Efek Penghambatan Siklus Sel dan Pemacuan Apoptosis Kombinasi Ekstrak Umbi Lapis Bawang Sabrang (Eleutherine Bulbosa (Mill.) Urb.) dan Doksorubisin pada Sel Kanker Payudara

Abstract

Sabrang onion bulbs (Eleutherine bulbosa (Mill.) Urb.) have been used for generations against various diseases such as breast and colon cancer, hypertension, hyperglycemia, hypercholesterolemia and stroke. This research was aimed to determine the cytotoxic effect of sabrang onion bulbs extract, its selectivity index, its effect when combined with doxorubicin, its cell cycle inhibition effect, its apoptosis triggering effect and its suppression of cyclin D1 and Bcl-2 expression in T47D cells. Extracts preparation was done using stepwise maceration method using solvents based on polarity level, which were n-hexane, ethylacetate and ethanol, and phytochemical screening along with extracts and simplex characterization were also done. The extracts were tested for their cytoxicity against T47D cells with MTT method, their selectivity index, combination of active extracts with doxorubicin. The cell cycle inhibition effect and apoptosis triggering effect were tested using flowcytometry method, and the suppression of cyclin D1 and Bcl-2 expression was tested using immunocytochemistry method. The result of simplex characterization, n-hexane extract of sabrang onion (NESO), ethylacetate extract of sabrang onion (EAESO) and ethanol extract of sabrang onion (EESO) were as such : water content 8.64%; 4.98%; 8.27% and 9.11%; levels of water-soluble extract Keyword(s) : sabrang onion, T47D, Vero, cytotoxic, MTT, CI (combination index), cell cycle, flowcytometry, apoptosis, immunocytochemistry, cyclin D1 and Bcl-2. 8.52%; 1.12%; 9,51% and 72.19%; levels of ethanol soluble extract 14.05%; 2.28%; 49.26% and 81.66%; total ash content 2.89%, 0.34%, 0.43% and 1.73% and acid insoluble ash content 0.59%; 0.06%; 0.07% and 0.20%, respectively. The phytochemical screening test results indicated that both simplex and extract contains alkaloid, flavonoid, glycoside, saponin, anthraquinone glycoside, tannin and triterpenoids/steroids. The cytotoxic test on T47D cells showed the IC50 values of NESO at 265.023 ug/ml, EAESO 147.124 ug/ml and EESO 3782.29 mg/ml. Tests using vero cells showed that EAESO was selective on T47D cells (SI >3). Furthermore, EAESO combined with doxorubicin showed strong synergistic effect. The optimal concentration combination that showed strong synergistic effect was found in the concentration of EAESO-doxorubicin 36.75 ug/ml - 1.0 ug/ml (1/4 IC50 - 1/2 IC50). The combination of EAESO and doxorubicin inhibited the cell cycle at G0-G1 phase with the percentage of 54.74% due to the suppression cyclin D1 expression, while the apoptosis test showed that the aforementioned combination triggered apoptosis at the percentage of 13.86% due to the suppression of Bcl-2 expression.

Bawang sabrang (Eleutherine bulbosa (Mill.) Urb.) sudah digunakan secara turun temurun untuk berbagai penyakit seperti kanker payudara dan kolon, hipertensi, diabetes mellitus, hiperkolesterol dan stroke. Penelitian ini bertujuan untuk mengetahui efek sitotoksik dari ekstrak umbi lapis bawang sabrang, indek selektivitas, efek kombinasinya dengan doksorubisin, siklus sel, apoptosis dan penekanan ekspresi siklin D1 dan Bcl-2 pada sel T47D dengan metode imunositokimia. Pembuatan ekstrak dilakukan dengan cara maserasi bertingkat menggunakan pelarut berdasarkan tingkat kepolarannya, n-heksan, etilasetat dan etanol serta dilakukan skrining fitokimia dan karakterisasi terhadap simplisia dan ekstrak. Ekstrak diuji sitotoksiknya terhadap sel T47D dengan menggunakan metode MTT, indeks selektivitas, kombinasi ekstrak aktif dengan doksorubisin, siklus sel, apoptosis dan penekanan ekspresi siklin D1 dan Bcl-2 pada sel T47D dengan metode imunositokimia. Hasil karakterisasi simplisia, ekstrak n-heksana bawang sabrang (ENBS), ekstrak etilasetat bawang sabrang (EEABS) dan ekstrak etanol bawang sabrang (EEBS) diperoleh kadar air berturut-turut 8,64%; 4,98%; 8,27% dan 9,11%; kadar sari larut air 8,52%; 1,12%; 9,51% dan 72,19%; kadar sari larut etanol 14,05%; 2,28%; 49,26% dan 81,66%; kadar abu total 2,89%; 0,34%; 0,43% dan 1,73% serta kadar abu yang tidak larut asam 0,59%; 0,06%; 0,07% dan 0,20%. Hasil skrining fitokimia simplisia dan ekstrak diperoleh golongan alkaloid, flavonoid, glikosida, saponin, antrakuinon glikosida, tannin dan triterpenoid/ steroid. Hasil pengujian sitotoksik larutan uji terhadap sel T47D memberikan nilai IC50 ENBS sebesar 265,023 μg/ml, EEABS 147,124 μg/ml dan EEBS 3.782,29 μg/ml. Pengujian dengan sel vero menunjukkan EEABS selektif terhadap sel T47D (IS>3). Selanjutnya EEABS dikombinasikan dengan doksorubisin terhadap sel T47D memberikan efek sinergis kuat. Konsentrasi kombinasi optimum yang memberikan efek sinergis kuat terdapat pada konsentrasi EEABS - doksorubisin 36,75 μg/ml – 1,0 μg/ml (1/4 IC50 – 1/2 IC50). EEABS dan kombinasinya dengan doksorubisin dilakukan uji siklus sel dengan metode flowsitometri, hasilnya menghambat siklus sel pada fase G0-G1 dengan presentase 54,74% disebabkan terjadi penekanan ekspresi siklin D1 sedangkan pengujian apoptosis menunjukkan mekanisme apoptosis dengan presentase 13,86% dikarenakan terjadi penekananan ekspresi Bcl-2.