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Quantitative Proteomic Analysis of Cellular Resistance to the Nanoparticle Abraxane Minzhi Zhao, Haiyun Li, Xiangli Bu, Chunni Lei, Qiaojun Fang, and Zhiyuan Hu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b03677 • Publication Date (Web): 01 Sep 2015 Downloaded from http://pubs.acs.org on September 3, 2015
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Quantitative Proteomic Analysis of Cellular Resistance to the Nanoparticle Abraxane Minzhi Zhao, Haiyun Li, Xiangli Bu, Chunni Lei, Qiaojun Fang* and Zhiyuan Hu*
CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology of China, Chinese Academy of Sciences, Beijing, 100190, China
Corresponding Author
[email protected];
[email protected] 1
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ABSTRACT Abraxane, an FDA-approved albumin-bound nanoparticle (NP) form of paclitaxel (PTX) to treat breast cancer and non-small cell lung cancer (NSCLC), has been demonstrated to be more effective than the original Taxol, the single molecule form. We have established a cell line from NSCLC A549 cells to be resistant to Abraxane. To further understand the molecular mechanisms involved in the NP drug resistance, global protein expression profiles of Abraxane sensitive (A549) and resistant cells (A549/Abr), along with the treatment of Abraxane, have been obtained by a quantitative proteomic approach. The most significantly differentially expressed proteins are associated with lipid metabolism, cell cycle, cytoskeleton, apoptosis pathways and processes, suggesting several mechanisms are working synergistically in A549 Abraxane-resistant cells. Overexpression of proteins in the lipid metabolism processes, such as E3 ubiquitin-protein ligase RNF139 (RNF139) and Hydroxymethylglutaryl-CoA synthase (HMGCS1), have not been reported previously in the study of paclitaxel resistance, suggesting possibly different mechanism between nanoparticle and single molecular drug resistance. In particular, RNF139 is one of the most up-regulated proteins in A549 Abraxane-resistant cell line, but remains no change when the resistant cells were further treated with Abraxane and down-regulated in the sensitive cells after 4 h treatment of Abraxane.
This study shows the use of a proteomic strategy to understand the unique response of
drug resistant cells to a nanoparticle therapeutic.
KEYWORDS: Abraxane
resistance; quantitative proteomics, nanoparticle drug; synergistic
mechanisms; E3 ubiquitin-protein ligase RNF139
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Abraxane is an albumin-bound nanoparticle (NP) form of paclitaxel (PTX) administered without the use of other excipients. It has been approved by FDA for use in patients with non-small cell lung cancer (NSCLC) and metastatic breast cancer.1-3 Abraxane was shown to be twice as effective as the conventional clinical formulation of PTX.4 The increased antitumor activity of Abraxane might be related to enhanced drug concentration in tumor through enhanced permeability and retention (EPR) effect of tumor.5, 6 However, whether albumin-bound PTX nanoparticle can address the drug resistance problem is currently unclear. 7 Drug resistance can be achieved as tumor cells inherently resist to the drug from the start, or the tumor cells initially respond to therapy, but eventually some of them acquire certain systematic changes through evolution.8 Studies have shown that drug resistance can be caused by more than one mechanisms, such as decreased drug uptake into cells and activation of DNA repair system. 9 So far, the mechanisms of drug resistance caused by PTX have been reported by many groups. Gercel-Taylor’s group studied the effect of paclitaxel treatments on chemosensitivity of ovarian cancer cells developed from a patient with stage IIIC disease and showed that the resistance came from alterations in the growth characteristics and overexpression of mutant p53 in the treated cells.10 Using Luria-Delbruck fluctuation analysis in the human uterine sarcoma cell line MES-SA as a model, Dumontet et al. showed that spontaneous mutational events underlie acquired drug resistance in these cells, and multidrug resistance (MDR) by activation of P-glycoprotein (P-gp, ABCB1, MDR1) is a common mechanism for cellular resistance to paclitaxel and other anticancer agents such as doxorubicin and vinblastine.11 Okada et al. and Parekh et al. demonstrated that taxane is a substrate for the drug resistance pump P-gp, which is able to confer resistance to a wide variety of naturally derived hydrophobic substances.12, 13 Therefore, the cells selected with taxane were found 3
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to have elevated levels of P-gp and to exhibit cross-resistance to a variety of other hydrophobic drugs.14 Other possible mechanisms of resistance to PTX include overexpression of Bcl-2,15 and alteration in microtubulin composition or dynamics.16-18 Since overexpression of P-gp is well-known to be responsible for multidrug resistance to PTX,19-21 inhibition of MDR1 is likely to have a significant improvement on PTX clinical application.
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However, it was shown that many P-gp
inhibitors failed to show efficacy during pre-clinical and clinical studies.23 Therefore, drug resistance is rather complicated that requires more study. Drug-resistant cells can display polymorphism in phenotype which is caused by several pathways altered simultaneously. In this scenario, large-scale profiling methods have the chance to explore the complexity of drug resistance and provide alternative targets for developing more effective anticancer therapies. Molecular profiling approaches based on proteomics have been widely applied to the identification of new markers of PTX resistance. Early study has found differentially expressed proteins among a sensitive and two paclitaxel resistant (induced and natural) ovarian cancer cell lines, and five of them were subjected to further validation.24 One study found that ERp57 plays an important role in chemoresistance mechanisms in ovarian cancer by modulating the attachment of microtubules to chromosomes following paclitaxel treatment through its interaction with class III beta-tubulin25. The expression pattern of different β-tubulin isotypes has been proposed as a mechanism of PTX resistance.26-28 Another report compared PTX-sensitive and resistant ovarian cancer cells with stepwisely increasing concentration of PTX over a period of 12 months. They found down-regulation of heterogeneous nuclear ribonucleoprotein A2/B1 isoform A2 (hnRNPA2) and GDP dissociation inhibitor 2 isoform 1 (GDI2) in resistant cells and confirmed ex vivo.29 The nucleus accumbens-1 (Nac-1), which belongs to the POZ/BTB (Pox virus and Zinc 4
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finger/Bric-a-brac tramtrack broad complex) transcription factor family, has been identified as a possible candidate chemoresistance gene in ovarian cancer.30-32 Several studies found the effects of mitochondria in mediating PTX induced apoptosis, and highlighted the role of voltage-dependent anion-selective channel protein (VDAC) in mediating chemosensitivity and resistance of tumor cells.32-36 We have previously established Abraxane-resistant NSCLC cell line (A549/Abr) from a drug sensitive cell line (A549) (Supplementary Fig. S2). According to Chauhan et al., Abraxane rapidly decreased its size from 130 nm to proximately 10 nm following dilution in plasma.
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It is also
reported that PTX existed as the albumin-bound form in tissue homogenate after treatment of Abraxane for 3 h.38 Since nanoparticles usually enter the cell by endocytosis, whether cells develop mechanisms specific to nanoparticle drug resistance except over-expression of ABCB1 is still unknown. To address these questions, we carried out the high throughput quantitative proteomics on the Abraxane-resistant NSCLC cell line.
RESULTS AND DISCUSSION Quantitative Proteomic analyses of A549 Abraxane-resistant and sensitive cells with or without treatment of Abraxane. To understand the mechanisms leading to Abraxane-resistant in A549/Abr cell line, global protein changes of A549 Abraxane-resistant cells, which were developed from long-time treatment of Abraxane, was compared to A549 sensitive cells. In order to understand the difference of protein expression patterns with short time treatment of Abraxane, we also study the proteomic profiles of both A549 Abraxane-resistant and sensitive cells after growing in 100nM of Abraxane for 4h. The IC50 values of Abraxane for A549 cells and A549/Abr cells were 5
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11.06±1.06nM and 1314.66±25.29nM, respectively. The resistance index (RI) of the A549/Abr cell line was 118.79, which indicated that an Abraxane-resistant cell line was successfully established. The growth curves of the parent cells and the drug-resistant cells showed that the proliferation rates of A549/Abr cells were lower than A549 cells. The growth curves of the parent cells and the drug-resistant cells showed that the proliferation rates of A549/Abr cells were lower than A549 cells (Fig. 2B). The population doubling time of A549 cells and A549/Abr cells was 49.56±5.53 h and 54.65±4.87 h, respectively (p
