 항암 효과가 있음을 확인할 수 있었으며 향후 cobrotoxin의 항암 효과를 실제 임상에 활용할 수 있게 되기를 기대한다.

• 대한의생명과학회지
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• 제19권2호
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• pp.118-123
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• 2013

Tumor necrosis factor-alpha (TNF-${\alpha}$) is a proinflammatory cytokine that mediates the inflammatory response and immune functions, and modulates the proliferation, differentiation and cell death of cancer cells. The differential functions of TNF-${\alpha}$ in various human cells due to the formation of different stimulating pathway upon the binding of TNF-${\alpha}$ to its receptors. In the present study, we examined the different effects of TNF-${\alpha}$ on the survival and apoptosis between normal granulocytes and human myeloid leukemia HL-60 cells. Although TNF-${\alpha}$ did not affect on the constitutive apoptosis of granulocytes, TNF-${\alpha}$ strongly induced the apoptosis of HL-60 cells in a dose- and a time-dependent manner. TNF-${\alpha}$-induced apoptosis was occurred via the activation of caspase 8, caspase 9 and caspase 3/7 and the induction of ROS production in HL-60 cells. Also, BAY-11-7085, a NF-${\kappa}B$ inhibitor, blocked the TNF-${\alpha}$-induced apoptosis in HL-60 cells. NF-${\kappa}B$ may be involved in TNF-${\alpha}$-induced apoptotic signaling pathway in HL-60 cells. These results suggest that TNF-${\alpha}$ activates apoptotic pathways and its process depends on cell type and many cellular factors. A better understanding of the differential effect of TNF-${\alpha}$ on cell apoptosis and survival may provide important information that can be used to elucidate the specific inhibitory effect of TNF-${\alpha}$ on the cancer dis.

• The Korean Journal of Physiology and Pharmacology
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• 제21권5호
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• pp.465-474
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• 2017

The aim of this study was to determine the raising anticancer effects of resveratrol (Res) on paclitaxel (PA) in non-small cell lung cancer (NSCLC) cell line A549. The $10{\mu}g/ml$ of Res had no effect on human fetal lung fibroblast MRC-5 cells or on A549 cancer cells and the 5 or $10{\mu}g/ml$ of PA also had no effect on MRC-5 normal cells. PA-L ($5{\mu}g/ml$) and PA-H ($10{\mu}g/ml$) had the growth inhibitory effects in NSCLC cell line A549, and Res increased these growth inhibitory effects. By flow cytometry experiment, after Res ($5{\mu}g/ml$)+PA-H ($10{\mu}g/ml$) treatment, the A549 cells showed the most apoptosic cells compared to other group treatments, and after additional treatment with Res, the apoptosic cells of both two PA concentrations were raised. Res+PA could reduce the mRNA and protein expressions of COX-2, and Res+PA could reduce the COX-2 related genes of VEGF, MMP-1, MMP-2, MMP-9, $NF-{\kappa}B$, Bcl-2, BclxL, procollagen I, collagen I, collagen III and CTGF, $TNF-{\alpha}$, $IL-1{\beta}$, iNOS and raise the TIMP-1, TIMP-2, TIMP-3, $I{\kappa}B-{\alpha}$, p53, p21, caspase-3, caspase-8, caspase-9, Bax genes compared to the control cells and the PA treated cells. From these results, it can be suggested that Res could raise the anticancer effects of PA in A549 cells, thus Res might be used as a good sensitizing agent for PA.

• Asian Pacific Journal of Cancer Prevention
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• 제17권9호
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• pp.4223-4231
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• 2016

Background: Development of new apoptosis-inducing drugs is a promising trend in anticancer therapy. For this purpose several formamidinoderivatives of doxorubicin were synthesized. The aim of our study was to investigate effects of the five formamidinodoxorubicins in the ES-2 human ovarian clear cell carcinoma line, for comparison with data obtained previously for SKOV-3 human ovarian adenocarcinoma cells, to answer the question of whether and to what extent the histological cell type is a possible determinant of sensitivity to tested anthracyclines. Materials and Methods: In our experimental work the following methods were used: spectrophotometric assays with MTT; fluorimetric assays - double staining with Hoechst 33258 and propidium iodide (PI), measurement of caspase-3, -8, -9 activity, intracellular accumulation of DOX and analogues, estimation of drug uptake, mitochondrial transmembrane potential; flow cytometry - phosphatidylserine (PS) externalization with annexin V-FITC and PI fluorochromes. Results: Effects of the derivatives of doxorubicin were partially linked with the specific type of cancer cell although intracellular accumulation and cellular uptake of DOX and derivatives were similar in both. All of the investigated derivatives were considerably more cytotoxic than DOX. Formamidinodoxorubicins were able to induce caspase-dependent apoptotic cell death in both cell types. Conclusions: All new formamidine derivatives of DOX were able to induce caspase - dependent apoptosis in human ovarian cancer cell lines SKOV-3 and ES-2. Obtained results suggested that formamidine derivatives of DOX may be promising candidates for the prospective chemotherapeutic agents for the two different histological subtypes of ovarian cancer.

• Asian Pacific Journal of Cancer Prevention
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• 제16권13호
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• pp.5471-5476
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• 2015

2-deoxy-D-Glucose (2DG) causes cytotoxicity in cancer cells by disrupting thiol metabolism. It is an effective component in therapeutic strategies. It targets the metabolism of cancer cells with glycolysis inhibitory activity. On the other hand, MLN4924, a newly discovered investigational small molecule inhibitor of NAE (NEDD8 activating enzyme), inactivates SCF E3 ligase and causes accumulation of its substrates which triggers apoptosis. Combination of these components might provide a more efficient approach to treatment. In this research, 2DG and MLN4924 were co-applied to breast cancer cells (MCF-7 and SKBR-3) and cytotoxic and apoptotic activity were evaluated the by Micro culture tetrazolium test (MTT), TUNEL and ELISA methods. Caspase3 and Bcl2 genes expression were evaluated by real time Q-PCR methods. The results showed that MLN4924 and MLN4924/2DG dose-dependently suppressed the proliferation of MCF7 and SKBR-3 cells. Cell survival of breast cancer cells exposed to the combination of 2DG/MLN4924 was decreased significantly compared to controls (p<0.05), while 2DG and MLN4924 alone had less pronounced effects on the cells. The obtained results suggest that 2DG/MLN4924 is much more efficient in breast cancer cell lines with enhanced cytotoxicity via inducing a apoptosis cell signaling gene, caspase-3.

• 동의생리병리학회지
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• 제18권1호
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• pp.200-205
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• 2004

Uterine leiomyoma is the most common tumor in the female genital tract. Although the tumor is benign, it is of paramount importance since it often causes profuse menstrual bleeding, pressure symptoms, and infertility. Nevertheless, the etiology and patholphysiology of this abnormality remain poorly understood. The traditional definitive treatment for uterine leiomyomas is hysterectomy and, even today, symptomatic leiomyomas are the leading cause of hysterectomy in Korea. Clearly, the development of a safe, effective, and nonsurgical method of treatment for leiomyoma would be of great benefit to many women. The present study was designed to investigate the effect of Rhubarb on apoptosis in uterine leiomyoma cells. Results demonstrate that Rhubarb inhibited cell growth in dose-dependent manner. Cell growth significantly decreased to 60% of control in the treatment of Rhubarb (300㎍/㎖). Associated with the decreased response, there was a concomitant and significant delay of subG1 8.32% above baseline in the treatment of Rhubarb (300㎍/㎖). The delay of subG1 showed a dose-dependent manner, as evidenced by the flow cytometry. The reduced cellular viability on exposure to Rhubarb may represent the induction of apoptosis, at least in part, as concomitantly evidenced by enhanced DNA fragmentation, PARP cleavage and caspase 9 and decreased pro-caspase 3. In addition, Rhubarb decreased clAP1 expression levels in dose-dependent manner. Talcen together, there results suggest that Rhubarb can produce a potent inhibition effect of apoptosis and implicate the delay of G1 phase in the cell cycle and pathways of caspase 3 and 9 in the mechanism underlying inhibitory apoptosis effect of Rhubarb.

• 동의생리병리학회지
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• 제24권6호
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• pp.942-949
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• 2010

Morus alba L., a kind of Oriental medicinal herbs, has been traditionally used to treat pulmonary asthma and congestion. According to recent studies, extracts of M. alba L. have showed anti-inflammatory, anti-oxidant, anti-tumor and hypoglycemic effects. However, the molecular mechanisms on how it acts as a death-inducer in cancer cells have not been fully understood. In this study, we investigated the cell death effects of methylene chloride extracts of M. alba L. (MEMA) in A549 human lung carcinoma cells. It was shown that MEMA induced the apoptotic cell death proved by increased sub-G1 phase cell population, apoptotic body formation and chromatin condensation. MEMA treatment induced the expression of death receptor-related proteins such as death receptor (DR) 4, DR5, Fas and FasL, which further triggered the activation of caspase-8 and the cleavage of Bid in a concentration-dependent manner. However, MEMA reduced anti-apoptotic Bcl-2 and Bcl-xL expression which contributed to the loss of mitochondrial membrane potential (MMP), and the activations of caspase-9 and caspase-3. Meanwhile, the morphological study indicated a characteristic finding of autophagy, such as the formation of autophagosomes in MEMA-treated cells. Furthermore, markers of autophagy, namely, the increased MDC-positive cells, conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II and increased beclin-1 accumulation, were observed. Taken together, these findings demonstrated that MEMA triggered both autophagy and apoptosis in A549 cancer cells. They might suggest that M. alba L. could be a prospective clinical application to treat human lung cancers.

• Journal of Acupuncture Research
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• 제24권2호
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• pp.1-14
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• 2007

목적 : 이 연구는 Vipera lebetina turanica의 사독약침파(蛇毒藥鍼波)(Snake venom toxin, SVT)이 in vitro에서 $NF-{\kappa}B$의 활성억제와 apoptosis 관련 단백질의 발현 조절을 통하여 세포자멸사(Apoptosis)를 유도하는지 in vivo에서 또한 전립선 암세포주인 PC-3 세포의 성장을 억제하는지 살펴보고자 하였다. 방법 : SVT를 처리한 후 PC-3의 성장억제를 관찰하기 위해 WST-1 assay, CCK-8 assay를 시행하였고，Apoptosis evaluation에는 DAPI, TUNEL staining assay를 시행하였으며，Apoptosis regulatory proteins의 변화 관찰에는 western blot analysis를 시행하였고，apoptosis와 연관된 $NF-{\kappa}B$의 활성 변화를 관찰하기 위해 EMSA시행하였으며，SVT의 핵내이동을 관찰하기 위해 Immunofluorescence Staining, Confocal immunocytochemistry를 시행하였으며，전립암세포의 종양형성에는 흉선을 제거한 쥐에 Tumorigenecity study를 시행하였다. 결과 : PC-3 세포에 SVT를 처리한 후，전립선 암세포의 성장，Apoptosis의 유발，Apoptosis관련 단백질의 발현，$NF-{\kappa}B$의 활성，SVT의 PC-3세포 핵내 이동여부 및 흉선제거 후 PC-3 세포를 이식한 쥐의 종양형성과정에 미치는 영향을 관찰하여 다음과 같은 결과를 얻었다. 1. PC-3 세포에서 SVT를 처리한 후 세포성장이 억제되고，세포자멸사가 유도되며，조절인자인 p53, caspase-3, -9는 증가되었고，Bcl-2는 감소되었다. 2. PC-3 세포에서 SVT를 처리한 후 $NF-{\kappa}B$의 활성이 유의하게 감소되었다. 3. DAPI로 염색된 상태에서 SVT가 PC-3 세포의 핵내로 이통되는 것이 관찰되었다. 4. 흉선제거 후 전립선 암세포주를 이식한 쥐에서 SVT를 피내로 주입한 결과 전립선암의 크기와 무게가 유의하게 감소하였다. 결론 : 이상의 결과는 SVT가 $NF-{\kappa}B$의 활성 억제를 통하여 인간 전립선암세포주인 PC-3의 세포자멸사를 유발함으로써 증식억제 효과가 있음을 입증한 것이며，이를 재확인한 생체 연구에서의 긍정적인 결과는 향후 SVT의 전립선암의 예방과 치료에 대한 효과적인 치료제 개발에 초석이 될 것으로 기대된다.