EFEKTIVITAS EKSTRAK AIR PURWACENG (Pimpinella alpina) PADA INDUKSI APOPTOSIS DAN EKSPRESI PROTEIN BERBAGAI GEN APOPTOSIS PADA SEL KANKER PARU A549

FARIZ IHSAN ARIFIANTO, dr. Setyo Purwono, Sp.PD

2014 | Skripsi | PENDIDIKAN DOKTER

Latar Belakang: Kanker paru merupakan kanker ke-3 terbanyak dan penyebab kematian utama pada pria akibat kanker. Kemoterapi kanker ini menimbulkan banyak efek samping sehingga perlu dicari agen kemoterapi alternatif yang memiliki aktivitas sitotoksik melalui mekanisme cell cycle arrest, menginduksi apoptosis, atau memicu ekspresi protein yang mampu menginduksi apoptosis. Purwaceng (Pimpinella alpina) mengandung bahan aktif Kumarin yang memiliki efek kemotoksik pada sel kanker. Peran Purwaceng (Pimpinella alpina) pada sitotoksik dan induksi apoptosis pada sel kanker paru masih belum jelas. Tujuan Penelitian: Untuk mengetahui efek sitotoksik dan kemampuan induksi apoptosis ekstrak air Pimpinella alpina terhadap sel kanker paru A549 melalui jalur ekspresi protein berbagai gen apoptosis. Metode Penelitian: Penelitian ini adalah eksperimental murni dengan sampel kultur sel kanker paru A549, di rentang dosis ekstrak purwaceng 1-10.000 μg/mL dan rentang dosis kontrol positif cisplatin 1-500 μg/mL dan waktu inkubasi 24-48 jam. Uji sitotoksik dilakukan terhadap sel kanker paru A549 yang telah diinkubasi selama 24 jam menggunakan larutan uji dan kontrol kemudian dibaca absorbansinya setelah pewarnaan MTT. Uji Induksi apoptosis dilakukan dengan pewarnaan etidium bromide-acridine orange dan pengamatan di mikroskop fluoresen. Uji ekspresi protein induksi apoptosis menggunakan pewarnaan imunohistokimia antibodi anti p53, Rb, Bcl-2, dan Caspase-9. Hasil Penelitian: Pada uji sitotoksik dengan inkubasi 24 jam didapatkan IC50 sebesar 3.862 μg/mL. Uji induksi apoptosis yang dideteksi menggunakan fluoresensi ethidium bromide-acridine orange mendapatkan nilai EC50 sebesar 5.271 μg/mL, dan ditandai dengan penurunan ekspresi Rb dan Bcl-2 serta peningkatan ekspresi p53 dan caspase-9. Statistik Chi-Square menunjukkan proporsi efek IC50 pada ekspresi gen berbeda secara bermakna (p<0.05) terhadap kontrol. Kesimpulan: Pimpinella alpina mempunyai efek sitotoksik terhadap sel kanker paru A549 dan menginduksi apoptosis melalui perubahan jalur apoptosis berupa penurunan ekspresi Rb dan Bcl-2 serta peningkatan p53 dan caspase-9.

Background: Lung cancer is the 3rd most frequent occurring cancer and the leading cause of cancer related death in men. Conventional treatment of lung cancer consists of multidrug chemotherapy. Although chemotherapy regiments has been proven to be effective, there are many highly undesirable side effects. Thus, an alternative chemotherapy agent is needed that has similar efficacy of conventional chemotherapy with minimal side effects. Alternative chemotherapy agent is an agent that has cytotoxic activity through the mechanism of cell cycle arrest, apoptosis induction capability, or trigger the expression of proteins that are able to induce apoptosis. Purwaceng (Pimpinella alpina) has Coumarin active compound that is known to have chemotoxic effect towards cancer cells. Role of Purwaceng (Pimpinella alpina) on the cytotoxic effect and induced apoptosis in lung cancer cells remains unclear. Objective: To determine the cytotoxic effects and apoptosis induction ability of water extracts of Pimpinella alpina for A549 lung cancer cells through apoptosis protein expression of various genes. Method: The study is purely experimental that used A549 lung cancer cells that were given a Purwaceng extract dose range of 1-10.000 μg/mL with 24-48 hours of incubation time. For comparison, a positive control containing cisplatin was used with a dose range of 1- 500 μg/mL. Cytotoxicity study were stained using MTT, where as apoptosis induction study used ethidium orange-acridine bromide. Protein expression of apoptosis induction study using anti-p53, Rb, Bcl-2, and Caspase-9 antibody immunohistochemitry staining. Result: The cytotoxicity results show incubation for 24 hours resulted in an IC50 of 3.862,00 μg/mL. Apoptosis induction study shows an EC50 value of 5.271,00 μg/mL, identified by the decrease in Rb and Bcl-2 expression and increase in p53 and caspase-9 expression. Chisquare statistical analysis shows proportionate effects of IC50 towards gene expression that are statistically significant (p<0.05) in comparison to negative control. Conclusion: This study shows Pimpinella alpina extract has cytotoxic and apoptosis inductive properties towards A549 lung cancer cell, identified by the decrease in Rb and Bcl-2 expression and increase in p53 and caspase-9 expression.

Kata Kunci : Pimpinella alpina, kanker paru, sitotoksik, apoptosis, ekpresi protein.