Quantitative proteomic analysis of enhanced cellular effects of electrochemotherapy with Cisplatin in triple-negative breast cancer cells

Due to the lack of the three main receptors, triple negative breast cancer (TNBC) is refractive to standard chemotherapy. Hence, alternate therapies are needed. TNBCs utilize glycolysis, which heightens their growth, proliferation, invasiveness, chemotherapeutic resistance and poor therapeutic response. This calls for novel therapeutic strategies to target these metabolic vulnerabilities present in TNBC. Electroporation-mediated chemotherapy, known as electrochemotherapy (ECT) is gaining momentum as an attractive alternative. However, its molecular mechanisms need better understanding. Towards this, label-free quantitative proteomics is utilized to gain insight into the anticancer mechanisms of ECT using electrical pulses (EP) and Cisplatin (CsP) on MDA-MB-231, human TNBC cells. The results indicate that EP + CsP significantly downregulated 14 key glycolysis proteins (including ENO1, LDHA, LDHB, ACSS2, ALDOA, and PGK1), compared to CsP alone. EP + CsP caused a switch in the metabolism with upregulation of 34 oxidative phosphorylation pathway proteins and 18 tricarboxylic acid (TCA) cycle proteins compared to CsP alone, accompanied by the upregulation of proteins linked to several metabolic reactions, which produce TCA cycle intermediates. Moreover, EP + CsP promoted multiple pathways to cause 1.3-fold increase in the reactive oxygen species concentration and induced apoptosis. The proteomics results correlate well with cell viability, western blot, and qPCR data. While some effects were similar for EP, more comprehensive and long-lasting effects were observed for EP + CsP, which demonstrate the potential of EP + CsP against TNBC cells.

Breast cancer is the second major cause of cancer death (after lung cancer) in women. Annually, one million new patients are estimated to be diagnosed with breast cancer 1 . Triple negative breast cancer (TNBC) contributes to about 15-20% of these cases. TNBC is a heterogeneous phenotype of the breast cancer, which lacks the expression of estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER2) receptors 2 . It has an aggressive clinical course and poor prognosis 3 with a short 5 year survival and increased 3 year recurrence rates 4 . There is a significantly increased likelihood of distant recurrence in TNBC (33.9%) compared to other breast cancers (20.4%) 3 . Aggressive and metastatic behavior of TNBC with distinct molecular signature and absence of targeted therapies pose a major challenge in the treatment. Recent reports suggest that TNBC harbor alterations in their metabolism, which correlate with the increased growth, proliferation, invasiveness, chemotherapeutic resistance and poor therapeutic response [4][5][6] . Particularly, TNBCs have elevated uptake and utilization of glucose and are highly dependent upon aerobic glycolysis 6,7 , while having disrupted mitochondrial function and oxidative phosphorylation (OXPHOS) 8,9 . The altered metabolism makes TNBC highly vulnerable to the inhibition of glycolysis, signifying that metabolic manipulation could be an effective strategy against TNBC. Consequently, the interest is growing in advancing new therapeutic strategies for TNBC patients when they cannot benefit from the current standard of treatment, including hormonal and trastuzumab therapies, because of the absence of target receptors 10 . The viable and non-viable cells were quantified using flow cytometry based on cell membrane permeability using two DNA binding dyes. The control cells (Ctrl) and the CsP treated cells had high viabilities of 96.3% and 96.9%, indicating poor cell death, while the EP and EP + CsP cells had lower viabilities of 27% and 28.9%, indicating no significant differences in cell viabilities of these two treatments. Figure 1b shows the metabolic activity of MDA-MB-231 cells at 4 h and 12 h of the treatments by quantifying ATP-independent substrate reducing potential. The results were normalized with the metabolic activity of Ctrl cells at 4 h (100%). The metabolic activity of Ctrl cells increased to 125% at 12 h. In the CsP treated samples, 83% and 106% of cells were metabolically active at 4 h and 12 h, respectively, showing no significant reduction in the metabolic activity (Fig. 1b). The EP treatment decreased the metabolic activity to 8.8% initially at 4 h, but the live cells revived back as indicated by a continued increase in the metabolic activity to 10.6% at 12 h and to 16% at 60 h (Table S1). For EP + CsP treatment, the fraction of metabolically active cells decreased significantly to 4.5% and 4.2% at 4 h and 12 h, respectively. The metabolic activity for EP + CsP treated cells showed a time dependent decrease as it reduced to 4% at 60 h (Table S1), which highlights a sustained effect of the EP + CsP treatment, contrary to EP.
Since in vivo application will involve both healthy and cancerous cells, we assessed the metabolic activity of non-tumorigenic mammary epithelial MCF10A cells (Fig. 1c). For EP treatment, the metabolic activity of www.nature.com/scientificreports www.nature.com/scientificreports/ MCF10A cells were 75% and 88% at 4 h and 12 h, respectively. For EP + CsP treatment, 65% and 51% of MCF10A cells were metabolically active at 4 h and 12 h, respectively. These results indicate that the effects of EP + CsP are specific to TNBC cells, as MCF10A cells maintained a significantly higher metabolic activity, compared to MDA-MB-231 cells.
overview of proteome analysis. Figure 2a shows the experimental workflow for proteome analysis.
LC-MS/MS data were collected using Q Exactive Orbitrap HF hybrid MS coupled with the UltiMate TM 3000 RSLCnano HPLC system. The tandem mass spectra were searched against the UniProt human protein database. Proteins identified in at least two of the three biological replicates at 1% FDR and with at least 2 MS/MS (spectral) counts were considered for further analysis, resulting in unambiguous identification of 2867 proteins/ protein families from 39622 peptides (Tables S2 and S3). Relative abundances of proteins in each sample were determined by label-free quantitation using LFQ values. Consistency of LFQ intensity is critical for the accurate measurement of protein abundances across multiple samples. The Boxplot (Fig. 2b) shows that the median and interquartile range of the triplicate samples were similar for each treatment, indicating the consistency of LC-MS/ MS measurements among the replicates. To further establish an experimentally supported inventory of differentially expressed proteins, we also measured correlations of protein intensities in triplicates of each treatment. As shown in Fig. 2c, we obtained a coefficient of determination (R 2 ) value of 0.99 for the pairwise comparisons of the Ctrl samples. Similar R 2 values were observed for other treatments as well (data not shown). The high R 2 values indicate high correlation and quality of the proteomics experiments.
We used LFQ values and MS/MS counts (LFQ ≠ 0 and MS/MS ≥ 2 for at least two of the three replicates) to classify the number of common and unique proteins present in each treatment. We identified 2311 proteins in control, 2477 in CsP, 2377 in EP, and 2457 in EP + CsP treated cells, of which 1959 (84.8% of the control) were commonly expressed in all three samples (Fig. 3a, Table S3). The number of uniquely identified proteins varied from 37 in Ctrl to 115 in CsP, 64 in EP, and 83 in EP + CsP samples. More proteins were identified in CsP than in the EP + CsP (2477 versus 2457) with more unique proteins, suggesting significant impact of ECT on cellular pathways through the increase or through the loss of many protein expressions (i.e., 302 proteins found in CsP were not found in EP + CsP). www.nature.com/scientificreports www.nature.com/scientificreports/ Analysis of differentially expressed proteins. The LFQ values of the common proteins in all four groups (1959) and common in two groups and unique in each group were used to perform statistical analysis and to detect significant differentially expressed proteins. Compared to the Ctrl, 46 proteins in the CsP, 364 proteins in the EP, and 512 proteins in the EP + CsP were upregulated (P < 0.05) (Table S4). Similarly, 36 proteins in the CsP, and 388 proteins in the EP and EP + CsP were downregulated (P < 0.05) (Table S4). When compared to CsP, 547 proteins were upregulated and 507 were downregulated in the EP + CsP treatment. Compared to EP, 13 proteins were upregulated and 16 were downregulated in the EP + CsP treatment. All proteins quantified in each treatment were visualized in Volcano plots depicting protein abundance changes between CsP versus Ctrl (Fig. 3b), EP + CsP versus Ctrl (Fig. 3c), and EP + CsP versus CsP (Fig. 3d). The differentially expressed proteins were clustered and visualized as a heatmap (Fig. 4a). Proteins within each treatment are clustered together and show consistent expression patterns. Between treatments, clusters of proteins for the Ctrl and CsP treatments are closer than the cluster of proteins for the EP and EP + CsP treatments, indicating the difference in protein expression induced by EP and EP + CsP treatments.
Further, we classified differentially expressed proteins using gene ontology (GO) annotation analysis for cellular component and molecular functions (Fig. 4b,c). EP affected a large number of proteins in the extracellular region, as well as in other cellular components, such as protein containing complexes, cell part, organelle (Fig. 4b). For EP + CsP, more proteins in membrane, cell part, organelle and macromolecular complexes assembly were upregulated. Molecular function analysis showed higher representation of proteins related to catalytic activity, binding, and structural molecule activity for both EP + CsP and EP (Fig. 4c). However, proteins in molecular function regulator, transcription regulator activity, and molecular transduction activity were uniquely affected for EP, and more proteins related to signal transduction, antioxidant activity, and receptor activity were uniquely affected for EP + CsP. A number of proteins involved in the translational activity were downregulated and more proteins involved in the transport activity were upregulated in the EP + CsP treatment compared to CsP and Ctrl treatments. www.nature.com/scientificreports www.nature.com/scientificreports/ Analysis of KEGG pathways for upregulated proteins (Fig. 5a) showed enrichment of OXPHOS, tricarboxylic acid (TCA) cycle, fatty acid metabolism, aromatic amino acid (tryptophan) metabolism, calcium and PPAR signaling in EP + CsP and EP as compared to the Ctrl or CsP. However, the number of upregulated proteins in these pathways were lower for EP compared to EP + CsP, indicating the additional effect of CsP with EP.
The lysosome, other glycan degradation, synthesis and degradation of ketone bodies, and ECM-receptor interaction pathways were upregulated only in EP, but N-Glycan biosynthesis, fatty acid metabolism, fatty acid elongation, glycerolipid metabolism, biosynthesis of unsaturated fatty acids, calcium signaling, and ribosome pathways were upregulated only in EP + CsP compared to the Ctrl or CsP (see Fig. S1a for more information). The expression of glutaminase (GLS) increased in EP + CsP and EP treatments compared to the Ctrl and CsP.
In contrast, pathways involved in cell proliferation, differentiation and migration were downregulated for both EP + CsP and EP (Fig. 5b). This included several signaling pathways, such as MAPK signaling, neurotrophin signaling, and VEGF signaling. The pathways, such as hippo signaling, and biosynthesis of amino acids were only enriched for downregulated proteins in EP + CsP from Ctrl or CsP. On the other hand, the pathways, such as, T cell receptor signaling, Toll-like receptor signaling, NOD-like receptor signaling, base excision repair, estrogen signaling, and pentose phosphate pathway were only enriched for proteins downregulated in EP from Ctrl or CsP.
The cell cycle pathway was downregulated only in EP + CsP, but base excision repair, the pathway involved in the DNA repair throughout the cell cycle was downregulated only in EP. This indicates that presence of CsP is required to impact the cell cycle, and EP alone only slows down the DNA repair process, which is also consistent with the anti-proliferative effect of CsP, which induces cell cycle arrest in cancer cells 35 . An important protein, proliferating cell nuclear antigen (PCNA), which is involved in the repair of DNA damage caused by CsP was significantly downregulated in EP + CsP and EP treatments compared to the CsP (EP + CsP vs CsP -↓2×, P = 0.0003; EP vs CsP -↓1.7×, P = 0.0071) and Ctrl (EP + CsP vs Ctrl -↓1.8×; P = 0.0001; EP vs Ctrl -↓1.5×, P = 0.012). The effect of EP + CsP were larger on PCNA compared to EP, and may help overcome the CsP resistance, as EP with CsP may increasingly stabilize CsP induced genomic DNA damage 36 .
Additionally, downregulated proteins included those in the regulation of actin cytoskeleton, focal adhesion, proteoglycans in cancer, purine metabolism, glycolysis/gluconeogenesis, amino acid biosynthesis, proteasomes, and ubiquitin mediated proteolysis. We observed downregulation of glycolysis pathway with 11 and 14 proteins in EP + CsP treatment compared to the Ctrl and CsP, respectively. For the EP treatment, while the glycolysis was not enriched for downregulated proteins from the Ctrl, but compared to the CsP, 12 downregulated proteins were enriched in glycolysis. For EP + CsP and EP treatments, the glucose metabolism proteins, notably www.nature.com/scientificreports www.nature.com/scientificreports/ fructose-bisphosphate aldolase A (ALDOA), alpha-enolase (ENO1), phosphoglycerate kinase 1 (PGK1), and lactate dehydrogenase isoforms A (LDHA) and B (LDHB) were all downregulated, compared to Ctrl and CsP, and acyl-CoA synthetase short-chain family member 2 (ACSS2), compared to CsP (Table S6). Figure 6(a-d) shows the list of top 10 upregulated and downregulated proteins and their expression levels (log2 fold change) in 4 different pairwise comparisons (CsP vs Ctrl; EP vs Ctrl, EP + CsP vs EP, EP + CsP vs CsP). The NipSnap homolog 2 (GBAS) was the most upregulated protein with a log2 fold change of 8.71 in CsP and catenin beta-1 (CTNNB1) was the most upregulated protein in EP with a log2 fold change of 10.42, compared to the Ctrl (Fig. 6a,b). Similarly, in the EP + CsP treatment glutamyl-tRNA (Gln) amidotransferase subunit C, mitochondrial (GATC) with a log2 fold change of 9.71, and titin (TTN) with a log2 fold change of 9.99 were the most upregulated proteins, compared to the EP and CsP treatment, respectively. (Fig. 6c,d).
The tubulin alpha-1A chain (TUBA1A) was the most downregulated protein with a log2 fold change of 10.99 in CsP and prefoldin subunit 1 (PFDN1) was the most downregulated protein with a log2 fold change of 9.07 in EP, compared to the Ctrl (Fig. 6a,b). Similarly, for the EP + CsP treatment, MHC class I antigen (HLA-C) with a log2 fold change of 9.27, and huntingtin-interacting protein (HYPK) with a log2 fold change of 9.20 were the most significantly downregulated proteins compared to the EP and CsP treatment, respectively (Fig. 6c,d).
Validation of proteomics results. To determine whether changes in the protein levels correspond to the changes at the transcript levels, we performed real time quantitative PCR (qPCR) experiments after 4 h of treatment for Ctrl, CsP and EP + CsP samples. Figure 7a shows mRNA level expressions of ENO1, LDHB, and GLS genes at 4 h of the treatments, and the comparison of the protein level changes observed at 4 h. The mRNA/protein levels were normalized with mRNA/protein expression levels of the Ctrl (level 1). The mRNA levels of ENO1 and LDHB genes decreased to 0.19 and 0.15 in EP + CsP as compared to the Ctrl, while they were 0.67 and 1.33 for CsP. In comparison, the protein expression of ENO1 and LDHB genes in EP + CsP decreased to 0.56 and 0.61 from the Ctrl, while they were 1.10 and 1.01 for CsP. Though we observed some minor up/down regulation in the mRNA levels of ENO1 and LDHB genes for CsP compared to Ctrl, no statistically significant difference was found, as also observed at the protein level. This validates our proteomics results and suggests that downregulation of ENO1 and LDHB is regulated at the transcript levels for EP + CsP. On the other hand, we observed ~3.5-fold increase in the mRNA level expression of GLS in EP + CsP and CsP, while it also increased by 2.08-fold from Ctrl and 1.8-fold from CsP, at the protein level. Overall, the mRNA level expressions of ENO1, LDHB and GLS correlate well with the protein level expressions.
We further validated the expression levels of GLS using the immunoblotting. Figure 7b shows the quantification of GLS expression for different treatments, normalized with β-tubulin and reported relative to the control. The representative immunoblots are shown in the inset. We observed that the GLS expression levels were ~1 for Ctrl and CsP treatments, not significantly different from each other. The EP + CsP treatment caused a 6-fold increase in the GLS expression levels from the control and CsP. This increase in GLS level in the immunoblotting correlates with the mRNA and protein level expressions for EP + CsP.
In addition, the reactive oxygen species (ROS) production levels in MDA-MB-231 cells were investigated following different treatments. Figure 7c shows the average luminescence (Lum) as relative light units (RLU), which www.nature.com/scientificreports www.nature.com/scientificreports/ The String Interaction analysis of the most significantly regulated proteins (|fold change| ≥ 2) but uniquely regulated in EP + CsP or EP, compared to CsP (e) Upregulated in EP + CsP vs CsP, (f) Upregulated in EP vs CsP, (g) Downregulated in EP + CsP vs CsP, (h) Downregulated in EP vs CsP. EP + CsP and EP protein expressions were compared to CsP and highly significantly regulated (|fold change| ≥ 2) proteins were identified: 339 in EP + CsP and 348 in EP. Out of these proteins, those commonly regulated in EP + CsP and EP were filtered out to obtain uniquely regulated proteins: 154 in EP + CsP and 164 in EP. These proteins were uploaded to STRING 76 to visualize the interaction and functional enrichment among these unique proteins with minimum required interaction score as medium confidence (0.4) and kmeans clustering (3 clusters). The nodes represent proteins, with color representing their localization. The colored nodes represent query proteins and first shell of interactions and white nodes represent second shell of interaction. Edges represent protein-protein www.nature.com/scientificreports www.nature.com/scientificreports/ directly corresponds to H 2 O 2 levels in MDA-MB-231 cells at 4 h. We observed a marginal increase in Lum to 3873 for CsP from 3852 in the Ctrl, suggesting that CsP alone did not cause a significant oxidative stress in TNBC cells at 4 h. The Lum was 4789 for EP and it was 5021 for EP + CsP, a 1.2 to 1.3-fold increase in H 2 O 2 levels compared to the cells treated with Ctrl and CsP. These results indicate that the EP + CsP and EP treatments increase ROS production in TNBC cells to cause oxidative stress inducing the cell death.
Cell death may take the form of apoptosis, a controlled and programmed cell death, or necrosis, a toxic and inflammatory process where the cell is a passive victim causing the expulsion of cellular constituents into the extracellular environment 37 . To understand the manner of cell death due to EP + CsP treatment, we stained the cells with Annexin V/7-Aminoactinomycin D (Annexin-V/7-AAD) and performed flowcytometry for quantitative analysis of live, early and late apoptosis, and necrotic cells for different treatment conditions (Fig. 7d). The EP and EP + CsP treatments reduced the fraction of live cells to 16 and 19% from 86% in Ctrl or CsP. The fraction of cells in the late apoptosis phase increased significantly to 69% from 6% in Ctrl and CsP. There was also an increase of necrotic cell population to 5% from 0.5% in the Ctrl and CsP. We observed that in EP + CsP, a total of 76% of cells were going through apoptosis, compared to only 5% undergoing necrosis. Similar behavior was also observed for the EP treated cells, where the apoptosis was prominent than necrosis with 73% of cells in late apoptosis, compared to 4% in necrosis. This indicates that EP + CsP and EP treatments induce apoptosis in majority of MDA-MB-231 cells, while causing necrosis in minority cell population to cause cell death. Thus, EP with and without CsP increases the production of ROS to cause oxidative stress and induces the apoptosis.

Discussion
The EP + CsP treatment altered the proteome landscape of MDA-MB-231 cells, compared to control, CsP, and EP only treatments. These cells are relatively resistant to commonly used chemotherapeutics (Cisplatin, Paclitaxel, and Doxorubicin) 38 , and are a standard in vitro model of highly invasive TNBC. The EP + CsP treatment caused effective cell death, while affecting cellular metabolism. The EP alone also compromised cell viability, and metabolic activity in these cells. However the cell viability recovered for EP only treatment. With EP + CsP treatment, the effects were long-lasting, and the cells remained metabolically inactive, indicating the superiority of the EP + CsP treatment. We also showed that the effects of EP + CsP are specific to TNBC cells as they do not affect non-tumorigenic mammary epithelial MCF10A cells, as much.
The proteomics results showed that EP + CsP affected not only the membrane proteins, but also proteins in multiple components, biological functions and processes demonstrating a cell-wide effect. The most prominent shift in cellular metabolism was observed for glycolysis, TCA cycle and OXPHOS. Glucose metabolism has been shown to influence proliferation and differentiation of cancer cells 39 . We observed downregulation of key glycolysis pathway proteins but upregulation of TCA cycle. Similar effects were also observed for EP. Eleven and fourteen of the 28 glycolytic enzymes identified in this study were downregulated in EP + CsP compared to Ctrl and CsP treatments. However, only twelve glycolytic enzymes were downregulated for EP compared to CsP and there was no enrichment of the glycolytic pathway compared to control.
The various key proteins of glucose metabolism that were downregulated include ACSS2, ALDOA, ENO1, PGK1, and LDH isoforms-LDHA and LDHB, for EP + CsP and EP. The expression of ACSS2 was reported to be inversely correlated with survival in a cohort of 154 cases of TNBC, indicating that it may be an effective anticancer target 40 . The inhibition of ALDOA was shown to break the feed-forward loop of glycolysis to inhibit the proliferation of cancer cells 41 . The ENO1 is a potential biomarker of TNBC, and its function in glycolysis is consistent with the distinct TNBC metabolism compared to other breast cancer subtypes 42,43 . The PGK1 is elevated in breast cancer tissues compared to normal tissues, and it's expression is dependent on the oxygen tension 44 . It has been shown to affect DNA replication and is found to be overexpressed in CsP-resistant ovarian cancer 45 . The LDH enzyme subunits A and B can catalyze the forward and backward conversion of pyruvate to lactate. The LDHA is an indicator of the breast cancer malignancy degree and its inhibition is shown to prevent TNBC brain metastasis 46 . The LDHB is shown to be specifically upregulated in basal-like/TNBC cell lines and tumors compared to luminal subtypes, and its expression results in a more comprehensive shift in TNBC tumor metabolism 47 . The increased expression of LDHB correlates to significantly poor clinical outcomes and is an essential gene for TNBC proliferation and survival in vitro and in vivo 47,48 . Recent reports suggest a novel, alternate hypothesis on metabolism in tumor cells, where hypoxic and glucose-deprived cells depend upon LDHB to utilize lactate secreted from the adjacent cells, which are undergoing aerobic glycolysis, in a phenomenon called "reverse Warburg effect" 47,49 . LDHB in tumor cells utilize lactate as energy source by converting it to pyruvate 50,51 , which feeds into the TCA cycle, indicating that the ECT of cells may also inhibit the reverse Warburg effect by downregulating LDHB expression. Considering this, the TNBC cells, which are distinctly wired for the dependence on glucose metabolism, are highly susceptible for its intervention 47 .
The ENO1 and LDHB mRNAs were downregulated in EP + CsP suggesting that the key glycolytic enzymes are regulated at the transcription level, which translates to proportional changes observed at the protein level. Increased expression in GLS at the mRNA level and protein level also appears to be consistent with the metabolic shift observed in this study. GLS converts glutamine to glutamate to generate α-ketoglutarate (αKG), which serves as a fuel to the TCA cycle.
While glycolysis was down, we observed upregulation of TCA cycle and OXPHOS pathway, indicating a switch in the metabolism from glycolysis to cellular respiration, with a larger dependency on oxidative energy associations. EP + CsP upregulated more proteins in the mitochondrion, but EP upregulated more proteins in the endoplasmic reticulum. In the downregulated proteins, EP + CsP affected more proteins in the cytosol, while EP affected a large number of proteins in the nucleus.
Significant difference between EP + CsP treated cells and Ctrl cells is indicated as **P < 0.005, ****P < 0.00005. Error bars are calculated using standard error. www.nature.com/scientificreports www.nature.com/scientificreports/ substrates for energy production for TNBC cells treated with EP + CsP and EP. The increased OXPHOS could also be attributed to the stress response as the cells try to generate ATP through alternate sources when the glycolysis is down. We observed an upregulation in several pathways, such as fatty acid degradation, fatty acid metabolism, fatty acid elongation, tryptophan metabolism, amino acid metabolism, glyoxylate and dicarboxylate metabolism which can produce pyruvate and TCA cycle intermediates like acetyl COA and oxaloacetate to replenish the TCA cycle. The increase in mitochondrial metabolic activities (OXPHOS and TCA Cycle) in response to the EP may enhance CsP induced cytotoxicity, as observed for the EP + CsP 52 .
Moreover, we observed an upregulation of peroxisome proliferator-activated receptor (PPAR) pathway proteins. The expression of long chain fatty acid transport protein 4 (SLC27A4/FATP4), which transports long-chain fatty acid through the membrane was upregulated for EP + CsP, compared to CsP. This indicates that EP application may increase cellular transport, not only through the membrane pore formation, but also via the upregulation of key transport proteins. The role of PPAR pathway is not only limited to the transport of the fatty acid, but it can also promote fatty acid degradation and fatty acid metabolism, which in turn can promote acetyl COA synthesis to replenish the TCA cycle.
The upregulation in the peroxisome pathway may also be linked to the TCA cycle, as it involves breaking down of the fatty acid to fuel the TCA cycle and to generate the cell membrane. Since, electroporation is a membrane phenomenon, the increased activity of the peroxisome pathway can also be towards the efforts to repair or regenerate the membrane following ECT. In addition, peroxisome pathway is involved to generate H 2 O 2 and to activate apoptosis 53 , correlating our results with previous studies 54,55 .
We also observed an upregulation of the calcium signaling pathway proteins only in EP + CsP treated cells from CsP, including the expression of voltage dependent anion channel proteins 1, 2, and 3 (VDCA1, VDCA2, VDCA3) and stromal interaction molecule 1 (STIM1). The VDCA channel proteins facilitate the diffusion of metabolites, ions (including calcium ions) and small hydrophilic molecules across outer mitochondrial membrane and regulate metabolism and mitochondria functions 56 . Also, VDAC1 expression increases with the increase in intracellular calcium concentration in response to apoptosis inducing agents 57 . STIM1 also works as a calcium sensor and is identified to be involved in calcium influx into the cells from the extracellular environment 58 . These proteins were specially affected for the EP + CsP, not for EP, indicating that ECT with cisplatin can facilitate the calcium transport into the cells, which is consistent with earlier finding, where they also used 100 µs pulses 59 . Calcium release from endoplasmic reticulum (ER) also causes the redistribution of the ER calcium sensor STIM1 60 . This links very well with the observed significant upregulation in protein processing in the ER pathway, which happens in response to the ER stress. It is previously shown that under stimulation, several unfolded and incompletely folded proteins accumulate in the ER lumen to trigger unfolded protein response (UPR), leading to ER stress and apoptosis 61 . This indicates that in the cells treated with EP + CsP, the increased intracellular calcium concentration due to the calcium influx from extracellular environment and its release from ER, can trigger apoptosis, under conditions with high ROS production 62 . This also correlate very well with the previous observations, where microsecond electrical pulses released calcium from the ER and other internal organelles 63 .
To confirm these observations, we further evaluated the H 2 O 2 levels and the apoptosis profile. We showed that the ROS production increases significantly in response to the EP + CsP and EP treatments, which may cause the oxidative stress in these cells to induce apoptosis. This correlates very well with our proteomics results as well as those reported previously by other researchers, who have shown that EP application can generate ROS, which can activate the apoptosis signaling pathways in cells 54,55,64,65 .
However, cancer cells harbor inherent resistance to ROS induced apoptosis 66 , which can significantly compromise the effectiveness of this EP-based therapy. Compared to EP, EP + CsP downregulated SLC1A5, which may sensitize MDA-MB-231 to ROS induced apoptosis 66 , and lead to a sustained effect of EP + CsP, as observed in this study. The RFC4, which promotes DNA double stranded break repair to cause resistance 67 , was also downregulated for EP + CsP from EP, indicating that DNA repair is slower in EP + CsP treated cells. In addition, the expression of G3BP2, which is implicated in the maintenance of breast tumor-initiating cells (TICs) 68 , was downregulated for EP + CsP, compared to EP. These results highlight the differences in the protein expression profiles for EP + CsP and EP treatments, and suggest that the effects of EP + CsP treatment were more comprehensive, compared to EP.
For treatment, cells were detached using trypsin, were centrifuged at 1000 rpm for 5 min at 4 °C and were resuspended in fresh media at 1 × 10 6 cells/mL. www.nature.com/scientificreports www.nature.com/scientificreports/ Drug. Cisplatin's (Sigma-Aldrich) stock solution was prepared by dissolving it in sterile double-distilled water.
The required volume from Cisplatin stock was added into cells to make 100 μM Cisplatin treatment concentration. electrical pulse application. Electroporation was performed using the BTX-ECM830 electroporator (Genetronics Inc., USA). Eight, unipolar, square-wave pulses of 1 Hz frequency at 1200 V/cm with 100 μs pulse duration were applied to 600 μL cells, suspended at 1 × 10 6 cells/mL in fresh-media with Cisplatin in BTX electroporation cuvettes (4 mm gap). Control and Cisplatin treatment did not receive any electrical pulse. Following treatment, cells were transferred to 6-well plates (600,000 cells/well) containing 2 mL of fresh-media and were cultured for 4 h for proteomics, flow cytometry, RT-qPCR, and immunoblotting experiments. flowcytometric assessment of cell viability and apoptosis. Following  immunoblotting. Following 4 h treatment, all cells were scraped, washed thrice with ice-cold 1 × PBS with centrifugation at 1,500 rpm at 4 °C, lysed using the RIPA buffer and sonication, as previously 69 . Lysates were cleared of debris with centrifugation at 14000 rpm. The Pierce ® BCA Assay (Thermo Fisher Scientific, USA) was used to estimate the protein concentration. The 5 × sample-buffer was added into protein (20 μg) from sample and heated to denature the protein. SDS-polyacrylamide gel electrophoresis was performed to separate the denatured proteins and were transferred onto a polyvinyl difluoride (PVDF) membrane for antibody staining and protein detection. The PVDF membrane was incubated overnight with 5% (w/v) nonfat dry milk at 4 °C for blocking. The blocked membrane was probed for proteins using primary antibodies for GLS (rabbit, PA5-35365; Thermo Fisher Scientific) and β-tubulin (mouse, E7; DSHB, University of Iowa). The proteins were detected using the appropriate Alexa Fluor ® secondary antibody (anti-rabbit, A-21109 or anti-mouse, A11375; Thermo Fisher Scientific). The blots were imaged at 680 nm for GLS and 790 nm for β-tubulin using the Li-Cor Odyssey infrared imaging System and were quantified using ImageJ.
Sample preparation for mass spectrometry analysis. Following 4 h treatment, all cells were scraped, washed thrice in ice-cold 1 × PBS with centrifugation at 1,000 rpm at 4 °C, and were re-suspended in 4 M urea.
Sample preparation was done, as previously 70  www.nature.com/scientificreports www.nature.com/scientificreports/ USA). Peptides elution was performed using 0.1% formic acid (FA) in 80% acetonitrile (ACN). Eluted peptides were vacuum dried and were re-suspended in 0.1% FA in 3% ACN. BCA assay was used to estimate peptide concentration and the concentration was adjusted to 0.5 µg/µL.
After 5 min, trap column was switched in-line with Acclaim ™ PepMap ™ RSLC C18 (75 µm × 15 cm, 3 μm 100 Å PepMap C18 medium, Thermo Fisher Scientific) analytical column and peptide were separated using 120 min LC gradient method at 35 °C. A 5-30% linear gradient of solvent B was run for 80 min, followed by 11 min of 45% solvent B and 2 min of 100% solvent B with an additional 7 min of isocratic flow. Solvent A was then applied at 95% for 20 min for column equilibration. A Top20 data-dependent MS/MS scan method was used to acquire MS data. Injection time was set to 100 milliseconds, resolution to 120,000 at 200 m/z, spray voltage of 2 eV and an AGC target of 1 × 10 6 for a full MS spectra scan with a range of 400-1650 m/z. High-energy C-trap dissociation was used to fragment precursor ions at a normalized collision energy of 27 eV. The MS/MS scans were acquired at a resolution of 15,000 at 200 m/z. The dynamic exclusion was set at 30 sec to circumvent repeated scanning of identical peptides. Data analysis. MaxQuant (v1.6.1.0) 71,72 was used to process MS/MS data against the Uniprot Homo sapiens fasta (http://www.uniprot.org; 26,446 entries as of October 05, 2018) concatenated with a common contaminants and a reverse-decoy database, as previously 73 . Cleavage enzymes were setup as Trypsin/P and LysC, with toleration of up to 2 missed cleavages. Mass error was set to 10 ppm and 20 ppm for precursor and fragment ions, respectively. Cysteine alkylation and methionine oxidation was set to fixed and variable modifications, respectively. The 0.01 was estimated false discovery rate threshold for both the peptides and the proteins levels. The peptide quantitation was conducted using "unique plus razor peptides". The non-unique peptides assigned to protein/protein group with most other peptides were razor peptides. Following MaxQuant processing, proteins with no MS/MS count and zero LFQ intensity were removed. The results were transferred to Data Analysis and Extension Tool (DAnTE) for Pearson correlations analysis. Proteins with LFQ≠0 and MS/MS ≥2 in at least two replicates for ≥1 of the treatments were retained for further analyses using a MATLAB script. Zero LFQ values were imputed with 281660, half of the lowest LFQ value (563320) observed across three treatments. LFQ intensities were log2 transformed and average was calculated for three replicates. The fold-change was calculated by subtracting the average log2 values [Δlog2 (LFQ intensity)] between proteins from each comparison group (CsP vs Ctrl; EP vs Ctrl; EP+CsP vs Ctrl; EP+CsP vs EP; EP vs CsP; EP+CsP vs CsP). Proteins with fold-change of |Δlog2| > 0.5, and P < 0.05 (Student's unpaired, two-tailed, t test) were significantly regulated. enrichment and string interaction analysis. Significantly regulated proteins were compared against the background of total 2867 quantified proteins to discover the pathway enrichment using KEGG database in DAVID 6.8 74 . Cellular localization and molecular functions of differentially expressed proteins was performed using PANTHER Classification System 75 against Homo sapiens database. STRING 76 was used to visualize the interaction and functional enrichment with minimum required interaction score as medium confidence (0.4) and kmeans clustering (3 clusters).
Statistical analysis. Repeated Measure ANOVA was used to derive statistical significance for cell metabolic activity, coupled with Tukey's multiple comparison test. MDA-MB-231 metabolic activity data was log transformed prior to statistical analysis to satisfy normality and homoscedasticity assumptions. Tukey's test tags each treatment with a letter or a group of letters to indicate their significance. The same letter or the same groups of letters indicate that they are not significantly different. The different letters or different group of letters indicate that they are significantly different (P < 0.05).
Student's unpaired, two-tailed, t-test was used for testing significance for log2 transformed proteomics data, and all other experimental data.
All experiments were performed in triplicates.

Data Availability
RAW proteomics data files, parameters used, and LC-MS/MS methodology and statistics have been deposited in the MassIVE public proteomics data repository (MassIVE ID: MSV000083360). The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.