Search

Rui Kuai,1,2,* Chitra Subramanian,3,* Peter T White,3,* Barbara N Timmermann,4 James J Moon,1,2,5 Mark S Cohen,3,6 Anna Schwendeman1,2 1Department of Pharmaceutical Sciences, College of Pharmacy, 2Biointerfaces Institute, University of Michigan, 3Department of Surgery, University of Michigan, Ann Arbor, MI, 4Department of Medicinal Chemistry, University of Kansas, Lawrence, KS, 5Department of Biomedical Engineering, 6Department of Pharmacology, University of Michigan, Ann Arbor, MI, USA *These authors contributed equally to this work Abstract: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy and has a 5-year survival rate of

OBJECTIVE: Scientific evidence is lacking for the antiarthritic efficacy of turmeric dietary supplements that are being promoted for arthritis treatment. Therefore, we undertook studies to determine the antiarthritic efficacy and mechanism of action of a well-characterized turmeric extract using an animal model of rheumatoid arthritis (RA). METHODS: The composition of commercial turmeric dietary supplements was determined by high-performance liquid chromatography. A curcuminoid-containing turmeric extract similar in composition to these supplements was isolated and administered intraperitoneally to female Lewis rats prior to or after the onset of streptococcal cell wall-induced arthritis. Efficacy in preventing joint swelling and destruction was determined clinically, histologically, and by measurement of bone mineral density. Mechanism of action was elucidated by analysis of turmeric's effect on articular transcription factor activation, microarray analysis of articular gene expression, and verification of the physiologic effects of alterations in gene expression. RESULTS: A turmeric fraction depleted of essential oils profoundly inhibited joint inflammation and periarticular joint destruction in a dose-dependent manner. In vivo treatment prevented local activation of NF-kappaB and the subsequent expression of NF-kappaB-regulated genes mediating joint inflammation and destruction, including chemokines, cyclooxygenase 2, and RANKL. Consistent with these findings, inflammatory cell influx, joint levels of prostaglandin E(2), and periarticular osteoclast formation were inhibited by turmeric extract treatment. CONCLUSION: These translational studies demonstrate in vivo efficacy and identify a mechanism of action for a well-characterized turmeric extract that supports further clinical evaluation of turmeric dietary supplements in the treatment of RA. [ABSTRACT FROM AUTHOR]

Green tea polyphenols have been shown to inhibit cancer in a variety of tumor models, including ultraviolet B (UVB)-induced non-melanoma skin cancer. In green tea extracts, the major dry mass constituent is the family of catechins, of which (-)-epigallocatechin-(3)-gallate (EGCG) is considered to be important for the chemopreventive activity. EGCG has been shown to hae antioxidant properties, but there has been little progress toward identifying the specific targets and mechanisms of its action. Using cultured human keratinocytes, we show that UVB-induced AP-1 activity is inhibited by EGCG in a dose range of 4.45 nM to 54.5 μM. EGCG is effective at inhibiting AP-1 activity when applied before, after or both before and after UVB irradiation. EGCG also inhibits AP-1 activity in the epidermis of a transgenic mouse model. This work begins to define a mechanism by which EGCG could be acting to inhibit UVB-induced tumor formation. [ABSTRACT FROM PUBLISHER]

Treatment regimens for head and neck squamous cell carcinoma (HNSCC) typically include cisplatin and radiotherapy and are limited by toxicities. We have identified naturally derived withalongolide A triacetate (WGA-TA) from Physalis longifolia as a lead compound for targeting HNSCC. We hypothesized that combining WGA-TA with cisplatin may allow for lower, less toxic cisplatin doses. HNSCC cell lines were treated with WGA-TA and cisplatin. After treatment with the drugs, the cell viability was determined by MTS assay. The combination index was calculated using CompuSyn. The expression of proteins involved in the targeting of translational initiation complex, epithelial to mesenchymal transition (EMT), and apoptosis were measured by western blot. Invasion and migration were measured using the Boyden-chamber assay. Treatment of MDA-1986 and UMSCC-22B cell lines with either WGA-TA or cisplatin alone for 72 h resulted in a dose dependent decrease in cell viability. Cisplatin in combination with WGA-TA resulted in significant synergistic cell death starting from 1.25 μM cisplatin. Combination treatment with WGA-TA resulted in lower cisplatin dosing while maintaining the downregulation of translational initiation complex proteins, the induction of apoptosis, and the blockade of migration, invasion, and EMT transition. These results suggest that combining a low concentration of cisplatin with WGA-TA may provide a safer, more effective therapeutic option for HNSCC that warrants translational validation.

Melanoma remains the most aggressive and fatal form of skin cancer, despite several FDA-approved targeted chemotherapies and immunotherapies for use in advanced disease. Of the 100 350 new patients diagnosed with melanoma in 2020 in the US, more than half will develop metastatic disease leading to a 5-year survival rate <30%, with a majority of these developing drug-resistance within the first year of treatment. These statistics underscore the critical need in the field to develop more durable therapeutics as well as those that can overcome chemotherapy-induced drug resistance from currently approved agents. Fortunately, several of the drug-resistance pathways in melanoma, including the proteins in those pathways, rely in part on Hsp90 chaperone function. This presents a unique and novel opportunity to simultaneously target multiple proteins and drug-resistant pathways in this disease via molecular chaperone inhibition. Taken together, we hypothesize that our novel C-terminal Hsp90 inhibitor, KU758, in combination with the current standard of care targeted therapies (e.g. vemurafenib and cobimetinib) can both synergize melanoma treatment efficacy in BRAF-mutant tumors, as well as target and overcome several major resistance pathways in this disease. Using in vitro proliferation and protein-based Western Blot analyses, our novel inhibitor, KU758, potently inhibited melanoma cell proliferation (without induction of the heat shock response) in vitro and synergized with both BRAF and MEK inhibitors in inhibition of cell migration and protein expression from resistance pathways. Overall, our work provides early support for further translation of C-terminal Hsp90 inhibitor and mitogen-activated protein kinase pathway inhibitor combinations as a novel therapeutic strategy for BRAF-mutant melanomas., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Withanolides are naturally derived heat shock protein 90 inhibitors that are potent in preclinical models of triple negative breast cancers. Conjugation to synthetic high-density lipoprotein nanoparticles improves solubility and targets delivery to the scavenger receptor B1. Triple negative breast cancers highly overexpress the scavenger receptor B1, and we hypothesize that encapsulation of the novel withalongolide A 4,19,27-triacetate by synthetic high-density lipoprotein will have enhanced efficacy against triple negative breast cancers in vivo., Methods: Validated human triple negative breast cancer cell lines were evaluated for the scavenger receptor B1 expression by quantitative polymerase chain reaction and Western blot. Withalongolide A 4,19,27-triacetate inhibitory concentration 50 values were obtained using CellTiter-Glo assays (Promega, Madison, WI, USA). The scavenger receptor B1-mediated drug uptake was evaluated in vitro under fluorescence microscopy and in vivo with IVIS imaging of mouse xenografts (MD-MBA-468LN). To evaluate drug efficacy, mice were treated with synthetic high-density lipoprotein alone, withalongolide A 4,19,27-triacetate alone, withalongolide A 4,19,27-triacetate synthetic high-density lipoprotein, and chemotherapy or Prussian blue stain (control)., Results: Triple negative breast cancer cell lines had greater scavenger receptor B1 expression by quantitative polymerase chain reaction and Western blot versus controls. Fluorescent-labeled synthetic high-density lipoprotein uptake was scavenger receptor B1-mediated in vitro, and in vivo tumor uptake using IVIS imaging demonstrated significantly increased tumor radiant efficiency versus control. Inhibitory concentration 50 for withalongolide A 4,19,27-triacetate-treated cells with or without synthetic high-density lipoprotein encapsulation were 70-fold to 200-fold more potent than synthetic high-density lipoprotein alone. In triple negative breast cancer mouse xenografts, treatment with synthetic high-density lipoprotein withalongolide A 4,19,27-triacetate resulted in a 54% decrease in tumor volume compared with the control or with synthetic high-density lipoprotein alone., Conclusion: The synthetic high-density lipoprotein withalongolide A 4,19,27-triacetate nanoconjugates are potent against triple negative breast cancers and show improved scavenger receptor B1-mediated targeting. Treatment with synthetic high-density lipoprotein-encapsulated withalongolide A 4,19,27-triacetate is able to significantly decrease the growth of tumor in mice compared with the control and has better efficacy than the current standard of care, warranting further evaluation as a novel therapeutic agent., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mimics should not exist without their models, yet often they do. In the system involving queen and viceroy butterflies, the viceroy is both mimic and co-model depending on the local abundance of the model, the queen. Here, we integrate population surveys, chemical analyses, and predator behavior assays to demonstrate how mimics may persist in locations with low-model abundance. As the queen becomes less locally abundant, the viceroy becomes more chemically defended and unpalatable to predators. However, the observed changes in viceroy chemical defense and palatability are not attributable to differing host plant chemical defense profiles. Our results suggest that mimetic viceroy populations are maintained at localities of low-model abundance through an increase in their toxicity. Sharing the burden of predator education in some places but not others may also lower the fitness cost of warning signals thereby supporting the origin and maintenance of aposematism., Competing Interests: The authors declare no competing interests.

We report relative bioactivities of extracts prepared from a large collection of plants from three national parks in Panama. Over 181 plants were collected, taxonomically identified and their detannified dichloromethane (DCM)-methanolic extracts were used for profiling selected bioactivities. Assays were performed to evaluate the antioxidant activity of the extracts for Antioxidant Response Element (ARE) induction, total non-enzymatic antioxidant potential, anti-inflammatory and anticancer properties. The high throughput analysis of 280 extracts resulted in identification of 57.5% of the extracts that could induce ARE at one or more concentrations tested, 93.5% that harbored total antioxidant capacity, and 2.1% of the extracts that showed lung cancer cell line-specific cytotoxicity. Data from our profiling experiments indicate that a large number of extracts could be a source for further isolation and chemical identification of compounds that could serve as leads for discovery of antioxidant, anticancer and anti-inflammatory agents to prevent or treat complex diseases like cancer and neurodegenerative disorders.

A major prediction of coevolutionary theory is that plants may target particular herbivores with secondary compounds that are selectively defensive. The highly specialized monarch butterfly ( Danaus plexippus ) copes well with cardiac glycosides (inhibitors of animal Na + /K + -ATPases) from its milkweed host plants, but selective inhibition of its Na + /K + -ATPase by different compounds has not been previously tested. We applied 17 cardiac glycosides to the D. plexippus -Na + /K + -ATPase and to the more susceptible Na + /K + -ATPases of two non-adapted insects ( Euploea core and Schistocerca gregaria ). Structural features (e.g., sugar residues) predicted in vitro inhibitory activity and comparison of insect Na + /K + -ATPases revealed that the monarch has evolved a highly resistant enzyme overall. Nonetheless, we found evidence for relative selectivity of individual cardiac glycosides reaching from 4- to 94-fold differences of inhibition between non-adapted Na + /K + -ATPase and D. plexippus -Na + /K + -ATPase. This toxin receptor specificity suggests a mechanism how plants could target herbivores selectively and thus provides a strong basis for pairwise coevolutionary interactions between plants and herbivorous insects.

Background: Pathways critical for neuroblastoma cancer stem cell function are targeted by 4,19,27-triacetyl withalongolide A (WGA-TA). Because neuroblastoma cells and their cancer stem cells highly overexpress the scavenger receptor class B type 1 receptor that binds to synthetic high-density lipoprotein, we hypothesized that a novel mimetic synthetic high-density lipoprotein nanoparticle would be an ideal carrier for the delivery of 4,19,27-triacetyl withalongolide to neuroblastoma and neuroblastoma cancer stem cells., Methods: Expression of scavenger receptor class B type 1 in validated human neuroblastoma cells was evaluated by quantitative polymerase chain reaction (qPCR) and Western blot. In vitro cellular uptake of synthetic high-density lipoprotein nanoparticles was observed with a fluorescence microscope. In vivo biodistribution of synthetic high-density lipoprotein nanoparticles was investigated with IVIS imaging. Self-renewal and migration/invasion were assessed by sphere formation and Boyden chamber assays, respectively. Viability was analyzed by CellTiter-Glo assay. Cancer stem cell markers were evaluated by flow cytometry., Results: qPCR and Western blot analysis revealed a higher level of scavenger receptor class B type 1 expression and drug uptake in N-myc amplified neuroblastoma cells. In vitro uptake of synthetic high-density lipoprotein was almost completely blocked by excess synthetic high-density lipoprotein. The synthetic high-density lipoprotein nanoparticles mainly accumulated in the tumor and liver, but not in other organs. Synthetic HDL-4,19,27-triacetyl withalongolide showed a 1,000-fold higher potency than the carrier (synthetic high-density lipoprotein) alone (P < .01) to kill neuroblastoma cells. Additionally, a dose-dependent decrease in sphere formation, invasion, migration, and cancer stem cell markers was observed after treatment of neuroblastoma cells with synthetic high-density lipoprotein-4,19,27-triacetyl withalongolide A., Conclusion: Synthetic high-density lipoprotein is a promising platform to improve the delivery of anticancer drug 4,19,27-triacetyl withalongolide A to neuroblastomas and neuroblastoma cancer stem cells through SR-B1 targeting in vitro and in vivo., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Despite recent advances in intensive chemotherapy treatments, long-term success is achieved in less than 30% of children with high-risk neuroblastoma (NB). Key regulatory pathways including the PI3K/Akt, mTOR and NF-κB are implicated in the pathogenesis of NB. Although drugs targeting these individual pathways are in clinical trials, they are not effective due to the activation of compensatory mechanisms. We have previously reported that natural novel withanolides from Physalis longifolia can potently inhibit these key regulatory pathways simultaneously. In the present study, we examined the efficacy and mechanisms through which novel withanolides and their acetate derivatives (WGA-TA and WGB-DA) from P.longifolia kill NB cells. The results from the study demonstrated that our novel acetate derivatives are highly effective in inhibiting the proliferation, shifting the cell cycle and inducing apoptosis in a dose dependent manner. Analysis of oncogenic pathway proteins targeted by withanolides indicated induction of heat shock response due to oxidative stress. Dose dependent decrease in clients of HSP90 chaperone function due to suppression of Akt, mTOR, and NF-κB pathways led to decrease in the expressions of target genes such as cyclin D1, N-myc and Survivin. Additionally, there was a dose dependent attenuation of the migration and invasion of NB cells. Furthermore, the lead compound WGA-TA showed significant reduction in tumor growth of NB xenografts. Taken together, these results suggest that withanolides are an effective therapeutic option against NBs., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Ginger and its extracts have been used traditionally as anti-inflammatory remedies, with a particular focus on the medicinal properties of its phenolic secondary metabolites, the gingerols. Consistent with these uses, potent anti-arthritic effects of gingerol-containing extracts were previously demonstrated by our laboratory using an experimental model of rheumatoid arthritis, streptococcal cell wall (SCW)-induced arthritis. In this study, anti-inflammatory effects of ginger's other secondary metabolites, the essential oils (GEO), which contain terpenes with reported phytoestrogenic activity, were assessed in female Lewis rats with SCW-induced arthritis. GEO (28 mg/kg/d ip) prevented chronic joint inflammation, but altered neither the initial acute phase of joint swelling nor granuloma formation at sites of SCW deposition in liver. Pharmacologic doses of 17-β estradiol (200 or 600 μg/kg/d sc) elicited the same pattern of anti-inflammatory activity, suggesting that GEO could be acting as a phytoestrogen. However, contrary to this hypothesis, GEO had no in vivo effect on classic estrogen target organs, such as uterus or bone. En toto, these results suggest that ginger's anti-inflammatory properties are not limited to the frequently studied phenolics, but may be attributable to the combined effects of both secondary metabolites, the pungent-tasting gingerols and as well as its aromatic essential oils.

A classic withanolide is defined as a highly oxygenated C28 ergostane-type steroid that is characterized by a C22-hydroxy-C26-oic acid δ-lactone in the nine-carbon side chain. Analysis of the reported (13)C NMR data of classic withanolides with hydroxy groups (C-14, C-17, and C-20) revealed that (1) a hydroxy (C-14 or C-17) substituent significantly alters the chemical shifts (C-7, C-9, C-12, and C-21) via the γ-gauche effect; (2) the chemical shift values (C-9, C-12, and C-21) reflect the orientation (α or β) of the hydroxy moiety (C-14 or C-17); (3) a double-bond positional change in ring A (Δ(2) to Δ(3)), or hydroxylation (C-27), results in a minuscule effect on the chemical shifts of carbons in rings C and D (from C-12 to C-18); and (4) the (13)C NMR γ-gauche effect method is more convenient and reliable than the traditional approach ((1)H NMR shift comparisons in C5D5N versus CDCl3) to probe the orientation of the hydroxy substituent (C-14 and C-17). Utilization of these rules demonstrated that the reported (13)C NMR data of withanolides 1a-29a were inconsistent with their published structures, which were subsequently revised as 1-16 and 12 and 18-29, respectively. When combined, this strongly supports the application of these methods to determine the relative configuration of steroidal substituents.

Background: Thyroid cancer stem cells (CSCs) with ALDH and CD44 markers contribute to tumor growth and aggressiveness. We hypothesized that novel HSP90 inhibitors (KU711, WGA-TA) and 17-AAG can effectively target the function of thyroid CSCs in vitro and prevent migration and invasion., Methods: Validated papillary (TPC1), follicular (FTC238,WRO), and anaplastic (ACT1) human thyroid cancer cell lines were treated with 3 HSP90 inhibitors. CSCs were quantified for aldehyde dehydrogenase by flow cytometry, CD44 expression by Western blot, and thyrosphere formation assay. Cellular pathway proteins were analyzed by Western blot and migration/invasion by Boyden-chambers., Results: WGA-TA and 17-AAG induced HSP70 compensation (not observed with KU711) on Western blot in all cell lines (>1,000 fold vs controls). Only WGA-TA degraded HSP90-Cdc37 complexing by 60-70% versus controls. Expression of HSP90 clients β-catenin, BRAF, Akt, and phospho-Akt were significantly inhibited by WGA-TA treatment (50-80%, 50-90%, >80%, and >90%) compared with controls, KU711, and 17-AAG treatment. KU711 and WGA-TA decreased CD44 expression in all cell lines (25-60% vs controls/17-AAG), decreased ALDEFLOR activity by 69-98% (P < .005), and decreased sphere formation by 64-99% (P < .05 each). Finally, cell migration was decreased by 31-98%, 100%, and 30-38%, and invasion by 75-100%, 100%, and 47% by KU711,WGA-TA, and 17-AAG treatment (P < .05) each, respectively., Conclusion: KU711 and WGA-TA are novel HSP90 inhibitors targeting CSC function and inhibiting cell migration/invasion in differentiated and anaplastic thyroid cancers, warranting further translational evaluation in vivo., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Four withanolides (1-4) and two sucrose esters (5, 6) were isolated from the aerial parts of Physalis neomexicana. The structures of 1-6 were elucidated through a variety of spectroscopic techniques. Cytotoxicity studies of the isolates revealed that 2 inhibited human breast cancer cell lines (MDA-MB-231 and MCF-7) with IC50 values of 1.7 and 6.3 μM, respectively.

An analytical method based on high-performance liquid chromatography-photodiode array detection was developed for the simultaneous determination of three anti-proliferative withanolides [withalongolide A ( 1: ), withaferin A ( 2: ) and withalongolide B ( 3: )] present in the aboveground biomass of the long-leaf groundcherry, Physalis longifolia. This method was achieved by biomass extraction followed by chromatographic separation on C18 column eluted with a gradient acetonitrile-water mobile phase. Calibration curves produced satisfactory linear regression (r(2) > 0.9995) for each examined sample. The method was also validated for accuracy, precision and limits of detection and quantification. Such an approach is applicable for the rapid detection and quantitative assessment of withanolides in various P. longifolia accessions., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Temozolomide (TMZ) has remained the chemotherapy of choice in patients with glioblastoma multiforme (GBM) primarily due to the lack of more effective drugs. Tumors, however, quickly develop resistance to this line of treatment creating a critical need for alternative approaches and strategies to resensitize the cells. Withaferin A (WA), a steroidal lactone derived from several genera of the Solanaceae plant family has previously demonstrated potent anti-cancer activity in multiple tumor models. Here, we examine the effects of WA against TMZ-resistant GBM cells as a monotherapy and in combination with TMZ. WA prevented GBM cell proliferation by dose-dependent G2/M cell cycle arrest and cell death through both intrinsic and extrinsic apoptotic pathways. This effect correlated with depletion of principle proteins of the Akt/mTOR and MAPK survival and proliferation pathways with diminished phosphorylation of Akt, mTOR, and p70 S6K but compensatory activation of ERK1/2. Depletion of tyrosine kinase cell surface receptors c-Met, EGFR, and Her2 was also observed. WA demonstrated induction of N-acetyl-L-cysteine-repressible oxidative stress as measured directly and through a subsequent heat shock response with HSP32 and HSP70 upregulation and decreased HSF1. Finally, pretreatment of TMZ-resistant GBM cells with WA was associated with O6-methylguanine-DNA methyltransferase (MGMT) depletion which potentiated TMZ-mediated MGMT degradation. Combination treatment with both WA and TMZ resulted in resensitization of MGMT-mediated TMZ-resistance but not resistance through mismatch repair mutations. These studies suggest great clinical potential for the utilization of WA in TMZ-resistant GBM as both a monotherapy and a resensitizer in combination with the standard chemotherapeutic agent TMZ.

Nine new withanolides (1-9), withahisolides A-I, were isolated along with nine known compounds (10-18) from the aerial parts of Physalis hispida. The structures of 1-9 were elucidated through a variety of spectroscopic techniques, while the structures of 1 and 2 were confirmed by X-ray crystallographic analysis. Compounds 1-3 are the first withanolides with nonaromatic six-membered ring D moieties. In addition, withanolide 8 represents a novel withanolide skeleton due to the absence of a C-13-C-17 bond within the steroidal nucleus.

Withanolides 2,3-dihydrotrechonolide A (1) and 2,3-dihydro-21-hydroxytrechonolide A (2) were isolated along with two known withanolides trechonolide A (3) and jaborosalactone 39 (4) from Jaborosa caulescens var. bipinnatifida (Solanaceae). The structures of 1-2 were elucidated through 2D NMR and other spectroscopic techniques. In addition, the structure of withanolide 1 was confirmed by X-ray crystallographic analysis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

To date, our work on solanaceous species (Datura wrightii, Jaborosa caulescens, Physalis hispida, Physalis longifolia, Vassobia breviflora and Withania somnifera) has resulted in the isolation of 65 withanolides, 31 of which were new, as well as the semi-synthesis of a further 30 withanolides. Structure identification and MTS assay-based antiproliferative evaluation of these 95 compounds revealed that a Δ(2)-1-oxo functionality in ring A, in conjunction with either a 5β,6β-epoxy or 5α-chloro-6β-hydroxy moiety in ring B, is the minimum structural requirement for withanolides to produce potent cytotoxic activity. Such structure-activity relationship analysis also revealed that oxygenation (the -OH or -OR groups) at C-4, 7, 11 and 12, as well as C-14 to C-28, did not contribute towards the observed antiproliferative activity. Herein, we present a complete overview of our work as it relates to the withanolides reported from 1965 to 2013.

Methanol solutions of the main withanolides (6-8) naturally present in Physalis longifolia yielded five artificial withanolides (1-5), including three new compounds (1-3). Withanolides 1 and 2 were identified as intramolecular Michael addition derivatives, while withanolides 3-5 were the result of intermolecular Michael addition. A comprehensive literature investigation was conducted to identify potential withanolide Michael addition artifacts isolated from Solanaceous species to date.

The natural product withaferin A exhibits potent antitumor activity and other diverse pharmacological activities. The recently discovered withalongolide A, a C-19 hydroxylated congener of withaferin A, was recently reported to possess cytotoxic activity against head and neck squamous cell carcinomas. Semisynthetic acetylated analogues of withalongolide A were shown to be considerably more cytotoxic than the parent compound. To further explore the structure-activity relationships, 20 new semisynthetic analogues of withalongolide A were synthesized and evaluated for cytotoxic activity against four different cancer cell lines. A number of derivatives were found to be more potent than the parent compound and withaferin A.

Objective: We sought to test the hypothesis that turmeric-derived curcuminoids limit reperfusion brain injury in an experimental model of stroke via blockade of early microvascular inflammation during reperfusion., Methods: Male Sprague Dawley rats subjected to MCAO/R were treated with turmeric-derived curcuminoids (vs. vehicle) 1 hour prior to reperfusion (300 mg/kg ip). Neutrophil adhesion to the cerebral microcirculation and measures of neutrophil and endothelial activation were assayed during early reperfusion (0-4 hours); cerebral infarct size, edema, and neurological function were assessed at 24 hours. Curcuminoid effects on TNFα-stimulated human brain microvascular endothelial cell (HBMVEC) were assessed., Results: Early during reperfusion following MCAO, curcuminoid treatment decreased neutrophil rolling and adhesion to the cerebrovascular endothelium by 76% and 67% and prevented >50% of the fall in shear rate. The increased number and activation state (CD11b and ROS) of neutrophils were unchanged by curcuminoid treatment, while increased cerebral expression of TNFα and ICAM-1, a marker of endothelial activation, were blocked by >30%. Curcuminoids inhibited NF-κB activation and subsequent ICAM-1 gene expression in HBMVEC., Conclusion: Turmeric-derived curcuminoids limit reperfusion injury in stroke by preventing neutrophil adhesion to the cerebrovascular microcirculation and improving shear rate by targeting the endothelium., (© 2013 John Wiley & Sons Ltd.)

Withaferin A (WA), a steroidal lactone derived from the plant Vassobia breviflora, has been reported to have anti-proliferative, pro-apoptotic, and anti-angiogenic properties against cancer growth. In this study, we identified several key underlying mechanisms of anticancer action of WA in glioblastoma cells. WA was found to inhibit proliferation by inducing a dose-dependent G2/M cell cycle arrest and promoting cell death through both intrinsic and extrinsic apoptotic pathways. This was accompanied by an inhibitory shift in the Akt/mTOR signaling pathway which included diminished expression and/or phosphorylation of Akt, mTOR, p70 S6K, and p85 S6K with increased activation of AMPKα and the tumor suppressor tuberin/TSC2. Alterations in proteins of the MAPK pathway and cell surface receptors like EGFR, Her2/ErbB2, and c-Met were also observed. WA induced an N-acetyl-L-cysteine-repressible enhancement in cellular oxidative potential/stress with subsequent induction of a heat shock stress response primarily through HSP70, HSP32, and HSP27 upregulation and HSF1 downregulation. Taken together, we suggest that WA may represent a promising chemotherapeutic candidate in glioblastoma therapy warranting further translational evaluation.

A new withanolide, named withawrightolide (1), and four known withanolides (2-5) were isolated from the aerial parts of Datura wrightii. The structure of compound 1 was elucidated through 2D NMR and other spectroscopic techniques. In addition, the structure of withametelin L (2) was confirmed by X-ray crystallographic analysis. Using MTS viability assays, withanolides 1-5 showed antiproliferative activities against human glioblastoma (U251 and U87), head and neck squamous cell carcinoma (MDA-1986), and normal fetal lung fibroblast (MRC-5) cells with IC50 values in the range between 0.56 and 5.6 μM.

Effects of curcuminoids on breast cancer cell secretion of the bone-resorptive peptide parathyroid hormone-related protein (PTHrP) and on lytic breast cancer bone metastasis were evaluated. In vitro, transforming growth factor (TGF)-β-stimulated PTHrP secretion was inhibited by curcuminoids (IC50 = 24 μM) in MDA-MB-231 human breast cancer cells independent of effects on cell growth inhibition. Effects on TGF-β signaling revealed decreases in phospho-Smad2/3 and Ets-1 protein levels with no effect on p-38 MAPK-mediated TGF-β signaling. In vivo, mice were inoculated with MDA-MB-231 cells into the left cardiac ventricle and treated ip every other day with curcuminoids (25 or 50 mg/kg) for 21 days. Osteolytic bone lesion area was reduced up to 51% (p < 0.01). Consistent with specific effects on bone osteolysis, osteoclast number at the bone-tumor interface was reduced up to 53% (p < 0.05), while tumor area within bone was unaltered. In a separate study, tumor mass in orthotopic mammary xenografts was also unaltered by treatment. These data suggest that curcuminoids prevent TGF-β induction of PTHrP and reduce osteolytic bone destruction by blockade of Smad signaling in breast cancer cells.

While the chemotherapeutic effect of curcumin, one of three major curcuminoids derived from turmeric, has been reported, largely unexplored are the effects of complex turmeric extracts more analogous to traditional medicinal preparations, as well as the relative importance of the three curcuminoids and their metabolites as anti-cancer agents. These studies document the pharmacodynamic effects of chemically-complex turmeric extracts relative to curcuminoids on human breast cancer cell growth and tumor cell secretion of parathyroid hormone-related protein (PTHrP), an important driver of cancer bone metastasis. Finally, relative effects of structurallyrelated metabolites of curcuminoids were assessed on the same endpoints. We report that 3 curcuminoid-containing turmeric extracts differing with respect to the inclusion of additional naturally occurring chemicals (essential oils and/or polar compounds) were equipotent in inhibiting human breast cancer MDA-MB-231 cell growth (IC50=10-16µg/mL) and secretion of osteolytic PTHrP (IC50=2-3µg/mL) when concentrations were normalized to curcuminoid content. Moreover, these effects were curcuminoid-specific, as botanically-related gingerol containing extracts had no effect. While curcumin and bis-demethoxycurcumin were equipotent to each other and to the naturally occurring curcuminoid mixture (IC50=58µM), demethoxycurcumin did not have any effect on cell growth. However, each of the individual curcuminoids inhibited PTHrP secretion (IC50=22-31µM) to the same degree as the curcuminoid mixture (IC50=16µM). Degradative curcuminoid metabolites (vanillin and ferulic acid) did not inhibit cell growth or PTHrP, while reduced metabolites (tetrahydrocurcuminoids) had inhibitory effects on cell growth and PTHrP secretion but only at concentrations ≥10-fold higher than the curcuminoids. These studies emphasize the structural and biological importance of curcuminoids in the anti-breast cancer effects of turmeric and contradict recent assertions that certain of the curcuminoid metabolites studied here mediate these anti-cancer effects.

Background: Sorafenib (SO), a multikinase-targeted inhibitor in clinical trials for papillary and anaplastic cancers, shows limited efficacy with moderate toxicity. Withaferin A (WA), a natural withanolide, shows potent preclinical anticancer activity in thyroid cancers through multiple cytotoxic mechanisms including heat-shock protein inhibition. We hypothesized that combination therapy (WA + SO) would have a synergistic effect against anaplastic and papillary carcinoma cells at lower sorafenib doses., Methods: Human papillary (BCPAP) and anaplastic (SW1736) thyroid cancer cell lines were evaluated after treatment with SO, WA, or their combination at different doses. Proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and trypan blue exclusion; apoptosis and cell-cycle arrest was measured by flow cytometry. Western analysis confirmed apoptosis (Poly ADP ribose polymerase [PARP] and caspase-3 cleavage) and Raf inhibition. Experiments were repeated in triplicate and were evaluated statistically with significance set at a P value of less than .05., Results: The concentration of drug at which 50% of the cells are inhibited (IC(50)) in BCPAP were 6.3 μmol/L (SO), .155 μmol/L (WA), and .055 μmol/L (IC(50)WA + 50% IC(50)SO), whereas in SW1736 cells the concentration was 7.6 μmol/L (SO), 2.5 μmol/L (WA), and 1.4 μmol/L (IC(50)WA + 50% IC(50)SO). Combination (WA + SO) at IC(50) decreased cell viability to 19% (from 50% individually). Apoptosis levels on flow cytometry in anaplastic cells increased significantly from 0% to 2% (SO or WA alone) to 89% (combo at IC(50), P < .001). Combination therapy apoptosis (PARP cleavage and caspase-3 inactivation) and BRAF/Raf-1 down-regulation were dose-dependent starting at 50% IC(50) levels. Cell-cycle modulation was significant with combination treatment (35% increase in G2 arrest at 50% IC(50)SO + WA and 70% increase at 75% IC(50)SO + WA; P < .01)., Conclusions: Combination therapy with sorafenib + withaferin showed synergistic efficacy in papillary and anaplastic cancers in vitro with significant induction of apoptosis. This combination achieved potent anticancer activity with lower overall doses of sorafenib, indicating a potential strategy to decrease sorafenib toxicity in future translational studies., (Copyright © 2012. Published by Elsevier Inc.)

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. While effective therapy exists for the primary tumor, there is a lack of effective treatment for metastatic disease currently. Natural withanolide withaferin A (WA) has shown efficacy in cancers demonstrating upregulation of pro-survival pathways. The purpose of the present study is to investigate the effect of WA as a potential therapeutic agent for UM in vitro as well as in vivo. UM cells were treated with WA and several cell-based assays, such as MTS, trypan blue exclusion assay, clonogenic, wound healing, cell cycle shift, annexin V/propidium iodide, and Western blot, were performed. In vivo experiments utilized the 92.1 cells in a xenograft murine model. WA inhibits cell proliferation of uveal melanoma cells with an IC50 of 0.90, 1.66, and 2.42 μM for OMM2.3, 92.1, and MEL290 cells, respectively. Flow cytometry analysis demonstrates G2/M cell cycle arrest and apoptosis at 1 μM WA in treated cells. WA induced apoptosis partly through the suppression of c-Met, Akt, and Raf-1 signaling activation. In vivo studies using WA reduced tumor growth in 100% of animals (p = 0.015). Our observation indicates that WA is a potent drug that inhibits cell proliferation, shifts cell cycle arrest, and induces apoptosis in multiple UM cell lines in vitro. WA-mediated apoptosis in UM cells is partly mediated though the suppression of c-Met and Akt activation. WA significantly decreases UM tumor growth in vivo and justifies further evaluation of this drug for the treatment of metastatic uveal melanoma.

As part of our ongoing effort to explore the chemical diversity of plants of the United States Midwest region, the isolation and identification of 13 pregnane glycosides named verticillosides A-M from Asclepias verticillata L. are reported. The structures of these compounds were elucidated by various spectroscopic techniques, including 1D and 2D NMR, IR, UV, and HRMS. The cytotoxicity of the isolates was evaluated against paired breast cell lines Hs578T (cancer) and Hs578Bst (normal), however, no significant growth inhibition was observed., (Copyright © 2012 Elsevier Ltd. All rights reserved.)