DETEKSI SINGLE NUCLEOTIDE POLYMORPHISM NQO1 P187S, p53 P72R, dan MDM2 T309G DENGAN TEKNIK PCR-RFLP DAN PCR-HRM PADA SAMPEL FORMALIN FIXED PARAFFIN-EMBEDDED (FFPE) PASIEN KANKER PAYUDARA

Detection test for Single Nucleotide Polymorphism (SNP) recently carried out by PCR-RFLP. This method needs post-PCR procedure to determine the result. Discovery of real time PCR lead to the new method for SNP detection, called PCR-HRM. This alternative method was being analyzed to find better detection method. Detection of SNP NQO1 P187S, p53 P72R, and MDM2 T309G was carried out because they are having a relation on the metabolism response on anthracycline chemotherapy. Formalin Fixed Paraffin-Embedded (FFPE) samples are routine samples that collected in cancer hospital. The riches of this samples can be used as useable diagnostic samples. Nevertheless, quality of DNA which is isolated from FFPE�s sample is poor because of formalin. were There two aims of this research. First was to know the benefit of FFPE sample�s in SNP detection. Second was to know the benefot of PCR-HRM and HRM�s profile in SNP NQO1 P187S, p53 P72R, and MDM2 T309G�s detection. FFPE�s samples were collected from operation. The samples were DNA�s isolated, and amplified using PCR (Polymerase Chain Reaction) for NQO1, p53, dan MDM2. Restriction Fragment Lenght Polymorphism (RFLP) was conducted using restriction enzymes HinfI, BstUI, dan MspA1I, respectively for NQO1, p53, dan MDM2. The second method was PCR-High Resolution Melting (PCR-HRM). This method was done using real time PCR Corbett Rotor Gene. The results was annalyzed using melting curve, normalization curve, and differentiation plot curve.The comparison between PCR-RFLP and PCR-HRM has been done to detect three SNP. Sequencing technique has been used as standart technique if there are any differences found between PCR-RFLP and PCR-HRM. There are 6 differences found in comparison between PCR-RFLP and PCR-HRM. The PCRRFLP �results showed 66,67% similarity with sequencing�s results. FFPE�s sample can be used for SNP NQO1 P187S, p53 P72R, and MDM2 T309G� detection. HRM�s profile can be analyzed using three curves. There are melting curve, normalization curve, and differentiation plot curve. The comparison between PCR-RFLP and PCR-HRM has done to detect three SNP. PCR-RFLP more reliable method than PCR-HRM if primer which is used not specially design for PCR-HRM. PCR-HRM is a faster method than PCR-RFLP for SNP NQO1 P187S, p53 P72R, and MDM2 T309G�s detection

PENINGKATAN SITOTOKSISITAS DOXORUBICIN SEBAGAI AGEN KEMOTERAPI OLEH SENYAWA ALFA TERPINEOL PADA SEL MCF-7

Alpha terpineol is a naturally alcoholic-monoterpene compound. This component is a part of essential oil extract in plants, such as Pines (Pinus merkusii). Alpha terpineol is a potential natural product with anticancer activity which is proven can decrease cells growth and induces cells death. Chemotherapeutic combination is a combined of a chemoprevention compound with chemotherapeutic agent to increase therapeutic effect and reducing toxicity. The purpose of this study is to determine the effect combination alpha terpineol with doxorubicin in breast cancer cell line MCF-7 The cytotoxic assay of alpha terpineol and doxorubicin were carried out by MTT method using alpha terpineol and doxorubicin concentration of 1,5625-50 µg/ml to determine IC50. The cytotoxic assay of their combination were carried out by MTT method using concentration were 1/2, 1/8 and 1/20 IC50 values. Apoptotic assay of their best combination concentration were conducted using flow cytometryAnnexin V-Pi. Alpha terpineol and doxorubicin showed cytotoxic effect on MCF-7 breast cancer cell with IC50 30 µg/ml and 2 µg/ml, respectively. Based on CI values, all combination concentration of alpha terpineol with doxorubicin showed strong synergistic effect (CI 0,1-0,3). Apoptotic assay by flow cytometry Annexin V-Pi showed alpha terpineol and doxorubicin combination caused 4,5% early apoptotic cell compared single doxorubicin. Based on this results, alpha terpineol is a potential compound to developed as co-chemoterapeutic agent. However, the molecular mechanism need to be explored further

PROFIL MicroRNA 451 (miR451) DAN P-GLYCOPROTEIN (PGP) PADA CELL LINE MCF-7 RESISTEN DOXORUBICIN (MCF-7/DOX)

Cancer is a deadly disease with high mortality and incidency. Doxorubicin (DOX) is anthracycline antitumor-antibiotic which used as chemotheurapeutic therapy against cancer. Some type of cancer cell was known to having low sensitivity or even resistant to various chemotherapy. In the previous study, microRNA451 (miR451) estimated play a role in inhibiting mechanism by post transcription regulation of P-glycoprotein-encoding gene as transport protein in chemotherapeutic resistance mechanism. The purpose of this study is to analyze miR451 and PGP expression profile on doxorubicin-resistant MCF-7 cell line (MCF-7/DOX) and to analyze relationship between miR451 expression level and PGP expression to obtain scientific information as a reference in development of microRNA research. This study was conducted in silico to analyze miR451 and MDR1 mRNA sequence interaction. Afterwards, the laboratory-experimental study was conducted in vitro to analyze miR451 expression in doxorubicin-resistant MCF-7 cell line (MCF-7/DOX). The methods that used in this study is the periodic doxorubicin treatment in parental MCF-7 cell line to obtain resistant-MCF-7 cell line. The resistance parameter that used in this study is IC 50 value in doxorubicin-treated MCF-7 is higher than parental MCF-7 in MTT assay. PGP expression was analyzed by immunocytochemistry and miR451 expression was analyzed by qRT PCR using U6 SnRNA as reference gene and UniSp6 as positive control. The result show that no miR451 amplification were detected in both parental MCF-7 and MCF-7/DOX in qRT PCR analysis, but based on immunocytochemistry assay, PGP expression was found on MCF-7/DOX cytoplasmic and cytoplasmic membrane. Based on this in vitro study, it can be concluded that there were PGP expression in MCF-7/DOX, but there were no miR451 expression in both MCF-7 and MCF-7/DOX. By resistance induction with doxorubicin treatment, the upregulation of PGP was occurred without influence of lost expression of miR451 in MCF-7/DOX

VARIASI DELESI 30 bp GEN LMP1 C-TERMINUS EBV PADA PENDERITA KARSINOMA NASOFARING

Epstein-Barr Virus (EBV) is one of the causes of nasopharyngeal carcinoma (NPC), which more than 90% the world populations are infected with EBV. LMP1 expression caused by EBV infection, in which LMP1 is oncogenic, because it can cause the transformation of normal cells to become cancerous. LMP1 gene has the ability to mediate transcription of NFκβ . Variation 30 bp deletion LMP1 C-terminus of EBV may alter the function of oncogenic signaling and increase potential. In Indonesia, information of genetic variation, especially variation of 30 bp deletion of the C-terminus of EBV LMP1 is still very limited, so it�s necessary to get data from other areas in order to understand the clinical pathogenesis of EBV infection. This study aimed to explore variations in the gene deletion of 30 bp EBV LMP1 C-terminus (nucleotides 168287-168256) of patients with NPC in Purwokerto and study the relationship with clinical stage and histopathological type. Samples were derived from 36 blood and 30 tissues in paraffin blocks examined DNA using Polymerase Chain Reaction (PCR). A total of 6 samples checked by sequencing to ensure the 30 bp deletion variant LMP1 C-terminus of EBV (nucleotides 168287- 168256). The results found no variation in the gene deletion of 30 bp EBV LMP1 C-terminus (nucleotides 168287-168256) either in 36 blood samples and 30 samples of tissue. Examination sequencing of 6 samples of blood and tissue suspected of having deletions, gave negative results. 30 bp deletion was not found

MEKANISME MOLEKULER KEMOPREVENTIF DAN ANTIKANKER SENYAWA AKTIF AKAR PASAK BUMI (Eurycoma longifolia Jack) Kajian In Vitro pada sel T47D dan In Vivo pada Kanker Payudara pada Tikus SD yang diinduksi DMBA

Breast Cancer incidence ranks the highest in Indonesia. DMBA (7,12- Dimethylbenz(a)anthrasen) is a carcinogen compound model to induce breast cancer. The breast cancer cell model used in this research is T47D. The genes which have roles in cancer incidence are p53, bcl-2, ras, p21, and kaspase. DNA repair is influenced by GADD45 gene. Cancer is also related with COX-2 induced inflammation. Pasak bumi (Eurycoma longifolia Jack) is one of the plants which is used as detoxification, free radicals antioxidant and anticancer medication. This research was started with bioassay guided isolation toward T47D and Vero cells expression. The extraction was conducted using ethanol, chloroform and water using maceration method. Ethanolic extract was fractionated with ethyl acetic. Ethyl acetic soluble fraction was isolated using chloroform-methanol-water (7:3:1) as mobile phase and columnar chromatography and preparative chromatography. The isolates tested were three dominant isolates which were isolate 1, isolate 2 and 3 which have Rf value of 0,91, 0,78 and 0,67 respectively on UV 366 detection. The most active isolate was elucidated with UV/vis, IR, H- NMR, C-NMR, LC-MS. The most active isolates also was examined its molecular mechanism through anti-inflammation (COX-2 inhibition), apoptosis induction (BCl2 inhibition and Kaspase-3 induction), as well as antiproliferation trough P53, P21, GADD45 expression induction and Ras reduction. Finally the most active isolate in molecular mechanism is used as the parameter of extraction standard which will be examined in vivo its chemopreventive effect on DMBA induced mouse. The results of the cytotoxity test on T47D cells shows that the IC50 value of ethanol extract = 167,7 ± 76,72 µg/mL, chloroform = 370,0 ± 70,4 µg/mL, water = -, ethyl asetic soluble fraction = 55,2 ± 31,6 µg/mL, non-soluble ethyl asetic fraction = 194,9 ± 7,8 µg/mL, isolat 1 = 10,6 ± 4,1 µg/mL, isolat 2 = 1391,4 ± 111,6 µg/mL, and isolat 3 = 474,1 ± 18,7 µg/mL. The cytotoxicity on Vero results on IC50 value of ethanol extract = 1791,1 ± 221,3 µg/mL, ethyl acetic soluble fraction = 1018,3 ± 47,9 µg/mL, and isolat 1= 465,6 ± 47,9µg/mL. Isolat 1 has structure with molecular weight of 637. Isolat 1 with 10 µg/mL compared with negative control decreases COX-2 expression by 78%, decreases Bcl-2 expression by 56