Changes in the Proteome Profile of A549 Cells Following Helichrysetin-Induced Apoptosis Suggest the Involvement of DNA Damage Response and Cell Cycle Arrest-Associated Proteins.

Our previous findings demonstrated that Helichrysetin possessed promising anti-cancer activity. It was able to induce apoptosis in the A549 cell line. However, its mechanism of action is unknown. The present study aimed to unravel possible underlying molecular mechanisms of helichrysetin-induced apoptosis in A549 (human lung carcinoma) cells using comparative quantitative proteomics (iTRAQ labeled), followed by an exhaustive bioinformatics analysis. Our results suggested that DNA damage response (DDR) and cell cycle arrest were responsible for lung cancer cell death with helichrysetin treatment. Among proteins that changed in abundance were Nrf2 and HMOX1. They are oxidative stress-related proteins and were increased in abundance. BRAT1 was also increased in abundance, suggesting an increase in DNA damage repair, indicating the occurrence of DNA damage due to oxidative stress. However, several essential DDR downstream proteins such as p-ATM, BRCA1, FANCD2, and Rb1 that would further increase DNA damage were found to be dramatically decreased in relative abundance. Cell cycle-related proteins, p53, p21, and cyclin D1, were increased while cyclin A, cyclin E, and cdk2 were decreased. This is predicted to facilitate S-phase arrest. Furthermore, excessive DNA damage and prolonged arrest would in turn result in the induction of mitochondrial-mediated apoptosis. Based on these observations, we postulate that the effects of helichrysetin were in part via the suppression of DNA damage response which led to DNA damage and prolonged cell cycle arrest. Subsequently, this event initiated mitochondrial-mediated apoptosis in A549 lung cancer cells.

Integrated omics-based pathway analyses uncover CYP epoxygenase-associated networks as theranostic targets for metastatic triple negative breast cancer.

BACKGROUND: Current prognostic tools and targeted therapeutic approaches have limited value for metastatic triple negative breast cancer (TNBC). Building upon current knowledge, we hypothesized that epoxyeicosatrienoic acids (EETs) and related CYP450 epoxygenases may have differential roles in breast cancer signaling, and better understanding of which may uncover potential directions for molecular stratification and personalized therapy for TNBC patients. METHODS: We analyzed the oxylipin metabolome of paired tumors and adjacent normal mammary tissues from patients with pathologically confirmed breast cancer (N = 62). We used multivariate statistical analysis to identify important metabolite contributors and to determine the predictive power of tumor tissue metabolite clustering. In vitro functional assays using a panel of breast cancer cell lines were carried out to further confirm the crucial roles of endogenous and exogenous EETs in the metastasis transformation of TNBC cells. Deregulation of associated downstream signaling networks associated with EETs/CYPs was established using transcriptomics datasets from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). Comparative TNBC proteomics using the same tissue specimens subjected to oxylipin metabolomics analysis was used as validation set. RESULTS: Metabolite-by-metabolite comparison, tumor immunoreactivity, and gene expression analyses showed that CYP epoxygenases and arachidonic acid-epoxygenation products, EET metabolites, are strongly associated with TNBC metastasis. Notably, all the 4 EET isomers (5,6-, 8,9-, 11,12-, and 14,15-EET) was observed to profoundly drive the metastasis transformation of mesenchymal-like TNBC cells among the TNBC (basal- and mesenchymal-like), HER2-overexpressing and luminal breast cancer cell lines examined. Our pathway analysis revealed that, in hormone-positive breast cancer subtype, CYP epoxygenase overexpression is more related to immune cell-associated signaling, while EET-mediated Myc, Ras, MAPK, EGFR, HIF-1α, and NOD1/2 signaling are the molecular vulnerabilities of metastatic CYP epoxygenase-overexpressing TNBC tumors. CONCLUSIONS: This study suggests that categorizing breast tumors according to their EET metabolite ratio classifiers and CYP epoxygenase profiles may be useful for prognostic and therapeutic assessment. Modulation of CYP epoxygenase and EET-mediated signaling networks may offer an effective approach for personalized treatment of breast cancer, and may be an effective intervention option for metastatic TNBC patients.

Phytoagent deoxyelephantopin derivative inhibits triple negative breast cancer cell activity by inducing oxidative stress-mediated paraptosis-like cell death.

Triple negative breast cancer (TNBC) is a highly metastatic cancer among the breast cancer subgroups. A thorny issue for clinical therapy of TNBC is lack of an efficient targeted therapeutic strategy. We previously created a novel sesquiterpene lactone analog (named DETD-35) derived from plant deoxyelephantopin (DET) which exhibits potent effects against human TNBC MDA-MB-231 tumor growth in a xenograft mouse model. Here we studied the mechanisms of both DET and DETD-35 against MDA-MB-231 cells. DETD-35 (3-fold decreased in IC50) exhibited better anti-TNBC cell activity than DET as observed through induction of reactive oxygen species production (within 2 h treatment) and damage to the ER structures, resulting in ER-derived cytoplasmic vacuolation and ubiquitinated protein accumulation in the treated cells. Intriguingly, the effects of both compounds were blockaded by pretreatment with ROS scavengers, N-acetylcysteine and reduced glutathione, and protein synthesis inhibitor, cycloheximide. Further, knockdown of MEK upstream regulator RAF1 and autophagosomal marker LC3, and co-treatment with JNK or ERK1/2 inhibitor resulted in the most significant attenuation of DETD-35-induced morphological and molecular or biochemical changes in cancer cells, while the inhibitory effect of DET was not influenced by MAPK inhibitor treatment. Therefore, DETD-35 exerted both ER stress-mediated paraptosis and apoptosis, which may explain its superior activity to DET against TNBC cells. Although the chemotherapeutic drug paclitaxel induced vacuole-like structures in MDA-MB-231 cells, no paraptotic cell death features were detected. This study provides a strategy for combating TNBC through sesquiterpene lactone analogs by induction of oxidative and ER stresses that cause paraptosis-like cell death.

Phytoagent Deoxyelephantopin and Its Derivative Inhibit Triple Negative Breast Cancer Cell Activity through ROS-Mediated Exosomal Activity and Protein Functions.

A novel plant sesquiterpene lactone derivative, DET derivative (DETD)-35, originating from parental deoxyelephantopin (DET) was previously observed to effectively suppress human triple negative breast cancer (TNBC) MDA-MB-231 cell activity and tumor growth in mice. In this study, the mechanisms underlying the activity of DETD-35 were elucidated. DET and DETD-35 induced reactive oxygen species (ROS) which caused structural damage and dysfunction of mitochondria and increased cytosolic calcium level, subsequently evoking exosome release from the cancer cells. Intriguingly, exosomes induced by both compounds had an atypical function. Cancer cell-derived exosomes commonly show metastatic potential, but upon DET/DETD-35 treatment exosomes showed anti-proliferative activity against MDA-MB-231 cells. Quantitative proteome analysis of TNBC cell-secreted exosomes showed that DET and DETD-35 attenuated the expression of proteins related to cell migration, cell adhesion, and angiogenesis. Furthermore, several exosomal proteins participating in biological mechanisms such as oxidative stress and decrease of transmembrane potential of mitochondria were found deregulated by treatment with either compound. Pretreatment with ROS scavenger, N-acetylcysteine, blockaded DET- or DETD-35-induced oxidative stress and calcium dependent exosome release mechanisms, and also reverted DET- or DETD-35-induced reprogramming exosomal protein expression profiles resulting in attenuation of exosomal toxicity against TNBC cell proliferation. In summary, this study shows that a plant-derived sesquiterpene lactone DET and its analog DETD-35 inhibitory TNBC cell activities through oxidative stress-induced cancer cell releasing exosomes in tandem with alteration of exosomal protein composition and functions. The findings of this study suggest that DETD-35 may be suitable for further development into an anti-TNBC drug.

Mechanistic Study of the Phytocompound, 2- ß -D-Glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne in Human T-Cell Acute Lymphocytic Leukemia Cells by Using Combined Differential Proteomics and Bioinformatics Approaches.

Bidens pilosa, a medicinal herb worldwide, is rich in bioactive polyynes. In this study, by using high resolution 2-dimensional gel electrophoresis coupled with mass spectrometry analysis, as many as 2000 protein spots could be detected and those whose expression was specifically up- or downregulated in Jurkat T cells responsive to the treatment with 2-ß-D-glucopyranosyloxy-1-hydroxytrideca-5,7,9,11-tetrayne (GHTT) can be identified. GHTT treatment can upregulate thirteen proteins involved in signal transduction, detoxification, metabolism, energy pathways, and channel transport in Jurkat cells. Nine proteins, that is, thioredoxin-like proteins, BH3 interacting domain death agonist (BID protein involving apoptosis), methylcrotonoyl-CoA carboxylase beta chain, and NADH-ubiquinone oxidoreductase, were downregulated in GHTT-treated Jurkat cells. Further, bioinformatics tool, Ingenuity software, was used to predict signaling pathways based on the data obtained from the differential proteomics approach. Two matched pathways, relevant to mitochondrial dysfunction and apoptosis, in Jurkat cells were inferred from the proteomics data. Biochemical analysis further verified both pathways involving GHTT in Jurkat cells. These findings do not merely prove the feasibility of combining proteomics and bioinformatics methods to identify cellular proteins as key players in response to the phytocompound in Jurkat cells but also establish the pathways of the proteins as the potential therapeutic targets of leukemia.

iTRAQ protein profile analysis of Arabidopsis roots reveals new aspects critical for iron homeostasis.

Iron (Fe) deficiency is a major constraint for plant growth and affects the quality of edible plant parts. To investigate the mechanisms underlying Fe homeostasis in plants, Fe deficiency-induced changes in the protein profile of Arabidopsis (Arabidopsis thaliana) roots were comprehensively analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification) differential liquid chromatography-tandem mass spectrometry on a LTQ-Orbitrap with high-energy collision dissociation. A total of 4,454 proteins were identified with a false discovery rate of less than 1.1%, and 2,882 were reliably quantified. A subset of 101 proteins was differentially expressed upon Fe deficiency. The changes in protein profiles upon Fe deficiency show low congruency with previously reported alterations in transcript levels, indicating posttranscriptional changes, and provide complementary information on Fe deficiency-induced processes. The abundance of proteins involved in the synthesis/regeneration of S-adenosylmethionine, the phenylpropanoid pathway, the response to oxidative stress, and respiration was highly increased by Fe deficiency. Using Fe-responsive proteins as bait, genome-wide fishing for partners with predictable or confirmed interologs revealed that RNA processing and ribonucleoprotein complex assembly may represent critical processes that contribute to the regulation of root responses to Fe deficiency, possibly by biasing translation efficiency.

Differential proteomic profiling identifies novel molecular targets of paclitaxel and phytoagent deoxyelephantopin against mammary adenocarcinoma cells.

A major germacranolide sesquiterpene lactone, deoxyelephantopin, identified from Elephantopus scaber L. (known as "Didancao" in Chinese medicine) showed significant antitumor growth and antimetastatic effect on murine mammary adenocarcinoma TS/A cells in vitro and in vivo in mice. Deoxyelephantopin exhibited a superior effect to that of the paclitaxel in prolonging median survival time of tumor-bearing animals in our recent study. To investigate the molecular mechanisms underlying the difference in efficacy between deoxyelephantopin and paclitaxel, we used 2-D DIGE and LC-ESI-MS/MS to profile proteins differentially expressed in the nucleus and cytoplasm of TS/A cells and used the MetaCore database to determine the functional protein networks affected by both treatments. Deoxyelephantopin and paclitaxel treatment produced regulation of molecules involved in proteolysis and calcium ion transport, suggesting the possible effects of both drugs on proteasome and endoplasmic reticulum machinery in TS/A cells. Western blot analysis of marker proteins (e.g., PDI, GRP78, TXND5, caspase-12, caspase-3 and PARP) further verified that induction of endoplasmic reticulum stress was associated with apoptosis induced by both deoxyelephantopin and paclitaxel, but only deoxyelephantopin inhibited proteasomal proteolysis in TS/A cells. The novel effects on targeting ER machinery and suppressing proteasome activity suggest the great potential of deoxyelephantopin for mammary cancer therapy.