Ekajayanti, PandePutuNovi (2015) Pengaruh Pemberian Fraksi Kloroform Scurrula Artropurpurea (Blume) Danser Terhadap Ekspresi Cytochrome–C Dan Apoptosis Pada Kultur Sel Hela. Magister thesis, Universitas Brawijaya.
Abstract
Kanker leher rahim atau ca serviks adalah tumor ganas primer yang berasal dari sel epitel skuamosa. Ca serviks merupakan kanker yang terjadi pada serviks atau leher rahim, suatu daerah pada organ reproduksi wanita yang merupakan pintu masuk ke arah rahim, letaknya antara rahim (uterus) dan liang senggama atau vagina. Hampir semua kasus kanker serviks (99%) berhubungan dengan infeksi genitalia yang disebabkan oleh Human Papillomavirus (HPV) yang merupakan infeksi virus terbanyak pada sistem reproduksi. HPV tipe 16 dan 18 memiliki gen yang setelah terintegrasi ke genom sel penjamu, mengkode protein yang menghambat atau menginaktifkan gen penekan tumor TP53 atau RB1 di sel epitel sasaran serta mengaktifkan gen yang terkait siklus sel, seperti siklin e sehingga terjadi proliferasi sel yang tidak terkendali serta penghambatan proses apoptosis sel pada kasus kanker serviks. Proses apoptosis sel tersebut saling terkait dalam siklus sel, dimana ditemukan beberapa kelompok gen seperti gen BCl – 2 dan MDM – 2. Gen BCL – 2 mengkode protein yang berperan sebagai anti – apoptosis, dimana protein ini bekerja untuk menekan fungsi protein BAX pada membran mitokondria karena protein BAX berperan untuk membuka Pt – pore sehingga Cytochrome – C keluar dari mitokondria. Cytochrome – C kemudian berikatan dengan Apaf – 1. Selanjutnya Apaf – 1 mengaktivasi kaspase kaskade, sehingga sel mengalami kematian (apoptosis). Strategi dalam terapi kanker dilakukan dengan berbagai cara antara lain penggantian tumor suppressor gene , menghambat proliferasi sel dan menginduksi apoptosis. Salah satu upaya pengobatan kanker tersebut dapat dilakukan dengan memanfaatkan senyawa alami yang terkandung dalam bahan hayati, salah satunya adalah benalu teh. S cur r ula atropurpureea ( Blume) Danser atau daun benalu teh merupakan salah satu species benalu yang ada di Indonesia mempunyai banyak manfaat untuk kesehatan. Fraksi kloroform daun benalu teh ini kaya akan senyawa flavonoid yang berpotensi sebagai antikanker. Adapun kandungan senyawa flavonoid tersebut antara lain flavanon, dihidroflavonol dan cathecin . Daun benalu teh dimaserasi bertingkat dengan pelarut N-heksan, kloroform dan etanol, lalu didapatkan fraksi kloroform yang paling poten menginduksi apoptosis pada sel HeLa dengan IC50 adalah 96,16 ppm. Penelitian ini bertujuan mengetahui pengaruh pemberian f raksi k loroform daun benalu teh ( S cur r ula artropurpurea ( Blume) Danser ) terhadap Ekspresi Cytochrome – C dan Apoptosis pada Kultur Sel HeLa . Sel HeLa dipapar dengan lima konsentrasi fraksi kloroform benalu teh yakni 25 µg/mL, 50 µg/mL, 100 µg/mL, 200 µg/mL, dan 400 µg/mL. Kemudian diinkubasi selama 24 jam. Ekspresi Cytochrome – C dideteksi dengan metode flow cytometry , sedangkan apoptosis sel HeLa dideteksi dengan metode TUNNEL (Apo-BrdU-IHC TM Kit, BioVision). Analisa data statistik menggunakan Oneway ANNOVA. Gambaran ekspresi cytochrome – c pada hasil pemeriksaan flowcytometry pada kelompok kontrol didapatkan rata-rata (58.983±3.031) lebih rendah daripada kelompok perlakuan. Pada kelompok perlakuan P1 yang diberikan dosis 25 µg/ml memiliki rata-rata ekspresi (66.428±2.982) yang tidak berbeda signifikan dengan kelompok perlakuan dosis 50 µg/ml (P2), 100 µg/ml (P3), dan 200 µg/ml (P4). Sedangkan gambaran apoptosis berdasarkan hasil pemeriksaan TUNNEL , pada kelompok kontrol didapatkan rata-rata (2.25±0.50). Pada kelompok perlakuan P2 yang diberikan dosis 50 µg/ml memiliki rata-rata (8.5±0.577) yang tidak berbeda signifikan dengan kelompok perlakuan dosis 100 µg/ml (P3), 200 µg/ml (P4) dan 400 µg/ml (P5). Hal ini menunjukkan bahwa terjadi peningkatan ekspresi cytochrome – c dan apoptosis yang diberikan perlakuan fraksi kloroform benalu teh ( Scurrula artropurpurea [ Blume ] Danser ) dengan 5 konsentrasi yang berbeda yaitu 25 µg/ml (P1), 50 µg/ml (P2), 100 µg/ml (P3), 200 µg/ml (P4) dan 400 µg/ml (P5) dibandingkan kontrol. Berdasarkan hasil uji one way anova pengaruh fraksi kloroform daun benalu teh (Scurrula artropurpurea [Blume] Danser) terhadap ekspresi cytochrome – c , didapatkan p value sebesar 0,000, lebih kecil daripada α = 0,05 (p 0,05). Artinya bahwa terdapat perbedaan yang signifikan pada ekspresi cytochrome – c akibat pemberian fraksi kloroform benalu teh (Scurrula artropurpurea [Blume] Danser) dengan konsentrasi yang berbeda. Berdasarkan hasil uji one way anova pengaruh fraksi kloroform daun benalu teh (Scurrula artropurpurea [Blume] Danser) terhadap apoptosis menggunakan Anova One Way , didapatkan p-value sebesar 0,000, lebih kecil daripada α = 0,05 (p 0,05). Artinya bahwa terdapat perbedaan yang signifikan apoptosis akibat pemberian fraksi kloroform benalu teh (Scurrula artropurpurea (Blume) Danser) dengan konsentrasi yang berbeda. Dari hasil yang diperoleh pada penelitian ini, didapatkan simpulan bahwa Fraksi Kloroform daun benalu teh ( S cur r ula artropurpurea ( Blume) Danser ) meningkatkan ekspresi Cytochrome – C dan Apoptosis pada
English Abstract
Cervix cancer is a primary malignant tumor which originates from squamous epithelia cells. It represents a cancer attacking on cervix. Indeed, cervix is a region in women reproductive organ where it becomes an entry gate into uterus. The position of cervix is between uterus and vagina. Almost all cases of cervix cancer (99%) are closely related to genitalia infection caused by Human Papilloma Virus (HPV). This virus is producing the greatest number of virus-based infection in the reproduction system. HPV type 16 and type 18 have genes which after integrating into host cellular genome, it will encode protein to impede or inactivate tumor suppressor genes TP53 or RB1 in target epithelia cells. Such HPV genes also activate genes that engaged within cellular cycle, including cyclin-e, and thus, cellular proliferation is beyond control and cellular apoptosis is hampered. It is evident in cervix cancer. Cellular apoptosis process is interdependent during cellular cycle. Several gene groups such as BCl-2 and MDM-2 are found. BCl-2 encodes the protein that functions as anti-apoptosis, and this protein works to suppress the function of protein BAX in mitochondria membrane because protein BAX opens Pt-pore such that Cytochrome-C leaves mitochondria. Cytochrome-C binds with Apaf-1. Subsequently, Apaf-1 will activate cascade caspace such that cells are subjected to death (apoptosis). Cancer therapy strategy may be conducted in various ways such as by replacing tumor suppressor gene , preventing cellular proliferation and inducing apoptosis. One effort for cancer medication is by using natural compound contained within biological materials, among other tea epiphyte. Scurrula artropurpurea [ Blume ] Danser or tea epiphyte is an epiphyte species in Indonesia but with various advantages for heath. Chloroform fraction of tea epiphyte leaf is enriched with flavonoid compound that is potential for anti-cancer. Flavonoid compound comprises of flavanon, dihydroflavanol and cathecin . Tea epiphyte leaf is gradually macerated with solvents such as N-hexane, chloroform and ethanol. This maceration has resulted in the most potential chloroform fraction in inducing apoptosis within HeLa cells. This chloroform fraction is IC50 with potentiality of 96.16 pm. The objective of research is to understand the influence of chloroform fraction of tea epiphyte leaf ( Scurrula artropurpurea [ Blume ] Danser ) on the expression of Cytochrome-C and Apoptosis in HeLa cell cultures. HeLa cell was exposed to five concentrations of tea epiphyte chloroform fraction, which are 25 m g/ml, 50 m g/ml, 100 m g/ml. The solution was incubated for 24 hours. The expression of Cytochrome-C was detected by flow cytometry method, while Apoptosis of HeLa cell was detected by TUNNEL method (APo-BrdU-IHC TM Kit, BioVision). Statistic data analysis is using One Way ANOVA. The description of Cytochrome-C expression as indicted by flow cytometry in control group was by average 58.983 ± 3.031, which was still lower than treatment group. Treatment Group P1 with dose 25 m g/ml had an average rate of Cytochrome-C expression for 66.428 ± 2.982, which seems not much different from treatment group with dose 50 m g/ml (P2), 100 m g/ml (P3), and 200 m g/ml (P4). Meanwhile, the description of Apoptosis, based on TUNNEL examination, in control group is by average 2.25 ± 0.50. Treatment Group P2 with dose 50 m g/ml had an average rate of Apoptosis of 8.5 ± 0.577 which is not significantly different from other treatment groups at dose 100 m g/ml (P3), 200 m g/ml (P4) and 400 m g/ml (P5). It showed that the increasing expression of Cytochrome-C expression and Apoptosis after treatment with chloroform fraction of tea epiphyte ( Scurrula artropurpurea [ Blume ] Danser ) in 5 different concentrations, including 25 m g/ml (P1), 50 m g/ml (P2), 100 m g/ml (P3), 200 m g/ml (P4) and 400 m g/ml (P5), is higher than control. In pursuance resulted of One Way ANNOVA over the influence of chloroform fraction of tea epiphyte ( Scurrula artropurpurea [ Blume ] Danser ) on Cytochrome-C expression, it seemed that p-value of 0.000 was obtained but smaller than a = 0.05 (p 0.05). It means that there was significant difference of Cytochrome-C expression due to the treatment of chloroform fraction of tea epiphyte ( Scurrula artropurpurea [ Blume ] Danser ) at different concentration. Based on the result of One Way Anova over the influence of chloroform fraction of tea epiphyte ( Scurrula atropurpurpurea [ Blume ] Danser ) on Apoptosis, the influence was proved by p-value of 0.000 which is still smaller than a = 0.05 (p 0.05). It means that there was significant difference of Apoptosis due to the treatment of chloroform fraction of tea epiphyte ( Scurrula artropurpurea [ Blume ] Danser ) at different concentration. Considering these results, it can be concluded that treatment with chloroform fraction of tea epiphyte leaf (
| Item Type: | Thesis (Magister) |
|---|---|
| Identification Number: | TES/615.321/EKA/p/2015/041503275 |
| Subjects: | 600 Technology (Applied sciences) > 615 Pharmacology and therapeutics > 615.3 Organics drugs |
| Divisions: | S2/S3 > Magister Kebidanan, Fakultas Kedokteran |
| Depositing User: | Sugiantoro |
| Date Deposited: | 15 May 2015 14:37 |
| Last Modified: | 15 May 2015 14:37 |
| URI: | http://repository.ub.ac.id/id/eprint/158027 |
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