In this study, the cytotoxicity of commonly used local anesthetics was evaluated on odontoblasts which are essential for pulpal homeostasis in vitro. Local anesthetics, such as articaine, bupivacaine, levobupivacaine, lidocaine, mepivacaine, prilocaine, and procaine, were tested on the odontoblast cell line, MDPC-23. The concentration-and time-dependent cytotoxic effects of local anesthetics on odontoblasts were measured by MTT assay. Among local anesthetics treated for 18 h, only bupivacaine significantly showed cell death in a concentration-(LC50=1.2mM) and time-dependent manner. To confirm cell death induced by bupivacaine, the observation of cell morphology and FACS using Annexin V and propidium iodide (PI) staining were performed. As a result of Annexin V and PI staining, as well as the morphological change, only bupivacaine induced apoptotic cell death on odontoblasts when compared with levobupivacaine and lidocaine. These results suggest that bupivacaine might affect normal pulpal integrity even after uneventful local anesthesia.

Cordyceps militaris is well known as a traditional herbal ingredient, which has been used for patients suffering from cancer in oriental medicine. In this study we have investigated the biochemical mechanisms of anti-proliferative effects by C. militaris extract(CBE) in human breast cancer MDA-MB-231 cells. It was found that CBE treatment induced chromatin condensation, mitochondrial energization, annexin V staining and sub-G1 phase DNA content. These indicators of apoptosis correlate with the mitochondrial dependent pathway, which results in the activation of caspase-3 activity. Both the cytotoxic effect by CBE treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, demonstrating the important role of caspase-3 in the observed cytotoxic effect. Co-treatment of CBE and LY294002, resulted in significantly induction of apoptosis. These results indicate that caspase-3 is a key regulator of apoptosis in response to CBE in human breast cancer MDA-MB-231 through down regulation of Akt, and that the C. militaris extract may therefore have therapeutic potential against human breast cancer.

Apoptos is s ignal- regulating kinase 1 (ASKl) is a rnitogen-activated protein kinase kinase kinase(MAP3K) 떼 th proapoptotic functlOn 1'he kinase activity of ASKl is stimulated by a variety of death signals , including 1'NFα • Fas ligation, reactive oxygen species, and antineoplastic agents, ASKl promotes cell death by activating the c- Jun N-termina l kinase/stress-activated protein kinase MKK4/MKK7-JNK/SAPK pathway and MKK3/ 1\αCK6-p38 pathway‘ ASKl activity is highly controlled in cells by multiple mechanisms, including phosphorylation, oligome ri zation, and protein- protein ll1 teractlOns Epigallocatechin-3-galla te(EGCG) is the major bioactive polyphenol present in green tea, It possesses anti-oxida nt , a nti - mutagenic‘ a n ti - prote이 ytic ， and anti-proliferative activity, In addition. it has been shown to inhibit cyclin activity, and inhibit cell cycle progression 1n the present study, we exarnined the effect of EGCG on ASKl- overexpressed cells , We expected that EGCG contributes to cell a poptos is by activating ASKl functlOn However, EGCG showed no suppressive effect on cell s urvival of ASKl-overexpressed cells and seemed to promote cell survlval Importantly, the EGCG treatment in creased Akt activity when cells expressed enough amount of ASKl protein, These results s uggest that the presence of ASKl may modify the inhibitory effect of EGCG on cell survival through Akt pathway,

Indirubin is the active ingredient 0 1' a traditional Chinese herbal medicine, Danggui Longhui Wan, used for the t reatment of chronic myelocytic leu kelma The author reports that novel indirubin derivative, 5'-nitro-indirubinox ime (5'-NIO) , has potent a nti -proliferative activity on human o1'al cancer cells , Treatment of KB cells with 5’ NIO s howed a s t rong cell growth inhibit ion during indicated time point , A typical apoptosis pattern was obtained with DNA fragmenta tion and i mmunofl uorescence analysis of annelxn-v-f!ous Western blot data revealed that p53 and p21CIP1/Waf- l level increased strongJy upon 5'-NIO treatment , The a uthor next tested whether 5'-NIO could activate apoptos is related proteins s uch as caspase-3/-7/-9, [n cells exposed to 5‘-NIO, activation of caspase-3 and -7 was observed, Interesti ngly‘ caspase-9 and cytochrome c cou ld s li ghtly activate in response to 5’-NIO. These results indicate that 5’-NIO strongly induces oral cancer cells apoptosis via a Mi tochondria-mediated cas pase cascade pathway, These observations together s uggest that 5’ NTO may have a possible therapeutic potentia[ to oral cancer

Chromosomal abnormality s uch as aneuploidy is one of the main factors to cause cancers This abnormality is caused by defects in centrosomal duplication‘ and most cancer cells have extra copies of centrosomes, r esulting in t he formation of multipolar spindles. Several kinases playing in mitotic phase have been implicated in regulating the centrosomal cycle‘ spindle checkpoint‘ and chromosome co ndensation and segregation. They also have Lhe ability to act as oncogenes. FOl studying the relationship between rnitotic kinase and oral cancers, the kinase activity of polo-like kinase-1, which is one of mitosis-specific kinases, is analyzed in oral carcinoma cells originated differently. Kinase activity was increased in these cancer cells compared to normal human gingival fibroblast primary cultured cells Moreover. the mitotic cell populations were a lso increased in these cell lines. Whereas the inhibition of Polo-like kinase-1 by C-terrninal domain in human gingival fibroblast cells induced multiploidy‘ the apoptotic cell population was increased in oral cancer cells overexpressed C-terminal domain 0 1' Polo- Ii ke kinase-1. These data suggested that polo-like kinase-1 might be involved in the on cogenic effect in oral cancer like other solid human carcinomas, and be target molecules for anti-cancer drug.

Many naturally occurring plant extracts are studied for their beneficial effects for health and particularly on cancer. Apoptosis, or programmed cell death, occurs in both normal and pathological conditions, including cancer Dysregulation of apoptosis allows transformed cells to continually and uninhibitedly enter the cell cycle, thus perpetuating the sequence of mutation, genomic instability and, finally, oncogenesis. To investigate the apoptosis-inducing effect of the extract of Fructus Trichosanthis (EFT) on leukemic HL-60 cells and its mechanism, HL-60 cells in vitro in culture medium were given different doses of the extract. The inhibitory rate of cells were measured by microculture tetrazolium assay, cell apoptotic rate was detected by flow cytometry, morphology of cell apoptosis was observed by DAPI fluorescence staining, and the activations of caspases and PARP were detected using Western blotting analysis. The extract could activate the caspase-3 and caspase-8, induce PARP cleavage, inhibit growth of HL-60 cells, and cause apoptosis significantly The suppression was in dose-dependent manner. Marked morphological changes of cell apoptosis including condensation of chromatin and nuclear fragmentation were observed clearly by DAPI fluorescence staining especially. These results will provide strong laboratory evidence of EFT for clinical treatment of acute leukemia.

Agaricus blazeiMurill is an edible mushroom distributed in Brazil and presently cultivated in other areas, including Korea, Japan, and China. Its chemical components, including steroids and lectin and various polysaccharides have been widely studied. For this, we used U937/vector and U937/Bcl-2 cells, which were generated by transfection of the cDNA of the Bcl-2 gene. As compared with U937/vector, U937/Bcl-2 cells exhibited a 4-fold greater expression of Bcl-2. Treatment with 0.5 or 4 mg/ml A. blazei Murill for 24 h produced morphological features of apoptosis in U937/vector cells, respectively. This was associated with caspase-3 activation and PARP degradation. In contrast, A. blazei Murill-induced caspase-3 activation and PARP degradation and apoptosis were significantly inhibited by z-DEVD-fmk in U937 cells. Bcl-2 overexpressing cells exhibited sustained caspase-3 activation and expression levels of the Bcl-2 proteins during A. blazei Murill-induced apoptosis. In addition, these findings indicate that Bcl-2 inhibits A. blazei Murill-induced apoptosis by a mechanism that interferes with Bcl-2 degradation and activity of caspase-3 that is involved in the execution of apoptosi.

본 연구는 배양 각막 상피세포에 anti - F AS and anti - F AS ligand antibody를 노출시 킨 후 세포고사 메커니즘을 결정하기 위해 시행하였다. 배양각막 상피세포에 antihuman FAS(N-18) goat polyclonal IgG를 50, 200, 500, 1,000 ng/m~ 또는 anti-human FAS ligand(C• 20) goat polyclonal IgG를 500 ng/m~으로 2 일과 4 일 동안 처 리 하였다. 주어진 기간 동안 배양한 후 배양액에 부유한 세포와 부착된 세포를 원심분리와 트립 신 처리 원섬분리를 이용하여 수확하였다. 각막상피세포에 대한 anti - F AS and antiF AS ligand antibody 의 영향을 알아보기 위해 Hoechst 33342 staining과 TUNEL stammg 방법을 이용하여 세포 세포고사 유도를 확인하였다. 세포막 수용체인 FAS protein의 발현을 알아보기 위해 ìmmunocytochemistry 를 시행하였다. anti-FAS antìbody를 처리한 군에서는 대조군에 비해 시간과 농도에 비례하여 후기 세포고사 소 견이 증가하였다. 그러나 anti - F AS ligand antibody를 처리한 군에서는 대조군과 차이 가 거의 없었다. 본 연구의 결과 FAS-FAS ligand system 이 각막상피세포에 발현되었 으며 이는 정상 각막 상피 생리 즉， 세포 탈피에 중요한 기능을 갖는 것으로 사료된다

본 연구는 활성화처리 방법 및 배양 조건이 돼지 단위발생란의 체외발달 및 apoptosis에 미치는 영향을 알아보기 위해 실시되었다. 도축장 유래 난소로부터 채취된 미성숙 난자를 42~44시간 동안 성숙배양한 후 사용하였다. Apoptosis는 TUNEL 방법을 사용하여 조사하였다. 실험 1에서는 성숙배양된 난자들을 electric pulse(1.2 kV/cm for 30μsec 2회, E), E + 6-dimethylaminopurine(6-DMAP) 또는 E + cycloheximide(CH) 방법으로 활성화 처리하여 PZM-3를 이용하여 5% CO2, 38.5℃에서 배양하였다. 실험 2에서는 전기자극을 이용하여 활성화처리된 난자들을 각각 PZM-3 또는 NCSU-23 배양액 내에서 배양하였다. 각 배양액 내의 난자들은 각각 20% O2 조건으로 나뉘어 배양하였다. E + 6-DMAP(36.5%) 또는 E + CH 구(32.5%)에서 E 구(27.7%)보다 유의적으로 높은 배반포 형성율을 보였다(P<0.05). 처리별 apoptosis 발생율은 각각 5.3%(E), 7.7%(6-DMAP) 및 7.1%(CH)였다. 실험 2에서는 PZM-3 구의 배반포 형성율이 NCSU-23 구에 비하여 산소분압조건과 관계없이 다소 높았다(28.2{sim}29.7% vs. 22.6~24.4%). PZM-3 및 20% O2 조건하에서 유의적으로 낮은 apoptosis 발생 비율을 나타냈다(9.2%, P<0.05). 그러므로 돼지 단위발생란을 chemical agent를 이용한 추가 활성화처리 후 PZM-3, 20% O2, 조건으로 배양하면 더 나은 배반포 발생율을 얻을 수 있다고 생각된다.

Resveratrol (3,4',5-trihydroxy-trans-stilbene), a naturally occuring polyphenol compound which present in the skin of grapes and red wine has been considered to posses chemopreventive and antioxidant properties. However, little is known about the cellular actions by which resveratrol mediates its therapeutic effects. In this study, the effect of resveratrol on cell proliferation and induction of apoptosis in human osteogenic sarcoma (HOS) cells was investigated. IC50 value was determined to be approximately 60μg/mℓ. Chromosomal DNA framgmentation analysis showed the appearance degraded DNA in time-and dose-dependent manner upon treatment of resveratrol. In order to observe the molecular mechanism involved in resveratrol-induced apoptosis, Western blot analysis was performed. We observed the decrease in the level of procaspase-3, the zymogen form of active caspase-3 in resveratrol-treated cells. This result implies that caspase-3 is activated upon treatment of resveratrol. The activation of caspase-3 was confirmed by the cleavage of poly(ADP-ribose) polymerase. Taken together, our data demonstrate that resveratrol has anti-proliferative effect on HOS cells and induced apoptosis through activation of caspase-3 and PARP cleavage.

본 연구는 topoisomerase inhibitors가 배양 각막 상피세포에서 세포고사를 유도하 는지를 조사기 위해 시행하였다. Topoisomerase inhibitors'{l camptothecin과 etoposide 를 제조사의 추천농도로 1-2 일 동안 배양하여 MTT assay를 이용하여 세포독성을 검 정하여 농도틀 정하고 세포고사를 확인하였다 세포고사의 형태적인 특징은 Hoechst 33342 staining, Annexin V - FITC/PI staining, DePsipher assay and CytoDEA TH staining을 이용하여 확인하였고 DNA fragmentation은 TUNEL파 agarose gel 전기 영동으로 확인하였다. Camptothecin 과 etoposide는 농도 의존성으로 세포고사룹 유또 하였는데 각막상피세포에서는 저l 조사의 추천농도보다 낮은 농도에서 세포고사플 유 도하여 다른 세포보다 민감한 것으로 나타났다- 이러한 결파로 볼 때 topOlsomerase l띠1ibitor는 각막상피세포에서 홀륭한 양성 대조군으로 활용될 수 있을 것으로 사료된다.

The purpose 01' pl'esent study was to examine the molecular events in apoptosis by CoCl2, mimicking hypoxic cond ition and recovering effects by LED ir l'adiation on Human SH-SY5Y neuroblastoma cells The SOUl'ce 0 1' light for ir l'adiation was a continuous-wave LED emitting at a wavelenl양h of 590 nm, and manufactured that ene rgy density was 5 mW!cm2 on sample surface, After ir l'adiation, cell viabi lity was measured with BrdU , cell morphol ogy was examined with Diff- Quik staining, cell signaling was monitored with various apoptosis-related molecules using RNase Pl'otection Assay(RPA) , W11en treated with CoC12, apoptotic induction was found in the SH-SY5Y cells in a concentration-dependent and time-dependent manner , Diff-Quik s taining was revealed that DNA fragmentation re presented apoptosis was examined in CoC12-tl'eated group, Moreover, RPA assay of SH-SY5Y cclls lIs ing val'iolls apoptosis-related molecllles showed that the apoptotic cell population was mcreased J-loweve. there was sorne signifïcant change in LED irradiatied cells aftel' treatement of CoC12 The main mechanism for Lhese a poptosis appearecl to be mito c hondriεt - m ecliated pathway, such as cytochrome- c‘ caspase-9, caspase-3, pro-apototic protein ßax, anti-apototic protein Bcl-2, and death receptor• mediated pathway, such as Fas, cas pase- 8, a ncl TNFRl These results demonstrate that CoCI2 induce apoptosis in SH-SY5Y via different dual apop tosis pathway through death receptor pathway as well as mitochondria- dependent pathway and LED irradiation can recl llces the CoCl2-induced apoptosis by blocking their internal signaling pathway

1'0 determine Lhe ll1echanism of cell c1eath incluced by iron chelators. we explored the pathways of the three structurally relatecl ll1 itogen-activatecl protein(MAP) kinase subfami li esduring iron cbelator- inclucecl apoptosis ancl differentiation of oral precancerous ancl cancel‘ cells. The iron chelator c1 eferoxamine(DFO) exertecl potent timeancl c1ose-c1epenclent inhi bitory effects on the growth of IHOK and HN4 cells The major mechanism of growth inhibition following DFO treatment was fOllncl to be apoptosis incluction. as assessecl by annexin V-FITC staining. cell cycle analysis‘ DNA lacldering, a ncl Hoechst staining. We report that DF'O s trongly activates the p38 MAP kinase and extracell ular signal- regu lated kinase(ERK). but c10es not activate the c-Jun N-terminal kin ase/ stl않s-activaLecl protein kinase(JNK/8APK) . Of the three MAP kinase blockers usecl‘ the selective p38 MAP kinase inhibitor 8ß203580 ancl ERK inhibitor PD98059 protected oral premaIignant ancl malignant cells againsL iron chelator- nclllced cell death. which incl icates that the p38 MAP kinase serves as a major mecliator 01' apoptos is induced by this iron chelator DFO also evoked the release of cytochrome c from mitochondria, and incluced the activation of caspase-3 ancl caspase-8 in oral cancer cells, which suggests that apoptosis occurs via the mi tochoncl ri on - mecl iaLed pathway. DFO enhanced the expression of Bax in IHOK ancl HN4 cells. consistent witll thei r p53 status Moreover. DFO downregulatecl the expression 01' Bcl-2 in oral cancer cells. which suggests that DFO- incluced apoptos is 01' oraJ cancer and precancerous cells may be mediatecl by an increase in the ratio of pro-apoptotic to anti-apoptotic proteins. ln terestingJy, trcatmcnt 01' IHOK ancl HN4 cel ls with 8B203580 abolishecl cytochrome c release‘ as wel l as the activation of caspase-3 and caspase-8. DFO suppressecl the expression of epithelial di ffe rentiation markers, such as involucrin, t ransglutaminase II. CK6. and CK19. ancl this suppression was blockecl by p38 ancl ERK MAP kinase ll1hlbltors The oral premalignant(IHOK) ancl malignant cell s(I-lN4) showed differential responses to DFO with respect to the expression of cel l cycle regulatory proteins. cell growth. ancl apoptosis. Coll ectively. the current study reveals that p38 MAP kinase plays an ill1 portant role in iron chela tor-mecliatecl cel l cleath and in the suppression of differentiation of oral premalignantandmalignanLcell s.by activating a c10wnstream apoptotic cascade that executes the ceIl c1eath pathway