Joomin Lee

Withaferin A inhibits activation of signal transducer and activator of transcription 3 in human breast cancer cells

We have shown previously that withaferin A (WA), a promising anticancer constituent of Ayurvedic medicine plant Withania somnifera, inhibits growth of human breast cancer cells in culture and in vivo in association with apoptosis induction. The present study builds on these observations and demonstrates that WA inhibits constitutive as well as interleukin-6 (IL-6)-inducible activation of signal transducer and activator of transcription 3 (STAT3), which is an oncogenic transcription factor activated in many human malignancies including breast cancer. The WA treatment (2 and 4 μM) decreased constitutive (MDA-MB-231) and/or IL-6-inducible (MDA-MB-231 and MCF-7) phosphorylation of STAT3 (Tyr(705)) and its upstream regulator Janus-activated kinase 2 (JAK2; Tyr(1007/1008)) in MDA-MB-231, which was accompanied by suppression of their protein levels especially at the higher concentration. Exposure of MDA-MB-231 or MCF-7 cells to WA also resulted in suppression of (i) transcriptional activity of STAT3 with or without IL-6 stimulation in both cells; (ii) dimerization of STAT3 (MDA-MB-231) and (iii) nuclear translocation of Tyr(705)-phosphorylated STAT3 in both cells. To our surprise, the IL-6-stimulation, either before or after WA treatment, did not have an appreciable effect on WA-mediated apoptosis in MDA-MB-231 or MCF-7 cell line. The IL-6-stimulated activation of STAT3 conferred a modest protection against WA-mediated suppression of MDA-MB-231 cell invasion. General implication of these findings is that WA can trigger apoptosis and largely inhibit cell migration/invasion of breast cancer cells even after IL-6-induced activation of STAT3, which should be viewed as a therapeutic advantage for this agent.

Growth Arrest by the Antitumor Steroidal Lactone Withaferin A in Human Breast Cancer Cells Is Associated with Down-regulation and Covalent Binding at Cysteine 303 of β-Tubulin

Background: The tubulin microtubule network remains an attractive anticancer target. Results: The antitumor steroidal lactone withaferin A (WA) down-regulates tubulin and binds to Cys303 of β-tubulin. Conclusion: Tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. Significance: Favorable safety and pharmacokinetic profiles merit clinical investigation of WA for prevention and/or treatment of breast cancer. Withaferin A (WA), a C5,C6-epoxy steroidal lactone derived from a medicinal plant (Withania somnifera), inhibits growth of human breast cancer cells in vitro and in vivo and prevents mammary cancer development in a transgenic mouse model. However, the mechanisms underlying the anticancer effect of WA are not fully understood. Herein, we report that tubulin is a novel target of WA-mediated growth arrest in human breast cancer cells. The G2 and mitotic arrest resulting from WA exposure in MCF-7, SUM159, and SK-BR-3 cells was associated with a marked decrease in protein levels of β-tubulin. These effects were not observed with the naturally occurring C6,C7-epoxy analogs of WA (withanone and withanolide A). A non-tumorigenic normal mammary epithelial cell line (MCF-10A) was markedly more resistant to mitotic arrest by WA compared with breast cancer cells. Vehicle-treated control cells exhibited a normal bipolar spindle with chromosomes aligned along the metaphase plate. In contrast, WA treatment led to a severe disruption of normal spindle morphology. NMR analyses revealed that the A-ring enone in WA, but not in withanone or withanolide A, was highly reactive with cysteamine and rapidly succumbed to irreversible nucleophilic addition. Mass spectrometry demonstrated direct covalent binding of WA to Cys303 of β-tubulin in MCF-7 cells. Molecular docking indicated that the WA-binding pocket is located on the surface of β-tubulin and characterized by a hydrophobic floor, a hydrophobic wall, and a charge-balanced hydrophilic entrance. These results provide novel insights into the mechanism of growth arrest by WA in breast cancer cells.

Critical role for reactive oxygen species in apoptosis induction and cell migration inhibition by diallyl trisulfide, a cancer chemopreventive component of garlic

Diallyl trisulfide (DATS) is a structurally simple but biologically active constituent of processed garlic with in vivo activity against chemically induced as well as oncogene-driven cancer in experimental rodents. This study offers novel insights into the mechanisms underlying anticancer effects of DATS using human breast cancer cells as a model. Exposure of human breast cancer cells (MCF-7 and MDA-MB-231) and a cell line derived from spontaneously developing mammary tumor of a transgenic mouse (BRI-JM04) to DATS resulted in a dose-dependent inhibition of cell viability that was accompanied by apoptosis induction. A non-tumorigenic normal human mammary cell line (MCF-10A) was resistant to growth inhibition and apoptosis induction by DATS. The DATS-induced apoptosis in MDA-MB-231, MCF-7, and BRI-JM04 cells was associated with reactive oxygen species (ROS) production as evidenced by fluorescence microscopy and flow cytometry using a chemical probe (MitoSOX Red). Overexpression of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) as well as Mn-SOD conferred significant protection against DATS-induced ROS production and apoptotic cell death in MDA-MB-231 and MCF-7 cells. Activation of Bak, but not Bax, resulting from DATS treatment was markedly suppressed by overexpression of Mn-SOD. The DATS treatment caused ROS generation, but not activation of Bax or Bak, in MCF-10A cells. Furthermore, the DATS-mediated inhibition of cell migration was partially but significantly attenuated by Cu,Zn-SOD and Mn-SOD overexpression in association with changes in levels of proteins involved in epithelial–mesenchymal transition. The DATS-mediated induction of heme oxygenase-1 was partially attenuated by overexpression of Mn-SOD. These results provide novel mechanistic insights indicating a critical role for ROS in anticancer effects of DATS.

Withaferin A inhibits experimental epithelial-mesenchymal transition in MCF-10A cells and suppresses vimentin protein level in vivo in breast tumors.

We have shown previously that withaferin A (WA), a bioactive component of the medicinal plant Withania somnifera, inhibits growth of cultured and xenografted human breast cancer cells and prevents breast cancer development and pulmonary metastasis incidence in a transgenic mouse model. The present study was undertaken to determine if the anticancer effect of WA involved inhibition of epithelial–mesenchymal transition (EMT). Experimental EMT induced by exposure of MCF‐10A cells to tumor necrosis factor‐α (TNF‐α) and transforming growth factor‐β1 (TGF‐β) was partially reversed by treatment with WA but not by its structural analogs withanone or withanolide A. Combined TNF‐α and TGF‐β treatments conferred partial protection against MCF‐10A cell migration inhibition by WA. Inhibition of TNF‐α and TGF‐β‐induced MCF‐10A cell migration by WA exposure was modestly attenuated by knockdown of E‐cadherin protein. MCF‐7 and MDA‐MB‐231 cells exposed to WA exhibited sustained (MCF‐7) or transient (MDA‐MB‐231) induction of E‐cadherin protein. On the other hand, the level of vimentin protein was increased markedly after 24 h treatment of MDA‐MB‐231 cells with WA. WA‐induced apoptosis was not affected by vimentin protein knockdown in MDA‐MB‐231 cells. Protein level of vimentin was significantly lower in the MDA‐MB‐231 xenografts as well as in MMTV‐neu tumors from WA‐treated mice compared with controls. The major conclusions of the present study are that (a) WA treatment inhibits experimental EMT in MCF‐10A cells, and (b) mammary cancer growth inhibition by WA administration is associated with suppression of vimentin protein expression in vivo.

Metabolic Alterations in Mammary Cancer Prevention by Withaferin A in a Clinically Relevant Mouse Model

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS This study reveals suppression of glycolysis in WA-mediated mammary cancer prevention in a clinically relevant mouse model.

Withaferin A causes activation of Notch2 and Notch4 in human breast cancer cells

Ayurvedic medicine plants continue to draw attention for the discovery of novel anticancer agents. Withaferin A (WA) is one such small-molecule constituent of the ayurvedic medicine plant Withania somnifera with efficacy against cultured and xenografted human breast cancer cells. However, the mechanism underlying anticancer effect of WA is not fully understood. This study was undertaken to determine the role of Notch signaling in anticancer effects of WA using human breast cancer cells as a model. Notably, Notch signaling is often hyperactive in human breast cancers. Exposure of MDA-MB-231 and MCF-7 human breast cancer cells to pharmacological concentrations of WA resulted in cleavage (activation) of Notch2 as well as Notch4, which was accompanied by transcriptional activation of Notch as evidenced by RBP-Jk, HES-1A/B, and HEY-1 luciferase reporter assays. On the other hand, WA treatment caused a decrease in levels of both transmembrane and cleaved Notch1. The WA-mediated activation of Notch was associated with induction of γ-secretase complex components presenilin1 and/or nicastrin. Inhibition of MDA-MB-231 and MDA-MB-468 cell migration resulting from WA exposure was significantly augmented by knockdown of Notch2 as well as Notch4 protein. Activation of Notch2 was not observed in cells treated with withanone or withanolide A, which are structural analogs of WA. The results of this study indicate that WA treatment activates Notch2 and Notch4, which impede inhibitory effect of WA on breast cancer cell migration.

Withaferin a suppresses estrogen receptor‐α expression in human breast cancer cells

We have shown previously that withaferin A (WA), a promising anticancer constituent of Ayurvedic medicine plant Withania somnifera, inhibits growth of MCF‐7 and MDA‐MB‐231 human breast cancer cells in culture and MDA‐MB‐231 xenografts in vivo by causing apoptosis. However, the mechanism of WA‐induced apoptosis is not fully understood. The present study was designed to systematically determine the role of tumor suppressor p53 and estrogen receptor‐α (ER‐α) in proapoptotic response to WA using MCF‐7, T47D, and ER‐α overexpressing MDA‐MB‐231 cells as a model. WA treatment resulted in induction as well as increased S15 phosphorylation of p53 in MCF‐7 cells, but RNA interference of this tumor suppressor conferred modest protection at best against WA‐induced apoptosis. WA‐mediated growth inhibition and apoptosis induction in MCF‐7 cells were significantly attenuated in the presence of 17β‐estradiol (E2). Exposure of MCF‐7 cells to WA resulted in a marked decrease in protein levels of ER‐α (but not ER‐β) and ER‐α regulated gene product pS2, and this effect was markedly attenuated in the presence of E2. WA‐mediated down‐regulation of ER‐α protein expression correlated with a decrease in its nuclear level, suppression of its mRNA level, and inhibition of E2‐dependent activation of ERE2e1b‐luciferase reporter gene. Ectopic expression of ER‐α in the MDA‐MB‐231 cell line conferred partial but statistically significant protection against WA‐mediated apoptosis, but not G2/M phase cell cycle arrest. Collectively, these results indicate that WA functions as an anti‐estrogen, and the proapoptotic effect of this promising natural product is partially attenuated by p53 knockdown and E2‐ER‐α.

Role of Mitogen-Activated Protein Kinases and Mcl-1 in Apoptosis Induction by Withaferin A in Human Breast Cancer Cells

Withaferin A (WA), a bioactive constituent of Ayurvedic medicine plant Withania somnifera, is a potent apoptosis inducer in cancer cells but the mechanism of cell death induction is not fully characterized. The present study was undertaken to determine the role of mitogen‐activated protein kinases (MAPK), including c‐jun NH2‐terminal kinase (JNK), extracellular signal‐regulated kinase (ERK), and p38 MAPK, and anti‐apoptotic protein myeloid cell leukemia‐1 (Mcl‐1) in regulation of WA‐induced apoptosis using human breast cancer cells. Exposure of MCF‐7 (estrogen responsive) and SUM159 (triple negative) human breast cancer cells to WA resulted in increased phosphorylation of ERK, JNK, and p38 MAPK, but these effects were relatively more pronounced in the former cell line than in SUM159. Overexpression of manganese‐superoxide dismutase conferred partial protection against WA‐mediated hyperphosphorylation of ERK, but not JNK or p38 MAPK. Cell death resulting from WA treatment in MCF‐7 cells was significantly augmented by pharmacological inhibition of ERK and p38 MAPK. Interestingly, the WA‐induced apoptosis in MCF‐7 cells was partially but significantly blocked in the presence of a JNK‐specific inhibitor. Pharmacological inhibition of ERK or JNK had no effect on WA‐induced apoptosis in SUM159 cells. The WA‐treated cells exhibited induction of long and short forms of Mcl‐1. RNA interference of Mcl‐1 alone triggered apoptosis. Furthermore, the WA‐induced cell death in MCF‐7 cells was modestly but significantly augmented by knockdown of the Mcl‐1 protein. These observations indicate that: MAPK have cell line‐specific role in cell death by WA, and Mcl‐1 induction confers modest protection against WA‐induced apoptosis.

Biomarkers of Phenethyl Isothiocyanate-Mediated Mammary Cancer Chemoprevention in a Clinically Relevant Mouse Model

BACKGROUND Phenethyl isothiocyanate (PEITC) is a natural plant compound with chemopreventative potential against some cancers and the ability to induce apoptosis in breast cancer cells. METHODS Female mouse mammary tumor virus-neu mice were fed a control AIN-76A diet (n = 35) or the same diet supplemented with 3 µmol PEITC/g diet (n = 33) for 29 weeks, at which time they were killed. Breast tissue sections were stained with hematoxylin and eosin for histopathological assessments, and incidence and size of macroscopic mammary tumors were assessed. Cell proliferation (Ki-67 staining), apoptosis (terminal deoxynucleotidyl transferase-mediated dUTP nick-labeling), and neoangiogenesis (CD31 staining) were determined in tumor sections. Plasma levels of transthyretin were measured in treated and control mice. Expression of proteins in mammary tumor sections was determined by immunohistochemistry. Proteomic profiling was performed by two-dimensional gel electrophoresis followed by mass spectrometry. All statistical tests were two-sided. RESULTS Administration of PEITC for 29 weeks was associated with 53.13% decreased incidence of macroscopic mammary tumors (mean tumor incidence, PEITC-supplemented diet vs control diet, 18.75% vs 40.00%, difference = -21.25%, 95% confidence interval [CI] = -43.19% to 0.69%, P = .07) and with a 56.25% reduction in microscopic mammary carcinoma lesions greater than 2 mm(2) (mean incidence, PEITC-supplemented diet vs control diet, 18.75% vs 42.86%, difference = -24.11%, 95% CI = -46.35% to -1.86%, P = .04). PEITC-mediated mammary cancer growth inhibition was not because of suppression of human epidermal growth factor receptor-2 expression but was associated with reduced cellular proliferation and neoangiogenesis, increased apoptosis, and altered expression of several proteins, including decreased ATP synthase in the tumor and increased plasma levels of transthyretin. CONCLUSIONS PEITC inhibits the growth of mammary cancers in a mouse model with similarities to human breast cancer progression. ATP synthase and transthyretin appear to be novel biomarkers associated with PEITC exposure.

Differential Effects of Phenethyl Isothiocyanate and D,L-Sulforaphane on TLR3 Signaling

Naturally occurring isothiocyanates (ITCs) from cruciferous vegetables are widely studied for their cancer chemopreventive effects. In this study, we investigated the effects of ITCs on TLR signaling, and found that the two most promising ITCs, phenethyl ITCs (PEITC) and D,L-sulforaphane (SFN), have differential effects on dsRNA-mediated innate immune signaling through TLR3. PEITC preferentially inhibited TLR3-mediated IFN regulatory factor 3 (IRF3) signaling and downstream gene expression in vivo and in vitro, whereas SFN caused inhibition of TLR3-mediated NF-κB signaling and downstream gene expression. Mechanistically, PEITC inhibited ligand (dsRNA)-dependent dimerization of TLR3, resulting in inhibition of signaling through IFN regulatory factor 3. In contrast, SFN did not disrupt TLR3 dimerization, indicating that it affects further downstream pathway resulting in NF-κB inhibition. To examine the biological significance of these findings in the context of antitumor activities of these compounds, we used two approaches: first, we showed that dsRNA-mediated apoptosis of tumor cells via TLR3 was inhibited in the presence of PEITC, whereas this response was augmented by SFN treatment; second, in a separate assay measuring anchorage-independent growth and colony formation by immortalized fibroblasts, we made similar observations. Again in this study, PEITC antagonized dsRNA-mediated inhibition of colony formation, whereas SFN enhanced the inhibition. These results indicate biologically relevant functional differences between two structurally similar ITCs and may provide important insights in therapeutic development of these compounds targeted to specific cancer.