• Journal of Acupuncture Research
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• v.24 no.2
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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.

• Journal of Chest Surgery
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• v.34 no.6
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• pp.447-453
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• 2001

Background: Although pulmonary resection is the standard approach for the management of pulmonary metastases from soft tissue sarcoma, most of them are unresectable and chemotherapy remains the only option. The effectiveness of the cytotoxic drugs may be limited by the toxicities that occur before the therapeutic dose is reached. The regional administration of doxorubicin using pulmonary arterial perfusion in a rodent model can produce 10 to 25 times higher concentrations in the lung than systemic administration with minimal systemic toxicities. However, it is unclear whether a high concentration of doxorubicin has beneficial effects for killing cancer cells. Material and Method: We studied this to evaluate the dose-dependent cytotoxic and apoptotic effects of doxorubicin on methylcholanthrene-induced rat fibrosarcoma(MCA) cells. This study examined the cytotoxicity and apoptosis-related gene expressions(Fas, FasL, Bax, caspase 1, caspase 2, caspase 8, Bcl-2, Bcl-xL, Bcl-xS) in MCA cells after 24 hours exposure to various concentrations of doxorubicin such as 1, 5, 10, 50, and 100 $\mu$M. Result: Dose-dependent cytotoxicity was observed after 24 hours exposure to doxorubicin. However, peak apoptosis after 24 hours exposure was observed at 5 $\mu$M of doxorubicin. Above 5 $\mu$M, apoptotic activity was decreased with dose-increment. All mRNA levels of apoptosis-related genes after 24 hours exposure were up-regulated above the control level at 1 $\mu$M of doxorubicin and then decreased by doxorubicin dose-increment except caspase 8, which showed higher levels than the control level at 5 $\mu$M. Apoptosis-related protein levels were highest at 1 $\mu$M of doxorubicin and then decreased by doxorubicin dose-increment. However, Bax and Bcl-xL proteins steadily showed higher levels than the control throughout the different concentrations of doxorubicin. Conclusion: These results suggest that apoptosis is the main cytotoxic mechanism in low concentrations of doxorubicin in MCA cells and apoptosis-related genes, such as Bax, caspase 8, and Bcl-xL, are involved. At high concentrations, doxorubicin still can kill MCA cells, even when apoptosis is inhibited, and have its propriety for achieving much cytotoxicity against MCA cells.

• Journal of Acupuncture Research
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• v.28 no.3
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• pp.101-110
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• 2011

목적 : 이 연구는 봉독이 NF-${\kappa}$B의 활성억제를 통하여 전립선 암세포주인 DU-145 세포의 성장을 억제하 는지를 확인하고 그 기전을 살펴보고자 하였다. 방법 : 봉독을 처리한 후 DU-145의 성장억제를 관찰하기 위해 WST-1 assay를 시행하였고, 세포자멸사의 관찰에는 DAPI staining assay를 통한 세포형태관찰을 시행하였으며, 염증관련유전자 발현 관찰에는 western blot analysis를 시행하였고, 세포자멸사와 연관된 NF-${\kappa}$B의 활성 변화를 관찰하기 위해 EMSA와 luciferase assay를 시행하였으며, DU-145에서 봉독과 NF-${\kappa}$B의 상호작용을 관찰하기 위해 transient transfection assay를 시행하여 세포생존율과 NF-${\kappa}$B의 활성 변동을 측정하였다. 결과 : DU-145 세포에서 봉독을 처리한 후 세포성장이 억제되었으며, 염증관련유전자 발현 및 NF-${\kappa}$B의 활성의 유의한 감소를 나타내었다. DU-145 세포에서 NF-${\kappa}$B의 p50와 IKK들을 치환하여 작용기를 없애고 봉독을 처리하였을 경우에도 세포활성 및 NF-${\kappa}$B의 활성의 유의한 감소를 나타내었다. 결론 : 이상의 결과는 봉독이 NF-${\kappa}$B의 활성 억제를 통하여 인간 전립선암세포주인 DU-145의 세포자멸사를 유발함으로써 증식억제 효과가 있음을 입증한 것으로 전립선암의 예방과 치료에 대한 효과적인 치료제 개발에 도움이 될 것으로 기대된다. 다만 그 기전에서 봉독은 기존연구와 같은 NF-${\kappa}$B p50 및 IKK들의 작용기와 상호작용 이외에 다른 기전이 관여되는 것으로 심화 연구를 요한다.

• Tuberculosis and Respiratory Diseases
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• v.63 no.3
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• pp.251-260
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• 2007

Purpose: An imbalance of cell proliferation and cell apoptosis is an important mechanism in carcinogenesis. Capase 3, survivin and p53 have been identified as important members of the apoptotic related proteins. This study evaluated the proliferating cell nuclear antigen(PCNA), apoptosis, apoptotic related protein such as capase 3, survivin and p53 using urethane-induced mouse lung carcinogenesis, which provides reproducible steps from hyperplasia to adenocarcinoma. Methods: Urethane was administered to the ICR mice through an intra-peritoneal injection, The mice were sacrificed at 5, 15, and 25 weeks after urethane intervention. The sequential morphological changes and immunohistochemical expression of PCNA, apoptosis, capase 3, survivin, and p53 were examined during mouse lung carcinogenesis. Results: During carcinogenesis, the sequential histological changes were observed from hyperplasia of type II pneumocytes, to anadenoma, and ultimately to an overt adenocarcinoma. The PCNA Labeling index (LI) was 9.6% in hyperplasia, 23.2% in adenoma, and 55.7% in adenocarcinoma, respectively. The apoptotic LI was 0.24% in hyperplasia, 1.25% in adenoma, and 5.27% in adenocarcinoma. A good correlation was observed between the PCNA LI and apoptotic LI. The expression of caspase 3 was remarkable- i.e., 46.7% in adenocarcinoma, in contrast to 15% in hyperplasia and 16% in adenoma. Survivin was detected weakly in the alveolar hyperplasia and showed an increasing expressional pattern in adenoma and adenocarcinoma. p53 expression was detected only in the adenocarcinoma lesions with an expression rate of 13.3%. The level of caspase 3 expression correlated with the increase in the apoptotic index. The positive expression of caspase 3 was associated with an increased apoptotic index. Conclusions: These results suggest that the PCNA LI and apoptotic LI might be useful markers for evaluating the risk of a malignant transformation. In addition, caspase, survivin and p53 might play a role in the early and late steges of urethane-induced mouse lung carcinogenesis.

• Applied Microscopy
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• v.37 no.4
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• pp.239-248
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• 2007

Alteration of temperature is one of cancer therapies. In general, severe hyperthermia(around $43^{\circ}C$) and hypothermia(around $18^{\circ}C$) trigger apoptosis through mitochondria, though the specific mechanism is still unknown. CC-t6 and GB-d1 cell lines, which were originally derived from human cholangiocarcinoma and gall bladder cancer, were established from a metastatic lymph node. To investigate the mechanism of metastatic cancer cell response to thermal stresses, hyperthermia($37^{\circ}C{\rightarrow}43^{\circ}C$) and hypothermia($37^{\circ}C{\rightarrow}17.4^{\circ}C$) were designed. Thermal stresses did not induce apoptosis but necrotic cell death. Any alterations of caspase-3, -9, cytochrome c, Bax, and Bcl-2 were not found in both hyperthermia and hypothermia exposed fells using western blot analysis. In the transmission electron microscopy, typical necrotic, but not apoptotic, changes were observed. These results suggest that temperature changes induce cell death through necrotic pathway in metastatic cancer in vitro, and it can be one of effective anticancer methods.

• Journal of Oral Medicine and Pain
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• v.34 no.3
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• pp.261-276
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• 2009

Lactacystin, a microbial natural product synthesized by Streptomyces, has been commonly used as a selective proteasome inhibitor in many studies. Proteasome inhibitors is known to be preventing the proliferation of cancer cells in vivo as well as in vitro. Furthermore, proteasome inhibitors, as single or combined with other anticancer agents, are suggested as a new class of potential anticancer agents. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying induction of apoptosis in SCC25 human tongue sqaumous cell carcinoma cell line treated with lactacystin. The viability of SCC25 cells, human normal keratinocytes (HaCaT cells) and human gingiva fibroblasts (HGF-1 cells), and the growth inhibition of SCC25 cells were assessed by MTT assay and clonogenic assay respectively. The hoechst staining, hemacolor staining and TUNEL staining were conducted to observe SCC25 cells undergoing apoptosis. SCC25 cells were treated with lactacystin, and Western blotting, immunocytochemistry, confocal microscopy, FAScan flow cytometry, MMP activity, and proteasome activity were performed. Lactacystin treatment of SCC25 cells resulted in a time- and does-dependent decrease of cell viability and a does-dependent inhibition of cell growth, and induced apoptotic cell death. Interestingly, lactacytin remarkably revealed cytotoxicity in SCC25 cells but not normal cells. And tested SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA contents, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, the up-regulation of Bax, and the activation of caspase-7, caspase-3, PARP, lamin A/C and DFF45 (ICAD). Flow cytometric analysis revealed that lactacystin resulted in G1 arrest in cell cycle progression which was associated with up-regulation in the protein expression of CDK inhibitors, $p21^{WAF1/CIP1}$ and $p27^{KIP1}$. We presented data indicating that lactacystin induces G1 cell cycle arrest and apoptois via proteasome, mitochondria and caspase pathway in SCC25 cells. Therefore our data provide the possibility that lactacystin could be as a novel therapeutic strategy for human tongue squamous cell carcinoma.

• Journal of the Korean Society of Radiology
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• v.10 no.6
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• pp.387-393
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• 2016

Tumor using the efficient concomitant radiotherapy and chemotherapy to remove, prior to surgery and, either reduce the size of the tumor after surgery, or was can be made smaller, Or excised tumor, in a way to be removed, most conventional surgical method is surgical excision surgery therapy. And methods reduce or tumor size, or smaller, chemotherapy can kill tumor is administered selectively anticancer agent which increases the radioactive susceptible to tumor cells, sensitive to susceptibility to radiation are those which make it possible to respond to, either TRAIL methods of various biological cytostatic can deform the protein, by deforming the structure of the protein help to cell death it is known. In this paper, the HCT-116 cells thought to be a cancer cell to analyze the interaction of TRAIL and radiation. Experimental results, single use of TRAIL and radiation, results were compared with the control group, it was found to have no significant effect on each cell proliferation and apoptosis. Conversely treated with TRAIL, when treated in parallel radiation, it was possible to know that the HCT-116 cells significantly apoptosis occurs, The proportion of G1 ratio G0 also was found to have increased. TRAIL conclusion is increased apoptosis radiation defensive cells can know that increased radiosensitivity, also possible to alter the cell cycle, reduce cell proliferation ability stepwise it was possible. TRAIL is increased apoptosis, decreased cell proliferative capacity, it is considered to be possible to use as a radiation sensitizer.

• Journal of Oral Medicine and Pain
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• v.36 no.1
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• pp.11-20
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• 2011

Valproic acid (VPA) is a well-known anticonvulsive agent and has been used in the treatment of epilepsy for almost 30 years. VPA emerged in 1997 as an antineoplastic agent. And it is known that antitmor activity of VPA is associated with its targeted at histone deacetylases. 17AAG, Inhibition of HSP90 leads to the proteasome degradation of the HSP90 client proteins, such as Akt, Raf/Ras, Erk, VEGF, cyclin D and p53, and causes potent antitumor activity. It is reported that 17AAG-induced HSP90 inhibition results in prevention of cell proliferation and induction of apoptosis in several types of cancer. This study was undertaken to investigate the synergistic apoptotic effect of co-treatment with the histone deacetylases inhibitor, VPA and the HSP90 inhibitor, 17AAG on human osteosarcoma (HOS) cells. Cell viability was evaluated by trypan-blue exclusion. Induction and augmentation of apoptosis were confirmed by Hoechst staining, flow cytometry (DNA hypoploidy and MMP change), Westen blot analysis and immunofluorescent staining. In this study, HOS cells co-treated with VPA and 17AAG showed several lines of apoptotic manifestation such as nuclear condensations, the reduction of MMP, the decrease of DNA content, the release of cytochrome c into cytosol, the translocation of AIF onto nuclei, and activation of caspase-3, caspase-7 and PARP whereas each single treated HOS cells did not. Although the single treatment of 1 mM VPA or 0.5 ${\mu}M$ 17AAG for 48 h did not induce apoptosis, the co-treatment with them induced prominently apoptosis. Therefore our data in this study provide the possibility that combination therapy with VPA and 17AAG could be considered as a novel therapeutic strategy for human osteosarcoma.

• Journal of the korean academy of Pediatric Dentistry
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• v.33 no.1
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• pp.13-24
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• 2006

Neuronal apoptotic events, consequently resulting in neuronal cell death, are occurred in hypoxic/ischemic condition. This cell death has been shown to be accompanied with the production of reactive oxygen species (ROS), which can attack cellular components such as nucleic acids, proteins and phospholipid. However, the underlying mechanisms of apoptosis induced in hypoxic/ischemic condition and its treatment methods are unsettled. Cobalt chloride $(CoCl_2)$ has been known to mimic hypoxic condition including the production of ROS. Epigallocatechin gallate (EGCG), a green tea polyphenol, has diverse pharmacologial activities in cell growth and death. This study was aimed to investigate the apoptotic mechanism by $CoCL_2$ and effects of EGCG on $CoCl_2-induced$ apoptosis in PC12 cells. Administration of $CoCl_2$ decreased cell survival in dose- and time-dependent manners and induced genomic DNA fragmentation. Treatment with $100{\mu}M$ EGCG for 30 min before PC12 cells were exposed to $150{\mu}M$ $CoCl_2$, being resulted in the cell viability and DNA fragmentation being rescued. $CoCl_2$ caused morphologic changes such as cell swelling and condensed nuclei whereas EGCG attenuated morphologic changes by $CoCl_2$. EGCG suppressed the apoptotic peak and a loss of ${\Delta}{\psi}_m$ induced by $CoCl_2$. $CoCl_2$ decreased Bcl-2 expression but Bax expression was not changed in $CoCl_2$- treated cells. EGCG attenuated the Bcl-2 underexpression by $CoCl_2$. $CoCl_2$ augumented the cytochrome c release from mitochondria into cytoplasm and increased caspase-8, -9 and caspase-3 activity a marker of the apoptotic executing stage. EGCG ameliorated the incruement in caspase-8, -9 and -3 activity, and cytochrome c release by $CoCl_2$ NAC (N-acetyl-cysteine), a scavenger of ROS, attenuated $CoCl_2-induced$ apoptosis in consistent with those of EGCG. These results suggest that $CoCl_2$ induces apoptotic cell death through both mitochondria- and death receptor-dependent pathway and EGCG has neuroprotective effects against $CoCl_2-induced$ apoptosis in PC12 cells.

• Journal of Life Science
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• v.19 no.8
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• pp.1047-1054
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• 2009

Oral squamous carcinoma (OSC) cells present resistance to chemotherapeutic agents-mediated apoptosis in the late stages of malignancy. Advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers. It was demonstrated here that Pseudomonas aeruginosa exotoxin A (PEA) significantly decreased the viability of chemoresistant YD-9 cells in the apoptosis mechanism. Apoptotic manifestations were evident through changes in nuclear morphology and generation of DNA fragmentation. PEA treatment induced caspase-3, -6 and -9 cleavage, and activation. These events preceded proteolysis of the caspase substrates poly (ADP-ribose) polymerase (PARP), DNA fragmentation factor 45 (DFF45), and lamin A in YD-9 cells. The reduction of mitochondrial membrane potential, release of cytochrome c and SmacjDlABLO from mitochondria to cytosol, andtranslocation of AlF into nucleus were shown. While p53, p21 and $14-3-3{\gamma}$ were upregulated, cyclin Band cdc2 were downregulated by PEA treatment. Taken together, PEA induces apoptosis in chemoresistant YD-9 cells via activation of caspases, mitochondrial events and regulation of cell cycle genes.