Mitochondrial proteomics with siRNA knockdown to reveal ACAT1 and MDH2 in the development of doxorubicin-resistant uterine cancer

Mitochondria are key organelles in mammary cells in responsible for a number of cellular functions including cell survival and energy metabolism. Moreover, mitochondria are one of the major targets under doxorubicin treatment. In this study, low-abundant mitochondrial proteins were enriched for proteomic analysis with the state-of-the-art two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assistant laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) strategy to compare and identify the mitochondrial protein profiling changes in response to the development of doxorubicin resistance in human uterine cancer cells. The mitochondrial proteomic results demonstrate more than fifteen hundred protein features were resolved from the equal amount pooled of three purified mitochondrial proteins and 101 differentially expressed spots were identified. In which, 39 out of these 101 identified proteins belong to mitochondrial proteins. Mitochondrial proteins such as acetyl-CoA acetyltransferase (ACAT1) and malate dehydrogenase (MDH2) have not been reported with the roles on the formation of doxorubicin resistance in our knowledge. Further studies have used RNA interference and cell viability analysis to evidence the essential roles of ACAT1 and MDH2 on their potency in the formation of doxorubicin resistance through increased cell viability and decreased cell apoptosis during doxorubicin treatment. To sum up, our current mitochondrial proteomic approaches allowed us to identify numerous proteins, including ACAT1 and MDH2, involved in various drug-resistance-forming mechanisms. Our results provide potential diagnostic markers and therapeutic candidates for the treatment of doxorubicin-resistant uterine cancer.


Introduction
Doxorubicin is one of primary anticancer drugs used in the treatment of a broad range of tumour types [1][2][3]. However, doxorubi-cin's precise anti-cancer mechanisms are complex; previous study showed that doxorubicin disturbs DNA replication by intercalation into DNA double strand and leads to the subsequent inhibition of DNA, RNA and proteins synthesis. Moreover, doxorubicin has been reported to inhibit topoisomerase 2 and leads to the breakage of DNA double strand which prevents DNA synthesis. In addition, doxorubicin directly damages cancer cells through disturbing the normal physiology of mitochondria resulting in cell apoptosis [4,5]. Nevertheless, doxorubicin-induced chemotherapy resistance has been widely reported in cancer chemotherapy such as breast cancer, lung cancer, leukaemia, osteosarcoma and uterine cancer [6][7][8][9][10], and is a primary therapeutic problem to the successful treatment of patients who receive cancer chemotherapy.
Chemotherapy resistance diminishes the anti-cancer effects of chemotherapy drugs used in the treatment of tumours. In clinical practice, chemotherapy resistance has been recognized as a severe problem when the concentrations of chemotherapy drugs approach at harmful and toxic dosages to kill tumours. Biological mechanisms associated with drug resistance have been described including the increased activities of plasma membrane drug efflux transporter such as adenosine triphosphate binding cassette-transporters, the enhanced detoxification enzymes such as glutathione reductase and the activation of cellular signalling pathways that direct cell survival and cell death [11]. However, not all of chemotherapy resistance can be completely explained by the elucidated mechanisms. Consequently, it is important to globally investigate the additional resistance mechanisms which have not yet to be clarified.
Because the development of chemotherapy resistance is multiple factors and related to the alterations in numerous proteins, it is advantageous to use proteomic strategies to globally determine chemotherapy resistance-related proteins. Our previous proteomic analysis has identified numerous proteins, including asparagine synthetase and membrane-associated progesterone receptor component 1, involved in a variety of drug-resistance-forming mechanisms [12]. In this study, to decrease complexity and discover low abundance proteins, subcellular proteomic analysis based on purification of mitochondrial proteins were performed to study the correlation between mitochondria and doxorubicin-induced chemotherapy resistance since mitochondria are crucial for the survival of resistance cancer cells and their defects contribute to the development of chemotherapy resistance [13,14]. To carry out an in vitro investigation into doxorubicin-resistance mechanisms in uterine cancer, increase our understanding of the molecular mechanisms involved, and identify potential chemotherapy resistance biomarkers with possible diagnostic or therapeutic applications, we established a serial of uterine sarcoma cancer lines, MES-SA, and its doxorubicin-resistant partners MES-SA/Dx-2 lM cells and MES-SA/Dx-8 lM cells as a model system to examine chemotherapy resistancedependent mitochondrial protein alterations via quantitative proteomic analysis with 2D-DIGE and MALDI-TOF mass spectrometry. This study also includes reports of studies that used siRNA silencing against selected identified proteins, ACAT1 and MDH2, to monitor and evaluate their potency against doxorubicin resistance.

Chemical and reagents
Generic chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), while reagents for 2D-DIGE were purchased from GE Healthcare (Uppsala, Sweden). All primary antibodies were purchased from Genetex (Hsinchu, Taiwan) and antimouse and anti-rabbit secondary antibod-ies were purchased from GE Healthcare. All the chemicals and biochemicals used in this study were of analytical grade.

Cell lines and cell cultures
The uterine sarcoma cancer line MES-SA was purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in McCoy's 5a modified medium containing 10% foetal bovine serum, Lglutamine (2 mM), streptomycin (100 lg/ml), penicillin (100 IU/ml) (all from Gibco-Invitrogen Corp., Paisley, UK). The doxorubicin-resistance lines MES-SA/Dx-2 lM and MES-SA/Dx-8 lM cell were both derived from MES-SA via stepwise increasing the doxorubicin concentrations in medium, and were maintained in the same medium and supplement with 0.2 lM and 0.8 lM doxorubicin respectively. All cells were incubated at 37°C in a humidified atmosphere containing 5% CO 2 . The IC 50 values for MES-SA and its doxorubicin-resistance lines MES-SA/Dx-2 lM and MES-SA/Dx-8 lM were 0.25 lM, 5.31 lM and 18.75 lM respectively.

Sample preparation for mitochondrial proteomic analysis
Mitochondria were isolated by using the mitochondrial isolation kit for mammalian cells (Millipore, Darmstadt, Germany) according to our previous report [15]. Briefly, following lysis of~2.5 9 10 7 of MES-SA, MES-SA/Dx-2 lM or MES-SA/Dx-8 lM, cell debris and nuclei were pelleted at 700 9 g, followed by centrifugation at 5000 9 g to pellet a mitochondrially enriched fraction. The crude mitochondria were washed in chilled 0.59 PBS and lysed in 2-DE lysis buffer containing 4% w/v CHAPS, 7 M urea, 2 M thiourea, 10 mM Tris-HCl, pH 8.3, 1 mM EDTA. Lysates were homogenized by passage through a 25-gauge needle for 10 times and the insoluble material removed by centrifugation at 13,000 r.p.m. for 30 min. at 4°C and protein concentrations were determined by using the coomassie protein assay reagent (Bio-Rad, Hercules, CA, USA).

MTT cell viability assay
The detailed MTT experimental procedure has been described in our previous study [12].
Mitochondrial membrane potential assay by JC-10 fluorescence and flow cytometry The mitochondrial membrane potentials of cultured cells were determined by using the fluorescent probe JC-10 (AAT Bioquest, Sunnyvale, CA, USA) following the manufacturer's recommendations. Briefly, cultured cells were exposed to 5 lM mitochondrial uncoupler carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) for 1 hr and then incubated in culture medium containing JC-10 for 1 hr at room temperature. The cells were washed with PBS and analysed by flow cytometry. Photomultiplier settings were adjusted to detect JC-10 monomer and aggregate fluorescence on the FL1 (525 nm) and FL2 (595 nm) detec- tors. The fluorescence ratio at these wavelengths was used to monitor changes in mitochondrial membrane potential.

Flow cytometry analysis for apoptosis detection
Annexin V/propidium iodide (PI) double assay was performed with the Annexin V, Alexa Fluor â 488 Conjugate Detection kit (Life Technologies, Paisley, UK). Briefly, 10 6 cells were resuspended in 500 ll binding buffer and stained with 5 ll Alexa Fluor 488 conjugated annexin V according to the manufacturer's instructions. 1 ll 100 lg/ml PI was added and mixed gently to incubate with cells for 15 min. at room temperature in the dark. After incubation period, samples were subjected to FCM analysis in 1 hr. by using BD Accuri C6 Flow Cytometry (BD Biosciences, San Jose, CA, USA). The data were analysed by using Accuri CFlow @ and CFlow Plus analysis software (BD Biosciences).

2D-DIGE, gel image analysis, protein staining, in-gel digestion and MALDI-TOF MS analysis
The detailed experimental procedures have been described in our previous publications [16][17][18]. Notably, peaks in the mass range of m/z 800-3000 were used to generate a peptide mass fingerprint that was searched against the Swiss-Prot/TrEMBL database (released on November 2011) with 533,049 entries by using Mascot software v2.3.02 (Matrix Science, London, UK). The parameters used for Mascot search are listed: Homo sapiens; tryptic digest with a maximum of 1 missed cleavage; carbamidomethylation of cysteine, partial protein N-terminal acetylation, partial methionine oxidation and partial modification of glutamine to pyroglutamate and a mass tolerance of 50 ppm. Identification was accepted based on significant MASCOT Mowse scores (P < 0.05), spectrum annotation and observed versus expected molecular weight and pI on 2-DE as well as at least 5 peptides in each identified protein.

Immunoblotting analysis
Immunoblotting analysis was used to validate the differential abundance of mass spectrometry identified proteins across MES-SA, MES-SA/Dx-2 lM and MES-SA/Dx-8 lM cells. The detailed experimental protocol has been described in our previous study [19]. All primary antibodies used for expression validation were purchased from Genetex.

siRNA design, construction and transfection
The siRNA against ACAT1 and MDH2 were synthesized by Invitrogen. The targeting sequences 5 0 -UAU UGU AGA CAU CAG UUA GCC CGU C-3 0 against ACAT1, and 5 0 -UAA CGA AGG AAC AUU CCA CAA CAC C-3 0 against MDH2 were designed and verified to be specific by Blast search against the human genome, and sequences of similar GC contents which do not match any known human coding sequence were used for negative control against ACAT1 and MDH2. Transfection was mediated with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instruction. Briefly, cells were transfected with 20 nM of ACAT1 siRNA and 20 nM of MDH2 siRNA or the corresponding control (pGCsi-control) in serum free medium containing Lipofectamine RNAi-MAX for 4 hrs followed by recovered in medium containing 10% FCS for 24 hrs. The efficiency of siRNA knockdown was monitored with immunoblotting by using primary antibodies against ACAT1 and MDH2.

Results
Development of doxorubicin-resistance uterine cancer lines   extraction. To examine the efficiency of mitochondrial protein extraction, immunoblotting analysis of the beta-tubulin (cytosolic marker) and VDAC2 (mitochondrial marker) levels were carried out and the results showed the mitochondrial fractions were significantly enriched (Fig. 2). The 2D-DIGE analysis shows that there is a significant protein expression profiling changes between the mitochondrial fraction and cytosolic fraction indicating the established mitochondrial enrichment is suitable for providing downstream mitochondrial proteome analysis (data not shown).
To identify altered abundance of mitochondrial proteins and relate it to the formation of chemotherapy drug resistance-induced by doxorubicin, the mitochondrial proteomic profiles of MES-SA, MES-SA/ Dx-2 lM and MES-SA/Dx-8 lM were analysed. The triplicates of the three different mitochondrial fractions from MES-SA, MES-SA/Dx-2 lM and MES-SA/Dx-8 lM cells were compared by 2D-DIGE to have a comprehensive overview of uterine cell drug resistance. After image analysis with DeCyder v7.0, more than 1704 protein spots were well-defined (Fig. 3). To reduce the intrinsic variability derived from protein samples and gel-to-gel variation, only those protein features appeared at least in all of the triplicate gel images were selected for statistical analysis. Furthermore, biological variation analysis of spots showing greater than 1.5-fold change in expression with a t-test score of less than 0.05 were visually checked before confirming the alterations for protein identification. MALDI-TOF MS identification revealed 101 unique differentially expressed proteins across MES-SA, MES-SA/Dx-2 lM and MES-SA/Dx-8 lM (data not shown). In which, 39 (39%) identified proteins were dominantly mitochondria-located proteins. Of the 39 mitochondrial proteins, 25 of these proteins were shown doxorubicin dose-dependent changes across MES-SA, MES-SA/Dx-2 lM and MES-SA/Dx-8 lM ( Fig. 4 and Table 1). In these mitochondrial proteins, most of them were involved in protein folding, signal transduction and redox regulation (Fig. 4). Importantly, a number of identified spots such as serine hydroxymethyltransferase and malate dehydrogenase have not been reported in drug-resistance associated study in our knowledge means these proteins are putative mitochondrial regulatory proteins in response to the development of drug resistance. As expect, some of well known resistance markers such as fumarate hydratase were also identified in this 2D-DIGE experiment [12].

Validation of characterized resistance-associated mitochondrial proteins via immunoblotting
The proteomic study identified a number of doxorubicin resistanceassociated mitochondrial proteins. It was essential to confirm the expression of these proteins by using independent biochemical experiments. To this end, we performed immunoblotting analyses to    To further verify the protein expression levels of unreported identified proteins across MES-SA, MES-SA/Dx-2 lM and MES-SA/Dx-8 lM, the expression of mitochondrial protein ACAT1 was examined and the immunofluorescence result demonstrated that ACAT1 was located in mitochondria due to the spatial overlapping of ACAT1 with mitotracker and its expression is up-regulated in MES-SA/Dx-2 lM and MES-SA/Dx-8 lM than in MES-SA implying ACAT1 is overexpressed in doxorubicin-induced drug resistant uterine cancer cells (Fig. 5B). Thus, ACAT1 is potentially to be a candidate for the diagnosis of doxorubicin-associated drug resistance in uterine cancer.
Notably, ACAT1, a major mitochondria-located protein, was dominantly distributed in the mitochondria of MES-SA, MES-SA/Dx-2 lM and MES-SA/Dx-8 lM cells and was gradually increasing its expression level in response to the drug resistance with a positive correlation (Fig. 5B). Further investigation demonstrated that the level of mitochondrialocated mitochondrial protein MDH2 was significantly increased in resistant line MES-SA/Dx-8 lM rather than in MES-SA or in MES-SA/ Dx-2 lM implying MDH2 is over-expressed in highly resistant uterine cancer cells (Fig. 5C). Thus, MDH2 is potentially to be a mitochondrial marker for the diagnosis of doxorubicin-associated drug resistance in uterine cancer. Importantly, MDH2 was dominantly distributed in the nucleus in both MES-SA and MES-SA/Dx-2 lM cells, but was gradually shifting to mitochondria in MES-SA/Dx-8 lM cells (Fig. 5C) implying the intracellular distribution of MDH2 is associated with doxorubicin-induced drug resistance.
Evaluation of the roles of ACAT1 and MDH2 on doxorubicin resistance in uterine cancer using siRNA knockdown We found that mitochondrial proteins, ACAT1 and MDH2 are overexpressed in the mitochondrial fractions of the doxorubicin-resistant  (Fig. 6D). Further study on MDH2 showed that treatment of MES-SA/Dx-8 lM cells with doxorubicin at the IC 50 doses increased the numbers of total apoptotic cells (LR + UR) from 13.4% to 23.8%, compared with increases ranging from 11% to 21.7% in MDH2 knockdown MES-SA/Dx-8 lM cells. These results indicated that MDH2 has no direct effect on the preventing doxorubicin-induced cell apoptosis in doxorubicin-resistant cells implying some other mechanisms other than apoptosis are contributed by MDH2 knockdown to regulate cell viability (Fig. 7D).

Discussion
A better understanding of chemotherapy-associated drug-resistance mechanisms is essential for offering opportunities in both drug-resistance detection and therapy. Current understanding demonstrated mitochondria is one of the major targets for chemotherapeutic drugs [14,20]. Additionally, mutations within the mitochondrial genomes are reported in most cancers, which are believed to promote chemoresistance [21]. So far, most of the functional mechanisms of chemoresistance were supposed from transcription-based researches, rather than from translation-level studies. Thus, in current study, we adopted a mitochondrial proteomic approach based on 2D-DIGE analysis coupled with MALDI-TOF/TOF MS to identify mitochondrial proteins which were differentially expressed in the doxorubicin-resistant human uterine cancer cell lines, MES-SA/Dx-2 lM and MES-SA/ Dx-8 lM, and its parental cell line MES-SA, to provide a broad view of protein expression and protein-protein interactions related to doxorubicin resistance in uterine cancer. The identified proteins might serve as potential resistant markers for prognosis or diagnosis of the uterine cancer patients to treatment with doxorubicin or other chemotherapy drugs such as 5-fluorouracil.
In current study, we observed 101 differentially expressed MALDI-TOF/TOF MS identified protein spots from isolated mitochondrial fractions across the 3 cell lines. In which, 39 out of the 101 proteins are mitochondria-located proteins. Although only 39% of the isolated proteins were assigned to be mitochondria located, the percentage is higher than the general total cellular protein analysis (~5% of total cellular proteins) [17,22,23]. Notably, part of cytosolic proteins are able to translocate into mitochondria but are assigned to be cytosolic proteins which might contribute to the low coverage rate of the mitochondrial fraction in total enriched proteins.
This study showed that ACAT1 and MDH2 proteins are potential in the formation of doxorubicin drug resistance. We validated the functions of ACAT1 and MDH2 in drug resistance, and found that their overexpressions contribute significantly to the development of doxorubicin resistance in MES-SA/Dx-2 lM and MES-SA/Dx-8 lM cells. Our studies also demonstrated that multiple mechanisms are correlated with the formation of chemotherapy resistance in uterine cancer, and that these different mechanisms might contribute to chemotherapeutic resistance in the treatment of uterine cancer.
ACAT1 is a mitochondrial enzyme participating in numerous pathways including fatty acid metabolism, synthesis and breakdown of ketone bodies, valine, leucine and isoleucine degradation, lysine degradation, tryptophan metabolism, pyruvate metabolism and butanoate metabolism [24][25][26]. Thus, ACAT1 plays an important role in the modulation of cell physiology. However, there is limited report demonstrated the potency of ACAT1 in either cancer research or chemotherapy resistance. Only one report shows that ACAT1 is a potential diagnostic cancer marker for renal carcinoma [27] with no report in drug resistant field. Our current results indicated that ACAT1 has profound effect in the formation of doxorubicin resistance and further implicated the complication between doxorubicin resistance and these metabolic pathways.
MDH2 is a citric acid cycle enzyme that catalyses the conversion of malate into oxaloacetate as well as involves in gluconeogenesis via reducing oxaloacetate into malate and traversing malate across the inner mitochondrial membrane. Additionally, MDH2 is one of the major players in malate-aspartate shuttle which can transfer electrons from cytosolic NADH to produce mitochondrial NADH and generate ATP via electron transport chain [28]. This implies up-regulation of MDH2 might increase cellular energy to supply biosynthesis of resistance-associated proteins and provide energy for P-glycoprotein to pump-out chemotherapeutic drugs. In addition, Liu et al.'s study revealed that MDH2 mediates prostate cancer resistance to docetaxel-chemotherapy through JNK pathway and MDH2 knockdown significantly enhanced the docetaxel sensitivity in prostate cancer cells. Their study also showed that MDH2 knockdown significantly reduced cellular ATP levels and increased the ADP/ATP and NAD + /NADH level as well as intracellular ROS concentration [29].
Drug resistant tumour cells have been reported to be more metastatic and invasive as well as increased wound healing ability, when compared with drug sensitive tumour cells. To study the effect of ACAT1 and MDH2 knockdown on wound healing of MES-SA, MES-SA/Dx-2 lM and MES-SA/Dx-8 lM, these cells were knockdown followed by performed wound closure experiments. The results indicated that a significant healing delay was observed in ACAT1 siRNA and MDH2 siRNA treated MES-SA/Dx-2 lM and MES-SA/Dx-8 lM cells. Compared to this condition, the restore of wound areas were found to reduce significantly when MES-SA cells were treated with either ACAT1 siRNA or MDH2 siRNA. These results implied that knockdown of ACAT1 and MDH2 has profound effect on doxorubicin resistance-associated wound healing (Fig. S1).
Mitochondrial hsp70 (HSPA9) is a crucial component in the import and folding of mitochondrial proteins and help to protect cells from stress including chemotherapeutic agents. In comparison with normal cells, most cancer cells overexpress mitochondrial hsp70 (HSPA9) at the basal level to resist to various damages at different stages of tumourigenesis and during anti-cancer treatment [30]. This is the reason why mitochondrial hsp70 (HSPA9) has been widely observed in cancer drug-resistance researches [31] as well as in aggressive cancer studies [32]. In current study, as expect mitochondrial hsp70 (HSPA9) is significantly overexpressed in doxorubicininduced drug resistance and might be further evaluated as a target to diminish drug resistance.
In this study, we also determined numerous potential biological functions of the identified proteins towards baseline resistance in human uterine cancer cells by using a Swiss-Prot search combined with KEGG pathway analysis. Our findings should be useful for a systematic study on the mechanisms of doxorubicin resistance in uterine cancer. Figure 8 shows a comparison of the expression profiles of the differentially expressed mitochondrial proteins for baseline resistance. Mitochondrial proteins known to regulate amino acid metabolism, electron transport, fatty acid metabolism, protein folding and TCA cycle are up-regulated in resistance cells, but not in sensitive cells, implying that doxorubicin-resistant cells have greater ability to maintain mitochondrial protein conformation, energy metabolism and biomacromolecule metabolism.
Protein species theory is proposed to be the smallest unit of proteome and believed the formation of different forms of proteins (such as post-translational modifications), each of which is able to have specific chemical structure and specific function. The original biosynthetic product of translation stands for a prototype protein species, the initial protein species. This initial protein species is possibly further biochemically processed, modified, or proteolytically and/or secreted out of the cell or transported to intracellular organelles, spliced or degraded. Each of these covalent modifications or processed of a protein leads to a new protein species [33,34]. In this study, some of identified proteins including HSP60, MDH2, mitochondrial inner membrane protein, stomatin-like protein 2 and peroxiredoxin-3 have been identified in multiple spots (Table 1) implying these proteins encounter differential processes/modifications and form new protein species against initial translational products.
To sum up, our current mitochondrial proteomic approaches allowed us to identify numerous proteins, including ACAT1 and MDH2 involved in various drug-resistance-forming mechanisms. Our results provide useful diagnostic markers and therapeutic candidates for the treatment of doxorubicin-resistant uterine cancer.