Efek Ekstrak Etanol dan Air Daun Belalai Gajah (Clinacanthus Nutans) terhadap Ekspresi Protein P53 Mouse Double Minute 2 Homolog dan AKt pada Sel Punca Kanker Payudara

Abstract

Background: Breast cancer is the most common cancer among women in Indonesia with the number of new cases reaching 48,998. The advancement of medical technology in the implementation of cancer patients has not been able to solve problems such as recurrence and resistance.One factor causing resistance and recurrence is the presence of breast cancer stem cells (BCSCs). Clinacanthus nutans (C.nutans) is a plant that is believed to have anti-cancer activity, but the effectiveness of this plant targeted on BCSCs has not been known to date. Our study aimed to analyse the cytotoxic effects of ethanol extracts of various concentrations and C.nutans water extract on the viability of BCSCs. Determining the most optimal IC50 values, determining the effects of C.nutans extract on cell cycles and cell apoptosis, assessing the influence of C.nutans extracts on proteins P53, MDM2 and AKt that are involved in the pathways of apoptosis and proliferation, and identifying active compounds. Method: In vitro experimental research post-test only control group. The research group was divided into eight groups: ethanol extract (EE) 100%,80%,60%,40%,20%, water extact of C.nutans, negative control and positive control. In this study, the identification of plants, the preparation of simplisia and extracts, the establishment of phytochemical screening and the total levels of phenols and flavonoids were performed. The cytotoxic effect is performed using MTT tests, cell cycle tests and apoptosis are performed with flowcytometry methods. Analysis of P53, MDM2 and AKt proteins was done using the Western- blot method. The cytotoxic effects of various groups on the viability of breast SPK were statistically evaluated using one-way anova on normal-distributed data and Kruskal-wallis test on non-normal-distributable data, p<0,05 values were considered statisticamente significant. Identification of active compounds in C.nutans extract was done using the LC-MS/QTOF method. Results: water content, water-soluble sulphur content, ethanol-solubility sulphure content, total ash content, acid insoluble ash levels are still within reasonable limits. In phytochemical screening, flavonoid compounds, glycosides and tannins were found in simplisia, ethanol extracts of various concentrations (100%,80%,60%,40%,20%) and water C.nutans. At the determination of total levels of phenols and phlavonoids, the highest concentrations were found on 100% C. nutans ethanol extract of 16.56±3.09 and 30.39±4.54 mg AGE/g extract. In the BCSCs validation test, a valid BCSCs was obtained with a CD44+/CD24- expression percentage of 92.4% and a mammosphere formed with diameter >60μm. We found a decrease in the viability of BCSCs after the administration of ethanol extract and C.nutans water with the smallest IC50 value found in C.nutans water extract of 16.16 μg/ml. In a cell cycle test, the 8 μg / ml dose of C. nutans water extracts were able to suppress cells in sub-phase G1 to 0.37 ± 0.12% (p < 0.05) whereas in phase G0/G1, a combination of 16μg /ml C. nutans water extract–Paclitaxel 16 nM was capable of suppressing up to 30.53 ±0.87% (p<0.05). In the G2/M phase the combination was found to increase the percentage of trapped cells to 40.43 ± 0.049% (P< 0.05). In the apoptosis test, a single dose of 8 μg/ml C.nutans water extract was already able to increase the number of BCSCs undergoing early apoptosis by 8.12±0.75% and increasingly. The combination of water extract C.nutans 16 μg/ml–Paclitaxel 16 nM was able to increase early apoptosis of BCSCs by 16.13±1.62% (p<0,05). Similarly, in the late apoptotic process, after administration of combination water extracted C.nutans 16μg/mL –Paclitaksel 16nM, an increase in the number of dead cells was found by 48.10±1.82% (P<0.05). In this study, we found that the water extract of C.nutans 8 μg/ml was capable of increasing the expression of protein P53 by 121,02±2,98% (p<0,05). This was also consistent with the MDM2 protein expression that decreased maximally after administration of C.nutans 8 μg/ml water extract to 81.81±12.85% (p<0,05. In the AKt protein expression test, the most decreasing AKt protein expressions were found in the C.nutans group of water extracts doses of 8μg/mL and 16μg /mL (p <0,05). In this study, five active compounds that have potential anti-cancer activity were identified, namely Isoorientin 2''-O-arabinoside,2-Hydroxyhexadecanoic acid,2- Aminohexadecanonic acid,Enigmol and Spisulosine. Conclusion:Ethanol and water extracts of C.nutans had a cytotoxic effect on BCSCs, meanwhile the potent cytotoxic effect belonged to water extract. The water extract of C. nutans worked synergistically with paclitaxel in intervening the pathway of proliferation and apoptosis of BCSCs.