1. Thitiporn Miiksombat 1, Narumol Bhummaphan 1, Teerakul Arpornsuwan 2 1 Student in Medical Technology Program, Faculty of Allied Health Sciences, Thammasat University 2 Department of medical Technology, Faculty of Allied Health Sciences, Thammasat University Abstract Safflower (Carthamus tinctorius Linn.) has been used as herbal for health. Its indication is used as Neurotropic, Cardiotropic, Hemopoetic, reduced Artherosclerosis and inhibited cancer cell growth. The present study was aimed to investigate the effect of the dichloromethane extract of Carthamus tinctorius Linn. on apoptosis of human colon carcinoma (SW 620 cell line) and underlying mechanism of herb-induced apoptosis. Apoptotic pathway involving Caspase 9 and p130 gene were investigated by RT-PCR technique. SW620 cell line was incubated with the dichloromethane fraction of C. tinctorius at 2 hr., 6 hr., 9 hr. and over night (18 hr.). The apoptotic gene expression in SW 620 treated cell line was investigated by RT-PCR. The PCR profile consisted of an initial denaturation at 95 o C for 2 min followed by 30 cycles of denaturation at 95 o C for 45 sec, 45 sec of annealing at 52 o C for Caspase 9 or at 51.5 C for p130 and extension at 72 o C for 45 sec. A final extension cycle at 72 o C for 10 min was completed before the reaction terminated by lowering the temperature to 4 o C. The effect of C. tinctorius would be further evaluated on Caspase 9 gene and p130 gene expression in SW 620 treated cell line. To investigate the optimum condition in Caspase9 and p130 genes mRNA expression in treated SW620 cell line (human colon carcinoma) by RT-PCR We would like to thank Academic research, Thammasat University for the financial support to this study. Thanks are also extended to Faculty of Allied Health Sciences, Thammasat University and Research & Development unit, Department of Medical Sciences, Ministry of Public Health The optimum condition of mRNA Caspase 9 and p130 gene expressions in SW 620 treated cell line using RT- PCR Objective Material & Method Cell culture (SW620 cell line) with dichloromethane extracted safflower, at 2 hr.,6 hr.,9 hr.,Overnight RNA extraction from human colon cancer cell (SW620 cell line) RT-PCR Optimization the appropriate condition in Caspase9 and p130 genes expression Results 981 bp p130 : Denaturation 95ºC 2 min Denaturation 95ºC 45 sec Annealing 51.5ºC 45 sec Extension 72ºC 45 sec 30 cycles Extension 72ºC 10 min 1 2 3 Line 1: MgCl 2 1 µl Line 2: MgCl 2 2 µl Caspase 9:Denaturation 95ºC 2 min Denaturation 95ºC 45 sec Annealing 52ºC 60 sec Extension 72ºC 45 sec 30 cycles Extention 72ºC 10 min Line 1: MgCl 2 1 µl Line 3: MgCl 2 3 µl 1 2 3 666 bp Conclusion & Discussion  p130 & Caspase9 gene may be involved in SW 620 apoptosis resulted in up-regulation of p130 & Caspase9 gene.  The level of mRNA expression p130 was detected at low level with dimers that may be cDNA degradation or Less of cDNA extraction.  The potential pro-apoptotic effect of C.tinctorius would be further evaluated on p130 & Caspase9 mRNA expression in SW620 treated cell line. References  Reed JC. Dysregulation of apoptosis in cancer. J Clin Oncol 1999;17:2941-53  Li P, Nijhawan D, Budihardjo I, et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex intiates an apoptotic protease cascade. Cell 1997;91:479-89  Claudio P. P., De Luca A., Howard C. M., Baldi A., Firpo E. J., Koff A., Paggi M. G., Giordano A. Functional analysis of pRb2/p130 interaction with cyclins. Cancer Res., 56: 2003-2008, 1996. Acknowledgement Introduction Figure 1: Caspase-9 plays an essential role in apoptosis during normal development. Figure 2: pRb2/p130 (Tumor suppressor gene) may regulate ER- transcription A healthy organism is an exquisitely integrated collection of differentiated cells, which maintain a balance between life and death. Apoptosis is the last phase of a cell's destiny. During apoptosis, the genome will fracture, the cell will shrink and part of the cell will disintegrate into smaller apoptosis bodies. Caspase-9 plays an essential role in apoptosis during normal development and pRb2/p130 (Tumor suppressor gene) may regulate ER- transcription Safflower (Carthamus tinctorius Linn.) has been used in Thailand, China, india as herbal tea for health. Its indication is used as neurotropic, cardiotropic, hemopoetic, reduced artherosclerosis.In 2003 was found that at 1% of concentration of safflower oil is suitable for inhibition in growth of colon cancer, Jing Lei Cheng,et al.(Japan),2003. Colon cancer starts in the large intestine or the rectum According to the American Cancer Society, colorectal cancer is one of the leading causes of cancer related deaths in the United States. In general treatments may include Chemotherapy, Surgery, Radiation therapy.