Objective: This study aim is to determine the effect of ethylacetate extract (EAE) of Moringa oleifera L. leaf and its combination with doxorubicin on cytotoxic effect, combination effect, cell cycle, apoptosis on MCF-7 cell lines. Methods: In vitro cytotoxic assays was determined by MTT (Microculture Tetrazolium Tehnique) assay, combined test was analysis with Compusyn software version 1, cell cycle inhibition and apoptosis were determined with Flowcytometry. Result: Cytotoxic activity of EAE with IC50 value 149.29 mg/mL, combination test of very strong synergistic at 1/8 IC50 EAE-1/4 IC50 doxorubicin concentration. The combination were caused cell accumulation in the G0/G1 phase, increased early and late apoptosis on MCF-7 cell lines. Conclusions: Based on the results combination giving a very strong synergistic effect. Therefore more research is needed on the mechanism
Keywords: Combination effect; Moringa oleifera L.; MCF-7; Doxorubicin; Ethylacetate
The therapy strategies available to cure breast cancer including cytotoxic agents (chemotherapy) have been considerable, but these cytotoxic agents have adverse effects on healthy tissue. One of the most widely used chemotherapy is doxorubicin [1], but its use provides side effects against normal tissue [1-3]. Dose reduction is can reduce the side effects of doxorubicin [4]. Therefore it becomes a challenge to improve clinical application of chemotherapeutic agents to be more effective. One of the strategies now gaining attention is the use of cochemotherapy which has a synergistic effect with chemotherapeutic agents to improve their efficacy and reduce their toxicity to healthy tissue. Therefore, research on chemoprevention agents has synergistic effects when combined with anticancer drugs.
One of the chemoprevention agents that can be used is the leaves of Moringa oleifera L., or better known as daun kelor in Indonesia. This plant is known to be potent anticancer by increasing apoptosis and inhibiting the proliferation of cancer cells [5-6]. Moringa leaves contain flavonoids (kaempferol, quercetin, myricetin), alkaloid (moringine), benzyl isothiocyanate (BITC), phenetyl isothiocyanate (PEITC) and glucosinolate which is particularly useful as cancer cell chemoprevention [6-9].
Previous research on the effect of ethanol extract of Moringa leaves gave a synergistic effect of increasing the cytotoxicity of doxorubicin in Hela cells, where this : Cytotoxic activity of EAE with IC50 value 149.29 g/mL, combination doxorubicin alone [10], while breast cancer cells had never been done. The aim of this study want to examine whether Moringa oleifera L. leaves has synergistic efficacy with chemotherapy agent doxorubicin so it could be decrease its effective dose which also reduces the toxicity of chemotherapy agent.
Fresh leaves of Moringa oleifera L. were collected from Bukit Batrem Village, East Dumai District, Riau Province, Indonesia. Moringa oleifera L. was identified in Herbarium Medanense (MEDA) University of Sumatera Utara no. 1785/MEDA/2017. Chemicals used were Doxorubicin (Ebewe), DMSO (Sigma), [3- (4,5- dimethylthiazole-2-yl) -2,5-diphenyl tetrazolium bromide] (MTT) (Sigma), Propidium Iodide kit (Biolegend), Annexin V (Biolegend).
The technique was used in graded maceration, 600 grams of leaf powder Moringa oleifera L. was macerated with nhexane (3x 6L), powder dried in air and re-extracted with ethylacetate (3x 6L), filtrate collected and filtered using filter paper Whatman no 42 (pore size 2.5 μm) and concentrated with rotary evaporator then inserted in freeze-dried to produced dry extract [11]. This study use ethylacetate as a solvent due to its characteristic as a semi polar solvent which can pull out polar and non polar compound
Phytochemical screening carried out on ethylacetate extract leaves of Moringa oleifera L includes examining the chemical secondary metabolites of alkaloids, flavonoids, glycosides, tannins, saponins, triterpenoids, and steroids.
The treatment of extract used several concentration series of 1000 μg / mL; 500 μg / mL; 250 μg / mL; 125 μg / mL; 62.5 μg / mL; 31.25 μg / mL and 15.625 μg / mL. The treatment of doxorubicin used several concentration series of 24.00 μg / mL, 12.00 μg / mL; 6.00 μg / mL; 3.00 μg / mL; 1.50 μg / mL; 0.75 μg / mL and 0.375 μg / mL.
MCF-7 cells and Vero cells used are from the Department of Parasitology Medical Faculty of Gadjah Mada University Yogyakarta. MCF-7 cells were grown on DMEM media and Vero cells with M199 media supplemented with 10% (Gibco) Fetal bovine, Penicillin 1% Streptomycin 1% (Gibco) and Fungizone 0.5% (Gibco) were incubated at 37°C, CO 5%. The inoculums 2 4 seeded on a 96 well plate (Iwaki), each well 1 x 10 cells/0.1 mL. Cell culture were incubated at 37°C, 5% CO for 24 hours. After 24 hours the media was discarded 2 and the cell plus EAE, doxorubicin, and its combination were incubated for 24 hours then the medium was removed and 0.5 mg / mL of MTT was added and incubated for 4 hours at 37°C, 5% CO after crystal 2.
formazan was formed and 10% SDS was added to dissolve the formazan crystals, then incubated for 24 hours at room temperature and shielded from light. The absorbance was measured with microplate reader at λ 595 nm [12]. The resulting absorbance was converted to a percentage of cell viability, then the selectivity index (IS) EAE was determined against MCF-7 cells. Further IC 50 single and combination treatment results were analyzed with Compusyn software version 1 to determine the Combination Index (CI) [13-14].
The equation to determine the viability of cells %Viability Absorbance of treatment– absorbance of medium = x100% absorbance of control cells − absorbance of medium The equation to determine selectivity index (SI) IC50 on Vero cells
5 MCF-7 cells (5x10 cells/mL) were seeded into 6-well plate and incubated for 24 hours then treated with EAE, doxorubicin, and its combination and then incubated for 24 hours. After 24 hours the media was moved into the conical tube and then into well plus trypsin 0,025%, then washed with 2x PBS, collected into the conical and centrifuged at 2500 rpm for 5 minutes. The supernatant was thrown away, in the pellet added cold ethanol 70% for 2 hours for cell fixation.Then added with PBS, centrifuged at 3000 rpm for 3 min, the supernatant was removed, in pellets was added PI kit (containing 40 μg /g/mL PI and RNAse 100 mL) and resuspended. Then mixture was incubated at 37°C for 30 minutes. The Sample was analyzed by FAC Scan Flowcytometer. Based on its DNA content the percentage of cell accumulation in the cell cycle (G1, S, and G2/M) were calculated using ModFit Lt.3.0 [15].
5 MCF-7 cells (5x10 cells/mL) were seeded into 6-well plate and incubated for 24 hours then treated with EAE, doxorubicin, and its combination and then incubating for 24 hours. After 24 hours the media is fed into the conical tube and then into well plus trypsin 0,025%.Then washed with PBS, collected into the conical and centrifuged at 2500 rpm for 5 minutes. The supernatant was thrown away, in pellets added PBS, the suspension was centrifuged at 3000 rpm for 3 min, the supernatant was removed and Annexin V kit added to the pellet and resuspended then incubated at 37°C for 30 minutes. The Sample was analyzed by FAC Scan Flowcytometer [12, 15].
Analyzing of the cytotoxic test data using SPSS 24 software by probit analysis and the combination test was analyzed with Compusyn Software version 1.
The extraction of 600 grams of Moringa dried leaf obtained 20.025 grams of thick extract and stored at 2- o 8 C.
Phytochemical screening result showed that ethylacetate extract leaves of Moringa oleifera L positively contains of flavonoids, saponins, tanins, glycosides and steroids/triterpenoid.
50 The cytotoxic effects of EAE, doxorubicin, and its combination against MCF-7 cells and their selectivity with Vero cells were determined by the MTT assay. In each EAE treatment, doxorubicin, and its combination were showed cell growth inhibition as indicated by IC 50 values. The IC50 value of EAE 149.29 g/mL and doxorubicin 5.80 μg/ml, and the combination was showed a higher inhibitory effect if it compare with the single treatment. The optimum combination index (very strong synergistic effect) showed in 1/8 IC value of EAE and 50 1/4 IC (18.66 μg/mL – 1.45 μg/mL) was categorized 50 with very strong synergistic effect (CI ˂ 0.1). These effects is related to cell cycle modulation and apoptotic induction.
The selectivity of EAE was determined with an executed cell viability assay on Vero cells. The single treatment of EAE was showed cytotoxicity effect on Vero cells with IC50 983.97 μg/ml. We were compared IC50 of EAE on Vero cells to MCF-7 cells to determine selectivity index (SI). Selectivity index of EAE was showed 6.59, SI>3 is supposed to be selective to MCF-7 cell lines. The result was showed that EAE is selective to MCF-7 cells instead of Vero cells [16].
The results of EAE cell viability values, doxorubicin, and their combinations were analyzed with Compusyn software version 1 which gave the result of the combined index (CI) value. The results obtained can be seen in table 1 and figure 1, where the grade of CI in each treatment with the value of CI > 1 antagonism, CI = 1 additive and CI < 1 synergism [13-14].
Doxorubicin is one of the chemotherapy agents with the potent activity of IC 5.8 μg/mL to decrease toxic effect 50 and prevent doxorubicin resistance to MCF-7 cells needed decrease dose of doxorubicin. In this study the combination of EAE and doxorubicin showed a very strong synergistic effect with CI value is 0.023 at the EAE-doxorubicin concentration (18.66 g/mL-1.45 g/mL). EAE increases the cytotoxic activity of doxorubicin in MCF-7 cells compared to single treatment. This synergistic effect is related to natural product contained in Moringa oleifera L. leaves such as flavonoid, isothiocyanate and glucosinolate is active compound to the induction of apoptosis and inhibition of cell cycle [6,9].
Determination of EAE, doxorubicin, and its combination GO-G1 in inhibiting cell cycle using flowcytometric method [15].
S-phase G2-M M1 The effect of EAE, Doxorubicin, and its combination are R1 M5 given in Figure 2, whereas with treatment of single doxorubicin at 2.90 μg/mL caused cell accumulation at File: KS SS .001 Total Ev ents: 20000 G0/G1 phase (55.00%), EAE at 74.63 μg/mL caused cell X Parameter: FL2-A FL2-Area (Linear) accumulationat G0/G1 phase (GO-G1 63.77Marker Ev ents)%, % Gated and % Total combination Mean CV Median File: 1/2 DOXO MCF7 SS.018 Total Ev ents: 20000 X Parameter: FL2-A FL2-Area (Linear) Marker Ev ents % Gated % Total Mean CV 50 All 5020 100.00 0.10 273.90 27.33 File: 1/2 DOXO MCF7 APOPT.004 Patient ID: 0409.18 50 50 Acquisition Date: 09-Apr-18 Gate: No Gate showed higher value compared than the cell control was 47.64%. These results indicate that EAE may increase the GO-G1 cytotoxic effect in the G /G phase.
G2-M0 1 S-phase Inhibition of the cell cycle by EAEM1 is probably caused by R1 M5 the effect of the active content contained in the leaves of Moringa oleifera L. that can inhibit the activation of Nuclear factor-kappa B (NF-kB). Which is NF-kB is a File: 1/2 EEADK MCF7 SS.003 Total Ev ents: 20000 X Parameter: FL2-A FL2-Area (Linear) transcription GO-G1factor that has a important role in cell Marker Ev ents % Gated % Total Mean CV Median File: KOMB MCF7 SS.001 Total Ev ents: 20000 X Parameter: FL2-A FL2-Area (Linear) Median Marker Ev ents % Gated % Total Mean CV Median 248.00 All 391 100.00 1.96 276.42 29.97 242.00 File: KOMB MCF7 APOP.002 Patient ID: 0419.18 Acquisition Date: 19-Apr-18 Gate: No Gate
Based on the results we obtained EAE had very strong synergistic effect with doxorubicin therefore potentially used as a co-chemotherapy agent for breast cancer therapy with its activity induces apoptosis, and inhibits cell cycle.
We gratefully thanks BPPSDM KEMENKES RI (Badan Pengembangan dan Pemberdayaan Sumber Daya Manusia Kementerian Kesehatan Republik Indonesia) through “Beasiswa Tugas Belajar 2016” for financial support in the study.