Cross-resistance to Vincristin and Etoposide in a sub line of the human breast
cancer T47D cells selected for Adriamycin-resistance
|Iranian Journal of
Pharmaceutical Research (2004) 2: 103-107
Received: August 2003
Accepted: June 2004
Copyright ? 2004 by School of Pharmacy
Shaheed Beheshti University of Medical Sciences and Health Services
Cross-resistance to Vincristin and Etoposide
in a sub line of the human breast cancer T47D cells selected for Adriamycin-resistance
Armaghan Emami-Forushania, Ebrahim Azizia*, Sirous Zeinalib, Seyed Naser Ostada
Research Lab., Department of Pharmacology and Toxicology, Faculty of
Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
bDepartment of Biotechnology, Pasteur Institute of
Iran, Tehran, Iran.
Breast cancer is one of the most
common malignancies among women. Although chemotherapy remains a major
therapeutic approach to treat cancers, drug therapy often fails for
several reasons, particularly the drug resistance. Resistance to
multiple chemotherapeutic agents is one of the most important problems
in the treatment of different types of cancers. Therefore, in this study
a resistant sub line of the human breast cancer T47D cells was isolated
in vitro by stepwise
exposure to increasing concentrations of Adriamycin (ADR) to compare the
characteristics of parent and resistant cells. We also evaluated the
phenomenon of cross-resistance to some other chemotherapeutic drugs. A
significant increase in doubling time of resistant cells, named T47D/ADR,
(94 h) was observed when compared to the parental T47D cells (50 h) that
indicates a relatively slow growth rate pattern of these cells. T47D/ADR
cells were 4 fold resistant to adriamycin and also showed
cross-resistance to vincristin (VCR, 3.5 fold) and to etoposide (VP-16,
5.5 fold) when compared to parent cells. Therefore, our results indicate
that T47D/ADR cells are also cross-resistant to structurally and
functionally different chemotherapeutic agents and can be used as a
model for studying molecular changes of drug resistance.
cancer; T47D cells; Adriamycin; Etoposide; Vincristin; Drug resistance.
In recent years, the incidence and
mortality of breast cancer among women worldwide have become the
most important medical issues. With progress in understanding of
the pathobiology of breast cancer, diagnosis and treatments have
improved (1, 2, 3).
Unfortunately, resistance to multiple
chemotherapeutic agents is a common problem in the treatment of
different types of cancers including breast cancer. This
resistance termed multidrug resistance (MDR) (4, 5), may be
intrinsic or acquired by tumor cells during or after treatment.
Several drugs including Adriamycin (ADR), Vincristin (VCR), and
Etoposide (VP16) with different structures and mechanisms of
antitumor actions fail to be effective due to MDR phenomenon
Adriamycin, an anthracycline antitumor
drug, is clinically active against many human malignancies
including breast cancer (10-12). Several mechanisms explain the
antitumor activity of ADR including DNA intercalation,
inhibition of topoisomerase IIa (TOPO IIa), interaction with membrane, and generation of
oxygen free radicals (13, 14). Vincristin is a vinca alkaloid
antitumor agent, which exerts its effects by binding to tubulin
and therefore inhibiting microtubule formation during mitosis
(6). Etoposide is a derivative of podophyllotoxin, which
inhibits TOPO IIa and is believed to cause breakdown of DNA (15).
Therefore, this study was conducted to
further elucidate the changes that occur in drug resistant cells
compared to parent cells. We isolated an Adriamycin-resistant
sub line of the human breast cancer T47D cells
in vitro by gradual exposure to increasing concentrations of
ADR. This sub line named T47D/ADR was then compared to the
parental cells with respect to the growth characteristics and
cross-resistance to VCR, and VP16.
Cell line and culture conditions
The human breast cancer T47D cell line (ATCC
HTB-133, USA) was obtained from Pasteur Institute Cell Bank of
IRAN (Tehran, IRAN). Cells were maintained in RPMI-1640 (Gibco,
USA) culture medium supplemented with 10% fetal bovine serum (Gibco,
USA) and 100 U/ml of penicillin and 100 ng/ml of streptomycin
(Sigma, UK) at 37?C in 5% CO2 incubator.
Establishment of an Adriamycin-resistant sub
An Adriamycin-resistant sub line was
isolated by continuous exposure of T47D cells to ADR (Adriblastina,
Italy) at concentrations starting from 1x 10-9 M and increasing
in a stepwise manner to 2.5?10-8 M within 9 months. Cells that
were capable of sustained growth in medium containing 1?10-8 M
of ADR were considered to be resistant to Adriamycin and are
referred to T47D/ADR cells here after. Cell viability was
determined after each step using trypan blue dye exclusion
Determination of growth characteristics of
T47D and T47D/ADR Cells
T47D and T47D/ADR cells were seeded in 24
well plates at 6?104 cells/well in 1 ml growth medium and
incubated in the presence or absence of ADR at 37?C in 5% CO2
incubator. After washing with PBS, the cells were trypsinized
and then counted using trypan blue dye exclusion method every 48
h for 11 days. The doubling time for each cell population was
then determined from its growth curve, in which each point was
the average determination of triplicate wells in three
Cytotoxicity and cross-resistance assay
The MTT (3-[4, 5-dimethylthiazol-2-yl]-2,
5-diphenyl tetrazolium bromide) based assay was performed by
seeding 2000 cells in 100 ml growth medium in the presence of increasing
concentrations of chemotherapeutic drugs (ADR: 0.1 to 100 nM,
VCR: 0.01 to100 nM, VP16: 0.1 to 1000 nM) into 96-well plates
and incubated at 37?C in 5% CO2 incubator for 96 hours. The
cells were then incubated with 25 ml MTT (5 mg/ml) at 37?C for 4 hours. After
dissolving the formazan crystals in 0.04 N HCl in isopropanol,
plates were read in a micro plate reader (Dynatech Lab Inc, USA)
at 570 nm. This experiment was performed in triplicate
determination and repeated three times.
SIGMASTAT? (Jandel Software, San Raphael,
CA) was used to perform statistical analysis of data. The
students t-test was used to examine the differences among
treatments. Mean differences with P values less than 0.05 were
considered to be significant.
Results and Discussion
Growth characteristics of T47D and T47D/ADR
The doubling time of the T47D/ADR cells
(94 h) increased significantly as compared with that of the
parental T47D cells (50 h). This indicates the slow growth rate
pattern of isolated resistant cells (Figure 1). The viability
assay also showed more than %95 viable cells in all steps of
Cytotoxicity of ADR, VCR, and VP16 on T47D
and T47D/ADR cells
The anti-proliferative effects of ADR, VCR
and VP16 on T47D cells and its resistant sub line measured by
MTT based assay indicate the cross-resistant properties of T47D/ADR
cells to VCR and also VP16 (Figure 2). The IC50 value for each drug determined from
corresponding survival curves was used to calculate the
fold-resistance to each drug by T47D/ADR compared to the
parental T47D cells (Table 1).
Many adriamycin-resistant cells exhibited
a high level of cross-resistance to other structurally unrelated
drugs that is called multidrug resistance (MDR) (4, 16, 17). Son
et al. showed that an ADR resistant human stomach-adenocarcinoma
cell line (MKN/ADR) had a high level of cross-resistance to
topoII-targeted drugs such as mitoxantron and etoposide but
showed no cross resistance to other chemotherapeutic agents such
as cisplatin, carboplatin and 5-FU. The doubling time of the MKN/ADR
cells (2.1 days) was more than the parent MKN cells. They
suggested that a quantitative reduction in topo IIa may contribute to the resistance of MKN cells
to ADR and other topoII-targeted drugs. It has also been
indicated that the differences in topo IIa expression was not the
reason for the difference in growth rate (10, 18).
In fact, one possible explanation for the
reduced proliferation rate and drug resistance would be a
reduction in expression of topo IIa which is required for DNA replication and has
been correlated with the cell proliferation (19-22). Reduced
expression of topo IIa has been implicated as a mechanism of
resistance to topo II inhibitors such as ADR, VP16, m-amsacrine
and mitoxantron (23, 24). Wosikowski et al. indicated that the
doubling time of resistant cells increased significantly
compared with parental cancer cells. In contrast to other
studies, they failed to show correlation between topo IIa expression and
proliferation rate of resistant cells (25).
In other study, an etoposide-resistant
breast cancer cell line (MCF7/VP16) was 28, 21 and 9 fold
resistant to VP16, Vm26 and doxorubicin, respectively. MCF7/VP16
cells also exhibited 2.8 and 5 fold resistance to mitoxantron
and vincristin but no cross-resistance to camptothecin, an
inhibitor of topo I. Their results showed that resistance to
epipodophyllotoxines in MCF7/VP16 cells is multi-factorial
involving reduction in intracellular drug concentration,
possibly MRP over expression, and also altered topo IIa drug sensitivity (15).
Another study showed a strong correlation
between the degree of P-gp expression and in vitro resistance to taxol
and adriamycin (26). Overexpression of P-gp or MRP levels may
reduce ADR and VCR accumulation and increase drug efflux. On the
other hand, reduction in the topo IIa protein levels
diminishes the main intracellular target of ADR and VP16 and
other topo IIa poisons. These changes may confer cross-resistance to
these agents (6,17).
Therefore, observed resistance in the
T47D/ADR cells apparently involves multiple mechanisms including
topo IIa, MDR1, and other genetic and epigenetic
alterations. Finally, it cannot be excluded that the cells
selected for resistance to chemotherapeutic drugs, in our study
as well as other similar studies, compose of a heterogeneous
population of cells each with its own distinct characteristics
and mechanism(s) of developing resistance to tested
chemotherapeutic drugs that needs to be further elucidated.
The authors are thankful to the office of
vice-chancellor for research of Tehran University of Medical
Sciences (TUMS) for the financial support of this project.
(1) Dos Santos Silva I,
Mangtani P, De Stavola BL, Bell J, Quinn M and Mayer D. Survival
from breast cancer among South Asian and non-South Asian women
resident in South East England.
Br. J Cancer (2003) 89: 508-12
(2) Engel J, Eckel R,
Kerr J, Schmidt M, Furstenberger G, Richter R, Sauer H, Senn HJ
and Holzel D. The process of metastasisation for breast cancer.
Eur J Cancer (2003)
(3) Piccart M, Parker LM
and Pritchard KI. Oestrogen receptor down regulation: an
opportunity for extending the window of endocrine therapy in
advanced breast cancer.
Ann. Oncol. (2003) 14: 1017-1025
(4) Li J, Xu LZ, He KL,
Guo WJ, Zeng YH, Xia P and Chen Y. Reversal effects of
nomegestrol acetate on multidrug resistance in adriamycin-resistant
MCF-7 breast cancer cell line.
Breast Cancer Res. (2001)
3 : 253-263
(5) Arancia G, Molinari
A, Calcabrini A, Meschini S and Cianfriglia M. Intracellular
P-glycoprotein in multidrug resistant tumor cells.
Ital. J. Anat. Embryol.
(2001) 106: 59-68
(6) Whelan RD, Waring CJ
and Hayes JD. Over expression of P-glycoprotein and
glutathione-s-transferase in MCF-7 cells selected for vincristin
resistance in vitro.
Int. J. Cancer (1992)
(7) Uchiyama-Kokubu N and
Watanabe T. Establishment and characterization of adriamycin-resistant
human colorectal adenocarcinoma HCT-15 cell lines with multidrug
Anticancer. Drugs (2001)
(8) Cre IA, Knight L, Di
Nicolantonio F, Sharma S and Gulliford T. Hemosensitization of
solid tumors by modulation of resistance mechanisms. Curr. Opin. Investig. Drugs (2002) 3: 634-40
(9) Faneyte IF, Kristel
PM and van de Vijver MJ. Determining MDR1/P-glycoprotein
expression in breast cancer.
Int. J. Cancer (2001) 93: 114-22
(10) Son YS, Suh JM, Ahn
SH, Kim JC, Yi Jy, Hur KC, Hong WS, Muller MT and Chung IK.
Reduced activity of topoisomerase IIa in an Adriamycin-resistant human
stomach - adenocarcinoma cell line.
Cancer Chemoth. Pharmacol. (1998)
(11) Callies S, de Alwis
DP, Wright JG, Sandler A, Burgess M and Aarons L. A population
pharmacokinetic model for doxorubicin and doxorubicinol in the
resence of a novel MDR modulator, zosuquidar trihydrochloride.Cancer.
Chemother. Pharmacol. (2003) 51: 107-18
(12) Larsen AK and
Skladanowski A. Cellular resistance to topoisomerase -targeted
drugs from drug uptake to cell death.
Biochim. Biophys. Acta (1998) 1400: 257-74
(13) Gewirtz DA. A
critical evaluation of the mechanisms of action proposed for the
Antitumor effects of the anthracycline antibiotics adriamycin
and daunorubicin. Biochem. Pharmacol. (1999) 17: 1311-1318
(14) Dunkern TR,
Wedemeyer I, Baumgartner M, Fritz G and Kaina B. Resistance of
p53 knockout cells to doxorubicin is related to reduced
formation of DNA strand breaks rather than impaired apoptotic
signaling. DNA Repair (2003)
(15) Schneider E, Horton
JK, Yang CH, Nakagawa M and Cowan KH. Multidrug
resistance-associated protein gene over expression and reduced
drug sensitivity of topoisomerase IIa in a human breast carcinoma MCF-7
cell line selected for etoposide resistance.
(1994) 54: 152-158
(16) Kakumoto M, Sakaeda
T, Takara K, Nakamura T, Kita T, Yagami T, obayashi H, Okamura N
and Okumura K. Effects of carvedilol on MDR1-mediated multidrug
resistance: comparison with verapamil.
(2003) 94: 81-6
(17) Kang MR and Chung IK.
Down regulation of topoisomerase IIa in human colorectal carcinoma
cells resistant to a Protoberberine alkaloid, berberrubine.
Mol. Pharmacol. (2002)
61 : 879-884
(18) McPherson JP, Brown
GA and Goldenberg GJ. Characterization of a DNA topoisomerase IIa
gene rearrangement in adriamycin resistant P388 leukemia:
expression of a fusion messenger RNA transcript encoding
and the retinoic acid receptor
Cancer Res. (1993)
(19) Heck MM and Earnshaw
WC. Topoisomerase II: a specific marker for cell proliferation.
J. Cell. Biol. (1986)
(20) Lynch BJ, Guinee DG
Jr and Holden JA. Human DNA topoisomerase IIa: a new marker of cell
proliferation in invasive breast cancer.
Hum. Pathol. (1997)
(21) Bildrici k, Tel N,
Ozalp SS, Yalcin OT and Yilmaz V. Prognostic significance of DNA
(Ki-S1) immunoexpression in endometrial carcinoma. Eur. J. Gynecol. Oncol. (2002) 23: 540-4
(22) Holden JA. DNA
topoisomerases as anticancer drug targets: from the laboratory
to the clinic. Curr. Med. Chem. Anti-Cancer
Agents (2001) 1: 1-25
(23) Matsumoto Y, Takano
H, Nagao S and Fojo T. Altered Topoisomerase IIa and Multidrug
Resistance-associated Protein Levels during Drug Selection:
Adaptations to Increasing Drug Pressure.
Jpn. J. Cancer Res. (2001) 92: 968-974
(24) Okada Y, Tosaka A,
Nimura Y, Kikuchi A, Yoshida S and Suzuki M. Atypical multidrug
resistance may be associated with catalytically active mutants
of human DNA topoisomerase II
Gene (2001) 272: 141-148.
(25) Wosikowski K,
Schuurhuis D, Kops JG, Saceda M and Bates SE. Altered gene
expression in drug-resistant human breast cancer cells. Clin. Cancer Res. (1997)
(26) Mechetner E,
Kyshtoobayeva A, Zonis S, Kim H, Stroup R, Garcia R, Parker RJ
and Fruehauf JP. Level of Multidrug Resistance (MDR1)
P-glycoprotein Expression by Human Breast Cancer Correlate with
Resistance to Taxol and Doxorubicin.
Clin. Cancer Res. (1998) 4: 389-398