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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (10): 1162-1170.doi: 10.3736/jcim20121014

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Synergistic effects of ethyl acetate fraction of Ficus septica Burm. f. and doxorubicin chemotherapy on T47D human breast cancer cell line

Agung E. Nugroho1,2, Adam Hermawan1,3, Dyaningtyas D. P. Putri1, Edy Meiyanto1,3, Lukman Hakim2 #br#   

  • Received:2012-03-08 Accepted:2012-04-09 Online:2012-10-20 Published:2018-10-15

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Objective: Previously, ethanolic extract of Ficus septica Burm. f. (Moraceae) leaves and its ethyl acetate soluble fraction (EASF) exhibited potent cytotoxic effects on T47D breast cancer cells. In the present study, we further investigated the effects of EASF of ethanolic extract of F. septica leaves in combination with doxorubicin on T47D breast cancer cell line in cytotoxicity, cell cycle arrest and apoptosis induction. .

Conclusion: The cytotoxic effect analysis on T47D cells was carried out using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Analysis of cell cycle distribution was performed using a flowcytometer and the data were analyzed using ModFit LT 3.0 program. Apoptosis assay was carried out by double staining method using ethydium bromide-acridin orange. The expression of cleaved-poly ADP-ribose polymerase in the T47D cell line was identified using immunohistochemical techniques.

Results: The combination of doxorubicin (2 to 8 nmol/L) with EASF (0.875 to 7 μg/mL) more potently inhibited cell growth than the single treatment of doxorubicin in T47D cells. In addition, the combination of doxorubicin and EASF could increase the incidence of cells undergoing apoptosis. EASF was found to improve cytotoxic effect of doxorubicin by changing the inhibition of cell cycle G2/M to G1 phase. The combination also exhibited a more intensive stimulatory effect on cleaved-PARP expression in T47D cells than the single treatment.

Conclusion: It is concluded that EASF may enhance doxorubicin activities in T47D cells by inducing apoptosis and cell cycle arrest.

Key words: Ficus, plant extracts, breast neoplasms, cell line, tumor, drug therapy, combination, apoptosis

"

EASF
(μg/mL)		 Cell viability (%)
Doxorubicin
(0 nmol/L)		 Doxorubicin
(2 nmol/L)		 Doxorubicin
(4 nmol/L)		 Doxorubicin
(6 nmol/L)		 Doxorubicin
(8 nmol/L)
0 100.00±0.40 57.19±3.75 46.25±9.59 39.33±10.09 39.93±9.78
0.875 46.44±10.62 33.27±4.25* 40.38±4.41 46.69±1.40 35.80±1.76
1.75 36.55±1.08 38.44±2.67* 29.79±3.97* 34.71±1.32 31.38±3.30
3.5 34.51±3.24 43.11±0.26* 28.05±5.18* 34.36±2.69 31.92±3.19
7 24.56±6.39 34.21±1.64* 25.91±2.93* 29.64±1.52 34.81±8.60

"

EASF (μg/mL) Combination index
Doxorubicin (2 nmol/L) Doxorubicin (4 nmol/L) Doxorubicin (6 nmol/L) Doxorubicin (8 nmol/L)
0.875 0.073 0.194 0.388 0.293
1.75 0.115 0.121 0.224 0.246
3.5 0.216 0.122 0.243 0.270
7 0.156 0.126 0.214 0.364

Figure 1

Combination effects of doxorubicin and EASF of Ficus septica on cell viability of T47D cells tested by MTT method (Light microscopy, ×100)Cell morphology was observed under an inverted light microscope after incubation with A: vehicle (control cells); B: EASF 7 μg/mL; C: doxorubicin 8 nmol/L; and D: combination of doxorubicin 8 nmol/L and EASF 7 μg/mL. Dead cells are pointed by black arrows. EASF: ethyl acetate soluble fraction; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide."

Figure 2

Apoptosis induction effects of doxorubicin, EASF and their combination on in T47D cells after 24 h incubation (Fluorosence microscopy, ×400) The cells were stained by etidium bromide-acridine orange and viewed under a fluorosence microscope. A: Control cells; B: EASF 7 μg/mL; C: Doxorubicin 8 nmol/L; D: Combination of doxorubicin 8 nmol/L and EASF 7 μg/mL. Viable and death cells are represented by green fluorescence. Orange fluorescence with DNA compact shows apoptosis (yellow arrow), while orange fluorescence with no DNA shows necrosis (white arrow). EASF: ethyl acetate soluble fraction. "

Figure 3

Cell cycle analysis using flowcytometryT47D cells were treated with doxorubicin, EASF and their combination for 24 h and stained by propidium iodide. A: Control cells; B: EASF 7 μg/mL; C: Doxorubicin 8 nmol/L; D: Combination of doxorubicin 8 nmol/L and EASF 7 μg/mL. EASF: ethyl acetate soluble fraction. "

Figure 4

Expression of cleaved-PARP in T47D cells (Light microscopy, ×400) Immunohistochemical method was used in the detection of cleaved-PARP. The cells were incubated in diaminobenzidine solution, and counterstained with Mayer’s haematoxylin. A: Control cells with antibody; B: Control cells without antibody; C: EASF 7 μg/mL; D: Doxorubicin 8 nmol/L; E: Combination of doxorubicin 8 nmol/L and EASF 7 μg/mL. Expression of cleaved-PARP protein is positively characterized by brown-stained cytoplasm of the cells. PARP: poly ADP-ribose polymerase; EASF: ethyl acetate soluble fraction. "

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