Elsevier

Biosensors and Bioelectronics

Volume 55, 15 May 2014, Pages 44-50
Biosensors and Bioelectronics

Frequency dependent impedimetric cytotoxic evaluation of anticancer drug on breast cancer cell

https://doi.org/10.1016/j.bios.2013.11.060Get rights and content

Highlights

  • Relative decrease of impedance is a measure of cytotoxic effect of ZD6474.

  • Cell death due to ZD6474 is inversely proportional to the impedance.

  • The quantitative relation between impedance and drug doses shows a negative correlation.

Abstract

The present work reports the impedance characteristics of MCF-7 cell lines treated with anticancer drug ZD6474 to evaluate the cytotoxic effect on cellular electrical behaviour using miniature impedance sensors. Four types of impedance sensing devices with different electrode geometries are fabricated by microfabrication technology. The frequency response characteristics of drug treated cells are studied to evaluate cytotoxic effect of ZD6474 and also to assess the frequency dependent sensitivity variation with electrode area. A significant variation in magnitude of measured impedance data is obtained for drug treated samples above 10 µM dose indicating prominent effect of ZD6474 which results in suppression of cell proliferation and induction of apoptosis process. The results obtained by impedimetric method are correlated well with conventional in vitro assays such as flow cytometry, cell viability assays and microscopic imaging. Finally an empirical relation between cell impedance, electrode area and drug dose is established from impedance data which exhibits a negative correlation between drug doses and impedance of cancer cells.

Introduction

Breast cancer stands second in cancer-related mortality of women. It originates in the epithelial cells of the terminal ductulo-lobular system of the breast tissue and is classified pathologically on the basis of morphological features of tumours. The majority of the breast carcinomas (about 40–75%) cannot be classified satisfactorily. Therefore they are termed as invasive ductal carcinoma (IDC) not otherwise specified (NOS). The specialized pathological subtypes of breast carcinoma include invasive lobular carcinoma (5–10%), medullary carcinoma (1–7%), mucinous carcinoma (2%), tubular carcinoma (<2%), and metaplastic carcinoma (<1%). Besides, cribriform, papillary, micropapillary, apocrine, glycogen-rich clear cell, lipid-rich, inflammatory, and adenoid cystic carcinomas are the rare form of specialized invasive carcinomas found in breast tissue (Fabbri et al., 2008, Viale, 2012). Although researchers are working for advanced screening programme and targeted drug delivery for combating this disease process, it is still a challenging issue to reduce the high mortality rate throughout the world (Nagalingam et al., 2012). Previously the conventional cell based assay was used to study the cell proliferation and viability which has been replaced by label free and non-invasive techniques in recent time. The different label-free technologies available to monitor cellular processes in real-time include electric cell–substrate impedance sensing (ECIS), quartz microbalance and optical light mode spectroscopy (Hong et al., 2011). Among them ECIS has been evolved as the most effective technique to measure the real time impedance of cultured cells on the surface of microelectrode which was first reported by Giaever and Keese (1984). At present ECIS is an established non-invasive electrochemical technique that has been successfully used to monitor adhesion, growth and differentiation of cells in real time (Chen et al., 2012, Hong et al., 2011, Janshoff et al., 2010, Park et al., 2011; Xiao and Luong, 2003, Xiao and Luong, 2010, Yu et al., 2011), cell migration (Burns et al., 1997, Chan et al., 2010, Hsu et al., 2010, Lee et al., 2004), morphological changes during apoptosis (Arndt et al., 2004, Yin et al., 2007), taste sensor (Ceriotti et al., 1991, Hui et al., 2013), cytotoxicity of several drugs (Asphahani and Zhang, 2007, Campbell et al., 2007, Ceriotti et al., 2007, Curtis et al., 2009, Klo et al., 2008, Male et al., 2010, Muller et al., 2011, Opp et al., 2009, Sun et al., 2013, Wang et al., 2013, Xiao and Luong, 2005, Xu et al., 2012) and stem cell research (Bagnaninchi and Drummond, 2011, Maercker et al., 2008, Park et al., 2011, Reitinger et al., 2012). However, further researches are required to explore the different technical aspects of ECIS methods.

In this paper, an attempt has been made to study the electrical impedance of cells treated in various doses of anticancer drug to correlate the electrical properties with their physiological conditions. ZD6474 has unique potential to combat breast cancer among the recently discovered various anticancer drugs. ZD6474 is a novel heteroaromatic-substituted anilinoquinazoline (Fig. S1), which acts as a potent and reversible inhibitor of Adenosine Tri Phosphate (ATP) (Hennequin et al., 2002). The inhibitory effect of ZD6474 on epidermal growth factor receptor (EGFR) tyrosine kinase has been reported earlier (Ciardiello and Tortora, 2001). ZD6474 inhibits two key pathways in tumour growth process. The first pathway targets indirectly by reducing vascular endothelial growth factor (VEGF)-dependent tumour angiogenesis and VEGF-dependent endothelial cell survival (Gille et al., 1997, Goldman et al., 1993, Perrotte et al., 1999, Salomon et al., 1995). The second pathway targets via inhibition of EGFR-dependent tumour cell proliferation and survival (Weber et al., 2003). However the impedimetric monitoring of cytotoxicty of ZD6474 has not been carried out till date.

In this paper, experiment has been carried out to understand the cellular behaviours of MCF-7 subjected to various drug doses of ZD6474 using fabricated ECIS devices. Among several breast cancer cell lines, MCF-7 cells (malignant and invasive) are widely used in experimental therapeutics as the cell line maintains ideal characteristics of mammary epithelium (Levenson and Jordan, 1997). The frequency response of electrical impedance of ZD6474 treated MCF-7 cells at 24 h has been investigated and compared with the results of standard techniques like MTT assay, flow cytometry, phase contrast and fluorescence imaging. In this experiment, additionally MCF-10A cells are used as negative control for an effective comparisoni of impedance data obtained for drug treated MCF-7 cells. MCF-10A cell is a non cancerous human breast cell line frequently used in scientific research for their high capacity to differentiate and proliferate in vitro. Efforts have also been given for quantitative estimation of the effect of drug dose on the cells using impedance data.

Section snippets

Materials and reagents

Pyrex glass wafers were purchased from Semiconductor wafer Inc., Taiwan. Polydimethylsiloxane (PDMS, Sylgard 184) was supplied by Dow Corning, Inc., Midland. SU8 was purchased from MicroChem, Newton. RPMI 1640 medium, foetal bovine serum, trypsin/EDTA solution, penicillin, and streptomycin were purchased from Himedia, India. Propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen, India. All other required reagents were supplied by Sigma-Aldrich, India.

Cell culture

Evaluation of cytotoxicty by ECIS methods

The different doses of ZD6474 are added with cells after 30 min inoculation of cultures on fabricated devices. The bode plots for the cytotoxic effects of ZD6474 on MCF-7 cells after 24 h are presented in Fig. 2 for Design 1 and Fig. S4 for Design 2, 3, and 4. The experimental data are fitted perfectly with the used equivalent circuit as described in Fig. S3. The results illustrate that the magnitude of the impedance value is inversely proportional to the WE area as its magnitude is highest for

Conclusions

This paper provides results about the cytotoxic effects of ZD6474 on MCF-7 cells in microfabricated devices using ECIS technique. The frequency dependent impedance data reveal that the cells treated above 10 µM drug dose show predominant reduction of its impedance magnitude as compared to the control. The decrease in magnitude of impedance value with the increase of drug doses demonstrates the dose dependent cytotoxic effect of ZD6474. Sensitivity of the devices decreases with the increase of

Acknowledgement

Authors would like to thank the staff members of MEMS Lab, IIT Kharagpur for microfabrication support and Indian Space Research Organisation for financial support to carry out the present work.

References (55)

  • S. Arndt et al.

    Biosens. Bioelectron.

    (2004)
  • C.E. Campbell et al.

    Biosens. Bioelectron.

    (2007)
  • L. Ceriotti et al.

    Biosens. Bioelectron.

    (2007)
  • S.-W. Chen et al.

    Biosens. Bioelectron.

    (2012)
  • G.-H. Hui et al.

    Biosens. Bioelectron.

    (2013)
  • R. Lind et al.

    Sens. Actuators B: Chem.

    (1991)
  • N.N. Mishra et al.

    Biosens. Bioelectron.

    (2005)
  • J. Muller et al.

    Biosens. Bioelectron.

    (2011)
  • D. Opp et al.

    Biosens. Bioelectron.

    (2009)
  • H.E. Park et al.

    J. Biomed. Biotechnol.

    (2011)
  • S. Reitinger et al.

    Biosens. Bioelectron.

    (2012)
  • D.S. Salomon et al.

    Crit. Rev. Oncol. Hematol.

    (1995)
  • X. Sun et al.

    Biosens. Bioelectron.

    (2013)
  • G. Viale

    Ann. Oncol.

    (2012)
  • L. Wang et al.

    Biosens. Bioelectron.

    (2008)
  • T. Wang et al.

    Biosens. Bioelectron.

    (2013)
  • C. Xiao et al.

    Toxicol. Appl. Pharmacol.

    (2005)
  • C. Xiao et al.

    Biosens. Bioelectron.

    (2010)
  • Y.C. Xu et al.

    Biosens. Bioelectron.

    (2012)
  • H. Yu et al.

    Biosens. Bioelectron.

    (2011)
  • H. Alborzinia et al.

    PLoS ONE

    (2011)
  • F. Asphahani et al.

    Analyst

    (2007)
  • P.O. Bagnaninchi et al.

    Proc. Natl. Acad. Sci. USA

    (2011)
  • L.J. Breckenridge et al.

    J. Neurosci. Res.

    (1995)
  • Brett, C.M.A., Brett, A.M.O., 1993. Electrochemistry-Priniciples. Methods, and Applications. Oxford University Press,...
  • A.R. Burns et al.

    J. Immunol.

    (1997)
  • L. Ceriotti et al.

    Gastroenterology

    (1991)
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