The improvement of doxorubicin activity on breast cancer cell lines by tangeretin through cell cycle modulation

RESEARCH ARTICLE

  The improvement of doxorubicin activity on breast cancer cell lines by tangeretin through cell cycle modulation

  Edy Meiyanto & Aditya Fitriasari & Adam Hermawan & Sendy Junedi & Ratna Asmah Susidarti Received: 24 May 2011 / Accepted: 10 June 2011 / Published online: 5 July 2011 # Institute of Oriental Medicine, Kyung Hee University 2011 Abstract Tangeretin, shows cytotoxic effect on COLO 205 colon cancer cells. Combination of tangeretin with tamox- ifen showed synergistic effect and increased the cancer cell sensitivity towards tamoxifen on T47D cells. However, the combination of tangeretin with chemotherapeutic agent doxorubicin on breast cancer cells have not been explored yet. Therefore, the aim of this research is to examine the improvement of cytotoxic effect of doxorubicin by tanger- etin through cell death induction and cell cycle modulation on MCF-7 and T47D cells. The cytotoxic effect of tangeretin, doxorubicin, and their combination on tested cells were carried out by using MTT assay. Cell cycle distribution was determined by flowcytometer FACS- Calibur and the flowcytometry data was analyzed using ModFit LT 3.0 program. Cell death assay were done by double staining method using ethydium bromide-acridin orange. Single treatment of tangeretin 5–100 μM did not show cytotoxic effect on MCF-7 and T47D cells. The combination of tangeretin 50 and 100 μM with doxorubicin 200 nM (MCF-7) and 7.5 nM (T47D) increased the cytotoxic effect of doxorubicin on both breast cancer cell lines. This improvement of cytotoxic effect is due to cell death induction and cell cycle modulation. Furthermore, single treatment of tangeretin showed cell death only on T47D cell and caused G1-phase arrest on MCF-7 cell and

  G2/M-phase arrest on T47D cell. While doxorubicin induced cell accumulation at G2/M phase in both cancer cell lines. However, combination of tangeretin and doxoru- bicin increased cell death on both cancer cell lines, compared with doxorubicin by itself. The combination also showed G1-phase arrest on MCF-7 cell and increased cell accumulation at G2/M phase on T47D cell. Based on this result, tangeretin is potential to be developed as co- chemotherapeutic agent for breast cancer by inducing apoptosis and cell cycle arrest. However, the molecular mechanism need to be explored further. Keywords Tangeretin . Co-chemotherapy . Breast cancer . Cell cycle arrest . Cell death Introduction Breast cancer is one of the death-cause cancer in the world (Jemal et al. ). Breast cancer is caused by the uncontrolled proliferation of breast cells which is happened because of their ability to avoid apoptotic mechanism (Yu et al. One drug for breast cancer therapy is doxorubicin. The using of doxorubicin has a lot of side effects and also resistance effects on breast cancer cell (Smith et al. Therefore, combination of doxorubicin with chemopreven- tive agent (co-chemotherapy) were needed to increase the activity of doxorubicin by inhibiting proliferation and inducing apoptosis of breast cancer cell.

• ) ^:

  One of the chemopreventive agents that has been reported to have antiproliferative effect is tangeretin, a polymethoxy flavone found in citrus species (Citrus aurantifolia). Tangeretin have been proven to inhibit the growth of estradiol-stimulated T47D cells (Van Slambrouck

  E. Meiyanto (

  A. Fitriasari :

  A. Hermawan : S. Junedi : R. A. Susidarti Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara, Yogyakarta 55281, Indonesia e-mail: meiyan_e@ugm.ac.id E. Meiyanto e-mail: meiyan_e@yahoo.com Orient Pharm Exp Med (2011) 11:183–190 DOI 10.1007/s13596-011-0016-4

  4 days treatment and cause G1-phase arrest on MCF-7 cells by 24 h, 48 h and 72 h treatment (Morley et al. ). Tangeretin has the smallest IC

  Treated cells abs Medium control abs Cells control abs Medium control abs

  cells/well) were transferred into 6-well plate (Iwaki, Japan) and incubated until the cells return to normal condition. Cells were treated by tangeretin, doxorubicin, and their combination, and incubated for 24 h. At the end of the incubation, the media containing free cells suspension were taken and transferred into 1.5 ml eppendorfs. The eppendorfs were centrifugated (2,000 rpm, 3 min) and the supernatant were removed. The cells in 6-well plate were added by phospate buffered saline (PBS), and the PBS were transferred into previous eppendorfs. The eppendorfs were centrifugated and the supernatant were removed again. This steps were repeated before the cells harvested by trypsin- EDTA 0.25% (Gibco, Invitrogen, Canada). Harvested cells were transferred into the eppendorfs and centrifugated (2,000 rpm, 30 s). The remaining cells in the 6-well plate were rinsed with PBS and transferred into the eppendorfs.

  5

  Flowcytometry assay Cells (5×10

  cells/well) were transferred to coverslips (Nunc, Denmark) in 24-well plate (Iwaki, Japan) and incubated for 24 h (50–60% confluent). Cells were treated by tangeretin, doxorubicin, and their combination, and incubated for 15 h. At the end of the incubation, coverslips containing cells were moved to object glass. Mixture solution of etidium bromide-acridine orange (Sigma, Sigma-Aldrich Corp, St. Louis, MO, USA) were added to the cells to form fluorescence cells. The fluorescence cells were examined immediately by fluorescence microscope (Zeiss MC80). Green fluorescence cells showed viable cells, while red fluorescence cells showed dead cells.

  4

  100% Cell death assay (double staining method) Cells (5×10

  Viable cells reacts with MTT to form purple formazan crystal. After 4 h, stopper sodium dodesil sulphate 10% in 0,1N sulphuric acid solution were added to dissolve formazan crystal. Cells were incubated over night and protected from light. Cells were shaken for 10 min before read by ELISA reader at λ 595 nm. The absorbance of each well converted to percentage of viable cells: % Viable cells ¼

  50

  cells/well) were transferred to 96-well plate (Iwaki, Japan) and incubated for 24 h (70–80% confluent). Cells were treated by tangeretin, doxorubicin, and their combination, and incubated for 24 h. At the end of the incubation, 5 mg/ml solution of MTT [3-(4,5-dimethylth- iazole-2-yl)-2,5-diphenyl tetrazolium bromide] (Sigma, Sigma-Aldrich Corp, St. Louis, MO, USA) were added to

  3

  Masashi Kawaichi (Nara Institute of Science and Technol- ogy (NAIST)), Nara, Japan. Cytotoxic assay (MTT assay) MCF-7 and T47D cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing Fetal Bovine Serum (FBS) 10% (v/v) (FBS qualified, Gibco, Invitro- gen™ USA) and penisillin-streptomisin 1% (v/v) (Gibco, Invitrogen Corporation, Grand Island, NY, 14072, USA). Cells (5×10

  Materials and methods Materials Tangeretin was obtained from Sigma Aldrich Chemie GmBH, Steinheim, Germany (Cat No. T8951) while doxorubicin was obtained from P.T. Ferron Par Pharmaceu- tical. A DMSO (Sigma Aldrich Chemie GmBH, Steinheim, Germany) solution of tangeretin was used for in vitro experiment by diluting desired consentration. The final DMSO concentration was not more than 1%. Cell lines MCF-7 and T47D cells were kindly provided by Prof.

  Those researches showed the potency of tangeretin as chemopreventive agent and became a basic for the development of tangeretin as co-chemotherapeutic agent to increase the cytotoxic activity and reduce the side effects of doxorubicin. Therefore, the purpose of this research is to examine the effect of tangeretin and its combination with doxorubicin on cell cycle and apoptosis of breast cancer cells.

  value on COLO 205 colon cancer cells, compared with the other flavonoid com- pounds, such as apigenin, kaempferol, myricetin, quercetin, luteolin, nobiletin, and rutin (Pan et al. ). Tangeretin also induce apoptosis on HL-60 leukemia cells (Hirano et al. ). The combination of tangeretin and tamoxifen in vitro increased the sensitivity of cancer cell synergistically towards tamoxifen (Bracke et al.

  The eppendorfs were centrifugated and the supernatant were removed. Pellet cells in eppendorfs washed by cold PBS and added by propidium iodide (PI) (Sigma Aldrich Chemie GmBH, Steinheim, Germany). The eppendorfs were wrapped in aluminum foil and incubated in 37°C for 10 min. After 10 min, cell suspension were homogenated and transferred into the flowcyto-tube to be analyzed by Fig. 1 Effect of tangeretin to the proliferation of MCF-7 (left) and T47D (right) breast cancer cells. The assay performed by ₃ incubating 5×10 cells/well with tangeretin (5–100 μM) for 24 h. After 24 h, cells were added by MTT reagent to cal- culate the absorbance which represent viable cells Result

  Induction of cell cycle arrest by tangeretin in MCF-7 and T47D cells Cytotoxicity of tangeretin

  Beside cell death induction, improvement of doxorubicin The direct cytotoxicity of tangeretin was assessed by MTT cytotoxic effect by tangeretin could also occurred through assay of MCF-7 and T47D breast cancer cell lines. Single cell cycle modulation. Cell cycle analysis of both breast treatment of tangeretin did not show cytotoxic effect on cancer cell lines showed that tangeretin and doxorubicin both cell lines, MCF-7 and T47D with cell viability more than 100% (Fig.

  This result showed that the using of

  tangeretin to the patient is probably safe without any toxicity to the cells. Surprisingly, combination of doxoru- bicin with tangeretin 5, 50, and 100 μM showed higher inhibition of cells growth than single treatment of doxoru- bicin in both breast cancer cell lines (Fig.

   ). On MCF-7

  cells tangeretin 50 μM and 100 μM decreased doxorubicin treated-cell viability from 66% to 42% and 66% to 31%, respectively. On T47D cells tangeretin 50 μM and 100 μM decreased doxorubicin treated-cell viability from 90% to 66% and 90% to 50%, respectively. This result suggested that this combination give improvement of doxorubicin cytotoxic effect. The decreasing cell viability possibly, could be occurred through inhibiting of cell proliferation and inducing cell death. Induction of cell death in MCF-7 and T47D cells by tangeretin and combination with doxorubicin The results from cytotoxic assay of tangeretin and doxoru- bicin were parallel with cell death assay using double staining method. Single treatment of doxorubicin and tangeretin in MCF-7 cells showed chromatin condensation and cell death induction, respectively. Combination of doxorubicin and tangeretin significantly increased the

  Fig. 2 Combination effect of doxorubicin and tangeretin to the incidence of cell death, compared to single treatment of proliferation of MCF-7 (a) and T47D (b) breast cancer cells. The doxorubicin (Fig.

   ). On T47D cells single treatment of ³

  assay performed by incubating 5×10 cell/wells with tangeretin, doxorubicin and tangeretin showed cell death induction, doxorubicin, and their combination on MCF-7 cell and T47D cell for while their combination increased the incidence of cell 24 h. After 24 h, cells were added by MTT reagent to calculate the absorbance which represent viable cells. Statistical analysis is death, compared to single treatment of doxorubicin (Fig.

   ).

  performed using analysis of variance (ANOVA) with Bonferroni’s test Orange fluorescence cells represent death cells that loss cell (SPSS release 17.0; SPSS Inc.,). Data were expressed as (Mean ± SD). membrane permeability and form apoptotic bodies that

• combination was considered significant to doxorubicin single

C D A B

  Fig. 3 Effect of tangeretin, doxorubicin, and their combina- tion on MCF-7 cell death. MCF-7 cell were treated by tangeretin, doxorubicin, and their combination for 15 h and stained by etidium bromide- acridine orange. a Cell control, b Tangeretin 100 μM, c Doxo- rubicin 200 nM, d Combination of tangeretin 100 μM and doxorubicin 200 nM. Viable cells give green fluorescence ( ), dead cells give orange fluorescence ( )

  A B C D

  Fig. 4 Effect of tangeretin, doxorubicin, and their combina- tion on T47D cell death. T47D cell were treated by tangeretin, doxorubicin, and their combina- tion for 15 h and stained by etidium bromide-acridine orange. a Cell control, b Tan- geretin 100 μM, c Doxorubicin 7.5 nM, d Combination of tan- geretin 100 μM and doxorubicin 7.5 nM. Viable cells give green fluorescence ( ), dead cells give orange fluorescence ( )

  Table 1 Cell cycle distribution Cell Treatment Concentration G1 (%) S (%) G2/M (%) after treatment of tangeretin, doxorubicin, and their combina-

  MCF-7 Control

  69.61

  22.75

  7.64

• – tion for 24 h

  Tangeretin 100 μM

  78.46

  13.95

  7.59 Doxorubicin 200 nM

  67.75

  2.84

  29.41 Tangeretin-Doxorubicin

  83.42

  0.00

  16.58 100 μM–200 nM

  42.19

  51.57

  6.24 Tangeretin 100 μM

• – T47D Control

  52.49

  21.16

  26.35 Doxorubicin 7.5 nM

  35.76

  38.78

  25.46 Tangeretin-Doxorubicin

  33.54

  19.69

  46.78 100 μM–7.5 nM have different cell cycle profile. Single treatment of tion of tangeretin and doxorubicin showed G1-phase arrest tangeretin induced cell accumulation at G1 phase on on MCF-7 cells and increased cell accumulation at G2/M MCF-7 cells and showed cell accumulation at G2/M phase phase on T47D cells (Table Figs.

   ). These results

  (G2/M-phase arrest) on T47D cells. While, doxorubicin suggested the domination of tangeretin in the improvement arrested both cell lines at G2/M-phase. However, combina- of doxorubicin cytotoxic effect.

  Fig. 5 MCF-7 cell cycle

  

A B

  modulation after treatment of tangeretin, doxorubicin, and

  Dip G1 : 69.61 % Dip G1 : 78.46 %

  1

  1

  their combination. MCF-7 cell

  Dip G2 : 7.64 % Dip G2 : 7.59 % 600 600

  were treated by tan3geretin,

  Dip S : 13.95 %

Dip S : 22.75 %

  doxorubicin, and their

  500 500

  combination for 24 h and stained by PI reagent before

  400 400 analyzed by flowcytometer.

  a Cell control, b Tangeretin

  Number Number

  100 μM, c Doxorubicin 300 300 200 nM, d Combination of tangeretin 100 μM and

  200 200

  doxorubicin 200 nM

  100 100 2 3

  2

  3

  4

  4

50 100 150 200 250

50 100 150 200 250

  Channels (FL2-A-FL2-Area) Channels (FL2-A-FL2-Area)

C D

  Dip G1 : 67.75 % Dip G1 : 83.42 % 600 600

  Dip G2 : 29.41 % Dip G2 : 16.58 % Dip S : 2.84 % Dip S : 0.00 % 500 500 400 400

  1

1 Number Number

  300 300 200 200 100 100

  3 2 2 3

  4

  4 50 100 150 200 250

50 100 150 200 250

  Channels (FL2-A-FL2-Area) Channels (FL2-A-FL2-Area)

  1. G1

  3. G2/M Fig. 6 T47D cell cycle modu-

  

A B

  1

  lation after treatment of tanger- etin, doxorubicin, and their

  Dip G1 : 42.19 % Dip G1 : 52.49 %

  1

  combination. T47D cell were

  Dip G2 : 6.24 % Dip G2 : 26.53 % 500 500

  treated by tangeretin, doxorubi-

  Dip S : 51.57 % Dip S : 21.16 %

  cin, and their combination for 24 h and stained by PI reagent

  400 400 r r

  before analyzed by flowcytom-

  e e b b

  eter. a Cell control, b Tangeretin

  m m 300 300

  100 μM, c Doxorubicin 7.5 nM,

  Nu Nu

  d Combination of tangeretin 100 μM and doxorubicin

  200 200

  2

  7.5 nM

  3

  2 100 100

  3

  4

  4 50 100 150 200 250 50 100 150 200 250

  Channels (FL2-A-FL2-Area) Channels (FL2-A-FL2-Area)

C D

  

Dip G1 : 35.76 %

Dip G1 : 33.54 %

Dip G2 : 25.46 %

  500 500 Dip G2 : 46.78 %

  

Dip S : 38.78 %

Dip S : 19.69 % 400 400 r r e e b b m m

  300 300 Nu Nu

  1

  1 200 200

  3 100 100

  4

  2

  4

  2

  3 50 100 150 200 250 50 100 150 200 250

  Channels (FL2-A-FL2-Area) Channels (FL2-A-FL2-Area)

  1. G1

  3. G2/M

  2. S

  4. SubG1

  Discussion Combination of tangeretin and doxorubicin showed cell death induction that supposed to be apoptosis. Previous study

  This study explored the effect of tangeretin alone and in mentioned that treatment of 30 μM tangeretin induced combination with doxorubicin on cytotoxicity, cell cycle, apoptosis of human neuroblastoma SH-SY5Y cells by and cell death induction of MCF-7 and T47D cells. Our reducing the mitochondrial membrane potential and elevated previous study showed that doxorubicin showed strong caspase-3 activity of non treatment cells, while combination of cytotoxic effect on MCF-7 and T47D cells with IC value tangeretin and nobiletin showed elevation of caspase-3

  50 of 467 and 15 nM, respectively (Hermawan et al.

  Junedi et al. ). The higher IC value on MCF-7 cells Tangeretin also inhibited the P-glycoprotein function and

  50

  is due to the characteristic of MCF-7 cells which is resistant significantly influenced the cell cycle, whereas they did not to doxorubicin by overexpressing anti-apoptotic protein induce apoptosis of MOLT4 Daunorubicin-Resistant T Bcl-2, P-glycoprotein, and phosphorilated Akt (Davis et al. Lymphoblastoid Leukemia Cells (Ishii et al. Chloro-

  

). form fraction of Citrus grandis leaf extract that contains

  However, T47D cells also showed resistance to doxorubicin tangeretin induced apoptosis showed by decreasing of the due to p53 mutation (Di Leo et al. Vayssade et al. protein levels of the precursors of caspase-9, -8, -3, and -7

  

. Thus tangeretin

T47D cells towards doxorubicin, the combination treatment probably also induced apoptosis of MCF-7 and T47D cells.

  There are two main mechanism of apoptosis, intrinsic and extrinsic pathway, through which apoptosis could be occurred in MCF-7 and T47D cells. These apoptotic mechanisms involve the expression of regulatory protein, such as p53, Bcl-2 family, and activation of caspase family (Kumar et al. ). MCF-7 cells express p53 and Bcl-2. Doxorubicin induces apoptosis through intrinsic mechanism by elevating p53 expression which is needed to induce the expression of pro-apoptosis protein (Bax) (Minotti et al. ). In addition, tangeretin increases the expression of p53 protein in colon cancer cell (Pan et al.

  Davis JM, Navolanic PM, Weinstein-Oppenheimer CR, Steelman LS, Wei H, Konopleva M, Blagosklonny MV, McCubrey JA (2003) Raf-1 and Bcl-2 induce distinct and common pathways that contribute to breast cancer drug resistance. Clin Cancer Res 9:1161–1170

  Kumar R, Vadlamudi RK, Adam L (2000) Apoptosis in mammary gland and cancer. Endocr Relat Cancer 7:257–269 Li X, Lu Y, Liang K, Liu B, Fan Z (2005) Differential responses to doxorubicin-induced phosphorylation and activation of akt in human breast cancer cells. Breast Cancer Res 7(5):R589– R597

  Kim H, Moon JY, Mosaddik A, Cho SK (2010) Induction of apoptosis in human cervical carcinoma HeLa cells by polymethoxylated flavone-rich Citrus grandis Osbeck (Dangyuja) leaf extract. Food Chem Toxicol 48:2435–2442

  Junedi S, Susidarti RA, Meiyanto E (2010) Naringenin Meningkatkan Efek Sitotoksik Doxorubicin Pada Sel Kanker Payudara T47D Melalui Induksi Apoptosis. Jurnal Ilmu Kefarmasian Indonesia 8 (2):85–90

  Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin.

  Ishii K, Tanaka S, Kagami K, Henmi K, Toyoda H, Kaise T (2010) Effects of naturally occurring polymethyoxyflavonoids on cell growth, p-glycoprotein function, cell cycle, and apoptosis of daunorubicin-resistant T lymphoblastoid leukemia cells. Cancer Invest 28:220–229

  Hirano T, Abe K, Gotoh M, Oka K (1995) Citrus flavone tangeretin inhibits leukaemic HL-60 cell growth partially through induction of apoptosis with less cytotoxicity on normal lymphocytes. Br J Cancer 72(6):1380–1388

  Hermawan A, Meiyanto E, Susidarti A (2010) Hesperidin Meningkatkan Aktivitas Sitotoksik Doxorubicin pada Sel MCF-7. Majalah Farmasi Indonesia 21(1):8–17

  Di Leo A, Tanner M, Desmed C, Paesman M, Cardoso F, Durbecq V, Chan S, Parren T, Aapro M, Sotiriou C, Piccart MJ, Larsimont D, Isola J (2007) 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. Ann Oncol 18:997–1003

  Bracke ME, Depypere HT, Boterberg T, Van Marck VL, Vennekens KM, Vanluchene E, Nuytinck M, Serreyn R, Mareel MM (1999) Influence of tangeretin on tamoxifen’s therapeutic benefit in mammary cancer. J Natl Cancer Inst 91(4):354–359

   ). Therefore, the increasing of cell death after

  References Akao Y, Itoh T, Ohguchi K, Iinuma M, Nozawa Y (2008) Interactive effects of polymethoxy flavones from citrus on cell growth inhibition in human neuroblastoma SH-SY5Y cells. Bioorg Med Chem 16:2803–2810

  Acknowledgement This work was supported by Program DIPA Universitas Gadjah Mada through Research Program Tim Hibah Pascasarjana Multitahun year 2009 and 2010.

  Conclusion This research shows that combination of tangeretin and doxorubicin synergically increases the cytotoxic effect of doxorubicin through cell death induction and cell cycle arrest. Based on this result, tangeretin is potential to be developed as co-chemotherapeutic agent for doxorubicin.

  This result showed the potency of tangeretin to be developed as co-chemotherapeutic agent for doxorubicin by inducing cell death and cell cycle arrest. The use of doxorubicin together with tangeretin is expected to increase the activity and reduce the side effects of doxorubicin. However, the molecular mechanism of cell death induction and cell cycle arrest by this combination need to be

  The significant increasing of cytotoxic effect after combination treatment of tangeretin and doxorubicin could also occurred through inhibition of multidrugs resistance (MDR) protein. Tanaka et al. ) reported that tangeretin increases vinblastine uptake by inhibiting P-gp efflux pump in Caco-2 cells. Those three mechanisms, MDR protein inhibition, apoptotic induction, cell cycle modulation are strengthened each other to form stronger cytotoxic effect on breast cancer cells.

  The different effect on MCF-7 and T47D cell cycle are occurred due to different mechanism action of tangeretin in both cells. Cell cycle arrest by tangeretin involves inhibi- tion of CDK2/CDK4 activity and induction of CDK inhibitor expression, such as p27 and p21, which are proved by Pan et al. on colorectal cancer (COLO 205), breast cancer (MDA-MB- 435 dan MCF-7), and colon cancer (HT-29). Doxorubicin inhibits the activity of cdc2 which is involved in G2-M phase transition (Ling et al. ). But the exact mecha- nism need to be explored further.

  While, T47D cells do not express p53. Therefore, apoptotic mechanism of T47D cells is occurred in p53- independent pathway. Van Slambrouck et al. ) showed that tangeretin inhibit ERK phosphorilation pathway which is involved in T47D cell proliferation. Perhaps, the increasing of apoptotic cell after combination treatment of tangeretin and doxorubicin on T47D cells also occurred through this mechanism. But the exact mechanism need to be explored more detail.

  combination treatment of tangeretin and doxorubicin on MCF-7 cells probably by apoptosis in p53-dependent pathway. The proposed mechanism is need to be explored further.

  Ling YH, el-Naggar AK, Priebe W, Perez-Soler R (1996) Cell cycle- p34cdc2/Cyclin B1 activity induced by doxorubicin in synchro- nized P388 cells. Mol Pharmacol 49(5):832–841 Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L (2004)

  Anthracyclins: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev 56:185–228 Morley K, Ferguson P, Korotpatnick J (2007) Tangeretin and nobiletin induce G1 cell cycle arrest but not apoptosis in human breast and colon cancer cells. Cancer Lett 251(1):168–178

  Pan MH, Chen WJ, Shiau SYL, Ho CT, Lin JK (2002) Tangeretin induces cell-cycle G ^{1 arrest through inhibiting cyclin-dependent} kinases 2 and 4 activities as well as elevating Cdk inhibitors p21 and p27 in human colorectal carcinoma cells. Carcinogenesis 23 (10):1677–1684

  Smith L, Watson MB, O’Kane SL, Drew MPJ, Lind MJ, Cawkwell L (2006) The analysis of doxorubicin resistance in human breast cancer cells using antibody microarrays. Mol Cancer Ther 5 (8):2115–2120

  Tanaka T, Makita H, Ohnishi M, Mori H, Satoh K, Hara A, Sumida T, Fukutani K, Tanaka T, Ogawa H (1997) Chemoprevention of 4- nitroquinoline 1-oxide-induced oral carcinogenesis in rats by flavonoids diosmin and hesperidin, each alone and in combina- tion. Cancer Res 57:246–252

  Valeria P, Barrera-Rodrigue R (2005) Changes in P-glycoprotein activity are mediated by the growth of a tumour cell line as multicellular spheroids. Cancer Cell Int 5(20), Summary

  Van Slambrouck S, Parmar VS, Sharma SK, De Bondt B, Foré F, Coopman P, Vanhoecke BW, Boterberg T, Depypere HT, Leclercq

  G, Bracke ME (2005) Tangeretin inhibits extracellular-signal- regulated kinase (ERK) phosphorylation. FEBS Lett 579 (7):1665–9

  Vayssade M, Haddada H, Faridoni-Laurens L, Tourpin S, Valent A, Bénard J, Ahomadegbe J (2005) p73 functionally replaces p53 in adriamycin-treated, p53-deficient breast cancer cells. Int J Cancer 116(6):860–869

  Yu B, Shen H, Gao F, Fan Y, Sun Z (2010) Expression of the apoptosis-related genes BCL-2 and BAD in human breast carcinoma and their associated relationship with chemosensitivity. J Exp Clin Cancer Res 29:107, pp 1–7