Camptothecin을 투여한 HL-60세포에서 topoisomerase, ras, myc 유전자의 발현 양상

정인철,이능주,이상욱,이송재,조무연 고신대학교 의학부 2004 高神大學校 醫學部 論文集 Vol.19 No.1

Background DNA topoismerases solve the topological problems associated with DNA replication, transcription recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. Camptothecin is an antitumor alkaloid that has been isolated from the Chinese tree, Camptotheca acuminata. DNA topoisomerase I has been firmly established as the molecular target of the camptothecin group of anticancer drugs. These drugs include camptothecin, topotecan, 9-amino-camptothecin, and irinotecan. Many of them are now in clinical trials and are showing activity against a wide variety of solid human malignancies. DNA chip microarray technology can be used to establish associations between characteristic gene expression patterns and molecular responses to drug therapy. This study was conducted to determine whether pretreatment of HL-60 human leukemia cells with the topoisomerase Ⅰ- directed drug camptothecin regulates topoisomerase, myc and ras gene expression and to investigate the activity of topoisomerase mediated by camptothecin in nuclear extract from HL-60 human leukemia cells. Methods We have conducted experiments on cell cytotoxicity in drug-treated cells, topoisomerase purification, enzyme assay using agarose gel electrophoresis, quantitative RT-PCR analysis, northern blotting techniques and oligo chip microarray analysis, respectively. Results Treatment of HL-60 cells with camptothecin resulted in marked inhibition of type Ⅰ topoisomerase activity and in suppression of c-myc gene from quantitative RT-PCR analysis and northern blotting techniques. In contrast, no significant changes were observed in the topoisomerase Ⅰexpression levels from RT-PCR analysis. In HL-60 cells treated with camptothecin, the expression of ras and topoisomerase Ⅲ β gene from oligo chip microarray analysis were increased over, but the expression of c-myc and topoisomerase Ⅱα gene were decreased over. Conclusion Our results suggest that topoisomerase Ⅰ is the target of camptothecin cytotoxicity but it dose not affect topoispmerase Ⅰ(type IB) expression, and the suppression of myc and topoisomerase Ⅱα mRNA expression by camptothecin is due to (I) a decrease in mRNA transcripts, and (2) an inhibition of cellular proliferation resulted from formation of a cleavable complex with drug.

Antitumor Effects of Camptothecin Combined with Conventional Anticancer Drugs on the Cervical and Uterine Squamous Cell Carcinoma Cell Line SiHa

하상원,김윤정,김원용,이정수 대한약리학회 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.2

Functional defects in mitochondria are involved in the induction of cell death in cancer cells. We assessed the toxic effect of camptothecin against the human cervical and uterine tumor cell line SiHa with respect to the mitochondria-mediated cell death process, and examined the combined effect of camptothecin and anticancer drugs. Camptothecin caused apoptosis in SiHa cells by inducing mitochondrial membrane permeability changes that lead to the loss of mitochondrial membrane potential, decreased Bcl-2 levels, cytochrome c release, caspase-3 activation, formation of reactive oxygen species and depletion of GSH. Combination of camptothecin with other anticancer drugs (carboplatin, paclitaxel, doxorubicin and mitomycin c) or signaling inhibitors (farnesyltransferase inhibitor and ERK inhibitor) did not enhance the camptothecin-induced cell death and caspase-3 activation. These results suggest that camptothecin may cause cell death in SiHa cells by inducing changes in mitochondrial membrane permeability, which leads to cytochrome c release and activation of caspase-3. This effect is also associated with increased formation of reactive oxygen species and depletion of GSH. Combination with other anticancer drugs (or signaling inhibitors) does not appear to increase the anti-tumor effect of camptothecin against SiHa cells, but rather may reduce it. Combination of camptothecin with other anticancer drugs does not seem to provide a benefit in the treatment of cervical and uterine cancer compared with camptothecin monotherapy.

Antitumor Effects of Camptothecin Combined with Conventional Anticancer Drugs on the Cervical and Uterine Squamous Cell Carcinoma Cell Line SiHa

Ha, Sang-Won,Kim, Yun-Jeong,Kim, Won-Yong,Lee, Chung-Soo The Korean Society of Pharmacology 2009 The Korean Journal of Physiology & Pharmacology Vol.13 No.2

Functional defects in mitochondria are involved in the induction of cell death in cancer cells. We assessed the toxic effect of camptothecin against the human cervical and uterine tumor cell line SiHa with respect to the mitochondria-mediated cell death process, and examined the combined effect of camptothecin and anticancer drugs. Camptothecin caused apoptosis in SiHa cells by inducing mitochondrial membrane permeability changes that lead to the loss of mitochondrial membrane potential, decreased Bcl-2 levels, cytochrome c release, caspase-3 activation, formation of reactive oxygen species and depletion of GSH. Combination of camptothecin with other anticancer drugs (carboplatin, paclitaxel, doxorubicin and mitomycin c) or signaling inhibitors (farnesyltransferase inhibitor and ERK inhibitor) did not enhance the camptothecin-induced cell death and caspase-3 activation. These results suggest that camptothecin may cause cell death in SiHa cells by inducing changes in mitochondrial membrane permeability, which leads to cytochrome c release and activation of caspase-3. This effect is also associated with increased formation of reactive oxygen species and depletion of GSH. Combination with other anticancer drugs (or signaling inhibitors) does not appear to increase the anti-tumor effect of camptothecin against SiHa cells, but rather may reduce it. Combination of camptothecin with other anticancer drugs does not seem to provide a benefit in the treatment of cervical and uterine cancer compared with camptothecin monotherapy.

Camptothecin을 투여한 HL-60 세포에서 topoisomerase, ras, myc 유전자의 발현 양상

정인철,이능주,이상욱,이송재,조무연 고신대학교(의대) 고신대학교 의과대학 학술지 2004 고신대학교 의과대학 학술지 Vol.19 No.1

Background : DNA topoisomerases solve the topological problems associated with DNA replication, transcription recombination, and chromatin remodeling by introducing temporary single- or double-strand breaks in the DNA. Camptothecin is an antitumor alkaloid that has been isolated from the Chinese tree, Camptotheca acuminata. DNA topoisomerase 1 has been firmly established as the molecular target of the camtothecin group of anticancer drugs. These drugs include camptothecin, topotecan, 9-amino-camptothecin, and irinotecan. Many of them are now in clinical trials and are showing activity against a wide variety of solid human malignancies. DNA chip microarray technology can be used to establish associations between characteristic gene expression patterns and molecular responses to drug therapy. This study was conducted to determine whether pretreatment of HL-60 human leukemia cells with the topoisomerase 1-directed drug camptothecin regulates topoisomerase, myc and ras gene expression and to investigate the activity of topoisomerase mediated by camptothecin in nuclear extract from HL-60 human leukemia cells. Methods : We have conducted experiments on cell cytotoxicity in drug-treated cells, topoisomerase purification, enzyme assay using agarose gel electrophoresis, quantitative RT-PCR analysis, northern blotting techniques and oligo chip microarray analysis, respectively. Results : Treatment of HL-60 cells with camtothecin resulted in marked inhibition of type 1 topoisomerase activity and in suppression of c-myc gene from quantitative RT-PCR analysis and northern blotting techniques. In contrast, no significant changes were observed in the topoisomerase 1 expression levels from RT-PCR analysis. In HL-60 cells treated with camptothecin, the expression of ras and topoisomerase 3b gene from oligo chip microarray analysis were increased over, but the expression of c-myc and topoisomerase 2a gene were decreased over. Conclusion : Our results suggest that topoisomerase 1 is the target of camptothecin cytotoxicity but it does not affect topoisomerase 1 (type 1b) expression, and the suppression of myc and topoisomerase 2a mRNA expression by camptothecin is due to (1) a decrease in mRNA transcripts, and (2) an inhibition of cellular proliferation resulted form formation of a cleavable complex with drug.

Topoisomerase inhibitor에 의한 각막상피세포의 세포고사 유도

김재민 대한시과학회 2005 대한시과학회지 Vol.7 No.1

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. This study was performed to examine the apoptosis induced in human comeal epithelial cells maintained in tissue culture by topoisomerase inhibitors. Topoisomerase inhibitors, camptothecin and etoposide at manufacturer-recommended concentration were added to the comeal epithelial cells for 24 or 48 hours. Cell viability was measured using MTT assay. The cells were then assessed for the level of apoptosis Morphologic features of apoptosis were demonstrated using Hoechst 33342 staining, Annexin V - FITC/PI staining, DePsipher assay and CytoDEA TH staining. DNA fragmentation was determined by means of an in situ cell detection procedure (TUNEL) or by electrophoresis on agarose gels. Camptothecin and etoposide induced apoptosis in HCE cells in a dose dependent manner. Camptothecin and etoposide induced apoptosis at lower than manufacturer recommended concentration. Camptothecin and etoposide elicited typical apoptotic morphologic changes. Topoisomerase inhibitors are more sensitive in apoptotic induction of HCE cells than other cells. These results indicate that topoisomerase inhibitors are able to play a role in good positive control of apoptotic inducers

Camptothecin activates SIRT1 to promote lipid catabolism through AMPK/FoxO1/ATGL pathway in C2C12 myogenic cells

Mei-Chen Lo,Jia-Yin Chen,Yung-Ting Kuo,Wei-Lu Chen,Horng-Mo Lee,Shyang-Guang Wang 대한약학회 2019 Archives of Pharmacal Research Vol.42 No.8

Caloric restriction activates sirtuin 1 (SIRT1)and induces a variety of metabolic effects that are beneficialfor preventing age-related disease. The present studyscreened a commercially available used drug library todevelop small molecule activators of SIRT1 as therapeuticsfor treatment of metabolic disorders. Using an in vitrofluorescence assay, the cancer therapeutic camptothecinincreased SIRT1 enzymatic activity by 5.5-fold, indicatingit to be a potent SIRT1 activator. Camptothecin also elevatedthe nicotinamide adenine dinucleotide (NAD)?/NADH ratio and increased SIRT1 protein levels in differentiatedC2C12 myogenic cells. Treatment of C2C12 myotubeswith camptothecin increased phosphorylation ofAMP-dependent kinase (AMPK) and acetyl-coenzyme Acarboxylase, caused nuclear translocation and deacetylationof forkhead box O1 (FoxO1), increased transcriptionand protein expression of adipose triglyceride lipase(ATGL), decreased the amount of intracellular oil droplets,and significantly increased b-oxidation of fatty acids. These in vitro data were confirmed in vivo as camptothecintreatment of C57BL/6J mice reduced fat and plasmatriglyceride levels. All of the above camptothecin-inducedalterations were attenuated by the SIRT1-specific inhibitornicotinamide and/or 6-[4-(2-piperidin-1-ylethoxy) phenyl]-3-pyridin-4-ylpyrazolo [1,5-a]pyrimidin (compound C). Thus, camptothecin activation of SIRT1 promotes lipidcatabolism through AMPK/FoxO1/ATGL signaling.

당뇨병성 망막증 발달에 있어서 포도당 농도에 의한 망막 혈관주위세포의 직접적 손상

신종욱,이지영,김정미,조인호 국립보건연구원 2000 국립보건원보 Vol.37 No.-

Camptothecin과 β -lapachone에 의한 혈관주위세포 사멸 기전 연구

이지영,신종욱,배성원,김정미,조계원,조인호 국립보건연구원 2000 국립보건원보 Vol.37 No.-

A simple, rapid and sensitive spectrofluorimetric method for the determination of camptothecin

Karwasara, Vijai Singh,Nahata, Alok,Dixit, Vinod Kumar 경희한의학연구센터 2012 Oriental Pharmacy and Experimental Medicine Vol.12 No.2

Camptothecin (CPT) represents a clinically useful class of anticancer agent. Proper identification and quantitation of the CPT in the plant extracts and in-vitro cell culture extracts is fundamental to assess the CPT content and its biosynthetic potential in plants. A simple, sensitive and rapid, spectrofluorimetric method has been developed and validated for the quantitative estimation of camptothecin. The method was validated in terms of linearity (2-20 ng/ml), precision (intra-day variation below 0.15, interday variation below 1.2), and accuracy (98.0 to 100.2%). The limit of detection and limit of quantification for CPT were found to be 0.10 ng/ml and 0.36 ng/ml, respectively. The developed spectrofluorimetric method provides a rapid and cost effective method for the routine analysis of CPT in plant extracts and tissue culture samples. The developed method was successfully used for the estimation of CPT in natural plant extracts and cell culture extracts. The Nothapodytes nimmoniana callus cells having nearly 3-fold higher CPT content over the leaf (0.005%) explant of the plant. The highest CPT content was found in the stem part (0.092%) followed by the fruit (0.088%). The method is simple, sensitive and precise; it can be used for the routine quality control testing of formulations containing CPT.

HL-60 세포에서 Camptothecin의 apoptosis 유도작용

김해종,천영진,김미영 대한약학회 1999 약학회지 Vol.43 No.3

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.