Alternating current electrical stimulation enhanced chemotherapy: a novel strategy to bypass multidrug resistance in tumor cells.
BACKGROUND
Tumor burden can be pharmacologically controlled by inhibiting cell division and by direct, specific toxicity to the cancerous tissue. Unfortunately, tumors often develop intrinsic pharmacoresistance mediated by specialized drug extrusion mechanisms such as P-glycoprotein. As a consequence, malignant cells may become insensitive to various anti-cancer drugs. Recent studies have shown that low intensity very low frequency electrical stimulation by alternating current (AC) reduces the proliferation of different tumor cell lines by a mechanism affecting potassium channels while at intermediate frequencies interfere with cytoskeletal mechanisms of cell division. The aim of the present study is to test the hypothesis that permeability of several MDR1 over-expressing tumor cell lines to the chemotherapic agent doxorubicin is enhanced by low frequency, low intensity AC stimulation.


METHODS
We grew human and rodent cells (C6, HT-1080, H-1299, SKOV-3 and PC-3) which over-expressed MDR1 in 24-well Petri dishes equipped with an array of stainless steel electrodes connected to a computer via a programmable I/O board. We used a dedicated program to generate and monitor the electrical stimulation protocol. Parallel cultures were exposed for 3 hours to increasing concentrations (1, 2, 4, and 8 microM) of doxorubicin following stimulation to 50 Hz AC (7.5 microA) or MDR1 inhibitor XR9576. Cell viability was assessed by determination of adenylate kinase (AK) release. The relationship between MDR1 expression and the intracellular accumulation of doxorubicin as well as the cellular distribution of MDR1 was investigated by computerized image analysis immunohistochemistry and Western blot techniques.


RESULTS
By the use of a variety of tumor cell lines, we show that low frequency, low intensity AC stimulation enhances chemotherapeutic efficacy. This effect was due to an altered expression of intrinsic cellular drug resistance mechanisms. Immunohistochemical, Western blot and fluorescence analysis revealed that AC not only decreases MDR1 expression but also changes its cellular distribution from the plasma membrane to the cytosol. These effects synergistically contributed to the loss of drug extrusion ability and increased chemo-sensitivity.


CONCLUSIONS
In the present study, we demonstrate that low frequency, low intensity alternating current electrical stimulation drastically enhances chemotherapeutic efficacy in MDR1 drug resistant malignant tumors. This effect is due to an altered expression of intrinsic cellular drug resistance mechanisms. Our data strongly support a potential clinical application of electrical stimulation to enhance the efficacy of currently available chemotherapeutic protocols.
Janigro
Damir
D
Division of Cerebrovascular Research, Cleveland Clinic Lerner College of Medicine, Cleveland, OH 44106, USA. janigrd@ccf.org
Perju
Catalin
C
Fazio
Vincent
V
Hallene
Kerri
K
Dini
Gabriele
G
Agarwal
Mukesh K
MK
Cucullo
Luca
L
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis; D000818:Animals; D000970:Antineoplastic Agents / Q000493:pharmacokinetics*; D004317:Doxorubicin / Q000493:pharmacokinetics*; D018432:Drug Resistance, Multiple; D004599:Electric Stimulation Therapy; D019450:Genes, MDR; D006801:Humans; D007150:Immunohistochemistry; D009369:Neoplasms / Q000188:drug therapy; D010539:Permeability; D051381:Rats; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000970:Antineoplastic Agents; D004317:Doxorubicin