Anticancer Effect by Combined Treatment of Artemisia annua L. Polyphenols and Docetaxel in DU145 Prostate Cancer Cells and HCT116 Colorectal Cancer Cells.

Docetaxel (DTX), a semi-synthetic analogue of paclitaxel (taxol), is known to exert potent anticancer activity in various cancer cells by suppressing normal microtubule dynamics. In this study, we examined how the anticancer effect of DTX is regulated by polyphenols extracted from Korean Artemisia annua L. (pKAL) in DU145 prostate cancer cells (mutant p53) and HCT116 colorectal cancer cells (wild-type p53). Here, we show that the anticancer effect of DTX was enhanced more significantly by pKAL in HCT116 cells than in DU145 cells via phase-contrast microscopy, CCK-8 assay, Western blot, and flow cytometric analysis of annexin V/propidium iodide-stained cells. Notably, mutant p53 was slightly downregulated by single treatment of pKAL or DTX in DU145 cells, whereas wild-type p53 was significantly upregulated by pKAL or DTX in HCT116 cells. Moreover, the enhanced anticancer effect of DTX by pKAL in HCT116 cells was significantly associated with the suppression of DTX-induced p53 upregulation, increase of DTX-induced phospho-p38, and decrease of DTX-regulated cyclin A, cyclin B1, AKT, caspase-8, PARP1, GM130, NF-κB p65, and LDHA, leading to the increased apoptotic cell death and plasma membrane permeability. Our results suggest that pKAL could effectively improve the anticancer effect of DTX-containing chemotherapy used to treat various cancers expressing wild-type p53.

Identification of novel membrane markers in circulating tumor cells of mesenchymal state in breast cancer.

Cancer metastasis is a major cause of cancer-related deaths worldwide. The ability to detect and monitor circulating tumor cells (CTCs) offers a promising approach to early detection and management of metastasis. Although studies on epithelial markers for CTC detection are actively underway, the discovery of mesenchymal markers has not been studied sufficiently. In this study, we developed a new pipeline to identify membrane markers in CTCs of mesenchymal state in breast cancer based on expression profiles of the 310 CTC samples. From the total CTC samples, only CTC samples in the mesenchymal state were collected by employing hierarchical clustering. In samples belonging to the mesenchymal state, we calculated the correlation coefficients between 1995 membrane genes and ZEB2, which was determined as the key mesenchymal signature, allowing the 84 positively correlated genes. Furthermore, to ensure clinical significance, Kaplan-Meier analysis were performed on the 124 breast cancer patients, resulting in the 14 genes predicting prognosis. By exploring genes commonly identified in the both analyses, F11R and PTGIR were characterized as membrane markers in CTCs of mesenchymal state in breast cancer, which were evaluated by enriched terms, literature evidence, and relevant molecular pathways. We expect that the results will be helpful to more effective strategies for metastasis management.

Artemisia annua L. Polyphenols Enhance the Anticancer Effect of ß-Lapachone in Oxaliplatin-Resistant HCT116 Colorectal Cancer Cells.

Recent studies suggest that the anticancer activity of ß-lapachone (ß-Lap) could be improved by different types of bioactive phytochemicals. The aim of this study was to elucidate how the anticancer effect of ß-Lap is regulated by polyphenols extracted from Korean Artemisia annua L. (pKAL) in parental HCT116 and oxaliplatin-resistant (OxPt-R) HCT116 colorectal cancer cells. Here, we show that the anticancer effect of ß-Lap is more enhanced by pKAL in HCT116-OxPt-R cells than in HCT116 cells via a CCK-8 assay, Western blot, and phase-contrast microscopy analysis of hematoxylin-stained cells. This phenomenon was associated with the suppression of OxPt-R-related upregulated proteins including p53 and ß-catenin, the downregulation of cell survival proteins including TERT, CD44, and EGFR, and the upregulation of cleaved HSP90, γ-H2AX, and LC3B-I/II. A bioinformatics analysis of 21 proteins regulated by combined treatment of pKAL and ß-Lap in HCT116-OxPt-R cells showed that the enhanced anticancer effect of ß-Lap by pKAL was related to the inhibition of negative regulation of apoptotic process and the induction of DNA damage through TERT, CD44, and EGFR-mediated multiple signaling networks. Our results suggest that the combination of pKAL and ß-Lap could be used as a new therapy with low toxicity to overcome the OxPt-R that occurred in various OxPt-containing cancer treatments.

Identification of Breast Cancer Metastasis Markers from Gene Expression Profiles Using Machine Learning Approaches.

Cancer metastasis accounts for approximately 90% of cancer deaths, and elucidating markers in metastasis is the first step in its prevention. To characterize metastasis marker genes (MGs) of breast cancer, XGBoost models that classify metastasis status were trained with gene expression profiles from TCGA. Then, a metastasis score (MS) was assigned to each gene by calculating the inner product between the feature importance and the AUC performance of the models. As a result, 54, 202, and 357 genes with the highest MS were characterized as MGs by empirical p-value cutoffs of 0.001, 0.005, and 0.01, respectively. The three sets of MGs were compared with those from existing metastasis marker databases, which provided significant results in most comparisons (p-value < 0.05). They were also significantly enriched in biological processes associated with breast cancer metastasis. The three MGs, SPPL2C, KRT23, and RGS7, showed highly significant results (p-value < 0.01) in the survival analysis. The MGs that could not be identified by statistical analysis (e.g., GOLM1, ELAVL1, UBP1, and AZGP1), as well as the MGs with the highest MS (e.g., ZNF676, FAM163B, LDOC2, IRF1, and STK40), were verified via the literature. Additionally, we checked how close the MGs were to each other in the protein-protein interaction networks. We expect that the characterized markers will help understand and prevent breast cancer metastasis.

ß-Lapachone Exerts Anticancer Effects by Downregulating p53, Lys-Acetylated Proteins, TrkA, p38 MAPK, SOD1, Caspase-2, CD44 and NPM in Oxaliplatin-Resistant HCT116 Colorectal Cancer Cells.

ß-lapachone (ß-Lap), a topoisomerase inhibitor, is a naturally occurring ortho-naphthoquinone phytochemical and is involved in drug resistance mechanisms. Oxaliplatin (OxPt) is a commonly used chemotherapeutic drug for metastatic colorectal cancer, and OxPt-induced drug resistance remains to be solved to increase chances of successful therapy. To reveal the novel role of ß-Lap associated with OxPt resistance, 5 µM OxPt-resistant HCT116 cells (HCT116-OxPt-R) were generated and characterized via hematoxylin staining, a CCK-8 assay and Western blot analysis. HCT116-OxPt-R cells were shown to have OxPt-specific resistance, increased aggresomes, upregulated p53 and downregulated caspase-9 and XIAP. Through signaling explorer antibody array, nucleophosmin (NPM), CD37, Nkx-2.5, SOD1, H2B, calreticulin, p38 MAPK, caspase-2, cadherin-9, MMP23B, ACOT2, Lys-acetylated proteins, COL3A1, TrkA, MPS-1, CD44, ITGA5, claudin-3, parkin and ACTG2 were identified as OxPt-R-related proteins due to a more than two-fold alteration in protein status. Gene ontology analysis suggested that TrkA, Nkx-2.5 and SOD1 were related to certain aggresomes produced in HCT116-OxPt-R cells. Moreover, ß-Lap exerted more cytotoxicity and morphological changes in HCT116-OxPt-R cells than in HCT116 cells through the downregulation of p53, Lys-acetylated proteins, TrkA, p38 MAPK, SOD1, caspase-2, CD44 and NPM. Our results indicate that ß-Lap could be used as an alternative drug to overcome the upregulated p53-containing OxPt-R caused by various OxPt-containing chemotherapies.

Identification of Growth Factors, Cytokines and Mediators Regulated by Artemisia annua L. Polyphenols (pKAL) in HCT116 Colorectal Cancer Cells: TGF-ß1 and NGF-ß Attenuate pKAL-Induced Anticancer Effects via NF-κB p65 Upregulation.

The anticancer effects of natural phytochemicals are relevant to the modulation of cytokine signaling pathways in various cancer cells with stem-like properties as well as immune cells. The aim of this study was to elucidate a novel anticancer mechanism of Artemisia annua L. polyphenols (pKAL) involved in the regulation of growth factors, cytokines and mediators in stem-like HCT116 colorectal cancer cells. Through RayBiotech human L-1000 antibody array and bioinformatics analysis, we show here that pKAL-induced anticancer effects are associated with downregulation of growth factor and cytokine signaling proteins including TGFA, FGF16, PDGFC, CCL28, CXCR3, IRF6 and SMAD1. Notably, we found that TGF-ß signaling proteins such as GDF10, ENG and TGFBR2 and well-known survival proteins such as NGF-ß, VEGFD and insulin were significantly upregulated by pKAL. Moreover, the results of hematoxylin staining, cell viability assay and Western blot analysis demonstrated that TGF-ß1 and NGF-ß attenuated pKAL-induced anticancer effects by inhibiting pKAL-induced downregulation of caspase-8, NF-κB p65 and cyclin D1. These results suggest that certain survival mediators may be activated by pKAL through the TGF-ß1 and NGF-ß signaling pathways during pKAL-induced cell death and thus, strategies to inhibit the survival signaling are inevitably required for more effective anticancer effects of pKAL.

Doxorubicin-Resistant TNBC Cells Exhibit Rapid Growth with Cancer Stem Cell-like Properties and EMT Phenotype, Which Can Be Transferred to Parental Cells through Autocrine Signaling.

Emerging evidence suggests that breast cancer stem cells (BCSCs), and epithelial-mesenchymal transition (EMT) may be involved in resistance to doxorubicin. However, it is unlear whether the doxorubicin-induced EMT and expansion of BCSCs is related to cancer dormancy, or outgrowing cancer cells with maintaining resistance to doxorubicin, or whether the phenotypes can be transferred to other doxorubicin-sensitive cells. Here, we characterized the phenotype of doxorubicin-resistant TNBC cells while monitoring the EMT process and expansion of CSCs during the establishment of doxorubicin-resistant MDA-MB-231 human breast cancer cells (DRM cells). In addition, we assessed the potential signaling associated with the EMT process and expansion of CSCs in doxorubicin-resistance of DRM cells. DRM cells exhibited morphological changes from spindle-shaped MDA-MB-231 cells into round-shaped giant cells. They exhibited highly proliferative, EMT, adhesive, and invasive phenotypes. Molecularly, they showed up-regulation of Cyclin D1, mesenchymal markers (ß-catenin, and N-cadherin), MMP-2, MMP-9, ICAM-1 and down-regulation of E-cadherin. As the molecular mechanisms responsible for the resistance to doxorubicin, up-regulation of EGFR and its downstream signaling, were suggested. AKT and ERK1/2 expression were also increased in DRM cells with the advancement of resistance to doxorubicin. Furthermore, doxorubicin resistance of DRM cells can be transferred by autocrine signaling. In conclusion, DRM cells harbored EMT features with CSC properties possessing increased proliferation, invasion, migration, and adhesion ability. The doxorubicin resistance, and doxorubicin-induced EMT and CSC properties of DRM cells, can be transferred to parental cells through autocrine signaling. Lastly, this feature of DRM cells might be associated with the up-regulation of EGFR.

Precise Characterization of Genetic Interactions in Cancer via Molecular Network Refining Processes.

Genetic interactions (GIs), such as the synthetic lethal interaction, are promising therapeutic targets in precision medicine. However, despite extensive efforts to characterize GIs by large-scale perturbation screening, considerable false positives have been reported in multiple studies. We propose a new computational approach for improved precision in GI identification by applying constraints that consider actual biological phenomena. In this study, GIs were characterized by assessing mutation, loss of function, and expression profiles in the DEPMAP database. The expression profiles were used to exclude loss-of-function data for nonexpressed genes in GI characterization. More importantly, the characterized GIs were refined based on Kyoto Encyclopedia of Genes and Genomes (KEGG) or protein-protein interaction (PPI) networks, under the assumption that genes genetically interacting with a certain mutated gene are adjacent in the networks. As a result, the initial GIs characterized with CRISPR and RNAi screenings were refined to 65 and 23 GIs based on KEGG networks and to 183 and 142 GIs based on PPI networks. The evaluation of refined GIs showed improved precision with respect to known synthetic lethal interactions. The refining process also yielded a synthetic partner network (SPN) for each mutated gene, which provides insight into therapeutic strategies for the mutated genes; specifically, exploring the SPN of mutated BRAF revealed ELAVL1 as a potential target for treating BRAF-mutated cancer, as validated by previous research. We expect that this work will advance cancer therapeutic research.

Activated ERK Signaling Is One of the Major Hub Signals Related to the Acquisition of Radiotherapy-Resistant MDA-MB-231 Breast Cancer Cells.

Breast cancer is one of the major causes of deaths due to cancer, especially in women. The crucial barrier for breast cancer treatment is resistance to radiation therapy, one of the important local regional therapies. We previously established and characterized radio-resistant MDA-MB-231 breast cancer cells (RT-R-MDA-MB-231 cells) that harbor a high expression of cancer stem cells (CSCs) and the EMT phenotype. In this study, we performed antibody array analysis to identify the hub signaling mechanism for the radiation resistance of RT-R-MDA-MB-231 cells by comparing parental MDA-MB-231 (p-MDA-MB-231) and RT-R-MDA-MB-231 cells. Antibody array analysis unveiled that the MAPK1 protein was the most upregulated protein in RT-R-MDA-MB-231 cells compared to in p-MDA-MB-231 cells. The pathway enrichment analysis also revealed the presence of MAPK1 in almost all enriched pathways. Thus, we used an MEK/ERK inhibitor, PD98059, to block the MEK/ERK pathway and to identify the role of MAPK1 in the radio-resistance of RT-R-MDA-MB-231 cells. MEK/ERK inhibition induced cell death in both p-MDA-MB-231 and RT-R-MDA-MB-231 cells, but the death mechanism for each cell was different; p-MDA-MB-231 cells underwent apoptosis, showing cell shrinkage and PARP-1 cleavage, while RT-R-MDA-MB-231 cells underwent necroptosis, showing mitochondrial dissipation, nuclear swelling, and an increase in the expressions of CypA and AIF. In addition, MEK/ERK inhibition reversed the radio-resistance of RT-R-MDA-MB-231 cells and suppressed the increased expression of CSC markers (CD44 and OCT3/4) and the EMT phenotype (ß-catenin and N-cadherin/E-cadherin). Taken together, this study suggests that activated ERK signaling is one of the major hub signals related to the radio-resistance of MDA-MB-231 breast cancer cells.

Analysis of the Effects of Nutrient Intake and Dietary Habits on Depression in Korean Adults.

While several studies have explored nutrient intake and dietary habits associated with depression, few studies have reflected recent trends and demographic factors. Therefore, we examined how nutrient intake and eating habits are associated with depression, according to gender and age. We performed simple and multiple regressions using nationally representative samples of 10,106 subjects from the Korea National Health and Nutrition Examination Survey. The results indicated that cholesterol, dietary fiber, sodium, frequency of breakfast, lunch, dinner, and eating out were significantly associated with depression (p-value < 0.05). Moreover, depression was associated with nutrient intake and dietary habits by gender and age group: sugar, breakfast, lunch, and eating out frequency in the young women's group; sodium and lunch frequency among middle-age men; dietary fibers, breakfast, and eating out frequency among middle-age women; energy, moisture, carbohydrate, lunch, and dinner frequency in late middle-age men; breakfast and lunch frequency among late middle-age women; vitamin A, carotene, lunch, and eating out frequency among older age men; and fat, saturated fatty acids, omega-3 fatty acid, omega-6 fatty acid, and eating out frequency among the older age women's group (p-value < 0.05). This study can be used to establish dietary strategies for depression prevention, considering gender and age.

Artemisia annua L. Polyphenol-Induced Cell Death Is ROS-Independently Enhanced by Inhibition of JNK in HCT116 Colorectal Cancer Cells.

c-Jun N-terminal kinase (JNK) is activated by chemotherapeutic reagents including natural plant polyphenols, and cell fate is determined by activated phospho-JNK as survival or death depending on stimuli and cell types. The purpose of this study was to elucidate the role of JNK on the anticancer effects of the Korean plant Artemisia annua L. (pKAL) polyphenols in p53 wild-type HCT116 human colorectal cancer cells. Cell morphology, protein expression levels, apoptosis/necrosis, reactive oxygen species (ROS), acidic vesicles, and granularity/DNA content were analyzed by phase-contrast microscopy; Western blot; and flow cytometry of annexin V/propidium iodide (PI)-, dichlorofluorescein (DCF)-, acridine orange (AO)-, and side scatter pulse height (SSC-H)/DNA content (PI)-stained cells. The results showed that pKAL induced morphological changes and necrosis or late apoptosis, which were associated with loss of plasma membrane/Golgi integrity, increased acidic vesicles and intracellular granularity, and decreased DNA content through downregulation of protein kinase B (Akt)/ß-catenin/cyclophilin A/Golgi matrix protein 130 (GM130) and upregulation of phosphorylation of H2AX at Ser-139 (γ-H2AX)/p53/p21/Bak cleavage/phospho-JNK/p62/microtubule-associated protein 1 light chain 3B (LC3B)-I. Moreover, JNK inhibition by SP600125 enhanced ROS-independently pKAL-induced cell death through downregulation of p62 and upregulation of p53/p21/Bak cleavage despite a reduced state of DNA damage marker γ-H2AX. These findings indicate that phospho-JNK activated by pKAL inhibits p53-dependent cell death signaling and enhances DNA damage signaling, but cell fate is determined by phospho-JNK as survival rather than death in p53 wild-type HCT116 cells.

Characterizing therapeutic signatures of transcription factors in cancer by incorporating profiles in compound treated cells.

MOTIVATION: Cancers are promoted by abnormal alterations in biological processes, such as cell cycle and apoptosis. An immediate reason for those aberrant processes is the deregulation of their involved transcription factors (TFs). Thus, the deregulated TFs in cancer have been experimented as successful therapeutic targets, such as RARA and RUNX1. This therapeutic strategy can be accelerated by characterizing new potential TF targets. RESULTS: Two kinds of therapeutic signatures of TFs in A375 (skin) and HT29 (colon) cancer cells were characterized by analyzing TF activities under effective and ineffective compounds to cancer. First, the therapeutic TFs (TTs) were identified as the TFs that are significantly activated or repressed under effective compared to ineffective compounds. Second, the therapeutically correlated TF pairs (TCPs) were determined as the TF pairs whose activity correlations show substantial discrepancy between the effective and ineffective compounds. It was facilitated by incorporating (i)compound-induced gene expressions (LINCS), (ii) compound-induced cell viabilities (GDSC) and (iii) TF-target interactions (TRUST2). As a result, among 627 TFs, the 35 TTs (such as MYCN and TP53) and the 214 TCPs (such as FOXO3 and POU2F2 pair) were identified. The TTs and the proteins on the paths between TCPs were compared with the known therapeutic targets, tumor suppressors, oncogenes and CRISPR-Cas9 knockout screening, which yielded significant consequences. We expect that the results provide good candidates for therapeutic TF targets in cancer. AVAILABILITY AND IMPLEMENTATION: The data and Python implementations are available at https://github.com/jmjung83/TT_and_TCP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

p53 Enhances Artemisia annua L. Polyphenols-Induced Cell Death Through Upregulation of p53-Dependent Targets and Cleavage of PARP1 and Lamin A/C in HCT116 Colorectal Cancer Cells.

Plant-derived natural polyphenols exhibit anticancer activity without showing any noticeable toxicities to normal cells. The aim of this study was to investigate the role of p53 on the anticancer effect of polyphenols isolated from Korean Artemisia annua L. (pKAL) in HCT116 human colorectal cancer cells. We confirmed that pKAL induced reactive oxygen species (ROS) production, propidium iodide (PI) uptake, nuclear structure change, and acidic vesicles in a p53-independent manner in p53-null HCT116 cells through fluorescence microscopy analysis of DCF/PI-, DAPI-, and AO-stained cells. The pKAL-induced anticancer effects were found to be significantly higher in p53-wild HCT116 cells than in p53-null by hematoxylin staining, CCK-8 assay, Western blot, and flow cytometric analysis of annexin V/PI-stained cells. In addition, expression of ectopic p53 in p53-null cells was upregulated by pKAL in both the nucleus and cytoplasm, increasing pKAL-induced cell death. Moreover, Western bot analysis revealed that pKAL-induced cell death was associated with upregulation of p53-dependent targets such as p21, Bax and DR5 and cleavage of PARP1 and lamin A/C in p53-wild HCT116 cells, but not in p53-null. Taken together, these results indicate that p53 plays an important role in enhancing the anticancer effects of pKAL by upregulating p53 downstream targets and inducing intracellular cell death processes.

Anthocyanins Isolated from Vitis coignetiae Pulliat Enhances Cisplatin Sensitivity in MCF-7 Human Breast Cancer Cells through Inhibition of Akt and NF-κB Activation.

Anthocyanins isolated from Vitis coignetiae Pulliat (Meoru in Korea) (AIMs) have various anti-cancer properties by inhibiting Akt and NF-κB which are involved in drug resistance. Cisplatin (CDDP) is one of the popular anti-cancer agents. Studies reported that MCF-7 human breast cancer cells have high resistance to CDDP compared to other breast cancer cell lines. In this study, we confirmed CDDP resistance of MCF-7 cells and tested whether AIMs can overcome CDDP resistance of MCF-7 cells. Cell viability assay revealed that MCF-7 cells were more resistant to CDDP treatment than MDA-MB-231 breast cancer cells exhibiting aggressive and high cancer stem cell phenotype. AIMs significantly augmented the efficacy of CDDP with synergistic effects on MCF-7 cells. Molecularly, Western blot analysis revealed that CDDP strongly increased Akt and moderately reduced p-NF-κB and p-IκB and that AIMs inhibited CDDP-induced Akt activation, and augmented CDDP-induced reduction of p-NF-κB and p-IκB in MCF-7 cells. In addition, AIMs significantly downregulated an anti-apoptotic protein, XIAP, and augmented PARP-1 cleavage in CDDP-treated MCF-7 cells. Moreover, under TNF-α treatment, AIMs augmented CDDP efficacy with inhibition of NF-κB activation on MCF-7 cells. In conclusion, AIMs enhanced CDDP sensitivity by inhibiting Akt and NF-κB activity of MCF-7 cells that show relative intrinsic CDDP resistance.

Polyphenols Extracted from Artemisia annua L. Exhibit Anti-Cancer Effects on Radio-Resistant MDA-MB-231 Human Breast Cancer Cells by Suppressing Stem Cell Phenotype, ß-Catenin, and MMP-9.

Artemisia annua L. has been reported to show anti-cancer activities. Here, we determined whether polyphenols extracted from Artemisia annua L. (pKAL) exhibit anti-cancer effects on radio-resistant MDA-MB-231 human breast cancer cells (RT-R-MDA-MB-231 cells), and further explored their molecular mechanisms. Cell viability assay and colony-forming assay revealed that pKAL inhibited cell proliferation on both parental and RT-R-MDA-MB-231 cells in a dose-dependent manner. The anti-proliferative effects of pKAL on RT-R-MDA-MB-231 cells were superior or similar to those on parental ones. Western blot analysis revealed that expressions of cluster of differentiation 44 (CD44) and Oct 3/4, matrix metalloproteinase-9 (MMP-9) and signal transducer and activator of transcription-3 (STAT-3) phosphorylation were significantly increased in RT-R-MDA-MB-231 cells compared to parental ones, suggesting that these proteins could be associated with RT resistance. pKAL inhibited the expression of CD44 and Oct 3/4 (CSC markers), and ß-catenin and MMP-9 as well as STAT-3 phosphorylation of RT-R-MDA-MB-231. Regarding upstream signaling, the JNK or JAK2 inhibitor could inhibit STAT-3 activation in RT-R-MDA-MB-231 cells, but not augmented pKAL-induced anti-cancer effects. These findings suggest that c-Jun N-terminal kinase (JNK) or Janus kinase 2 (JAK2)/STAT3 signaling are not closely related to the anti-cancer effects of pKAL. In conclusion, this study suggests that pKAL exhibit anti-cancer effects on RT-R-MDA-MB-231 cells by suppressing CD44 and Oct 3/4, ß-catenin and MMP-9, which appeared to be linked to RT resistance of RT-R-MDA-MB-231 cells.

Morin enhances auranofin anticancer activity by up-regulation of DR4 and DR5 and modulation of Bcl-2 through reactive oxygen species generation in Hep3B human hepatocellular carcinoma cells.

Evidence suggests that auranofin (AF) exhibits anticancer activity by inhibiting thioredoxin reductase (TrxR). Here, in this study, we have investigated the synergistic effects of AF and morin and their mechanism for the anticancer effects focusing on apoptosis in Hep3B human hepatocellular carcinoma cells. We assessed the anticancer activities by annexin V/PI double staining, caspase, and TrxR activity assay. Morin enhances the inhibitory effects on TrxR activity of AF as well as reducing cell viability. Annexin V/PI double staining revealed that morin/AF cotreatment induced apoptotic cell death. Morin enhances AF-induced mitochondrial membrane potential (ΔΨm) loss and cytochrome c release. Further, morin/AF cotreatment upregulated death receptor DR4/DR5, modulated Bcl-2 family members (upregulation of Bax and downregulation of Bcl-2), and activated caspase-3, -8, and -9. Morin also enhances AF-induced reactive oxygen species (ROS) generation. The anticancer effects results from caspase-dependent apoptosis, which was triggered via extrinsic pathway by upregulating TRAIL receptors (DR4/DR5) and enhanced via intrinsic pathway by modulating Bcl-2 and inhibitor of apoptosis protein family members. These are related to ROS generation. In conclusion, this study provides evidence that morin can enhance the anticancer activity of AF in Hep3B human hepatocellular carcinoma cells, indicating that its combination could be an alternative treatment strategy for the hepatocellular carcinoma.

Deconvoluting essential gene signatures for cancer growth from genomic expression in compound-treated cells.

MOTIVATION: Essential gene signatures for cancer growth have been typically identified via RNAi or CRISPR-Cas9. Here, we propose an alternative method that reveals the essential gene signatures by analysing genomic expression profiles in compound-treated cells. With a large amount of the existing compound-induced data, essential gene signatures at genomic scale are efficiently characterized without technical challenges in the previous techniques. RESULTS: An essential gene is characterized as a gene presenting positive correlation between its down-regulation and cell growth inhibition induced by diverse compounds, which were collected from LINCS and CGP. Among 12 741 genes, 1092, 1 228 827 962, 1 664 580 and 829 essential genes are characterized for each of A375, A549, BT20, LNCAP, MCF7, MDAMB231 and PC3 cell lines (P-value ≤ 1.0E-05). Comparisons to the previously identified essential genes yield significant overlaps in A375 and A549 (P-value ≤ 5.0E-05) and the 103 common essential genes are enriched in crucial processes for cancer growth. In most comparisons in A375, MCF7, BT20 and A549, the characterized essential genes yield more essential characteristics than those of the previous techniques, i.e. high gene expression, high degrees of protein-protein interactions, many homologs and few paralogs. Remarkably, the essential genes commonly characterized by both the previous and proposed techniques show more significant essential characteristics than those solely relied on the previous techniques. We expect that this work provides new aspects in essential gene signatures. AVAILABILITY AND IMPLEMENTATION: The Python implementations are available at https://github.com/jmjung83/deconvolution_of_essential_gene_signitures. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Publisher Correction: CODA: Integrating multi-level context-oriented directed associations for analysis of drug effects.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Petri net-based prediction of therapeutic targets that recover abnormally phosphorylated proteins in muscle atrophy.

BACKGROUND: Muscle atrophy, an involuntary loss of muscle mass, is involved in various diseases and sometimes leads to mortality. However, therapeutics for muscle atrophy thus far have had limited effects. Here, we present a new approach for therapeutic target prediction using Petri net simulation of the status of phosphorylation, with a reasonable assumption that the recovery of abnormally phosphorylated proteins can be a treatment for muscle atrophy. RESULTS: The Petri net model was employed to simulate phosphorylation status in three states, i.e. reference, atrophic and each gene-inhibited state based on the myocyte-specific phosphorylation network. Here, we newly devised a phosphorylation specific Petri net that involves two types of transitions (phosphorylation or de-phosphorylation) and two types of places (activation with or without phosphorylation). Before predicting therapeutic targets, the simulation results in reference and atrophic states were validated by Western blotting experiments detecting five marker proteins, i.e. RELA, SMAD2, SMAD3, FOXO1 and FOXO3. Finally, we determined 37 potential therapeutic targets whose inhibition recovers the phosphorylation status from an atrophic state as indicated by the five validated marker proteins. In the evaluation, we confirmed that the 37 potential targets were enriched for muscle atrophy-related terms such as actin and muscle contraction processes, and they were also significantly overlapping with the genes associated with muscle atrophy reported in the Comparative Toxicogenomics Database (p-value < 0.05). Furthermore, we noticed that they included several proteins that could not be characterized by the shortest path analysis. The three potential targets, i.e. BMPR1B, ROCK, and LEPR, were manually validated with the literature. CONCLUSIONS: In this study, we suggest a new approach to predict potential therapeutic targets of muscle atrophy with an analysis of phosphorylation status simulated by Petri net. We generated a list of the potential therapeutic targets whose inhibition recovers abnormally phosphorylated proteins in an atrophic state. They were evaluated by various approaches, such as Western blotting, GO terms, literature, known muscle atrophy-related genes and shortest path analysis. We expect the new proposed strategy to provide an understanding of phosphorylation status in muscle atrophy and to provide assistance towards identifying new therapies.

Author Correction: CODA: Integrating multi-level context-oriented directed associations for analysis of drug effects.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.