Mass spectrometry-based proteomic analysis to characterize cisplatin induced early signaling events in head and neck squamous cell carcinoma.

Cisplatin is the commonly used chemotherapeutic drug in treatment of various cancers. However, development of resistance towards cisplatin results in tumor recurrence. Here, we aim to understand the mechanisms of action of cisplatin and emergence of resistance to cisplatin using mass spectrometry-based proteomic approach. A panel of head and neck squamous cell carcinoma (HNSCC) cell lines were treated with cisplatin at respective IC50 for 24 h and label-free mass spectrometry analysis was carried out. Proteomic analysis of A253, FaDu, Det562 and CAL27 cell lines upon cisplatin treatment resulted in the identification of 5,060, 4,816, 4,537 and 4,142 proteins, respectively. Bioinformatics analysis of differentially regulated proteins revealed proteins implicated in DNA damage bypass pathway, translation and mRNA splicing to be enriched. Further, proteins associated with cisplatin resistance exhibited alterations following short-term cisplatin exposure. Among these, class III tubullin protein (TUBB3) was found to be upregulated in cisplatin-treated cells compared to untreated cells. Western blot analysis confirmed the elevated expression of TUBB3 in cells treated with cisplatin for 24 h, and also in cisplatin resistant HNSCC cell lines. This study delineates the early signaling events that enable HNSCC cells to counteract the cytotoxic effects of cisplatin and facilitate the development of resistance.

Exploration of Nitrotyrosine-Containing Proteins and Peptides by Antibody-Based Enrichment Strategies.

Nitrotyrosine, or 3-nitrotyrosine, is an oxidative post-translational modification induced by reactive nitrogen species. Although nitrotyrosine is considered a marker of oxidative stress and has been associated with inflammation, neurodegeneration, cardiovascular disease, and cancer, identification of nitrotyrosine-modified proteins remains challenging owing to its low stoichiometric levels in biological samples. To facilitate a comprehensive analysis of proteins and peptides containing nitrotyrosine, we optimized an immunoprecipitation-based enrichment workflow using a cell line model. The identification of proteins and peptides containing nitrotyrosine residues was carried out after peroxynitrite treatment of cell lysates, which generated modified nitrotyrosine residues on susceptible sites on proteins. We evaluated the efficacy of enriching nitrotyrosine-modified proteins and peptides by employing four different commercially available monoclonal antibodies directed against nitrotyrosine. LC-MS/MS analysis resulted in the identification of 1377 and 1624 nitrotyrosine-containing peptides from protein- and peptide-based enrichment experiments, respectively. Although the yield of nitrotyrosine-containing peptides was higher in experiments where peptides rather than proteins were enriched, we found a substantial proportion (37-65%) of identified nitrotyrosine-containing peptides contained nitrotyrosine at the N-terminus. However, in protein-based immunoprecipitation <9% of nitrotyrosine-containing peptides had nitrotyrosine modification at the N-terminus of the peptide. Overall, our study resulted in the identification of 2603 nitrotyrosine-containing peptides of which >2000 have not previously been reported. We synthesized 101 novel nitrotyrosine-containing peptides identified in our analysis and analyzed them by LC-MS/MS to validate our findings. We have confirmed the validity of 70% of these peptides, as they demonstrated a similarity score exceeding 0.7 when compared to peptides identified through experimental methods. Finally, we also validated the presence of nitrotyrosine modification on PKM and EF2 proteins in peroxynitrite-treated samples by immunoblot analysis. The large catalog presented in this study along with the workflow should facilitate the investigation of nitrotyrosine as an oxidative modification in a variety of settings in greater detail.

Automated Sample Preparation Workflow for Tandem Mass Tag-Based Proteomics.

Although tandem mass tag (TMT)-based isobaric labeling has become a powerful approach for multiplexed protein quantitation, automating the workflow for this technique has not been easy to achieve for widespread adoption. This is because preparation of TMT-labeled peptide samples involves multiple steps ranging from protein extraction, denaturation, reduction, and alkylation to tryptic digestion, desalting, labeling, and cleanup, all of which require a high level of proficiency. The variability resulting from multiple processing steps is inherently problematic, especially with large-scale clinical studies that involve hundreds of samples where reproducibility is critical for quantitation. Here, we sought to compare the performance of a recently introduced platform, AccelerOme, for an automated proteomic workflow employing TMT labeling with the manual processing of samples. Cell pellets were prepared and subjected to a 16-plex experiment using an automated platform and a conventional manual protocol. Single-shot liquid chromatography with tandem mass spectrometry analysis revealed a higher number of proteins and peptides identified using the automated platform. Efficiency of tryptic digestion, alkylation, and TMT labeling were similar in both manual and automated processes. In addition, comparison of quantitation accuracy and precision showed similar performance in an automated workflow compared to manual sample preparation by an expert. Overall, we demonstrated that the automated platform performs at a level similar to a manual process performed by an expert for TMT-based proteomics. We anticipate that this automated workflow will increasingly replace manual pipelines and has the potential to be applied to large-scale TMT-based studies, providing robust results and high sample throughput.

Urinary Proteomics for Discovery of Gastric Cancer Biomarkers to Enable Precision Clinical Oncology.

For precision in clinical oncology practice, detection of tumor-derived peptides and proteins in urine offers an attractive and noninvasive alternative for diagnostic or screening purposes. In this study, we report comparative quantitative proteomic profiling of urine samples from patients with gastric cancer and healthy controls using tandem mass tags-based multiplexed mass spectrometry approach. We identified 1504 proteins, of which 246 were differentially expressed in gastric cancer cases. Notably, ephrin A1 (EFNA1), pepsinogen A3 (PGA3), sortilin 1 (SORT1), and vitronectin (VTN) were among the upregulated proteins, which are known to play crucial roles in the progression of gastric cancer. We also found other overexpressed proteins, including shisa family member 5 (SHISA5), mucin like 1 (MUCL1), and leukocyte cell derived chemotaxin 2 (LECT2), which had not previously been linked to gastric cancer. Using a novel approach for targeted proteomics, SureQuant, we validated changes in abundance of a subset of proteins discovered in this study. We confirmed the overexpression of vitronectin and sortilin 1 in an independent set of urine samples. Altogether, this study provides molecular candidates for biomarker development in gastric cancer, and the findings also support the promise of urinary proteomics for noninvasive diagnostics and personalized/precision medicine in the oncology clinic.

Optimizing single cell proteomics using trapped ion mobility spectrometry for label-free experiments.

Although single cell RNA-seq has had a tremendous impact on biological research, a corresponding technology for unbiased mass spectrometric analysis of single cells has only recently become available. Significant technological breakthroughs including miniaturized sample handling have enabled proteome profiling of single cells. Furthermore, trapped ion mobility spectrometry (TIMS) in combination with parallel accumulation-serial fragmentation operated in data-dependent acquisition mode (DDA-PASEF) allowed improved proteome coverage from low-input samples. It has been demonstrated that modulating the ion flux in TIMS affects the overall performance of proteome profiling. However, the effect of TIMS settings on the analysis of low-input samples has been less investigated. Thus, we sought to optimize the conditions of TIMS with regard to ion accumulation/ramp times and ion mobility range for low-input samples. We observed that an ion accumulation time of 180 ms and monitoring a narrower ion mobility range from 0.7 to 1.3 V s cm-2 resulted in a substantial gain in the depth of proteome coverage and in detecting proteins with low abundance. We used these optimized conditions for proteome profiling of sorted human primary T cells, which yielded an average of 365, 804, 1116, and 1651 proteins from single, five, ten, and forty T cells, respectively. Notably, we demonstrated that the depth of proteome coverage from a low number of cells was sufficient to delineate several essential metabolic pathways and the T cell receptor signaling pathway. Finally, we showed the feasibility of detecting post-translational modifications including phosphorylation and acetylation from single cells. We believe that such an approach could be applied to label-free analysis of single cells obtained from clinically relevant samples.

A High-Throughput Workflow for FFPE Tissue Proteomics.

Laser capture microdissection (LCM) has become an indispensable tool for mass spectrometry-based proteomic analysis of specific regions obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples in both clinical and research settings. Low protein yields from LCM samples along with laborious sample processing steps present challenges for proteomic analysis without sacrificing protein and peptide recovery. Automation of sample preparation workflows is still under development, especially for samples such as laser-capture microdissected tissues. Here, we present a simplified and rapid workflow using adaptive focused acoustics (AFA) technology for sample processing for high-throughput FFPE-based proteomics. We evaluated three different workflows: standard extraction method followed by overnight trypsin digestion, AFA-assisted extraction and overnight trypsin digestion, and AFA-assisted extraction simultaneously performed with trypsin digestion. The use of AFA-based ultrasonication enables automated sample processing for high-throughput proteomic analysis of LCM-FFPE tissues in 96-well and 384-well formats. Further, accelerated trypsin digestion combined with AFA dramatically reduced the overall processing times. LC-MS/MS analysis revealed a slightly higher number of protein and peptide identifications in AFA accelerated workflows compared to standard and AFA overnight workflows. Further, we did not observe any difference in the proportion of peptides identified with missed cleavages or deamidated peptides across the three different workflows. Overall, our results demonstrate that the workflow described in this study enables rapid and high-throughput sample processing with greatly reduced sample handling, which is amenable to automation.

Four-dimensional proteomics analysis of human cerebrospinal fluid with trapped ion mobility spectrometry using PASEF.

A quadrupole time-of-flight mass spectrometer coupled with a trapped ion mobility spectrometry (timsTOF) operated in parallel accumulation-serial fragmentation (PASEF) mode has recently emerged as a platform capable of providing four-dimensional (4D) features comprising of elution time, collision cross section (CCS), mass-to-charge ratio, and intensity of peptides. The PASEF mode provides ∼100% ion sampling efficiency both in data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes without sacrificing sensitivity. In addition, targeted measurements using PASEF integrated parallel reaction monitoring (PRM) mode have also been described. However, only limited number of studies have used timsTOF for analysis of clinical samples. Although Orbitrap mass spectrometers have been used for biomarker discovery from cerebrospinal fluid (CSF) in a variety of neurological diseases, these Orbitrap-derived datasets cannot readily be applied for driving experiments on timsTOF mass spectrometers. We generated a catalog of peptides and proteins in human CSF in DDA mode on a timsTOF mass spectrometer and used these data to build a spectral library. This strategy allowed us to use elution times and ion mobility values from the spectral library to design PRM experiments for quantifying previously discovered biomarkers from CSF samples in Alzheimer's disease. When the same samples were analyzed using a DIA approach combined with a spectral library search, a higher number of proteins were identified than in a library-free approach. Overall, we have established a spectral library of CSF as a resource and demonstrated its utility for PRM and DIA studies, which should facilitate studies of neurological disorders.

Regional heterogeneity in mitochondrial function underlies region specific vulnerability in human brain ageing: Implications for neurodegeneration.

Selective neuronal vulnerability (SNV) of specific neuroanatomical regions such as frontal cortex (FC) and hippocampus (HC) is characteristic of age-associated neurodegenerative diseases (NDDs), although its pathogenetic basis remains unresolved. We hypothesized that physiological differences in mitochondrial function in neuroanatomical regions could contribute to SNV. To investigate this, we evaluated mitochondrial function in human brains (age range:1-90 y) in FC, striatum (ST), HC, cerebellum (CB) and medulla oblongata (MD), using enzyme assays and quantitative proteomics. Striking differences were noted in resistant regions- MD and CB compared to the vulnerable regions- FC, HC and ST. At younger age (25 ± 5 y), higher activity of electron transport chain enzymes and upregulation of metabolic and antioxidant proteins were noted in MD compared to FC and HC, that was sustained with increasing age (≥65 y). In contrast, the expression of synaptic proteins was higher in FC, HC and ST (vs. MD). In line with this, quantitative phospho-proteomics revealed activation of upstream regulators (ERS, PPARα) of mitochondrial metabolism and inhibition of synaptic pathways in MD. Microtubule Associated Protein Tau (MAPT) showed overexpression in FC, HC and ST both in young and older age (vs. MD). MAPT hyperphosphorylation and the activation of its kinases were noted in FC and HC with age. Our study demonstrates that regional heterogeneity in mitochondrial and other cellular functions contribute to SNV and protect regions such as MD, while rendering FC and HC vulnerable to NDDs. The findings also support the "last in, first out" hypothesis of ageing, wherein regions such as FC, that are the most recent to develop phylogenetically and ontogenetically, are the first to be affected in ageing and NDDs.

An insight in proteome profiling of Tuta absoluta larvae after entomopathogenic fungal infection.

Tuta absoluta (L.) (Lepidoptera: Gelechiidae), a major pest of solanaceous plant species, causes serious losses in the agriculture sector around the globe. For better pest management, entomopathogenic fungi such as Beauveria bassiana and Purpureocillium lilacinum, play an efficient role in suppressing the pest population. The present study was carried out to analyse the effects post fungal infections through proteome profiling using an Orbitrap Fusion Tribrid mass spectrometer. A total of 2,201 proteins were identified from the fourth instar larvae of T. absoluta, of which 442 and 423 proteins were significantly dysregulated upon infection with P. lilacinum and B. bassiana respectively. The potential proteins related to immune systems as well as detoxification processes showed significant alterations after post fungal infection. Studies on T. absoluta proteomics and genomics as well as the consequences of entomopathogenic fungal infection on the immune response of this insect could provide an initial framework for exploring more fungus-host interactions for the development of better strategies for integrated pest management.

Age-Associated Molecular Changes in Human Hippocampus Subfields as Determined by Quantitative Proteomics.

The hippocampus demonstrates age-associated changes in functions, neuronal circuitry, and plasticity during various developmental stages. On the contrary, there is a significant knowledge gap on age-associated proteomic alterations in the hippocampus subfields. Using tandem mass tag-based high-resolution mass spectrometry and quantitative proteomics, we report here age-associated changes in the human hippocampus at the subregional level. We used formalin-fixed paraffin-embedded hippocampal tissue sections from a total of 12 healthy individuals, with 3 individuals from each of the 4 different age groups, specifically, 1-10, 21-30, 31-40, and 81-90 years. We found that lysosome and oxidative phosphorylation were the pathways enriched in the 81- to 90-year age group. On the contrray, nervous system development, synaptic plasticity and transmission, messenger RNA (mRNA) splicing, and electron transport chain (ETC) complex-I activity were the enriched biological processes observed in the younger age groups. In a hippocampus subfield context, our topline findings on age-associated proteome changes include altered expression of proteins associated with adult neurogenesis with age in the dentate gyrus and increased expression of immune response-associated proteins with age in certain cornu ammonis sectors of the hippocampus. Signal peptide analysis predicted hippocampal proteins with secretory potential. While these new findings warrant replication in larger study samples, the current data contribute to (1) our understanding of the molecular basis of proteomic changes across various age groups in hippocampus subfields in healthy individuals, and (2) the design and interpretation of future research on the age-associated neurodegenerative disorders.

Cerebrospinal fluid lipidomics for biomarkers of Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia and is associated with serious neurologic sequelae resulting from neurodegenerative changes. Identification of markers of early-stage AD could be important for designing strategies to arrest the progression of the disease. The brain is rich in lipids because they are crucial for signal transduction and anchoring of membrane proteins. Cerebrospinal fluid (CSF) is an excellent specimen for studying the metabolism of lipids in AD because it can reflect changes occurring in the brain. We aimed to identify CSF lipidomic alterations associated with AD, using untargeted lipidomics, carried out in positive and negative ion modes. We found CSF lipids that were significantly altered in AD cases. In addition, comparison of CSF lipid profiles between persons with mild cognitive impairment (MCI) and AD showed a strong positive correlation between the lipidomes of the MCI and AD groups. The novel lipid biomarkers identified in this study are excellent candidates for validation in a larger set of patient samples and as predictive biomarkers of AD through future longitudinal studies. Once validated, the lipid biomarkers could lead to early detection, disease monitoring and the ability to measure the efficacy of potential therapeutic interventions in AD.

Whole-Exome Sequencing Analysis of Oral Squamous Cell Carcinoma Delineated by Tobacco Usage Habits.

Oral squamous cell carcinoma (OSCC) is a common cancer of the oral cavity in India. Cigarette smoking and chewing tobacco are known risk factors associated with OSCC. However, genomic alterations in OSCC with varied tobacco consumption history are not well-characterized. In this study, we carried out whole-exome sequencing to characterize the mutational landscape of OSCC tumors from subjects with different tobacco consumption habits. We identified several frequently mutated genes, including TP53, NOTCH1, CASP8, RYR2, LRP2, CDKN2A, and ATM. TP53 and HRAS exhibited mutually exclusive mutation patterns. We identified recurrent amplifications in the 1q31, 7q35, 14q11, 22q11, and 22q13 regions and observed amplification of EGFR in 25% of samples with tobacco consumption history. We observed genomic alterations in several genes associated with PTK6 signaling. We observed alterations in clinically actionable targets including ERBB4, HRAS, EGFR, NOTCH1, NOTCH4, and NOTCH3. We observed enrichment of signature 29 in 40% of OSCC samples from tobacco chewers. Signature 15 associated with defective DNA mismatch repair was enriched in 80% of OSCC samples. NOTCH1 was mutated in 36% of samples and harbored truncating as well as missense variants. We observed copy number alterations in 67% of OSCC samples. Several genes associated with non-receptor tyrosine kinase signaling were affected in OSCC. These molecules can serve as potential candidates for therapeutic targeting in OSCC.

Proteomic and phosphoproteomic profiling of shammah induced signaling in oral keratinocytes.

Shammah is a smokeless tobacco product often mixed with lime, ash, black pepper and flavorings. Exposure to shammah has been linked with dental diseases and oral squamous cell carcinoma. There is limited literature on the prevalence of shammah and its role in pathobiology of oral cancer. In this study, we developed a cellular model to understand the effect of chronic shammah exposure on oral keratinocytes. Chronic exposure to shammah resulted in increased proliferation and invasiveness of non-transformed oral keratinocytes. Quantitative proteomics of shammah treated cells compared to untreated cells led to quantification of 4712 proteins of which 402 were found to be significantly altered. In addition, phosphoproteomics analysis of shammah treated cells compared to untreated revealed hyperphosphorylation of 36 proteins and hypophosphorylation of 83 proteins (twofold, p-value ≤ 0.05). Bioinformatics analysis of significantly altered proteins showed enrichment of proteins involved in extracellular matrix interactions, necroptosis and peroxisome mediated fatty acid oxidation. Kinase-Substrate Enrichment Analysis showed significant increase in activity of kinases such as ROCK1, RAF1, PRKCE and HIPK2 in shammah treated cells. These results provide better understanding of how shammah transforms non-neoplastic cells and warrants additional studies that may assist in improved early diagnosis and treatment of shammah induced oral cancer.

Proteomic Alterations Associated with Oral Cancer Patients with Tobacco Using Habits.

Tobacco abuse is a major risk factor associated with the development of oral squamous cell carcinoma. Differences in molecular aberrations induced by tobacco exposure by chewing or smoking form are not well studied in case of oral cancer. We used tandem mass tag-based quantitative proteomic approach to delineate proteomic alterations in oral cancer patients based on their history of tobacco using habits (patients who chewed tobacco, patients who smoked tobacco, and those with no history of tobacco consumption). Our data identified distinct dysregulation of biological processes and pathways in each patient cohort. Bioinformatics analysis of dysregulated proteins identified in our proteomic study revealed dysregulation of collagen formation and antigen processing/presentation pathway in oral cancer patients who smoked tobacco, whereas proteins associated with the process of keratinization showed enrichment in patients who chewed tobacco. In addition, we identified overexpression of proteins involved in immune pathways and downregulation of muscle contraction-mediated signaling events in all three cohorts, irrespective of tobacco using habits. This study lays the groundwork for identification of protein markers that may aid in identification of high-risk patients for cancer development based on the history of tobacco exposure habits.

Signaling alterations in oral keratinocytes in response to shisha and crude tobacco extract.

BACKGROUND: Tobacco consumption in smoking and non-smoking forms has been consequential in the rise of oral cancer cases. Among different forms, epidemiological studies from Middle Eastern countries and rural parts of northern India have reported increasing association of oral cancer with waterpipe (hookah) smoking. However, molecular mechanisms and role played by waterpipe smoking in the onset of oral carcinogenesis remains unexplored. METHODS: In this study, immortalized normal human oral keratinocytes were chronically treated with extracts of two varieties of waterpipe tobacco-crude tobacco and processed shisha. Phenotypic changes and molecular aberrations were examined using cell culture-based assays and mass spectrometry-based quantitative proteomic analysis, respectively. Bioinformatics analysis was utilized to analyze proteomics data and identify dysregulated pathways. RESULTS: Our data indicate that chronic treatment with waterpipe tobacco extracts increased proliferation, invasion, migration, and significant dysregulation of protein expression in oral keratinocytes. Altered expression of proteins involved in interferon signaling pathway were observed with both varieties of tobacco. Overexpression of cholesterol metabolism and vesicle-mediated transport proteins were identified exclusively in cells treated with crude tobacco extract. Bioinformatics analyses revealed different oncogenic response in oral cells based on the type of waterpipe tobacco used. CONCLUSIONS: This study may serve as a useful resource in understanding the early onset of oral cancer attributed to waterpipe smoking.

A network map of thrombopoietin signaling.

Thrombopoietin (THPO), also known as megakaryocyte growth and development factor (MGDF), is a cytokine involved in the production of platelets. THPO is a glycoprotein produced by liver and kidney. It regulates the production of platelets by stimulating the differentiation and maturation of megakaryocyte progenitors. It acts as a ligand for MPL receptor, a member of the hematopoietic cytokine receptor superfamily and is essential for megakaryocyte maturation. THPO binding induces homodimerization of the receptor which results in activation of JAKSTAT and MAPK signaling cascades that subsequently control cellular proliferation, differentiation and other signaling events. Despite the importance of THPO signaling in various diseases and biological processes, a detailed signaling network of THPO is not available in any publicly available database. Therefore, in this study, we present a resource of signaling events induced by THPO that was manually curated from published literature on THPO. Our manual curation of thrombopoietin pathway resulted in identification of 48 molecular associations, 66 catalytic reactions, 100 gene regulation events, 19 protein translocation events and 43 activation/inhibition reactions that occur upon activation of thrombopoietin receptor by THPO. THPO signaling pathway is made available on NetPath, a freely available human signaling pathway resource developed previously by our group. We believe this resource will provide a platform for scientific community to accelerate further research in this area on potential therapeutic interventions.

Cigarette smoke and chewing tobacco alter expression of different sets of miRNAs in oral keratinocytes.

Carcinogenic effect of tobacco in oral cancer is through chewing and/or smoking. Significant differences exist in development of oral cancer between tobacco users and non-users. However, molecular alterations induced by different forms of tobacco are yet to be fully elucidated. We developed cellular models of chronic exposure to chewing tobacco and cigarette smoke using immortalized oral keratinocytes. Chronic exposure to tobacco resulted in increased cell scattering and invasiveness in immortalized oral keratinocytes. miRNA sequencing using Illumina HiSeq 2500 resulted in the identification of 10 significantly dysregulated miRNAs (4 fold; p ≤ 0.05) in chewing tobacco treated cells and 6 in cigarette smoke exposed cells. We integrated this data with global proteomic data and identified 36 protein targets that showed inverse expression pattern in chewing tobacco treated cells and 16 protein targets that showed inverse expression in smoke exposed cells. In addition, we identified 6 novel miRNAs in chewing tobacco treated cells and 18 novel miRNAs in smoke exposed cells. Integrative analysis of dysregulated miRNAs and their targets indicates that signaling mechanisms leading to oncogenic transformation are distinct between both forms of tobacco. Our study demonstrates alterations in miRNA expression in oral cells in response to two frequently used forms of tobacco.

Altered signaling associated with chronic arsenic exposure in human skin keratinocytes.

Modulation of signaling pathways upon chronic arsenic exposure remains poorly studied. Here, we carried out SILAC-based quantitative phosphoproteomics analysis to dissect the signaling induced upon chronic arsenic exposure in human skin keratinocyte cell line, HaCaT. We identified 4171 unique phosphosites derived from 2000 proteins. We observed differential phosphorylation of 406 phosphosites (twofold) corresponding to 305 proteins. Several pathways involved in cytoskeleton maintenance and organization were found to be significantly enriched (p<0.05). Our data revealed altered phosphorylation of proteins associated with adherens junction remodeling and actin polymerization. Kinases such as protein kinase C iota type (PRKCI), mitogen-activated protein kinase kinase kinase 1 (MAP3K1), tyrosine-protein kinase BAZ1B (BAZ1B) and STE20 like kinase (SLK) were found to be hyperphosphorylated. Our study provides novel insights into signaling perturbations associated with chronic arsenic exposure in human skin keratinocytes. All MS/MS data have been deposited to the ProteomeXchange with identifier PXD004868.

Gold nanoparticles-conjugated quercetin induces apoptosis via inhibition of EGFR/PI3K/Akt-mediated pathway in breast cancer cell lines (MCF-7 and MDA-MB-231).

Epidermal growth factor plays a major role in breast cancer cell proliferation, survival, and metastasis. Quercetin, a bioactive flavonoid, is shown to exhibit anticarcinogenic effects against various cancers including breast cancer. Hence, the present study was designed to evaluate the effects of gold nanoparticles-conjugated quercetin (AuNPs-Qu-5) in MCF-7 and MDA-MB-231 breast cancer cell lines. Borohydride reduced AuNPs were synthesized and conjugated with quercetin to yield AuNPs-Qu-5. Both were thoroughly characterized by several physicochemical techniques, and their cytotoxic effects were assessed by MTT assay. Apoptotic studies such as DAPI, AO/EtBr dual staining, and annexin V-FITC staining were performed. AuNPs and AuNPs-Qu-5 were spherical with crystalline nature, and the size of particles range from 3.0 to 4.5 nm. AuNPs-Qu-5 exhibited lower IC50 value compared to free Qu. There was a considerable increase in apoptotic population with increased nuclear condensation seen upon treatment with AuNPs-Qu-5. To delineate the molecular mechanism behind its apoptotic role, we analysed the proteins involved in apoptosis and epidermal growth factor receptor (EGFR)-mediated PI3K/Akt/GSK-3ß signalling by immunoblotting and immunocytochemistry. The pro-apoptotic proteins (Bax, Caspase-3) were found to be up regulated and anti-apoptotic protein (Bcl-2) was down regulated on treatment with AuNPs-Qu-5. Additionally, AuNPs-Qu-5 treatment inhibited the EGFR and its downstream signalling molecules PI3K/Akt/mTOR/GSK-3ß. In conclusion, administration of AuNPs-Qu-5 in breast cancer cell lines curtails cell proliferation through induction of apoptosis and also suppresses EGFR signalling. AuNPs-Qu-5 is more potent than free quercetin in causing cancer cell death, and hence, this could be a potential drug delivery system in breast cancer therapy.

Quercetin reverses EGF-induced epithelial to mesenchymal transition and invasiveness in prostate cancer (PC-3) cell line via EGFR/PI3K/Akt pathway.

Epidermal growth factor (EGF) plays an important role in metastasis and tumorigenesis of prostate cancer. Epithelial-mesenchymal transition (EMT) is a process in tumor progression during which cancer cells undergo dramatic changes acquiring highly invasive properties. The purpose of this study was to determine the effect of quercetin on EGF-induced EMT in prostate cancer (PC-3) cell line. Quercetin, a plant flavonoid, prevented EGF-induced invasion and migration of PC-3 cells. The protein and mRNA expressions of E-cadherin and N-cadherin were studied by immunocytochemistry, Western blotting and real-time polymerase chain reaction. Quercetin prevented EGF-induced expression of N-cadherin and vimentin and increased the expression of E-cadherin in PC-3 cells, therefore preventing EGF-induced EMT. EGF-induced cell adhesion proteins, intercellular adhesion molecule and vascular cell adhesion molecule were significantly decreased by quercetin treatment. Furthermore, mRNA and protein expressions of Snail, Slug and Twist showed that quercetin significantly decreased EGF-induced expressions of Snail, Slug and Twist. The protein expressions of epidermal growth factor receptor (EGFR)/phosphatidylinositide 3-kinases (PI3K)/Akt/extracellular signal-regulated kinase (ERK)1/2 pathway showed that quercetin prevents EGF-induced EMT via EGFR/PI3k/Akt/ERK1/2 pathway and by suppressing transcriptional repressors Snail, Slug and Twist in PC-3 cells. Thus, it is concluded from the present study that quercetin may prevent cancer metastasis by targeting EMT.