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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. "

[1] Hanahan D, Weinberg RA . The hallmarks of cancer[J]. Cell, 2000,100(1):57-70
doi: 10.1016/S0092-8674(00)81683-9
[2] Rang HP, Dale MM, Ritter JM, Moore PK . Pharmacology.5th ed[M]. London: Churchill Livingstone, 2003, 693-698
[3] World Health Organization. Cancer. (2012-02)[ 2012-02-15]. Cancer. (2012-02) [2012-02-15]. .
[4] Jemal A, Siegel R, Xu J, Ward E . Cancer statistics, 2010[J]. CA Cancer J Clin, 2010,60(5):277-300
doi: 10.3322/caac.20073
[5] Stuckey A . Breast cancer: epidemiology and risk factors[J]. Clin Obstet Gynecol, 2011,54(1):96-102
doi: 10.1097/GRF.0b013e3182080056
[6] Mazumdar M, Fournier D, Zhu DW, Cadot C, Poirier D, Lin SX . Binary and ternary crystal structure analyses of a novel inhibitor with 17β-HSD type 1: a lead compound for breast cancer therapy[J]. Biochem J, 2009,424(3):357-366
doi: 10.1042/BJ20091020
[7] Jin H, Zangar RC . Protein modifications as potential biomarkers in breast cancer[J]. Biomark Insights, 2009, 4191-200
doi: 10.4137/BMI.S2557 pmid: 2805424
[8] Tjindarbumi D, Mangunkusumo R . Cancer in Indonesia, present and future[J]. Jpn J Clin Oncol, 2002,32(Suppl):S17-S21
doi: 10.1093/jjco/hye123 pmid: 11959873
[9] Todorova I, Simeonova G, Simeonov R, Dinev D . Efficacy and toxicity of doxorubicin and cyclophosphamide chemotherapy in dogs with spontaneous mammary tumours[J]. Trakia J Sci, 2005,3(5):51-58
[10] Greidanus J, Willemse PH, Uges DR, Oremus ET, De Langen ZJ, De Vries EG . Continuous infusion of low-dose doxorubicin, epirubicin and mitoxantrone in cancer chemotherapy: a review[J]. Pharm Weekbl Sci, 1988,10(6):237-245
[11] Ficus septica Burm . F. on human breast cancer T47D cell lines[J]. Int J Phytomedicine, 2011,3(2):216-226
[12] Mosmann T . Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[J]. J Immunol Methods, 1983,65(1-2):55-63
doi: 10.1016/0022-1759(83)90303-4
[13] Chou TC, Talalay P . Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors[J]. Adv Enzyme Regul, 1984, 2227-55
[14] Hermawan A, Meiyanto E, Susidarti RA . Hesperidin increases cytotoxic activity of doxorubicin on MCF-7 cells[J]. Indones J Pharm, 2010,21(1):8-17
[15] Meiyanto E, Fitriasari A, Hermawan A, Junedi S, Susidarti RA . The improvement of doxorubicin activity on breast cancer cell lines by tangeretin through cell cycle modulation[J]. Orient Pharm Exp Med, 2011,11(3):183-190
doi: 10.1007/s13596-011-0016-4
[16] Haryanti S, Junedi S, Meiyanto E . Ethanolic extract of Hedyotis corymbosa L. increases cytotoxic activity of doxorubicin on MCF-7 breast cancer cell[J]. Indones J Biotech, 2009,14(1):1146-1154
[17] Kumala S, Septiseptyana EP, Meiyanto E . Cytotoxic effect of secondary metabolites produced by endopytic fungi 1.3.11, 1.1.6 and 1.2.6 isolated from the fruit of “Tanaman Buah Makassar”(Brucea janvanica L. Merr) on in-vitro T47D and MCF-7 intact cells and identification of the fungus 1.3.11 by ribosomal DNA sequence analysis[J]. Int J Pharm Pharm Sci, 2010,2(2):80-83
[18] Nurrochmad A, Lukitaningsih E, Meiyanto E . Anticancer activity of rodent tuber(Thyphonium flagelliforme(lodd.) Blume) on human breast cancer T47D cells[J]. Int J Phytomedicine, 2011,3(1):138-146
[19] Meiyanto E, Handayani S, Susidarti RA, Jenie RI . Chloroform fraction of Areca(Areca catechu L.) induces apoptosis and decreases Bcl-2 expression on MCF-7 cells[J]. Artocarpus, 2008,8(2):55-59
[20] Meiyanto E, Handayani S, Jenie RI . Snake beans(Vigna sinensis(L) Savi ex Hassk) extract increases breast epithelial cells proliferation[J]. Indones J Pharm, 2008,19(4):191-197
[21] Pebriana RB, Wardhani BWK, Widayanti E, Wijayanti NLS, Wijayanti TR, Riyanto S, Meiyanto E . Apoptotic effect of Kenikir leaves(Cosmos caudatus Kunth.) methanolic extract on breast cancer cell line[J]. Pharmacon, 2008,9(1):21-26
[22] Di Leo , Tanner M,Desmedt C,Paesmans M,Cardoso F,Durbecq V,Chan S,Perren T,Aapro M,Sotiriou C,Piccart MJ,Larsimont D,Isola J; TAX 303 translational study team.p-53 gene mutations as a predictive marker in a population of advanced breast cancer patients randomly treated with doxorubicin or docetaxel in the context of a phase Ⅲ clinical trial[J]. Ann Oncol, 2007,18(6):997-1003
doi: 10.1093/annonc/mdm075
[23] Vayssade M, Haddada H, Faridoni-Laurens L, Tourpin S, Valent A, Bénard J, Ahomadegbe JC . P73 functionally replaces p53 in adriamycin-treated, p53-deficient breast cancer cells[J]. Int J Cancer, 2005,116(6):860-869
doi: 10.1002/ijc.21033 pmid: 15849742
[24] Rudin CM, Thompson CB . Apoptosis and disease: regulation and clinical relevance of programmed cell death[J]. Annu Rev Med, 1997, 48267-281
doi: 10.1146/annurev.med.48.1.267 pmid: 9046961
[25] Grimm D, Wehland M, Pietsch J, Infanger M, Bauer J . Drugs interfering with apoptosis in breast cancer[J]. Curr Pharm Des, 2011,17(3):272-283
doi: 10.2174/138161211795049723 pmid: 21348828
[26] Kumar R, Vadlamudi RK, Adam L . Apoptosis in mammary gland and cancer[J]. Endocr Relat Cancer, 2000,7(4):257-269
doi: 10.1677/erc.0.0070257 pmid: 11174847
[27] Fan TJ, Han LH, Cong RS, Liang J . Caspase family proteases and apoptosis[J]. Acta Biochim Biophys Sin (Shanghai), 2005,37(11):719-727
doi: 10.1111/j.1745-7270.2005.00108.x pmid: 16270150
[28] Sodhi RK, Singh N, Jaggi AS . Poly(ADP-ribose) polymerase-1(PARP-1) and its therapeutic implications[J]. Vascul Pharmacol, 2010,53(3-4):77-87
doi: 10.1016/j.vph.2010.06.003
[29] Peralta-Leal A, Rodríguez MI, Oliver FJ . Poly(ADP-ribose) polymerase-1(PARP-1) in carcinogenesis: potential role of PARP inhibitors in cancer treatment[J]. Clin Transl Oncol, 2008,10(6):318-323
doi: 10.1007/s12094-008-0207-8
[30] Boulares AH, Yakovlev AG, Ivanova V, Stoica BA, Wang G, Iyer S, Smulson M . Role of poly(ADP-ribose) polymerase(PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells[J]. J Biol Chem, 1999,274(33):22932-22940
doi: 10.1074/jbc.274.33.22932
[31] Rios J, Puhalla S . PARP inhibitors in breast cancer: BRCA and beyond[J]. Oncology (Williston Park), 2011,25(11):1014-1025
doi: 10.1038/leu.2011.139 pmid: 22106552
[32] Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L . Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity[J]. Pharmacol Rev, 2004,56(2):185-229
doi: 10.1124/pr.56.2.6
[33] Kim JB, Yu JH, Ko E, Lee KW, Song AK, Park SY, Shin I, Han W, Noh DY . The alkaloid berberine inhibits the growth of Anoikis-resistant MCF-7 and MDA-MB-231 breast cancer cell lines by inducing cell cycle arrest[J]. Phytomedicine, 2010,17(6):436-440
doi: 10.1016/j.phymed.2009.08.012
[34] Wu PL, Rao KV, Su CH, Kuoh CS, Wu TS . Phenanthroindolizidine alkaloids and their cytotoxicity from the leaves of Ficus septica. Heterocycles. 2002; 57(12):2401-2408.
doi: 10.3987/COM-02-9615
[35] Ueda JY, Takagi M , Shin-ya K. Aminocaprophenone- and pyrrolidine-type alkaloids from the leaves of Ficus septica.[J] Nat Prod. 2009; 72(12):2181-2183.
doi: 10.1021/np900580f pmid: 19938815
[36] Damu AG, Kuo PC, Shi LS, Li CY, Kuoh CS, Wu PL, Wu TS . Phenanthroindolizidine alkaloids from the stems of Ficus septica.[J] Nat Prod. 2005; 68(7):1071-1075.
doi: 10.1021/np050095o pmid: 16038551
[37] Damu AG, Kuo PC, Shi LS, Li CY, Su CR, Wu TS . Cytotoxic phenanthroindolizidine alkaloids from the roots of Ficus septica. Planta Med. 2009; 75(10):1152-1156.
doi: 10.1055/s-0029-1185483 pmid: 19296431
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