Ultrasound-Induced New Cellular Mechanism Involved in Drug Resistance

et al. (2012) Ultrasound-Induced New Cellular Mechanism Involved in Drug Resistance. PLoS ONE 7(12): e48291. doi:10.1371/journal.pone.0048291 Ultrasound-Induced New Cellular Mechanism Involved in Drug Resistance Mariame A. Hassan 0 Yukihiro Furusawa 0 Masami Minemura 0 Natalya Rapoport 0 Toshiro Sugiyama 0 Takashi Kondo 0 Yiqun G. Shellman, University of Colorado, United States of America 0 1 Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan, 2 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt, 3 Department of Gastroenterology and Hematology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan, 4 Department of Bioengineering, University of Utah , Salt Lake City, Utah , United States of America The acoustic effects in a biological milieu offer several scenarios for the reversal of multidrug resistance. In this study, we have observed higher sensitivity of doxorubicin-resistant uterine sarcoma MES-SA/DX5 cells to ultrasound exposure compared to its parent counterpart MES-SA cells; however, the results showed that the acoustic irradiation was genotoxic and could promote neotic division in exposed cells that was more pronounced in the resistant variant. The neotic progeny, imaged microscopically 24 hr post sonication, could contribute in modulating the final cell survival when an apoptotic dose of doxorubicin was combined with ultrasound applied either simultaneously or sequentially in dual-treatment protocols. Depending on the time and order of application of ultrasound and doxorubicin in combination treatments, there was either desensitization of the parent cells or sensitization of the resistant cells to doxorubicin action. - Funding: Financial support for this study was provided by a Grant-in-Aid for Scientific Research (B) (22390229), Japan Society for the Promotion of Science (to TK), (C) (No. 20590765) (to MM) and by research grant from International Association of Sensitization for Cancer Treatment (to MA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Multidrug resistance (MDR) is a unique inherent or induced system for protection by which cancer cells can experience reduced cytotoxicity in response to a wide range of chemotherapeutics. The non-specificity of this system, that is acquiring cross resistance to various unrelated drugs, undermines the outcomes of chemotherapy [1]. MDR comprises different mechanisms, the most common of which involves the reduction of intracellular drug accumulation. This occurs through the expression of membrane proteins that can extrude the internalized drug molecules before they can even reach the cytoplasm [2]. These proteins are energypowered transporters belonging to the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily proteins. Mammalian P-glycoprotein (P-gp) was the first identified member of this family and is present at varying levels in every human tissue [3]. As mentioned earlier, the ABC transporters pump a broad spectrum of substrates which hardly share a common structural lead or action. However, the knowledge of membrane composition and how it impacts the internalization of exogenous molecules into cells suggests that pump substrates might share a degree of lipophilicity as a common trait. In fact, all P-gp substrates are lipophilic compounds which can readily cross the cell membrane in absence of the efflux proteins. Based on this, it might be also expected that P-gp-expressing cells might have different membrane properties to provide an optimal phase balance for their efficient functioning [4]. If the intracellular concentration of P-gp substrates is a result of the equilibrium attained between drug uptake by passive diffusion and drug efflux by these multi-drug transporters, then, modulating one of these two factors can result in increasing the intracellular accumulation of these compounds. Not only is this step indispensable in overcoming resistance attributable to P-gp expression, but also indispensable in tumor cells possessing other resistance pathways (e.g. drug inactivation). In general, increasing the intracellular concentration of drugs serves in surpassing the threshold of cells to reverse their toxicity efficiently. The basic strategies in Pgp-mediated MDR reversal sought the inactivation of the efflux proteins, either directly through the use of inhibitors [5] or indirectly through ATP depletion or membrane fluidization [6,7]. Projecting the knowledge of Ultrasound (US) interactions with biological systems on MDR reversal, we can predict numerous scenarios of sensitization. The most prominent effect of US is its ability to (transiently) permealize cell membranes to P-gp substrates through sonoporation [8]. Also, US-induced hyperthermia, due to the partial absorption of acoustic energy, can increase the accumulation of drugs probably due to (transient) membrane fluidization that might affect the functioning of the efflux pumps [9,10]. Acoustic effects are not limited to cellular membranes; however, they extend to intracellular targets including mitochondria, endoplasmic reticulum and the nuclear territory. The impact of US hits on intracellular targets manifests as increased intracellular oxidative stress, induction of apoptosis [11,12,13], alteration in gene expression levels, and DNA damage [14,15,16]. Although these manifestations correlated with increased cell killing in many studies, reflecting the potential of US as an adjuvant tool in cancer eradication and further supporting the rationale of employing US in MDR reversal, there were occasions in which the enhancement of cell killing was not satisfactory, especially for solid tumor-derived (adherent) cancer cell lines [17,18]. The decade-old studies on the use of US in MDR reversal showed in some cases higher sensitivity of drugresistant cells to US exposure [8,19,20]. This interesting and important, and yet unexplained, finding never correlated to the number of studies on this approach nor to the body of knowledge accumulated over these years on the underlying mechanisms. We have also noticed that the relatively successful trials were reported on a limited number of cancer cell lines (e.g. human hepatocarcinoma and ovarian carcinoma). The somewhat stymied progression in this issue implies that the final outcome has not been always encouraging or plausible. In an effort to define the impact of US in MDR, we planned this study to evaluate the differential sensitivities of drug-sensitive uterine sarcoma cell line (MES-SA) and its doxorubicin (Dox)resistant variant (MES-SA/DX5 cells) as a new model cell line to US exposure from different analytical perspectives. Despite the observed higher sensiti (...truncated)