Multiple effects of electroporation on the adhesive behaviour of breast cancer cells and fibroblasts

Cancer Cell International

Rumiana D Tzoneva¹  Iana H Tsoneva¹  Viktoria N Pehlivanova¹ 

[1] Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113, Bulgaria

关键词: Electroporation;    Actin cytoskeleton;    Fibroblasts;    Breast cancer cells;   

Others  :  795204
DOI  :  10.1186/1475-2867-12-9

 received in 2012-01-11, accepted in 2012-03-22,  发布年份 2012

【 摘 要 】

Background

Recently electroporation using biphasic pulses was successfully applied in clinical developments for treating tumours in humans and animals. We evaluated the effects of electrical treatment on cell adhesion behaviour of breast cancer cells and fibroblasts. By applying bipolar electrical pulses we studied short- and long-lived effects on cell adhesion and survival, actin cytoskeleton and cell adhesion contacts in adherent cancer cells and fibroblasts.

Methods

Two cancer cell lines (MDA-MB-231 and MCF-7) and one fibroblast cell line 3T3 were used. Cells were exposed to high field intensity (200 - 1000 V/cm). Cell adhesion and survival after electrical exposure were studied by crystal violet assay and MTS assay. Cytoskeleton rearrangement and cell adhesion contacts were visualized by actin staining and fluorescent microscope.

Results

The degree of electropermeabilization of the adherent cells elevated steadily with the increasing of the field intensity. Adhesion behaviour of fibroblasts and MCF-7 was not significantly affected by electrotreatment. Interestingly, treating the loosely adhesive cancer cell line MDA-MB-231 with 200 V/cm and 500 V/cm resulted in increased cell adhesion. Cell replication of both studied cancer cell lines was disturbed after electropermeabilization. Electroporation influenced the actin cytoskeleton in cancer cells and fibroblasts in different ways. Since it disturbed temporarily the actin cytoskeleton in 3T3 cells, in cancer cells treated with lower and middle field intensity actin cytoskeleton was well presented in stress fibers, filopodia and lamellipodia. The electrotreatment for cancer cells provoked preferentially cell-cell adhesion contacts for MCF-7 and cell-ECM contacts for MDA-MB- 231.

Conclusions

Cell adhesion and survival as well as the type of cell adhesion (cell-ECM or cell-cell adhesion) induced by the electroporation process is cell specific. The application of suitable electric pulses can provoke changes in the cytoskeleton organization and cell adhesiveness, which could contribute to the restriction of tumour invasion and thus leads to the amplification of anti-tumour effect of electroporation-based tumour therapy.

【 授权许可】

   
2012 Pehlivanova et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 13.

Figure 2.

【 参考文献 】

• [1]Mir LM, Orlowski S: Mechanisms of electrochemotherapy. Adv Drug Deliv Rev 1999, 35:107-118.
• [2]Sersa G, Cufer T, Cemazar M, Rebersek M, Zvonimir R: Electrochemotherapy with bleomycin in the treatment of hypernephroma metastasis: case report and literature. Tumori 2000, 86:163-165.
• [3]Tsoneva I, Nikolova B, Georgieva M, Genova M, Tomov T, Rols MP, Berger MR: Induction of apoptosis by electrotransfer of positively charged proteins as Cytochrome C and Histone H1 into cells. Biochim Biophys Acta 2005, 1721:55-64.
• [4]Neumann E, Kakorin S, Tsoneva I, Nikolova B, Tomov T: Calcium mediated DNA adsorption to yeast cells and kinetiks of cell transformation by electroporation. Biophys J 1996, 71:868-877.
• [5]Rols MP, Teissie J: Experimental-evidence for the involvement of the cytoskeleton in mammalian-cell electropermeabilization. Biochim Biophys Acta 1992, 1111:45-50.
• [6]Rols MP: Electropermeabilization, a physical method for the delivery of therapeutic molecules into cells. Biochim Biophys Acta Biomembr 2006, 1758:423-428.
• [7]Jarm T, Cemazar M, Miklavcic D, Sersa G: Antivascular effects of electrochemotherapy: implications in treatment bleeding metastases. Expert Rev Anticancer Ther 2010, 10:729-746.
• [8]Tekle E, Astumian RD, Chock PB: Electroporation by using bipolar oscillating electric field: an improved method for DNA transfection of NIH 3 T3 cells. Proc Natl Acad Sci USA 1991, 88:4230-4234.
• [9]Kotnik T, Mir LM, Flisar K, Puc M, Miklavcic D: Cell membrane electropermeabilization by symmetrical bipolar rectangular pulses Part I. Increased efficiency of permeabilization. Bioelectrochemistry 2001, 54:83-90.
• [10]Kotnik T, Pucihar G, Rebersˇek M, Miklavcˇic D, Mir ML: Role of pulse shape in cell membrane electropermeabilization. Biochim Biophys Acta 2003, 1614:193-200.
• [11]Daskalov I, Mudrov N, Peycheva E: Exploring new instrumentation parameters for electrochemotherapy. IEEE Eng Med Biol Mag 1999, 18:62-66.
• [12]Peycheva E, Daskalov I, Tzoneva I: Electrochemotherapy of mycosis fungoides by interferon. Bioelectrochemistry 2007, 70:283-286.
• [13]Spugnini EP, Vincenzi B, Citro G, Tonini G, Dotsinsky I, Mudrov N, Baldi A: Electrochemotherapy for the treatment of squamous cell carcinoma in cats: A preliminary report. Vet J 2009, 179:117-120.
• [14]Davalos RV, Mir LM, Rubinsky B: Tissue ablation with irreversible electroporation. Ann Biomed Eng 2005, 33:223-31.
• [15]Byrne CM, Thomson JF: Role of electrochemotherapy in the treatment of metastatic melanoma and other metastatic and primary skin tumors. Expert Rev Anticancer Ther 2006, 6:671-678.
• [16]Sersa G, Miklavcic D, Cemazar M, Rudolf Z, Pucihar G, Snoj M: Electrochemotherapy in treatment of tumours. Eur J Surg Oncol 2008, 34:232-240.
• [17]Peycheva E, Daskalov I: Electrochemotherapy of skin tumours: comparison of two electroporation protocols. J BUON 2004, 9:47-50.
• [18]Cemazar M, Miklavcic D, Scancar J, Dolzan V, Golouh R, Sersa G: Increased platinium accumulation in SA-1 tumor cells after in vivo electrochemotherapywith cisplatin. Br J Cancer 1999, 79:1386-1391.
• [19]Onik G, Rubinsky B, Mikus P: Irreversible electroporation: implications for prostate ablation. Technol Cancer Res Treat 2007, 6:1533-0346.
• [20]Davalos RV, Mir LM, Rubinsky B: Tissue ablation with irreversible lectroporation. Ann Biomed Eng 2005, 33:223-23.
• [21]Blangero C, Rols MP, Teissie J: Cytoskeletal reorganization during electric fieldinduced fusion of Cinese hamster ovary cells grown in monolayers. Biochim Biophys Acta 1989, 981:295-302.
• [22]Rosazza C, Escoffre JM, Zumbusch A, Rols MP: The actin cytoskeleton has an active role in the electrotransfer of plasmid DNA in mammalian cells. Mol Ther 2011, 19:913-921.
• [23]Titushkin I, Cho M: Regulation of cell cytoskeleton and membrane mechanics by electric field: role of linker proteins. Biophys J 2009, 96:717-728.
• [24]Wang E, Zhao M, Forrester JV, McCaig CD: Bi-directional migration of lens epithelial cells in a physiological electrical field. Exp Eye Res 2003, 76:29-37.
• [25]Kanthou C, Kranjc S, Sersa G, Tozer G, Zupanic A, Cemazar M: The endothelial cytoskeleton as a target of electroporation-based therapies. Mol Cancer Ther 2006, 5:3145-3152.
• [26]Xiao D, Tang L, Zeng C, Wang J, Luo X, Yao C, Sun C: Effect of actin cytoskeleton disruption on electric pulse-induced apoptosis and electroporation in tumour cells. Cell Biol Int 2011, 35:99-104.
• [27]Kanduser M, Miklavcic D, Pavlin M: Mechanisms involved in gene electrotransfer using high- and low-voltage pulses-an in vitro study. Bioelectrochemistry 2009, 74:265-71.
• [28]Yizraeli ML, Weihs D: Time-Dependent Micromechanical Responses of Breast Cancer Cells and Adjacent Fibroblasts to Electric Treatment. Cell Biochem Biophys 2011, 61:605-18.
• [29]Oelz D, Schmeiser C, Small JV: Modeling of the actin-cytoskeleton in symmetric lamellipodial fragments. Cell Adh Migr 2008, 2:117-126.
• [30]Burridge K, Kelly T, Nuckolls G, Tumer C: Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Dev Biol 1988, 4:487-525.
• [31]O'Connor TP, Bentley D: Accumulation of actin in subsets of pioneer growth cone filopodia in response to neural and epithelial guidance cues in situ. J Cell Biol 1993, 123:935-948.
• [32]Mizutani K, Miki H, He H, Maruta H, Takenawa T: Essential role of neural Wiskott- Aldrich syndrome protein in podosome formation and degradation of extracellular matrix in src-transformed fibroblasts. Cancer Res 2002, 62:669-674.
• [33]Gimona M, Buccione R, Courtneidge SA, Linder S: Assembly and biological role of podosomes and invadopodia. Curr Opin Cell Biol 2008, 20:235-241.
• [34]Fish S, Ruoslahti E: Integrins and anoikis. Curr Opin Cell Biol 1997, 9:7001-7006.
• [35]Glukhova M, Koteliansky V, Sastre X, Thiery J: Adhesion systems in normal breast and in invasive breast carcinoma. Am J Pathol 1995, 146:706-716.
• [36]Palovuori R: Regulation of cell-cell adhesion and actin cytoskeleton in nontransformed and transformed epithelial cells. In Ph.D. Thesis. University of Oulu, Department of pathology; 2003.
• [37]Charalabopopulus K, Pignetti M: Adhesion molecules and cancer. Arch Hellenic Med 2001, 18:16-19.
• [38]Sadlonova A, Novak Z, Johnson MR, Bowe DB, Gault SR, Page GP, Thottassery JV, Welch DR, Andra R, Frost AR: Breast fibroblasts modulate epithelial cell proliferation in three-dimensional in vitro co-culture. Breast Cancer Res 2005, 7:R46-59. BioMed Central Full Text
• [39]Kunz-Schughart LA, Knuechel R: Tumor-associated fibroblasts (part I): active stromal participants in tumor development and progression? Histol Histopath 2002, 17:599-621.
• [40]Tyan SW, Kuo WH, Huang CK, Pan CC, Shew JY, Chang KJ, Lee EY, Lee WH: Breast Cancer Cells Induce Cancer-Associated Fibroblasts to Secrete Hepatocyte Growth Factor to Enhance Breast Tumorigenesis. PLoS One 2011, 6:e15313.
• [41]Orimo A, Gupta PB, Sgro DC, Arenzana-Seisdedos F, Delaunay T, Naeem R, Carey VJ, Richardson AL, Weinberg RA: Stromal Fibroblasts Present in Invasive Human Breast Carcinomas Promote Tumor Growth and Angiogenesis through Elevated SDF- 1/CXCL12 Secretion. Cell 2005, 121:335-348.
• [42]Daskalov I, Bankov S: Electrical stimulation of innervated muscles. J Clin Eng 1997, 22:383-390.
• [43]Rols M, Teissie J: Electropermeabilization of mammalian cells to macromolecules: control by pulse duration. Biophys J 1998, 75:1415-1423.
• [44]Hernandez JL, Coil T, Ciudad J: A highly efficient electroporation method for the transfection of endothelial cells. Angiogenesis 2004, 7:235-241.
• [45]Cemazar M, Jarm T, Miklavcic D, Lebar A, Ihan A, Kopitar N, Grego S: Effect of electric-filed intensity on electropermeabilization and electrosensitivity of various tumor-cell lines in vitro. Electromagn Biol Med 1998, 17:263-272.
• [46]Wong AT, Gumbiner BM, Sadlonova A: Adhesion-independent mechanism for suppression of tumor cell invasion by E-cadherin. J Cell Biol 2003, 161:1191-1203.
• [47]Woodhouse E, Chuaqui R, Liotta L: General mechanism of metastasis. Cancer (Phila.) 1997, 80:1529-1537.
• [48]Yamaguchi H, Lorenz M, Kempiak S, Sarmiento C, Coniglio S, Symons M, Segall J, Eddy R, Miki H, Takenawa T, Condeelis J: Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin. J Cell Biol 2005, 168:441-452.
• [49]Harkin D, Hay E: Effects of electroporation on the tubulin cytoskeleton and directed migration of corneal fibroblast cultured within collagen matrices. Cell Motil Cytoskeleton 1996, 35:345-357.
• [50]Cho M, Thatte HS, Silvia MT, Golan DE: Transmembrane calcium influx induced by ac electric fields. FASEB J 1999, 13:677-683.
• [51]Sauer H, Stanelle R, Heschler J, Wartenberg M: The DC electrical-field-induced Ca2+ response and growth stimulation of multicellular tumor spheroids are mediated by ATP release and purinergic receptor stimulation. J Cell Sci 2002, 11:3265-3273.
• [52]Understanding the migration of cancer cells [http://www.sciencedaily.com/releases/2008/06/080623105027.htm] webcite
• [53]Wang F, Hansen R, Radisky D, Yoneda T, Barcellos-Hoff M, Petersen O, Turley E, Bissell M: Phenotypic Reversion or Death of Cancer Cells by Altering Signaling Pathways in Three-Dimensional Contexts. J Natl Cancer Inst 2002, 94:1494-1503.
• [54]Bakin A, Safina A, Rinehart C, Daroqui C, Darbary H, Helfman D: A critical role of tropomyosins in TGF-beta regulation of the actin cytoskeleton and cell motility in epithelial cells. Mol Biol Cell 2004, 15:4682-4694.