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ISSN: 20880197
e-ISSN: 2355-8989
Anis Fauzia, Umi Any Tiyas Wati, Cahyaning Gita Rizkita, Eka Wahyuni Nurul
Qoriah, Ibrahim Arifin*
Abstract
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rinsed with PBS and then transferred to the Test solution is added to the wells and
same conical. Centrifuged by 600 rpm for 5 incubated for 24 hours. Conical transferred to
minutes and the supernatant was discarded. the cell media and the wells are washed with
The precipitated cells were washed with cold 500 mL of PBS, then transferred to the same
PBS 500 mL and centrifuged by 600 rpm for conical and harvesting with the addition of
5 minutes and then PBS discarded, added 500 0.25% trypsin-EDTA as much as 150
mL of 70% alcohol and stored conical at a mL/well and incubated 3 minutes. Media
temperature of 370C for 30 minutes and then culture is added 1000 mL/well and cells
centrifuged by 600 rpm for 5 minutes and the resuspended. Cells centrifuged by 600 rpm
alcohol is removed, add 500l PBS and for 5 minutes, the media removed. The cell
centrifuged by 2000 rpm for 3 minutes. sediment was dissolved in buffer kit annexin
Flowcytometry reagent is added and allowed V-FLOUS. The cell suspension was
to stand 30 minutes in conical wrapped in homogenized and incubated for 15 min at
aluminum foil. The cell suspension is room temperature in conical wrapped in
transferred to flowcyto-tube and analyzed by aluminum foil. Cells are then transferred to
FACS Calibur flowcytometer program to flowcyto-tube and analyzed (Gattenlohner, et
determine the profile of the cell cycle (Ayers, al., 2009).
et al., 2011).
Analysis of Cytotoxicity Assay Data
Observation of Apoptosis using Data absorbance or OD (Optical
Flowcytometry Dencity) of ELISA reader is converted to
A number of 5 x 105 cells/well percent of cell viability using the following
transferred into a 6-well plate, incubated in a formula:
5% CO2 incubator at 37 C for overnight.
(1)
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which provide IC50 of 778,05 g /ml (Fita, lowering T47D cells viability is presented in
2015), whereas doxorubicin cytotoxicity give Figure 1, while the morphology of breast
IC50 value of 141.055 nM. Cytotoxicity cancer cells after treatment with doxorubicin
profile of MLE which shows its ability in and MLE is shown in Figure 2.
Figure 1. The ability of MLE to decrease the viability T47D cell. Methanol extract kenikir leaves
inhibits the growth of T47D breast cancer cell gives IC50 value of 504,840 g/ml.
Cytotoxicity combination assay was and doxorubicin was lowered to under its IC50
conducted to know the effectivy of the respectively so can suppress the side effects
combination MLE and doxorubicin in of doxorubicin. The results show the
inhibiting the growth of T47D cells. In the Combination Index of MLE and doxorubicin
combination assay, the concentration of MLE as shown in Table 1.
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Table 1. Combination Index (CI) of MLE and Doxorubicin in T47D breast cancer cells.
MLE
Concentration Doxorubicin concentration (nM)
(g/ml) 71 47,00 35,25 23,50
252,50 0,6 1,0 0,6 0,7
Combination Index in Table 1 shows has a greater cytotoxic effect than the sole
that the combination MLE of 252,50 g/ml effect of each compound. The best synergistic
with doxorubicin of 71 nM and 35,25 nM; the effect is shown by the combination MLE of
combination MLE of 126,25 g/ml and 84,17 g/ml and doxorubicin of 23,50 nM
doxorubicin of 23,50 nM; and the with CI value of 0,5. Doxorubicin and MLE
combination MLE of 84.17 g/ml and combination effects in T47D breast cancer
doxorubicin of 47,00 nM and 23,50 nM, cells is shown in Figure 3.
provides a synergistic effect which means it
Figure 3. Combination effects of MLE and doxorubicin in T47D breast cancer cells.
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Table 2. The distribution of T47D cells in cell cycle phases after treatment
Sample G1 phase (%) S phase (%) G2/M phase (%)
Kontrol Sel 32,78 30,24 28,08
MLE 84,17 g/ml 35,24 21,74 44,29
Doxorubicin 23,5 nM 46,57 16,45 39,10
MLE 84,17 g/ml + Doxorubicin 23,5
nM 30,37 26,76 50,20
Table 3. Death Percentage Cells after Treatment in Breast Cancer Cells T47D
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