• Title, Summary, Keyword: PARP fragmentation

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Expression of Poly (ADP-ribose) Polymerase During Apoptosis Induced by Ultraviolet Radiation in HeLa $S_3$ Cells

  • Chang, Jeong-Hyun;Kwon, Heun-Young
    • Biomedical Science Letters
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    • v.12 no.4
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    • pp.349-354
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    • 2006
  • Induction of apoptosis allows the organism to get rid of abnormal cells and also of tumor cells. Understanding the mechanism involved in Ultraviolet radiation (UV) induced apoptosis may improve its therapeutic efficacy. In this study, we present expression of poly (ADP-ribose) polymerase (PARP) during apoptosis induced by UV in HeLa $S_3$ cells. Four different assays were performed in this study: morphological assessment of apoptotic cells and cell viability, DNA fragmentation analysis by agarose gel electrophoresis, quantitative assay of fragmented DNA, and expression of PARP by the western blot analysis. The percentages of apoptotic HeLa $S_3$ cells irradiated with $75J/m^2$ UV was increased continuously from 3 hrs incubation. DNA ladder pattern was appeared at 6 hrs. The amount of nucleosomal DNA fragments in cells treated UV increased from 3 to 12 hrs incubation and gradually decreased. The cleavage of PARP in HeLa $S_3$ cells irradiated with UV was induced, and the cleavage of PARP was more delayed in the cells pretreated with $5J/m^2$ UV and subsequently irradiated with $75J/m^2$ UV. than that in the cells only irradiated with $75J/m^2$ UV. Thus these data suggest that the cleavage of PARP relates with DNA fragmentation associated with apoptosis.

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Induction of Apoptosis by Camptothecin in HL-60 Cells (HL-60 세포에서 Camptothecin의 apoptosis 유도작용)

  • 김해종;천영진;김미영
    • YAKHAK HOEJI
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    • v.43 no.3
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    • pp.385-390
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    • 1999
  • Camptothecin (CPT) has been known to induce apoptosis in various cancer cell lines. To examine the intracellular apoptotic death signal initiated by CPT, we investigated the possible connection between caspase-3 activation and GSH depletion during CPT-induced apoptosis in HL-60 cells. Treatment of cells with $1{\;}{\mu}M$ CPT induced PARP cleavage accompanied by DNA fragmentation. z-VAD-fmk, a caspase-3 inhibitor, blocked the CPT-induced DNA fragmentation. Pretreatment of cells with N-acetylcysteine, a precursor of GSH biosynthesis, failed to inhibit CPT-induced PARP celavage and DNA gragmenatation. No significant changes in GSH depletion is not essential for caspase activation during CPT-induced apoptosis. We also investigated whether CPT-induced apoptosis is associated with changes of the levels of Bax and Bcl-2, two proteins involved in the control of apoptosis. Bcl-2 levels exhibited a late decrease compared with the kinetics of DNA fragmentation, whereas Bax levels increased more rapidly after CPT treatment. These results suggest that Bax plays more important role than Bcl-2 in inducing DNA fragmentation and may function upsteam of proteolytic activation of caspase-3 pathway in CPT-induced apoptosis.

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Ultraviolet Radiation-Induced Apoptosis is Inversely Correlated with the Expression Level of Poly(ADP-ribose) Polymerase

  • Oh, Kyu Seon;Lee, Dong Wook;Chang, Jeong Hyun;Moon, Yong Suk;Um, Kyung ll
    • Animal cells and systems
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    • v.5 no.1
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    • pp.77-83
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    • 2001
  • The present study was conducted to elucidate whether the expression level of poly(ADP-ribose) polymerase (PARP) is related to the ultraviolet radiation (UV)-induced apoptosis. After treatment of the mammalian cell lines HeLa S3 and Chinese hamster ovary (CHO) with 50 J/m2 UV, induction of apoptosis was determined by several means during 24 h post-incubation. Incidence of apoptosis was much lower in CHO than HeLa S3 cells based on the percentage of apoptotic cells in terms of morphological changes in nucleus or direct counting of viable cells and qualitative or quantitative DNA fragmentation. Interestingly, when the expression level of PARP was measured by western blotting, the amounts of PARP that was retained at each time point inversely correlated with the incidences of apoptosis in these cells. Concomitant with generation of the 85 kDa fragment, 116 kDa PARP disappeared in HeLa S3 within 6 h after UV treatment, whereas a fair amounts of 116 kDa band was still retained in CHO cells at 36 h post-incubation. This inverse relationship was also observed in the adaptive response system, in which cells weve treated with a high dose of UV after pretreatment with a low dose. As expected, typical adaptive responses appeared in CHO cells but not in HeLa cells, showing greater cell viability and lesser DNA fragmentation. During the adaptive response in CHO cells, PARP was expressed at much higher level compared to the single, high dose-treated cells. Interestingly, even though PARP was induced at 6 h post-incubation In both cell types, its expression was more prominent in CHO cells. Thus, our data indicate that the retained level of intact PARP against UV damage inversely correlates with incidence of apoptosis in mammalian cells, and also suggest that a machinery to protect the PARP degradation against UV damage exists in CHO but not in HeLa S3 cells.

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The Essential Oil of Artemisia iwayomogi Kitamura Induces Apoptosis on Human Oral Epidermoid Carcinoma Cells

  • Jeong, Mi-Ran;Cha, Jeong-Dan;Lee, Kyung-Yeol;Kil, Bong-Seop;Han, Jong-Hyun;Lee, Young-Eun
    • Food Science and Biotechnology
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    • v.16 no.4
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    • pp.531-536
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    • 2007
  • The aerial part of Artemisia iwayomogi Kitamura has traditionally been used for inflammation, infectious disease, cancer, pyretic, diuretic, liver protective effect, and choleretic purposes in Korea. We investigated that the essential oil induces apoptosis in KB cell as evidenced by Hoechst-33258 dye staining, flow cytometry (cell cycles), and DNA fragmentation for nuclear condensation and Western blotting for activation of caspases-3, -8, -9, Bax, Bcl-2, cytochrome c, and poly (ADP-ribose) polymerase (PARP) cleavage. In the present study, we found that the essential oil could induce apoptosis in KB cells, as characterized by DNA fragmentation, activation of caspase-3, -8, and -9, and PARP cleavage. The efficacious induction of apoptosis was observed as a dose-dependent. The essential oil-induced apoptotic cell death was accompanied by up-regulation of Bax and down-regulation of Bcl-2. The essential oil also caused the loss of mitochondrial membrane potential and cytochrome c release from mitochondria to cytosol. These findings indicate that mitochondrial pathways might be involved in the essential oil-induced apoptosis and enhance our understanding of the anticancer function of the essential oil in herbal medicine.

Effect of Butanol Fraction of Mylabris phalerata on Induction of Apoptosis in U937 cells (반묘 BuOH층의 U937 세포주에 대한 apoptosis유도 효과)

  • 허정은;윤택준;이종수;정진홍;김성훈
    • YAKHAK HOEJI
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    • v.45 no.5
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    • pp.484-490
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    • 2001
  • Mylabris phalerata(MP) is an insect that has been used for the treatment of cancer in oriental medicine. To evaluate the anticancer activity of Mylabris phalerata, We measured the cytotoxicity of Mylabris phalerata solvent fractions such as MC, EA, BuOH and residual layers on U937, human monocytic leukemia cells. Of those fractions BuOH layer of Mylabris phalerata was the most effective with ID$_{50}$ of 140$\mu\textrm{g}$/ml. It effectively caused DNA fragmentation from the concentration of 50$\mu\textrm{g}$/ml, showed apoptotic nucleus by tenets assay and expressed apototic portion stained by Annexin-V. It also induced the activation of caspase-3 and cleavage of the substrate poly (ADP-ribose) polymerase (PARP). These results suggest BuOH layer of Mylabris phalerata exerts anticancer activity by induction of apoptosis via activation of caspase-3 protease.e.

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An Experimental Study on Apoptosis of cultivated Wild Ginseng Distilled Herbal Acupuncture by Concentration Level (농도별(濃度別) 산양산삼(山養山蔘) 증류약침(蒸溜藥鐵)의 Apoptosis에 관(關)한 실험적(實驗的) 연구(硏究))

  • Cho, Hee-Chul;Lee, Sun-Gu;Kwon, Ki-Rok
    • Journal of Pharmacopuncture
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    • v.7 no.2
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    • pp.5-17
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    • 2004
  • Objective : In order to measure the efficacy of cultivated wild ginseng distilled herbal acupuncture by concentration level, we've treated A549 human lung cancer lines with different concentrations of cultivated wild ginseng distilled herbal acupuncture and examined mRNA and proteins which take parts in apoptosis. Methods : A549 human lung cancer lines were treated with various concentration levels of cultivated wild ginseng distilled herbal acupuncture and cell toxicity was carefully examined. From the analysis of DNA fragmentation, RT-PCR and Western blot, manifestation of mRNA and proteins which are associated with apoptosis were inspected. Results : The following results were obtained on apoptosis of A549 human lung cancer lines after administering various concentration levels of cultivated wild ginseng distilled herbal acupuncture. 1. Measuring cell toxicity of lung cancer cells, strong cell toxicity was detected at high concentration level(1000ul, 1200ul), but no consistent concentration dependent reliance was detected. 2. Through DNA fragmentation, we were able to confirm cell destruction in all groups. 3. Experiment groups treated with cultivated wild ginseng distilled herbal acupuncture showed inhibition of Bcl-2 and COX-2 at mRNA and Protein level, whileas increase of Bax was shown. 4. Manifestation of p21, p53, Cyclin E, and Cyclin Dl were confirmed in all groups. 5. Extrication of Cytochrome C was detected at all groups, as well as increased activity of the enzyme caspase-3 and caspase-9, and PARP fragmentation were confirmed. Conclusion : According to the results, we can carefully deduce cell destruction of A549 human lung cancer lines were induced by Apoptosis. At the fixed level, cultivated wild ginseng distilled herbal acupuncture showed decrease of Bcl-2 and COX-2, as well as increase of Bax. Since cultivated wild ginseng distilled herbal acupuncture increases manifestation of p21, p53, Cyclin E, and Cyclin Dl, it affects cellular cycle and through these phenomena, we can consider extrication of Cytochrome C, increase of caspase, and PARP fragmentation are the results.

An Experimental Study on Apoptosis of Cultivated Wild Ginseng Distilled Herbal Acupuncture by controlled pH and Electrolyte (pH 및 전해질 조절 산양산삼(山養山蔘) 증류약침(蒸溜藥鍼)의 Apoptosis에 관(關)한 실험적(實驗的) 연구(硏究))

  • Chu, Ching sheng;Lee, Sun-gu;Kwon, Ki-rok
    • Journal of Acupuncture Research
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    • v.21 no.6
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    • pp.1-17
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    • 2004
  • Objective : To compare and examine how adjustment of pH and electrolyte can affect the efficacy of cultivated wild ginseng distilled herbal acupuncture, we've administered pure cultivated wild ginseng distilled herbal acupuncture and pH and electrolyte adjusted cultivated wild ginseng distilled herbal acupuncture on A549 human lung cancer lines. Then mRNA and proteins which take parts in apoptosis were examined. Methods : Pure cultivated wild ginseng distilled herbal acupuncture treated group was set as the control group and pH and electrolyte adjusted cultivated wild ginseng distilled herbal acupuncture groups were administered on A549 human lung cancer lines. Cell toxicity was carefully examined and from the analysis of DNA fragmentation, RT-PCR, and Western blot, manifestation of mRNA and proteins which are associated with apoptosis were inspected. Results : The following results were obtained on apoptosis of A549 human lung cancer lines after administering pH and electrolyte adjusted cultivated wild ginseng distilled herbal acupuncture. 1. Measuring cell toxicity of lung cancer cells, higher cell toxicity was detected at pH and electrolyte adjusted groups and the results were concentration dependent. 2. Through DNA fragmentation, we were able to confirm cell destruction in all groups. 3. Experiment groups treated with cultivated wild ginseng distilled herbal acupuncture showed inhibition of Bcl-2 and COX-2 at mRNA and Protein level, whileas increase of Bax was shown. 4. Manifestation of p21, p53, Cyclin E, and Cyclin D1 were confirmed in all groups. 5. Extrication of Cytochrome C was detected at all groups, as well as increased activity of the enzyme caspase-3 and caspase-9, and PARP fragmentation were confirmed. Conclusions : From the above results, we can carefully deduce cell destruction of A549 human lung cancer lines were induced by Apoptosis. At the same concentration level, cell destruction efficacy was better with adjusted pH and electrolyte. Cultivated wild ginseng distilled herbal acupuncture also showed decrease of Bcl-2 and COX-2, as well as increase of Bax. Since cultivated wild ginseng distilled herbal acupuncture increases manifestation of p21, p53, Cyclin E, and Cyclin D1, it affects cellular cycle and through these phenomena, we can consider extrication of Cytochrome C, increase of caspase, and PARP fragmentation are the results.

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Induction of Apoptosis in Human Oral Epidermoid Carcinoma Cells by Essential Oil of Chrysanthemum boreale Makino

  • Cha, Jeong-Dan;Jeong, Mi-Ran;Lee, Young-Eun
    • Food Science and Biotechnology
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    • v.14 no.3
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    • pp.350-354
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    • 2005
  • The effect of the essential oil obtained from Chrysanthemum boreale Makino on the apoptosis of KB cells was investigated. Cytotoxicity and cellular DNA content were analyzed by MTT assay, flow cytometry, agarose gel electrophoresis, and Hoechst 33258 staining. The caspase-3 and poly (ADP-ribose) polymerase (PARP) proteins were estimated by Western blotting method. The various cytotoxic effects of the essential oil which are hallmarks of apoptosis, including DNA fragmentation, apoptotic body formation, and sub-G1 DNA content, all progressed in a dose-dependent manner. Treatment with an apoptosis-inducing concentration of the essential oil caused rapid and transient induction of caspase 3 activity. Further, the efficacious induction of PARP cleavage and caspase-3 activation was observed at an essential oil concentration of 0.1 and 0.2 mg/mL for 12 hr.

Inhibitory Effect of Apoptosis of Human Astrocytes by Juniper Oil (신경교(神經膠) 성상세포(星狀細胞)에서 쥬니퍼오일에 의한 세포자멸사(細胞自滅死)의 억제(抑制) 효과(效果))

  • Kim Tae-Heong;Kim Tae-Heon;Lee Sung-Ryull;Lyu Yeoung-Su
    • Journal of Oriental Neuropsychiatry
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    • v.11 no.2
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    • pp.1-9
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    • 2000
  • In previous studies, heat shock has been reported to induce the apoptosis or programmed cell death through the activation of caspase-3. 1 investigated the effect of juniper pure essential oil on the heat shock-induced apoptosis in human astrocyte cell line CCF-STTGI. Treatment of the astrocytes with heat shock markedly induced apoptotic cell death. However, pretreatment of the astrocytes with juniper oil ingibited the heat shock-induced apoptosis. To determine whether juniper inhibits the heat shock-induced activation of these apoptotic proteases, activation of CPP32 was assessed by Western blotting. Consistent with flow cytometry. DNA fragmentation and giemsa staining, heat shock-induced activation of CPP32 was blocked by juniper oil. Poly(ADP-ribose) polymerase (PARP), cysteine protease substrates were fragmented as a consequence of apoptosis by heat shock. Juniper oil inhibited the PARP fragmentation. This juniper oil also inhibited the heat shock-induced activation of caspase-3. These results suggest that juniper oil may modulate the apoptosis through the activation of the interleukin-1-converting enzyme-like protease.

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Inhibitory Effect of Lemon Oil on Apoptosis in Astrocytes (신경교(神經膠) 성상세포(星狀細胞)에서 레몬오일에 의한 세포자멸사(細胞自滅死)의 억제효과(抑制效果))

  • Kim, Jun-Han;Kim, Tae-Heon
    • Journal of Oriental Neuropsychiatry
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    • v.11 no.1
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    • pp.37-46
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    • 2000
  • We investigated the effects of lemon pure essential oils on the heat shock-induced apoptosis in human astrocyte cell line CCF-STTG1. In previous studies, hear shock has been reported to induce the apoptosis or programmed cell death through the activation of caspase-3. Treatment of CCF-STTG1 cells with heat shock markedly induced apoptotic cell death as determined by flow cytometry. Interestingly, pretreatment of CCF-STTG1 cells with lemon pure essential oils inhibited the heat shock-induced apoptosis. Lemon also inhibited the heat shock-induced apoptosis in primary cultured rat astrocytes. To determine whether lemon inhibits the heat shock-induced activation of these apoptotic proteases, activation of CPP32 was assessed by Western blotting. Consistent with flow cytometry, DNA fragmentation and giemsa staining, heat shock-induced activation of CPP32 was blocked by lemon pure essential oil. PARP, cysteine protease substrates were fragmented as a consequence of apoptosis by heat shock. Lemon oil inhibited the PARP fragmentation. This essential oil also inhibited the heat shock-induced activation of caspase-3. These results suggest that lemon pure essential oils may modulate the apoptosis through the activation of the ICE-like caspases.

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