withanolides and Lung-Neoplasms

The anticancer activities of Withaferin-A (Wi-A) and Withanone (Wi-N) from Ashwagandha and Caffeic Acid Phenethyl Ester (CAPE) from honeybee propolis have been well documented. Here, we examined the binding potential of these natural compounds to inhibit the constitutive phosphorylation of epidermal growth factor receptors (EGFRs). Exon 20 insertion mutants of EGFR, which show resistance to various FDA approved drugs and are linked to poor prognosis of lung cancer patients, were the primary focus of this study. Apart from exon 20 insertion mutants, the potential of natural compounds to serve as ATP competitive inhibitors of wildtype protein and other common mutants of EGFR, namely L858R and exon19del, were also examined. The potential of natural compounds was compared to the positive controls such as erlotinib, TAS6417 and poziotinib. Similar to known inhibitors, Wi-A and Wi-N could displace and binds at the ATP orthosteric site of exon19del, L858R and exon20, while CAPE was limited to wildtype EGFR and exon 20 insertion mutants only. Moreover, the binding free energy of the natural drugs against EGFRs was also comparable to the positive controls. This computational study suggests that Wi-A and Wi-N have potential against multiple mutated EGFRs, warranting further

Topics: Adenosine Triphosphate; Antineoplastic Agents; Caffeic Acids; Computational Biology; Computer Simulation; Dimerization; ErbB Receptors; Erlotinib Hydrochloride; Exons; Gene Deletion; Gene Expression Regulation, Neoplastic; Humans; Indolizines; Ligands; Lung Neoplasms; Molecular Docking Simulation; Molecular Dynamics Simulation; Mutation; Phenylethyl Alcohol; Quinazolines; Withanolides

Inflammation in the tumor microenvironment is positively correlated with cancer progression and metastasis as well as the risk of thromboembolism in lung cancer patients. Here we show, in human non-small cell lung cancer (NSCLC) cell lines, the master inflammatory cytokine tumor necrosis factor (TNF-α) induced tissue factor expression and procoagulant activity, and these effects were potently inhibited by 4β-hydroxywithanolide E (4HW), a natural compound isolated from Physalis peruviana. Furthermore, combination of 4HW and TNF-α caused synergistic cytotoxicity against NSCLC cells by inducing caspase-dependent apoptosis. The underlying mechanism by which 4HW reverses the procoagulant effect of TNF-α but enhances its cytotoxic effect appears to be due to inhibition of NF-κB, which is a key switch for both inflammation-induced coagulation and cell survival. Our results suggest that 4HW may have a potential application for treating inflammation-derived cancer progression and cancer-associated hypercoagulable state.

Topics: A549 Cells; Antineoplastic Agents; Apoptosis; Blood Coagulation; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Humans; Lung Neoplasms; Microscopy, Fluorescence; Physalis; Real-Time Polymerase Chain Reaction; Tumor Necrosis Factor-alpha; Withanolides

Cancer, an uncontrolled proliferation syndrome, is treated with synthetic chemotherapeutic drugs that are associated with severe adverse effects. Development and application of new natural compounds is warranted to deal with the exponentially increasing incidence of cancer worldwide. Keeping selective toxicity to cancer cells as a priority criterion, we developed a combination of Cucurbitacin B and Withanone, and analyzed its anticancer potential using non-small cell lung cancer cells. We demonstrate that the selective cytotoxicity of the combination, called CucWi-N, to cancer cells is mediated by induction of cellular senescence that was characterized by decrease in Lamin A/C, CDK2, CDK4, Cyclin D, Cyclin E, phosphorylated RB, mortalin and increase in p53 and CARF proteins. It compromised cancer cell migration that was mediated by decrease in mortalin, hnRNP-K, vascular endothelial growth factor, matrix metalloproteinase 2, and fibronectin. We provide in silico, molecular dynamics and experimental data to support that CucWi-N (i) possesses high capability to target mortalin-p53 interaction and hnRNP-K proteins, (ii) triggers replicative senescence and inhibits metastatic potential of the cancer cells, and (iii) inhibits tumor progression and metastasis in vivo. We propose that CucWi-N is a potential natural anticancer drug that warrants further mechanistic and clinical studies.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Carrier Proteins; Cell Cycle Checkpoints; Cell Line, Tumor; Cellular Senescence; Drug Combinations; Female; Guanylate Kinases; HSP70 Heat-Shock Proteins; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Molecular Docking Simulation; Signal Transduction; Triterpenes; Tumor Suppressor p53-Binding Protein 1; Withanolides

Withaferin-A (Wi-A) has been shown to possess anticancer activity. Molecular mechanism(s) of its action has not been fully resolved. We recruited low dose of Wi-A that caused slow growth arrest in cancer cells and was relatively safe for normal cells. Consistently, we detected nuclear translocation of nuclear factor kappa B (NFκB) and activation of p38MAPK selectively in cancer cells. Bioinformatics analyses revealed that Wi-A did not disrupt IKKα/IKKβ-Nemo complex that regulates NFκB activity. However, it caused moderate change in the conformation of IKKβ-Nemo interacting domain. Experimental data revealed increased level of phosphorylated IκBα in Wi-A-treated cells, suggesting an activation of IKK complex that was supported by nuclear translocation of NFκB. Molecular docking analysis showed that Wi-A did not disrupt; however, decreased the stability of the NFκB-DNA complex. It was supported by downregulation of DNA-binding and transcriptional activities of NFκB. Further analysis revealed that Wi-A caused upregulation of CARF (collaborator of ARF) demonstrating an activation of DNA damage oxidative stress response in both cancer and normal cells. In line with this, upregulation of p21WAF1, p16INK4A, and hypophosphorylated pRB and induction of senescence were observed demonstrating that Wi-A-induced senescence is mediated by multiple pathways in which CARF-mediated DNA damage and oxidative stress play a major role.

Topics: Apoptosis; Blotting, Western; Cell Survival; Cellular Senescence; Computational Biology; DNA Damage; DNA-Binding Proteins; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Immunoprecipitation; Lung Neoplasms; Molecular Docking Simulation; NF-kappa B; Signal Transduction; Transcription Factors; Tumor Cells, Cultured; Withanolides

Adenylate kinase 4 (AK4) has been identified as a biomarker of metastasis in lung cancer. However, the impacts of AK4 on metabolic genes and its translational value for drug repositioning remain unclear.. Ingenuity upstream analyses were used to identify potential transcription factors that regulate the AK4 metabolic gene signature. The expression of AK4 and its upstream regulators in lung cancer patients was examined via immunohistochemistry. Pharmacological and gene knockdown/overexpression approaches were used to investigate the interplay between AK4 and its upstream regulators during epithelial-to-mesenchymal transition (EMT). Drug candidates that reversed AK4-induced gene expression were identified by querying a connectivity map. Orthotopic xenograft mouse models were established to evaluate the therapeutic efficacy of drug candidates for metastatic lung cancer.. We found that HIF-1α is activated in the AK4 metabolic gene signature. IHC analysis confirmed this positive correlation, and the combination of both predicts worse survival in lung cancer patients. Overexpression of AK4 exaggerates HIF-1α protein expression by increasing intracellular ROS levels and subsequently induces EMT under hypoxia. Attenuation of ROS production with N-acetylcysteine abolishes AK4-induced invasion potential under hypoxia. Pharmacogenomics analysis of the AK4 gene signature revealed that withaferin-A could suppress the AK4-HIF-1α signaling axis and serve as a potent anti-metastatic agent in lung cancer.. Overexpression of AK4 promotes lung cancer metastasis by enhancing HIF-1α stability and EMT under hypoxia. Reversing the AK4 gene signature with withaferin-A may serve as a novel therapeutic strategy to treat metastatic lung cancer.

Topics: A549 Cells; Adenocarcinoma of Lung; Adenylate Kinase; Animals; Carcinoma, Non-Small-Cell Lung; Epithelial-Mesenchymal Transition; Follow-Up Studies; Gene Knockdown Techniques; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Oxidative Stress; Reactive Oxygen Species; Transfection; Tumor Hypoxia; Withanolides; Xenograft Model Antitumor Assays

In pathway-targeted cancer drug therapies, the relatively rapid emergence of drug-tolerant persisters (DTPs) substantially limits the overall therapeutic benefit. However, little is known about the roles of DTPs in drug resistance. In this study, we investigated the features of epidermal growth factor receptor-tyrosine kinase inhibitor-induced DTPs and explored a new treatment strategy to overcome the emergence of these DTPs. We used two EGFR-mutated lung adenocarcinoma cell lines, PC9 and II-18. They were treated with 2 μM gefitinib for 6, 12, or 24 days or 6 months. We analyzed the mRNA expression of the stem cell-related markers by quantitative RT-PCR and the expression of the cellular senescence-associated proteins. Then we sorted DTPs according to the expression pattern of CD133 and analyzed the features of sorted cells. Finally, we tried to ablate DTPs by glucose metabolism targeting therapies and a stem-like cell targeting drug, withaferin A. Drug-tolerant persisters were composed of at least two types of cells, one with the properties of cancer stem-like cells (CSCs) and the other with the properties of therapy-induced senescent (TIS) cells. The CD133

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cellular Senescence; Drug Resistance, Neoplasm; ErbB Receptors; Flow Cytometry; Gefitinib; Glucose; Humans; Lung Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Targeted Therapy; Neoplastic Stem Cells; Phloretin; Polymerase Chain Reaction; Protein Kinase Inhibitors; Quinazolines; Withanolides; Xenograft Model Antitumor Assays

The signal transducers and activators of transcription 3 (STAT3) signaling pathway plays critical roles in the pathogenesis and progression of various human cancers, including non-small cell lung cancer (NSCLC). In this study, we aimed to evaluate the therapeutic potential of physalin A, a bioactive withanolide derived from Physalis alkekengi var. francheti used in traditional Chinese medicine, was evaluated in human NSCLC cells. Its and determined whether it effect oninhibited both constitutive and induced STAT3 activity, through repressing the phosphorylation levels of JAK2 and JAK3, resulting in anti-proliferation and pro-apoptotic effects on NSCLC cells was also determined, and. theThe antitumor effects of physalin A were also validated usingin an in vivo mouse xenograft models of NSCLC cells. Physalin A had anti-proliferative and pro-apoptotic effects in NSCLC cells with constitutively activated STAT3; it also suppressed both constitutive and induced STAT3 activity by modulating the phosphorylation of JAK2 and JAK3. Furthermore, physalin A abrogated the nuclear translocation and transcriptional activity of STAT3, thereby decreasing the expression levels of STAT3, its target genes, such as Bcl-2 and XIAP. Knockdown of STAT3 expression by small interfering RNA (siRNA) significantly enhanced the pro-apoptotic effects of physalin A in NSCLC cells. Moreover, physalin A significantly suppressed tumor xenograft growth. Thus, as an inhibitor of JAK2/3-STAT3 signaling, physalin A, has potent anti-tumor activities, which may facilitate the development of a therapeutic strategy for treating NSCLC.

Topics: A549 Cells; Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Janus Kinase 2; Janus Kinase 3; Lung Neoplasms; Male; Medicine, Chinese Traditional; Mice; Mice, Inbred BALB C; Phosphorylation; Plant Preparations; Proto-Oncogene Proteins c-bcl-2; RNA Interference; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Transcription, Genetic; Withanolides; X-Linked Inhibitor of Apoptosis Protein; Xenograft Model Antitumor Assays

Physalin A (PA) is an active withanolide isolated from Physalis alkekengi var. franchetii, a traditional Chinese herbal medicine named Jindenglong, which has long been used for the treatment of sore throat, hepatitis, and tumors in China. In the present study, we firstly investigated the effects of PA on proliferation and cell cycle distribution of the human non-small cell lung cancer (NSCLC) A549 cell line, and the potential mechanisms involved. Here, PA inhibited cell growth in dose- and time-dependent manners. Treatment of A549 cells with 28.4 μM PA for 24 h resulted in approximately 50 % cell death. PA increased the amount of intracellular ROS and the proportion of cells in G2/M. G2/M arrest was attenuated by the addition of ROS scavenger NAC. ERK and P38 were triggered by PA through phosphorylation in a time-dependent manner. The phosphorylation of ERK and P38 were not attenuated by the addition of NAC, but the use of the p38 inhibitor could reduce, at least in part, PA-induced ROS and the proportion of cells in G2/M. PA induces G2/M cell cycle arrest in A549 cells involving in the p38 MAPK/ROS pathway. This study suggests that PA might be a promising therapeutic agent against NSCLC.

Topics: Acetylcysteine; Carcinoma, Non-Small-Cell Lung; Cell Division; Cell Line, Tumor; G2 Phase; Humans; Lung Neoplasms; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Reactive Oxygen Species; Withanolides

To explore the effect of Withaferin A on A549 cellular proliferation and apoptosis in non-small cell lung cancer (NSCLC).. NSCNC cell line A549 was selected to explore the effect of Withaferin A on A549 cellular proliferation, apoptosis and the PI3K/Akt signal pathway capable of regulating tumor biological behavior by assessment of cellular proliferation, cellular apoptotic rates and cellular cycling as well as by immuno-blotting.. Withaferin A could inhibit A549 cellular proliferation and the control rate was dosage-dependent (P<0.05), which also increased time-dependently with the same dosage of Withaferin A (P<0.05). The apoptotic indexes in A549 cells treated with 0, 2.5, 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A for 48 h were significantly different (P<0.05). In addition, the apoptotic rates of each group in both early and advanced stages were higher than those in 0 μmol·L-1 (P<0.05), which were evidently higher after 48 h than those after 24 h (P<0.05). A549 cells treated by Withaferin A for 48 h were markedly lower in Bcl-2 level and obviously higher in Bax and cleaved caspase-3 levels than those treated by 0 μmol·L-1 Withaferin A (P<0.05), and there were significant differences among 5, 10 and 20 μmol·L-1 Withaferin A (P<0.05). The ratios of A549 cells treated by Withaferin A for 48 h in G0/G1 stage were higher than those in 0 μmol·L-1 , while those in S and G2/M stages were obviously lower than those in G2/M stage, and there were significant differences in 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A (P<0.05). Additionally, p-Akt/Akt values were in reverse association with dosage, and the differences were significant (P<0.05).. Withaferin A can inhibit the proliferation and apoptosis of A549 cells by suppressing activation of the PI3K/Akt pathways.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Line, Tumor; Cell Proliferation; Humans; Lung Neoplasms; Mitosis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Withanolides

Efficacy of withaferin A (WA), an Ayurvedic medicine constituent, for prevention of mammary cancer and its associated mechanisms were investigated using mouse mammary tumor virus-neu (MMTV-neu) transgenic model.. Incidence and burden of mammary cancer and pulmonary metastasis were scored in female MMTV-neu mice after 28 weeks of intraperitoneal administration with 100 µg WA (three times/week) (n = 32) or vehicle (n = 29). Mechanisms underlying mammary cancer prevention by WA were investigated by determination of tumor cell proliferation, apoptosis, metabolomics, and proteomics using plasma and/or tumor tissues. Spectrophotometric assays were performed to determine activities of complex III and complex IV. All statistical tests were two-sided.. WA administration resulted in a statistically significant decrease in macroscopic mammary tumor size, microscopic mammary tumor area, and the incidence of pulmonary metastasis. For example, the mean area of invasive cancer was lower by 95.14% in the WA treatment group compared with the control group (mean = 3.10 vs 63.77 mm2, respectively; difference = -60.67 mm2; 95% confidence interval = -122.50 to 1.13 mm2; P = .0536). Mammary cancer prevention by WA treatment was associated with increased apoptosis, inhibition of complex III activity, and reduced levels of glycolysis intermediates. Proteomics confirmed downregulation of many glycolysis-related proteins in the tumor of WA-treated mice compared with control, including M2-type pyruvate kinase, phospho glycerate kinase, and fructose-bisphosphate aldolase A isoform 2.. This study reveals suppression of glycolysis in WA-mediated mammary cancer prevention in a clinically relevant mouse model.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cluster Analysis; Electrophoresis, Gel, Two-Dimensional; Female; Glycolysis; Immunohistochemistry; In Situ Nick-End Labeling; Lung Neoplasms; Mammary Neoplasms, Experimental; Mammary Tumor Virus, Mouse; Medicine, Ayurvedic; Mice; Mice, Transgenic; Random Allocation; Withanolides

Withaferin A (WFA) is purified from the plant Withania somnifera and inhibits the vimentin cytoskeleton. Vimentin overexpression in cancer correlates with metastatic disease, induction of epithelial to mesenchymal transition and reduced patient survival. As vimentin functions in cell motility, we wanted to test the hypothesis that WFA inhibits cancer metastasis by disrupting vimentin function. These data showed that WFA had weak cytotoxic and apoptotic activity at concentrations less than or equal to 500 nM, but retained potent anti-invasive activity at these low doses. Imaging of breast cancer cell lines revealed that WFA induces perinuclear vimentin accumulation followed by rapid vimentin depolymerization. A concomitant induction of vimentin ser56 phosphorylation was observed, which is consistent with vimentin disassembly. Structure activity relationships were established using a set of chemically modified WFA analogs and showed that the predicted vimentin-binding region of WFA is necessary to induce vimentin ser56 phosphorylation and for its anti-invasive activity. Pharmacokinetic studies in mice revealed that WFA reaches peak concentrations up to 2 μM in plasma with a half-life of 1.36 hr following a single 4 mg/kg dose. In a breast cancer metastasis mouse model, WFA showed dose-dependent inhibition of metastatic lung nodules and induced vimentin ser56 phosphorylation, with minimal toxicity to lung tissue. Based upon these studies, we conclude that WFA is a potent breast cancer anti-metastatic agent and the anti-metastatic activity of WFA is, at least in part, mediated through its effects on vimentin and vimentin ser56 phosphorylation.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Movement; Female; Humans; Liver Neoplasms; Lung Neoplasms; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serine; Vimentin; Withanolides

The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown.. Herein, we isolated the main pure compound, 4beta-Hydroxywithanolide (4betaHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug.. It was shown that DNA damage was significantly induced by 1, 5, and 10 microg/mL 4betaHWE for 2 h in a dose-dependent manner (p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4betaHWE in both dose- and time-dependent manners (p < 0.05 and 0.001 for 24 and 48 h, respectively). The half maximal inhibitory concentrations (IC50) of 4betaHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 microg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4betaHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 microg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 microg/mL for 24 h.. In this study, we demonstrated that golden berry-derived 4betaHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer.

Topics: Antineoplastic Agents; Apoptosis; Cell Division; Cell Line, Tumor; Cell Nucleus; Comet Assay; DNA Damage; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; G2 Phase; Humans; Inhibitory Concentration 50; Lung Neoplasms; Physalis; Plant Extracts; Withanolides

Phytochemical studies on the aerial parts of Withania somnifera L. Dunal. (Solanaceae) led to the isolation of a chlorinated steroidal lactone (27-acetoxy-4β,6α-dihydroxy-5β-chloro-1-oxowitha-2,24-dienolide), a diepoxy withanolide (5β,6β,14α,15α-diepoxy-4β,27-dihydroxy-1-oxowitha-2,24-dienolide), and withaferin A. Their structures were elucidated by using spectroscopic techniques. All three compounds exhibited a growth inhibition and cytotoxic activity against human lung cancer cell line (NCI-H460), with withaferin A being the most potent (GI(50)=0.18 μg/mL and LC(50)=0.45 μg/mL) among three compounds tested.

Topics: Antineoplastic Agents, Phytogenic; Drug Screening Assays, Antitumor; Humans; Hydrocarbons, Chlorinated; Lung Neoplasms; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Pakistan; Plants, Medicinal; Stereoisomerism; Withania; Withanolides