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

Abstract

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 4-6. 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 ...

High-throughput, Label-Free Quantitative Proteomic Studies of the Anticancer Effects of Electrical Pulses with Turmeric Silver Nanoparticles: an in vitro Model Study

“…The peroxisome pathway, which involves breaking down the FA for the membrane regeneration can also generate H 2 O 2 to trigger apoptosis 56 . We also measured the H 2 O 2 levels, validating that TurNP + EP treatment causes the oxidative stress in these cells to cause cell death, correlating well with previous results 20,55 .…”

“…The increased OXPHOS could also be a stress response as cells try to generate energy through alternate sources upon downregulation of glycolysis. Increased OXPHOS activity can also generate ROS to activate cell death pathways, as we showed previously for EP application with cisplatin and curcumin in MDA-MB-231 cells 20,55 . The peroxisome pathway, which involves breaking down the FA for the membrane regeneration can also generate H 2 O 2 to trigger apoptosis 56 .…”

“…Eight, 600-1200 V/cm, 100 μs unipolar, square wave pulses at 1 Hz repetition rate were applied using a BTX-ECM830 electroporator (Genetronics Inc., USA). For this, 600 μL cell suspension (1 × 10 6 cells/mL) with or without TurNP in BTX electroporation cuvettes (4 mm gap) was used, as previously 20,55 . No electrical pulses were applied to Ctrl and TurNP.…”

“…Viability assay. Following treatment, cells were transferred to 96-well plates (MDA-MB-231: 20,000 cells and MCF 10 A: 10,000 cells (as suggested by manufacturer's protocol)) with fresh-media, as previously 20,55 . The cells were incubated for 12 h to assess the metabolic activity using RealTime-Glo MT Cell Viability Assay (Promega, USA), as per manufacturer's protocol.…”

“…Lum value of sample Lum value of control 100 (1) Hydrogen peroxide assessment. MDA-MB-231 cells were transferred to 96-well plates (20,000 cells/well) after treatment, with 60 μL fresh-media and were incubated for 12 h, as described previously 20,55 . At 7 h, 20 μL of H 2 O 2 substrate solution from ROS-Glo H 2 O 2 assay kit (Promega) was added to cells and incubated for 5 h more.…”