Increasing medicinal and phytochemical compounds of coneflower (Echinacea purpurea L.) as affected by NO3-/NH4+ ratio and perlite particle size in hydroponics.

Medicinal plants are considered as one of the most important sources of chemical compounds, so preparing a suitable culture media for medicinal plant growth is a critical factor. The present study is aimed to improve the caffeic acid derivatives and alkylamides percentages of Echinacea purpurea root extract in hydroponic culture media with different perlite particle size and NO3-/NH4+ ratios. Perlite particle size in the growing media was varied as very coarse perlite (more than 2 mm), coarse perlite (1.5-2 mm), medium perlite (1-1.5 mm), fine perlite (0.5-1 mm), and very fine perlite (less than 0.5 mm) in different ratios to peat moss (including pure perlite, 50:50 v/v, 30:70 v/v, and pure peat moss). Two NO3-/NH4+ ratios (90:10 and 70:30) were tested in each growing media. All phytochemical analyses were performed according to standard methods using high performance liquid chromatography (HPLC). It was found that the E. purpurea grown in the medium containing very fine-grade perlite with 50:50 v/v perlite to peat moss ratio had the maximum caffeic acid derivatives, including chicoric acid (17 mg g-1 DW), caftaric acid (6.3 mg g-1 DW), chlorogenic acid (0.93 mg g-1 DW), cynarin (0.84 mg g-1 DW), and echinacoside (0.73 mg g-1 DW), as well as, alkylamides (54.21%). The percentages of these phytochemical compounds increased by decreasing perlite particle size and increasing of NO3-/NH4+ ratio. The major alkylamide in the E. purpurea root extract was dodeca-2E, 4E, 8Z-10 (E/Z)-tetraenoic acid isobutylamide in all treatments, ranging from 31.12 to 54.21% of total dry weight. It can be concluded that optimizing hydroponic culture media and nutrient solution has significant effects on E. purpurea chemical compounds.

Optimizing hydroponic culture media and NO3-/NH4+ ratio for improving essential oil compositions of purple coneflower (Echinacea purpurea L.).

Medicinal plants represent a valuable commodity due to beneficial effects of their natural products on human health, prompting a need for finding a way to optimize/increase their production. In this study, a novel growing media with various perlite particle size and its mixture with peat moss was tested for hydroponic-based production of Echinacea purpurea medicinal plant under greenhouse conditions. The plant growth parameters such as plant height, total fresh leave weight, fresh root weight, total biomass, total chlorophyll, leaf area, and essential oil compositions were assessed. Perlite particle size in the growing media was varied from very coarse (more than 2 mm) to very fine (less than 0.5 mm), and the ratio between perlite and peat moss varied from 50:50 v/v to 30:70 v/v. In addition, two nitrate (NO3-) to ammonium (NH4+) ratios (90:10 and 70:30) were tested for each growing media. The medium containing very fine-grade perlite and 50:50 v/v perlite to peat moss ratio was found to be most optimal and beneficial for E. purpurea performance, resulting in maximal plant height, fresh and dry weight, leaf surface area, and chlorophyll content. It was also found that an increase in NO3-/NH4+ ratio caused a significant increase in plant growth parameters and increase the plant essential oil content. The major terpene hydrocarbons found in extract of E. purpurea with the best growth parameters were germacrene D (51%), myrcene (15%), α-pinene (12%), ß-caryophyllene (11%), and 1-Pentadecene (4.4%), respectively. The percentages of these terpene hydrocarbons were increased by increasing of NO3-/NH4+ ratio. It can be concluded that decreasing the perlite particle size and increasing the NO3-/NH4+ ratio increased the plant growth parameters and essential oil compositions in E. purpurea.

Application of diatomite for sorption of Pb, Cu, Cd and Zn from aqueous solutions: kinetic, thermodynamic studies and application of response surface methodology (RSM).

Contamination of water and soil with toxic metals is a serious environmental issue. To study the Pb, Cu, Cd, and Zn sorption behavior by diatomite, batch experiments were carried out with increasing levels of initial concentration (0-200 mg/L) under different contact times (0-360 min) and temperatures (283, 293, 303, and 313 K). The effects of concentration (0-200 mg/L), pH (3-6), and ionic strength (0.01-0.06 mol/L) on the sorption were modeled using response surface methodology (RSM). Results showed that adsorption data were well-fitted by the Langmuir equation. The sorption of metals intensified by increasing initial concentration and pH but ionic strength had inverse effect. High value for R2 (0.99) and adjusted R2 (0.99) showed that the removal of ions can be described by response surface method. One-way ANOVA showed (p-value < 0.0001) that quadratic model is the best model for determining the interaction of variables. The values of the sorption energy parameter from Dubinin-Radushkevich model (E < 8 kJ K-1  mol-1 ) and negative values of ∆G showed that the sorption of the metals was physical and spontaneous. The positive values of enthalpy (ΔH) indicated that the sorption reaction of metals was endothermic at 283-313 K. PRACTITIONER POINTS: Applications of diatomite increased the sorption of Pb, Cd, Zn, and Cu from aqueous solutions. Diatomite, as low-cost adsorbent, had significant potential to sorption of ions. The sorption of heavy metals by adsorbent intensified by increasing initial concentration and pH but ionic strength had inverse effect. High value for R2 (0.99) and adj-R2 (0.99) showed that removal of metals can be described by response surface method (RSM) and the initial concentration of metal was the most significant factor.

Improving growth properties and phytochemical compounds of Echinacea purpurea (L.) medicinal plant using novel nitrogen slow release fertilizer under greenhouse conditions.

Medicinal plant production is most important than other agricultural plants due to their phytochemical compounds effects on human health. Paying attention to plant nutrition requirement is so important. In order to assess the effect of nitrate (NO3-) dosage supplies from two types of fertilizers on growth and phytochemical properties of Echinacea purpurea rhizomata cum radicibus, an experiment with completely simple design was carried out under greenhouse conditions. Two types of fertilizers (new invented nitrogen (N) slow release fertilizer and urea chemical fertilizer) at three dosages (50, 100, and 150 mM) were applied. Plant growth parameters and total phenolic (TPC), total flavonoids (TFC), polysaccarides content, essential oil content, caffeic acid derivatives, and anti-radical scavenging activities of E. purpurea were assessed. The results showed the significant (p ≤ 0.01) differences among treatments, both in growth and phytochemical properties. Using of N slow release, especially in 150 mM dosage, significantly increased all the plant growth and phytochemical properties. The dried E. purpurea rhizomata cum radicibus contained more caftaric acid (max 12.56 mg g-1 DW) and chicoric acid (max 7.56 mg g-1 DW) than other derivatives. Despite the impact of heavy metals on yield and growth of E. purpurea, the concentration of all heavy metals and micronutrients (boron (B), cadmium (Cd), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), and zinc (Zn)) in studied soil and fertilizer samples was less than United States Environmental Protection Agency (USEPA) limits of contamination. Based on the results, using of N slow release fertilizers can improve phytochemical properties of the plant due to its polymeric structure and can be a suitable substitution of chemical fertilizers, especially in medicinal plants growth.

Distribution of phosphorous pools in western river sediments of the Urmia Lake basin, Iran.

Impact of anthropogenic loading of phosphorous (P) to an aquatic ecosystem can be qualitatively assessed by measuring the buildup and distribution of P in sediments and by differentiating bioavailable and recalcitrant P pools. Distribution of P pools in sediments is affected by the physico-chemical properties including specific elements, particle size distribution, pH, electrical conductivity (EC), and carbonate content. We applied X-ray fluorescence and scanning electron microscopy (SEM) methods to characterize sediments from western rivers in the Urmia Lake basin in Iran with a particular focus on properties that are relevant to P speciation. Phosphorous pools were sequentially extracted into operationally defined exchangeable (EXCH-P), iron and aluminum oxide-bound (Fe/Al-P), calcium-bound (Ca-P), and residual (RES-P) P pools. In river sediments, the size of P pool was found to be in the order of Ca-P > RES-P > Fe/Al-P > EXCH-P indicating small fraction of bioavailable P pool and Ca-P minerals being the most dominant P sink. Carbonate-related properties had an inverse relationship with bioavailable P pools in the river sediments studied. The principal component analysis (PCA) of the sequential extraction data with sediment properties revealed that four principal components described 82.7% of total variation. Similarly, particle size-related properties were found to have the highest eigenvalues in the first PC. Electron diffraction spectra (EDS) and X-ray fluorescence (XRF) analyses showed a largely uniform distribution of P in the upstream sediment. However, limited evidence of local enrichment of P with Fe, Al, and Ca contents was observed in the downstream river sediments. Correlation of Fe/Al-P pool size with Al2O3 and SiO2 contents indicated that P was associated with Al oxide and clay minerals in the sediment matrix. Overall, the results from this study provide insights into the variability of upstream and downstream river processes and their relationship with P pools with regard to their bioavailability. These results are expected to be useful in assessing the potential impact of P loading on the aquatic ecosystem in the Urmia Lake basin.

Potential Anticancer Properties and Mechanisms of Action of Withanolides.

Plant-based Ayurvedic medicine has been practiced in India for thousands of years for the treatment of a variety of disorders. They are rich sources of bioactive compounds potentially useful for prevention and treatment of cancer. Withania somnifera (commonly known as Ashwagandha in Ayurvedic medicine) is a widely used medicinal plant whose anticancer value was recognized after isolation of steroidal compounds withanolides from the leaves of this shrub. Withaferin A is the first member of withanolides to be isolated, and it is the most abundant withanolide present in W. somnifera. Its cancer-protective role has now been established using chemically induced and oncogene-driven rodent cancer models. The present review summarizes the key preclinical studies demonstrating anticancer effects of withaferin along with its molecular targets and mechanisms related to its anticancer effects. Anticancer potential of other withanolides is also discussed.

A multi-targeted approach to suppress tumor-promoting inflammation.

Cancers harbor significant genetic heterogeneity and patterns of relapse following many therapies are due to evolved resistance to treatment. While efforts have been made to combine targeted therapies, significant levels of toxicity have stymied efforts to effectively treat cancer with multi-drug combinations using currently approved therapeutics. We discuss the relationship between tumor-promoting inflammation and cancer as part of a larger effort to develop a broad-spectrum therapeutic approach aimed at a wide range of targets to address this heterogeneity. Specifically, macrophage migration inhibitory factor, cyclooxygenase-2, transcription factor nuclear factor-κB, tumor necrosis factor alpha, inducible nitric oxide synthase, protein kinase B, and CXC chemokines are reviewed as important antiinflammatory targets while curcumin, resveratrol, epigallocatechin gallate, genistein, lycopene, and anthocyanins are reviewed as low-cost, low toxicity means by which these targets might all be reached simultaneously. Future translational work will need to assess the resulting synergies of rationally designed antiinflammatory mixtures (employing low-toxicity constituents), and then combine this with similar approaches targeting the most important pathways across the range of cancer hallmark phenotypes.

Broad targeting of resistance to apoptosis in cancer.

Apoptosis or programmed cell death is natural way of removing aged cells from the body. Most of the anti-cancer therapies trigger apoptosis induction and related cell death networks to eliminate malignant cells. However, in cancer, de-regulated apoptotic signaling, particularly the activation of an anti-apoptotic systems, allows cancer cells to escape this program leading to uncontrolled proliferation resulting in tumor survival, therapeutic resistance and recurrence of cancer. This resistance is a complicated phenomenon that emanates from the interactions of various molecules and signaling pathways. In this comprehensive review we discuss the various factors contributing to apoptosis resistance in cancers. The key resistance targets that are discussed include (1) Bcl-2 and Mcl-1 proteins; (2) autophagy processes; (3) necrosis and necroptosis; (4) heat shock protein signaling; (5) the proteasome pathway; (6) epigenetic mechanisms; and (7) aberrant nuclear export signaling. The shortcomings of current therapeutic modalities are highlighted and a broad spectrum strategy using approaches including (a) gossypol; (b) epigallocatechin-3-gallate; (c) UMI-77 (d) triptolide and (e) selinexor that can be used to overcome cell death resistance is presented. This review provides a roadmap for the design of successful anti-cancer strategies that overcome resistance to apoptosis for better therapeutic outcome in patients with cancer.

Cancer prevention and therapy through the modulation of the tumor microenvironment.

Cancer arises in the context of an in vivo tumor microenvironment. This microenvironment is both a cause and consequence of tumorigenesis. Tumor and host cells co-evolve dynamically through indirect and direct cellular interactions, eliciting multiscale effects on many biological programs, including cellular proliferation, growth, and metabolism, as well as angiogenesis and hypoxia and innate and adaptive immunity. Here we highlight specific biological processes that could be exploited as targets for the prevention and therapy of cancer. Specifically, we describe how inhibition of targets such as cholesterol synthesis and metabolites, reactive oxygen species and hypoxia, macrophage activation and conversion, indoleamine 2,3-dioxygenase regulation of dendritic cells, vascular endothelial growth factor regulation of angiogenesis, fibrosis inhibition, endoglin, and Janus kinase signaling emerge as examples of important potential nexuses in the regulation of tumorigenesis and the tumor microenvironment that can be targeted. We have also identified therapeutic agents as approaches, in particular natural products such as berberine, resveratrol, onionin A, epigallocatechin gallate, genistein, curcumin, naringenin, desoxyrhapontigenin, piperine, and zerumbone, that may warrant further investigation to target the tumor microenvironment for the treatment and/or prevention of cancer.

Synthesis and Cytotoxicity of Semisynthetic Withalongolide A Analogues.

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.

Cytotoxicity of withaferin A in glioblastomas involves induction of an oxidative stress-mediated heat shock response while altering Akt/mTOR and MAPK signaling pathways.

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 novel combination of withaferin A and sorafenib shows synergistic efficacy against both papillary and anaplastic thyroid cancers.

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.

Novel withanolides target medullary thyroid cancer through inhibition of both RET phosphorylation and the mammalian target of rapamycin pathway.

BACKGROUND: Despite development of current targeted therapies for medullary thyroid cancer (MTC), long-term survival remains unchanged. Recently isolated novel withanolide compounds from Solanaceae physalis are highly potent against MTCs. We hypothesize that these withanolides uniquely inhibit RET phosphorylation and the mammalian target of rapamycin (mTOR) pathway in MTC cells as a mechanism of antiproliferation and apoptosis. METHODS: MTC cells were treated with novel withanolides and MTC-targeted drugs. In vitro studies assessed cell viability and proliferation (MTS; trypan blue assays), apoptosis (flow cytometry with Annexin V/PI staining; confirmed by Western blot analysis), long-term cytotoxic effects (clonogenic assay), and suppression of key regulatory proteins such as RET, Akt, and mTOR (by Western blot analysis). RESULTS: The novel withanolides potently reduced MTC cell viability (half maximal inhibitory concentration [IC(50)], 270-2,850 nmol/L; 250-1,380 nmol/L for vandetanib; 360-1,640 nmol/L for cabozantinib) with induction of apoptosis at <1,000 nmol/L of drug. Unique from other targeted therapies, withanolides suppressed RET and Akt phosphorylation and protein expression (in a concentration- and time-dependent manner) as well as mTOR activity and translational activity of 4E-BP1 and protein synthesis mediated by p70S6kinase activation at IC(50) concentrations. CONCLUSION: Novel withanolides from Physalis selectively and potently inhibit MTC cells in vitro. Unlike other MTC-targeted therapies, these compounds uniquely inhibit both RET kinase activity and the Akt/mTOR prosurvival pathway. Further translational studies are warranted to evaluate their clinical potential.

Minor withanolides of Physalis longifolia: structure and cytotoxicity.

In our recent publication on bioactive guided isolation of compounds from Physalis longifolia (Solanaceae) novel anti-proliferative agents withalongolides A (4) and B (5), and their highly cytotoxic analogues, withalongolide A 4,19,27-triacetate (4a) and withalongolide B 4,19-diacetate (5a) were elucidated. In this study, the two lead compounds (4, 5) were re-isolated in gram quantities for the purpose of further analogue preparation and in vivo testing that would continue to probe structure-activity relationships. During this process, two additional withanolides, named withalongolides O (1) and P (2), were elucidated. Their structures were determined by spectroscopic techniques with 1 being subsequently confirmed by X-ray crystallographic analysis. Utilizing a MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] viability assay, withalongolide O (1) and its 4,7-diaceatate (1a), both containing the functionalities of Δ(2)-1-oxo- in A ring, a 5ß,6ß-epoxy in B ring, and a lactone ring in the nine-carbon side chain, exhibited potent cytotoxicity against human head and neck squamous cell carcinoma (JMAR and MDA-1986), melanoma (B16F10 and SKMEL-28), and normal fetal lung fibroblast (MRC-5) cells with IC(50) values in the range between 0.15 and 2.95 µM. In addition, the previously reported α orientation of 7-acetate group in acnistins C and D should be revised to the ß orientation on the basis of NMR data comparison.

Natural withanolide withaferin A induces apoptosis in uveal melanoma cells by suppression of Akt and c-MET activation.

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.

Inhibition of cell growth and induction of apoptosis in ovarian carcinoma cell lines CaOV3 and SKOV3 by natural withanolide Withaferin A.

OBJECTIVE: Withaferin A, a natural withanolide, has shown anti-cancer properties in various cancers including breast cancer, but its effects in ovarian cancer remain unexplored. Notch 1 and Notch3 are critically involved in ovarian cancer progression. We decided to examine the effects of Withaferin A in ovarian carcinoma cell lines and its molecular mechanism of action including its regulation of Notch. METHODS: The effects of Withaferin A were examined in CaOV3 and SKOV3 ovarian carcinoma cell lines using MTS assay, clonogenic assay, annexin V/propidium iodide flow cytometry, and cell cycle analysis. Western analysis was conducted to examine the molecular mechanisms of action. RESULTS: Withaferin A inhibited the growth and colony formation of CaOV3 and SKOV3 cells by inducing apoptosis and cell cycle arrest. These changes correlated with down-regulation of Notch1, Notch3, cdc25C, total and phosphorylated Akt, and bcl-2 proteins. CONCLUSIONS: Withaferin A inhibits CaOV3 and SKOV3 ovarian carcinoma cell growth, at least in part by targeting Notch1 and Notch3.

Novel C-terminal Hsp90 inhibitor for head and neck squamous cell cancer (HNSCC) with in vivo efficacy and improved toxicity profiles compared with standard agents.

BACKGROUND: Current therapies for HNSCC, especially platinum agents, are limited by their toxicities and drug resistance. This study evaluates a novel C-terminal Hsp90 inhibitor (CT-Hsp90-I) for efficacy and toxicity in vitro and in vivo in an orthotopic HNSCC model. Our hypothesis is that C-terminal inhibitors exhibit improved toxicity/efficacy profiles over standard therapies and may represent a novel group of anticancer agents. METHODS: MDA-1986 HNSCC cells were treated with doses of 17-AAG or KU363 (a CT-Hsp90-I) and compared for antiproliferation by GLO-Titer and trypan blue exclusion and for apoptosis by PARP cleavage and caspase-3 inactivation by Western analysis. In vivo studies in Nu/Nu mice examined an orthotopic model of MDA-1986 cells followed by drug dosing intraperitoneally for a 21-day period (mg/kg/dose: cisplatin = 3.5, low-dose KU363 = 5, high-dose KU363 = 25, 17-AAG = 175). Tumor size, weight, and toxicity (body score) were measured 3×/week. RESULTS: The IC(50) levels for KU363 = 1.2-2 µM in MDA-1986. KU363 induces apoptosis at 1 µM with cleavage of PARP and inactivation of caspase-3 levels after 24 h. Client proteins Akt and Raf-1 were also downregulated at 1-3 µM of drug. In vivo, 100% of controls had progressive disease, while 100% of cisplatin animals showed some response, all with significant systemic toxicity. High-dose KU363 showed 88% of animals responding and low-dose KU363 showed 75% responding. KU363 animals showed significantly less toxicity (P < 0.01) than cisplatin or 17-AAG. CONCLUSION: This novel CT-Hsp90-I KU363 manifests potent anticancer activity against HNSCC, showing excellent in vivo efficacy and reduced toxicity compared with standard agents justifying future translational evaluation.

Withaferin a induces proteasome-dependent degradation of breast cancer susceptibility gene 1 and heat shock factor 1 proteins in breast cancer cells.

The purpose of this study was to examine the regulation of prosurvival factors heat shock factor 1 (HSF1) and breast cancer susceptibility gene 1 (BRCA1) by a natural withanolide withaferin A (WA) in triple negative breast cancer cell lines MDA-MB-231 and BT20. Western analysis was used to examine alternations in HSF1 and BRCA1 protein levels following WA treatment. A protein synthesis inhibitor cycloheximide and a proteasome inhibitor MG132 were used to investigate the mechanisms of HSF1 and BRCA1 regulation by WA. It was found that WA induced a dose-dependent decrease in HSF1 and BRCA1 protein levels. Further analysis showed that levels of HSF1 and BRCA1 proteins decreased rapidly after WA treatment, and this was attributed to WA-induced denaturation of HSF1 and BRCA1 proteins and subsequent degradation via proteasome-dependent, and protein-synthesis dependent mechanism. In summary, WA induces denaturation and proteasomal degradation of HSF1 and BRCA1 proteins. Further studies are warranted to examine the contribution of HSF1 and BRCA1 depletion to the anticancer effects of WA in breast cancer.

Anti-proliferative withanolides from the Solanaceae: a structure-activity study.

As part of our search for bioactive compounds from plant biodiversity, 29 withanolides (1, 3-6, 9, 12-18, and 20-35) were recently isolated from three members of the Solanaceae: Physalis longifolia, Vassobia breviflora, and Withania somnifera. Six derivatives (2, 7, 8, 10, 11, and 19) were prepared from these naturally occurring withanolides. All compounds (1-35) were evaluated for in vitro anti-proliferative activity against an array of cell lines [melanoma cell lines (B16F10, SKMEL28); human head and neck squamous cell carcinomas (HNSCC) cell lines (JMAR, MDA1986, DR081-1); breast cancer cell line (Hs578T), and non-malignant human cell line (MRC5)]. This led to the discovery of 15 withanolides, with IC50 values in the range of 0.067-17.4 µM, including withaferin A 1, withaferin A 4,27-diacetate 2, 27-O-glucopyranosylwithaferin A 3, withalongolide H 4, withalongolide C 5, withalongolide A 6, withalongolide A 4,27-diacetate 7, withalongolide A 4,19,27-triacetate 8, withalongolide B 9, withalongolide B 4-acetate 10, withalongolide B 4,19-diacetate 11, withalongolide D 16, withalongolide E 17, withalongolide G 21, and 2,3-dihydrowithaferin A 3-O-sulfate 22). In order to update the growing literature on withanolides and their activities, we summarized the distribution, structural types and anti-proliferative activities for all published withanolides to date. The structure-activity relationship analysis (SARA) confirmed the importance of the presence of a Δ2-1-oxo- functionality in ring A, a 5ß,6ß-epoxy or 5α-chloro-6ß-hydroxy groupings in ring B, and nine carbon side chain with a lactone moiety for cytotoxic activity. Conversely, the SARA indicated that the -OH or -OR groups at C-4, 7, 11, 12, 14, 15, 16, 17, 18, 19, 20, 23, 24, 27, 28 were not contributors to the observed anti-proliferative activity within the systems analyzed.

Cytotoxic withanolide constituents of Physalis longifolia.

Fourteen new withanolides, 1-14, named withalongolides A-N, respectively, were isolated from the aerial parts of Physalis longifolia together with eight known compounds (15-22). The structures of compounds 1-14 were elucidated through spectroscopic techniques and chemical methods. In addition, the structures of withanolides 1, 2, 3, and 6 were confirmed by X-ray crystallographic analysis. Using a MTS viability assay, eight withanolides (1, 2, 3, 7, 8, 15, 16, and 19) and four acetylated derivatives (1a, 1b, 2a, and 2b) showed potent cytotoxicity against human head and neck squamous cell carcinoma (JMAR and MDA-1986), melanoma (B16F10 and SKMEL-28), and normal fetal fibroblast (MRC-5) cells with IC50 values in the range between 0.067 and 9.3 µM.