Resveratrol Analogues Research Articles

Production of highly bioactive resveratrol analogues pterostilbene and piceatannol in metabolically engineered grapevine cell cultures.

SummaryGrapevine stilbenes, particularly trans‐resveratrol, have a demonstrated pharmacological activity. Other natural stilbenes derived from resveratrol such as pterostilbene or piceatannol, display higher oral bioavailability and bioactivity than the parent compound, but are far less abundant in natural sources. Thus, to efficiently obtain these bioactive resveratrol derivatives, there is a need to develop new bioproduction systems. Grapevine cell cultures are able to produce large amounts of easily recoverable extracellular resveratrol when elicited with methylated cyclodextrins and methyl jasmonate. We devised this system as an interesting starting point of a metabolic engineering‐based strategy to produce resveratrol derivatives using resveratrol‐converting enzymes. Constitutive expression of either Vitis vinifera resveratrol O‐methyltransferase (Vv ROMT) or human cytochrome P450 hydroxylase 1B1 (Hs CYP1B1) led to pterostilbene or piceatannol, respectively, after the engineered cell cultures were treated with the aforementioned elicitors. Functionality of both gene products was first assessed in planta by Nicotiana benthamiana agroinfiltration assays, in which tobacco cells transiently expressed stilbene synthase and Vv ROMT or Hs CYP1B1. Grapevine cell cultures transformed with Vv ROMT produced pterostilbene, which was detected in both intra‐ and extracellular compartments, at a level of micrograms per litre. Grapevine cell cultures transformed with Hs CYP1B1 produced about 20 mg/L culture of piceatannol, displaying a sevenfold increase in relation to wild‐type cultures, and reaching an extracellular distribution of up to 45% of total production. The results obtained demonstrate the feasibility of this novel system for the bioproduction of natural and more bioactive resveratrol derivatives and suggest new ways for the improvement of production yields.

Piceatannol increases the expression of hepatocyte growth factor and IL-10 thereby protecting hepatocytes in thioacetamide-induced liver fibrosis.

Piceatannol is a polyphenolic analog of resveratrol that selectively inhibits the non-receptor tyrosine kinase-Syk. This study investigates the potential ability of piceatannol to attenuate liver fibrosis and protect hepatocytes from injury. Thioacetamide was injected in adult male mice (100 mg/kg, i.p., 3 times/week) for 8 weeks. Piceatannol (1 or 5 mg/kg per day) was administered by oral gavage during the last 4 weeks. Liver function biomarkers, tissue malondialdehyde (MDA), cytokeratin-18 (CK18), hepatocyte growth factor (HGF), and interleukin-10 (IL-10) were measured. Necroinflammation, fibrosis, expression of transforming growth factor (TGF)-β1, and α-smooth muscle actin (SMA) were scored by histopathological examination and immunohistochemistry. Obtained results showed ability of piceatannol (1 mg/kg) to restore liver function and reduce inflammation. It significantly (p < 0.001) reduced MDA, CK18, TGF-β1, and α-SMA expression, and increased HGF and IL-10. It can be concluded that piceatannol at low dose can inhibit TGF-β1 induced hepatocytes apoptosis and exerts an anti-inflammatory effect attenuating fibrosis progression.

Dietary pterostilbene is a novel MTA1-targeted chemopreventive and therapeutic agent in prostate cancer.

Overexpression of the epigenetic modifier metastasis-associated protein 1 (MTA1) is associated with aggressive human prostate cancer. The purpose of this study was to determine MTA1- targeted chemopreventive and therapeutic efficacy of pterostilbene, a natural potent analog of resveratrol, in pre-clinical models of prostate cancer. Here, we show that high levels of MTA1 expression in Pten-loss prostate cooperate with key oncogenes, including c-Myc and Akt among others, to promote prostate cancer progression. Loss-of-function studies using human prostate cancer cells indicated direct involvement of MTA1 in inducing inflammation and epithelial-to-mesenchymal transition. Importantly, pharmacological inhibition of MTA1 by pterostilbene resulted in decreased proliferation and angiogenesis and increased apoptosis. This restrained prostatic intraepithelial neoplasia (PIN) formation in prostate-specific Pten heterozygous mice and reduced tumor development and progression in prostate-specific Pten-null mice. Our findings highlight MTA1 as a key upstream regulator of prostate tumorigenesis and cancer progression. More significantly, it offers pre-clinical proof for pterostilbene as a promising lead natural agent for MTA1-targeted chemopreventive and therapeutic strategy to curb prostate cancer.

Resveratrol analogue 4,4'-dihydroxy-trans-stilbene potently inhibits cancer invasion and metastasis.

We investigated the preventive effects of resveratrol analogue 4,4′-dihydroxy-trans-stilbene (DHS) on cancer invasion and metastasis. Two different in vivo approaches of mouse and zebrafish lung cancer invasion models were employed in our study. The in vitro results showed that DHS displays potent inhibition on anchorage-dependent or -independent cell growth of LLC cells, leading to impairment of the cell cycle progression with reduction of cell numbers arresting at the G1 phase, an evident accumulation of pre-G1 events correlated with apoptotic behaviour. In addition, DHS induces a marked inhibition of LLC cell migration and matrigel invasion. In a murine lung cancer model, tumour volume, cell proliferation, and tumour angiogenesis were significantly inhibited by DHS. Importantly, liver metastatic lesions were significantly reduced in DHS-treated mice. Similarly, DHS significantly inhibits lung cancer cell dissemination, invasion and metastasis in a zebrafish tumour model. These findings demonstrate that DHS could potentially be developed as a novel therapeutic agent for treatment of cancer and metastasis.

A Comprehensive Review on the Chemotherapeutic Potential of Piceatannol for Cancer Treatment, with Mechanistic Insights.

Cancer is a diverse class of diseases characterized by uncontrolled cell growth that constitutes the greatest cause of mortality and morbidity worldwide. Despite steady progress, the treatment modalities of cancer are still insufficient. Several new concepts have emerged for therapeutic intervention in malignant diseases with the goal of identifying specific targets and overcoming resistance against current cytotoxic therapies. Many studies have reported the remarkable and significant properties of dietary plant polyphenols such as curcumin, resveratrol, flavopiridol, indirubin, magnolol, piceatannol, parthenolide, epigallocatechin gallate, and cucurbitacin as anticancer agents known for their pleiotropic effects on cancer, immune cells, and inflammation. Piceatannol, an analogue and metabolite of resveratrol, is a natural stilbene commonly found in grape skins and wine. Compared to resveratrol, this molecule exhibits superior bioactivities as an inhibitor of COX-1/2 and the CSN-associated kinase. Piceatannol is thought to be a potent natural compound with many therapeutic effects, such as the prevention of hypercholesterolemia, arrhythmia, atherosclerosis, angiogenesis, and cardiovascular diseases. It also demonstrates vasorelaxation, antioxidant, and anticancer activities. This comprehensive review summarizes the current data regarding the mechanisms of action of piceatannol, its chemopreventive properties, and its possible therapeutic potential against various types of human cancer.

The Scavenging of DPPH, Galvinoxyl and ABTS Radicals by Imine Analogs of Resveratrol.

Resveratrol (3,5,4′-trihydroxystilbene) is a phytoalexin produced by plants. Resveratrol is known for its anti-cancer, antiviral and antioxidant properties. We prepared imine analogs of resveratrol ((hydroxyphenyliminomethyl)phenols) and tested their antioxidant activity. All prepared resveratrol analogs were able to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH), galvinoxyl radical (GOR) and 2,2′-azino-bis(3-ethylbenzothiazoline)-6-sulphonic acid (ABTS) radicals. The antioxidant activity efficiency correlated with the number and position of hydroxyl groups. The most effective antioxidants were resveratrol analogs containing three hydroxyl groups in the benzylidene part of their molecules. These results provide new insights into the relationship between the chemical structure and biological activity of resveratrol analogs.

Abstract B17: Epigenetic regulation of NOTCH oncogenic signaling in breast cancer

Abstract Alterations in DNA methylation occur at different stages of cancer, including initiation, and may underlie up-regulation of genes with oncogenic functions. The NOTCH pathway is often overactive in breast cancer and plays roles in cancer development and progression. It is therefore a possible target for anti-cancer strategies. However, the mechanisms of NOTCH regulation in mammary carcinogenesis remain unclear which hinders the development of effective approaches to target this oncogenic pathway. Interestingly, certain dietary compounds such as polyphenols with a stilbenoid structure suppress the NOTCH signals in cancer and were shown in our study to modify epigenetic marks in genes positively regulating the pathway, including MAML2. In the present study, using two polyphenols, resveratrol from grapes and pterostilbene from blueberries, we investigate the implication of DNA methylation in regulation of NOTCH in breast cancer. Non-invasive MCF10CA1h and invasive MCF10CA1a human breast cancer cell lines were used as an experimental model. Following genome-wide DNA methylation analysis with Illumina 450K BeadChip array, pyrosequencing and QPCR were performed to assess methylation and expression of MAML2 and NOTCH target genes. Chromatin immunoprecipitation (ChIP) was applied to determine binding of epigenetic enzymes to MAML2 regulatory region. Depletion with siRNA was applied to establish functional link between MAML2 silencing and NOTCH activity. We found 4,293 CpG sites differentially methylated upon 9-day treatment of MCF10CA1h breast cancer cells with 15µM resveratrol as compared to untreated cells (0.05&amp;lt;differential methylation&amp;lt;-0.05, p&amp;lt;0.03, Wilcoxon rank-sum test). Among these changes, 3,508 CpGs corresponding to 1,707 genes were hypermethylated and functionally associated with oncogenic signaling pathways, including NOTCH. As the array data indicated, resveratrol led to re-methylation of enhancer region of MAML2 that is a coactivator of NOTCH target genes. The 15-20% increase in methylation of MAML2 was confirmed by pyrosequencing in breast cancer cells with both high and low invasive potential. Pterostilbene, an analog of resveratrol, exerted similar effects on MAML2 methylation state at 10µM concentration. Along with methylation of the MAML2 enhancer region, the compounds decreased MAML2 expression by 30-60%. Stronger MAML2 down-regulation was achieved in invasive cancer cells as compared to non-invasive cells after exposure to either resveratrol or pterostilbene. Epigenetic silencing of MAML2 was associated with downregulation of NOTCH target genes, including HES1 (30-50%), HEY1 (30-60%), and NOTCH1 (40-65%). Depletion of MAML2 with siRNA mimicked polyphenols' effects leading to suppression of NOTCH pathway. It supports a functional link between MAML2 and NOTCH signaling. The most profound effects were observed in invasive MCF10CA1a cells where further analyses revealed 2-fold increase in trimethylation of H3K27 (repressive histone mark) and 30% decrease in acetylation of H3K9 (activating mark) at MAML2 enhancer region upon exposure to resveratrol. The condensed chromatin structure was associated with binding of DNMT3B to the tested fragment of MAML2 upon polyphenol treatment whereas no binding was detected in untreated cells. It suggests the role of DNMT3B in increased methylation of MAML2, which is consistent with increased DNMT3B expression upon treatment with polyphenols. Our results establish a role for epigenetic modifications in regulation of NOTCH oncogenic signaling in breast cancer. It constitutes a novel insight into regulation of oncogenic signals in cancer and provides support for epigenetic-targeting strategies as an effective anti-cancer approach. This study was supported by the Purdue University Center for Cancer Research, Indiana CTSI (UL1TR001108), and Women's Global Health Institute granted to BS. Citation Format: Katarzyna Lubecka, Lucinda Kurzava, Kirsty Flower, Hannah Buvala, Dorothy Teegarden, Ignacio Camarillo, James M. Flanagan, Barbara Stefanska. Epigenetic regulation of NOTCH oncogenic signaling in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B17.

Abstract 40: Methylated resveratrol analogue DMU-212 undergoes metabolic activation to DMU-214 in ovarian A-2780 and SKOV-3 cancer cells

Abstract Resveratrol (3,4',5-trans-trihydroxystilbene) is a naturally occurring phytoalexin which shows a wide spectrum of biological activity. Although the anticancer effects of resveratrol have been revealed, its clinical application is limited because of a low bioavailability and a rapid clearance from the circulation. The structural alteration of the stilbene motif of resveratrol has been found to be a promising strategy for generation of several synthetic analogues with improved pharmacokinetic parameters. The substitution of hydroxyl groups of resveratrol for methoxy groups may increase the molecule stability, making it less susceptible to phase II conjugation reactions in vivo. The structure-activity studies have revealed that introduction of additional methoxy groups resulted in an increased cytotoxic properties of resveratrol analogues. Furthermore, the methoxy groups at positions 3,5- and 3,4,5- of the trihydroxystilbene scaffold have been identified as crucial for pro-apoptotic activity of the compound. To date few reports have demonstrated the antiproliferative effect of DMU-212 (3,4,4',5-tetramethoxystilbene) in transformed fibroblasts, prostate, liver, colon, cervical, breast and ovarian cancer cells. It has been shown that DMU-212 is biotransformed to four metabolites: 3'-hydroxy-3,4,5,4'-tetramethoxystilbene (DMU-214), 4'-hydroxy-3,4,5-trimethoxystilbene (DMU-281), 4-hydroxy-3,5,4'-trimethoxystilbene (DMU-291), and 3-hydroxy-4,5,4'-trimethoxystilbene (DMU-807). However, if these metabolites retain the biological activity of DMU-212 remains unknown. Therefore, we evalueted the cytotoxic activity of these four metabolites of DMU-212 and the induction of apoptosis in ovarian A-2780 and SKOV-3 cancer cell lines. CYP1A1 and CYP1B1 are known to catalyze the conversion of estradiol to 4-hydroxyestradiol. Hence, the compounds such as resveratrol or DMU-212, which are structurally similar to the endogenous oestrogen estradiol, have been suggested to undergo metabolism by these enzymes. Surprisingly, DMU-212 has been shown to entirely inhibit CYP1B1 in ovarian A-2780 cancer cell line. Since CYP1B1 has been suggested to be involved in DMU-212 biotransformation, it can be hypothesised that one or more metabolites of DMU-212 regulate the expression of CYP1B1 by the feedback inhibition. To have an insight into DMU-212 metabolites' role in the expression of isoenzymes of CYP1 family we assessed CYP1A1 and CYP1B1 mRNA and protein levels in metabolites-treated A-2780 and SKOV-3 cancer cells. The inhibitory effect of four metabolites of DMU-212 was investigated in ovarian A-2780 and SKOV-3 cancer cell lines by MTT assays. The induction of apoptosis was assayed by Cell Death Detection ELISAPLUS test, Caspase-Glo® 3/7 luminescent test and flow cytometry analysis. The expression of CYP1A1 and CYP1B1 mRNA and protein levels was investigated by RT-qPCR and Western blott, respectively. From among four metabolites of DMU-212, DMU-214 exhibited the highest cytotoxic activity (IC50 A-2780=13.26±2.11 nM; IC50 SKOV-3=29.21±3.17nM). Furthermore, DMU-214 has been revealed to exert more pronounced inhibitory effects than the prototype compound, DMU-212. In addition, we observed that the stronger cytotoxic activity of DMU-214 evoked its greater pro-apoptotic effect. DMU-214 caused an increase in nucleosomes level in A-2780 and SKOV-3 cells, EF= 15.89 and 7.91, respectively. Exposure of either cell line to DMU-214 resulted in a statistically significant decrease in CYP1A1 and CYP1B1 mRNA and protein levels as compared to control. We showed for the first time that DMU-212 undergoes metabolic activation to DMU-214 in ovarian A-2780 and SKOV-3 cancer cell lines. Since DMU-214 has been observed to decrease expression of CYP1A1 and CYP1B1 in A-2780 and SKOV-3 cells our results provide a new insight into the concept of their feedback inhibition by the compound tested. Citation Format: Sylwia Borys, Malgorzata Kucinska, Dorota Trzybulska, Mariusz Kaczmarek, Marcin Wierzchowski, Marek Murias, Jadwiga Jodynis-Liebert, Hanna Piotrowska. Methylated resveratrol analogue DMU-212 undergoes metabolic activation to DMU-214 in ovarian A-2780 and SKOV-3 cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 40.

Tetrahydroxystilbene Glucoside Inhibits Excessive Autophagy and Improves Microvascular Endothelial Dysfunction in Prehypertensive Spontaneously Hypertensive Rats.

Autophagy exists in vascular endothelial cells, but the relationship between autophagy and blood vessel dysfunction in hypertension remains elusive. This study aimed to investigate role of autophagy in vascular endothelial dysfunction in prehypertension and hypertension and the underlying mechanisms involved. Furthermore, we sought to determine if and how tetrahydroxystilbene glucoside (TSG), a resveratrol analogue and active ingredient of Polygonum multiflorum Thunb used for its cardiovascular protective properties in traditional Chinese medicine, influences vascular endothelial function. Male spontaneously hypertensive rats (SHRs) aged 4 weeks (young) and 12 weeks (adult) were studied and the vascular function of isolated aorta and mesenteric artery was assessed in vitro. Compared with Wistar Kyoto rats (WKY), young and adult SHRs showed endothelial dysfunction of the aorta and mesenteric artery, along with decreased pAkt, pmTOR, and autophagic marker protein p62 and increased LC3 II/I in microvascular but not aortic tissues. TSG administration for 14 days significantly improved mesenteric vascular endothelial function, increased levels of pAkt and pmTOR, and decreased autophagy. Pretreatment of young SHRs with the mTOR inhibitor rapamycin blocked the antiautophagic and vasodilative effects of TSG. Moreover, TSG significantly activated Akt-mTOR signaling in HUVECs and reduced the autophagic levels in vitro, which were almost completely blocked by rapamycin. In summary, mesenteric endothelial dysfunction in prehypertensive SHRs was at least partly attributable to excessive autophagy in vascular tissues. TSG partly restored microvascular endothelial dysfunction through activating the Akt/mTOR pathway, which consequently suppressed autophagy, indicating that TSG could be potentially applied to protect vascular function against subclinical changes in prehypertension.

Synthesis, Antitubercular and Leishmanicidal Evaluation of Resveratrol Analogues

In this paper we continue our efforts in the search for new Schiff bases as resveratrol analogues as promising antitubercular and antileishmanial agents. Compounds were evaluated in vitro against Mycobacterium tuberculosis and Leishmania species. Compounds showed varying activity against promastigotes of all Leishmania species tested (concentration leading to reduction of 50% of parasite growth-IC50 values ranging from 1.60 to 15.53 µg mL-1). Majority of compounds exhibited good results against Leishmania species with IC50 values below 10.00 µg mL-1, with no cytotoxicity against macrophages. One of the analogues showed the best activity against amastigotes of L. amazonensis (IC50 = 5.73 µg mL-1). Furthermore, the compounds showed comparable or better effect than drugs commonly used in tuberculosis treatment such as Cycloserine®. The analogue that was the most active for M. tuberculosis had minimal inhibitory concentration MIC90 = 0.78 µg mL-1. The in vitro cytotoxicity on Vero cells (CC50), as well as the selectivity index (SI = CC50/MIC) were also evaluated, and showed that the synthesized analogues are not cytotoxic.

Stimulation of Eryptosis, the Suicidal Erythrocyte Death by Piceatannol

Background/Aims: Piceatannol, an analog and metabolite of resveratrol, is effective against various disorders including malignancy. It is in part effective by triggering suicidal death or apoptosis of tumor cells. Cellular mechanisms mediating the proapoptotic effect of Piceatannol include mitochondrial depolarization and cytochrome c release. Erythrocytes lack mitochondria but may nevertheless enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the triggering of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress and ceramide formation. The present study explored, whether Piceatannol induces eryptosis and, if so, to shed some light on the cellular mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, reactive oxygen species (ROS) formation from 2',7'-dichlorodihydrofluorescein (DCF) diacetate-dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemoglobin concentration in the supernatant was taken as measure of hemolysis. Results: A 48 hours exposure of human erythrocytes to Piceatannol (10 - 20 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased DCFDA-fluorescence, significantly increased ceramide abundance, but did not significantly increase Fluo3-fluorescence. Removal of extracellular Ca²⁺ slightly blunted but did not abolish the effect of Piceatannol on annexin-V-binding and forward scatter. Piceatannol (20 µM) significantly augmented the increase of annexin-V-binding, but significantly blunted the decrease of forward scatter following treatment with the Ca²⁺ ionophore ionomycin. Conclusions: Piceatannol triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca²⁺ and involving oxidative stress and ceramide formation.

Therapeutic Potential and Molecular Targets of Piceatannol in Chronic Diseases.

Piceatannol (3,3',4,5'-tetrahydroxy-trans-stilbene; PIC) is a naturally occurring stilbene present in diverse plant sources. PIC is a hydroxylated analog of resveratrol and produced from resveratrol by microsomal cytochrome P450 1A11/2 and 1B1 activities. Like resveratrol, PIC has a broad spectrum of health beneficial effects, many of which are attributable to its antioxidative and anti-inflammatory activities. PIC exerts anticarcinogenic effects by targeting specific proteins involved in regulating cancer cell proliferation, survival/death, invasion, metastasis, angiogenesis, etc. in tumor microenvironment. PIC also has other health promoting and disease preventing functions, such as anti-obese, antidiabetic, neuroptotective, cardioprotective, anti-allergic, anti-aging properties. This review outlines the principal biological activities of PIC and underlying mechanisms with special focus on intracellular signaling molecules/pathways involved.

Encapsulation of piceatannol, a naturally occurring hydroxylated analogue of resveratrol, by natural and modified cyclodextrins.

In this work, an in-depth study of the interaction between piceatannol (a type of stilbene with high biological activity) and different natural and modified cyclodextrins (CDs) is made, using steady state fluorescence. This bioactive molecule forms a 1 : 1 complex with all the natural (α-CD, β-CD and γ-CD) and modified (HP-β-CD, HE-β-CD and M-β-CD) CDs tested. Among natural CDs, the interaction of piceatannol with β-CD was the most efficient. However, the modified CDs showed higher encapsulation constants (KF) than β-CD, except M-β-CD; the highest KF being found for HP-β-CD (14 048 ± 702 M(-1)). The encapsulation of piceatannol in the internal cavity of CDs showed a strong dependence on pH and temperature. The interaction between HP-β-CD and piceatannol was less effective in the pH region where the stilbene begins to suffer the deprotonation of its hydroxyl group. Moreover, the values of KF decreased as the system temperature increased. To obtain information on the mechanism involved in the piceatannol affinity for CD, the thermodynamic parameters of the complexation (ΔH°, ΔS° and ΔG°) were studied, the results showed a negative entropy (-3.7 ± 0.2 J mol(-1) K(-1)), enthalpy (-24.6 ± 1.2 kJ mol(-1)) and Gibbs free energy change at 25 °C (-23.5 ± 1.2 J mol(-1)). Finally, molecular docking calculations provided further insights into how the different interactions influence the complexation constant. A high degree of correlation was observed between the computed scores and experimental values.

Pterostilbene Inhibits the Growth of Human Esophageal Cancer Cells by Regulating Endoplasmic Reticulum Stress

Background/Aims: Pterostilbene (PTE), a natural dimethylated resveratrol analog from blueberries, is known to have diverse pharmacological activities, including anticancer properties. In this study, we investigated the anticancer activity of PTE against human esophageal cancer cells both in vitro and in vivo and explored the role of endoplasmic reticulum (ER) stress (ERS) signaling in this process. Methods: Cell viability, the apoptotic index, Caspase 3 activity, adhesion, migration, reactive oxygen species (ROS) levels, and glutathione (GSH) levels were detected to explore the effect of PTE on human EC109 esophageal cancer cells. Furthermore, siRNA transfection and a chemical inhibitor were employed to confirm the role of ERS. Results: PTE treatment dose- and time-dependently decreased the viability of human esophageal cancer EC109 cells. PTE also decreased tumor cell adhesion, migration and intracellular GSH levels while increasing the apoptotic index, Caspase 3 activity and ROS levels, which suggest the strong anticancer activity of PTE. Furthermore, PTE treatment increased the expression of ERS-related molecules (GRP78, ATF6, p-PERK, p-eIF2α and CHOP), upregulated the pro-apoptosis-related protein PUMA and downregulated the anti-apoptosis-related protein Bcl-2 while promoting the translocation of cytochrome c from mitochondria to cytosol and the activation of Caspase 9 and Caspase 12. The downregulation of ERS signaling by CHOP siRNA desensitized esophageal cancer cells to PTE treatment, whereas upregulation of ERS signaling by thapsigargin (THA) had the opposite effect. N-Acetylcysteine (NAC), a ROS scavenger, also desensitized esophageal cancer cells to PTE treatment. Conclusions: Overall, the results indicate that PTE is a potent anti-cancer pharmaceutical against human esophageal cancer, and the possible mechanism involves the activation of ERS signaling pathways.

Determination of pinostilbene in rat plasma by LC–MS/MS: Application to a pharmacokinetic study

Pinostilbene (3-methoxyresveratrol or trans-3,4′-dihydroxy-5-methoxystilbene) is a naturally occurring monomethylether analogue of resveratrol (trans-3,5,4′-trihydroxystilbene) that exhibits various pharmacological activities. To further examine its medicinal potential, a sensitive LC–MS/MS method was developed and validated for the determination of pinostilbene in rat plasma. Heavy Isotope labelled resveratrol was used as an internal standard. The ESI was operated in its negative ion mode while pinostilbene and resveratrol were measured by multiple reaction monitoring (MRM) using precursor-to-product ion transitions of m/z 241→181 and m/z 233→191, respectively. This LC–MS/MS method had excellent selectivity, sensitivity (LLOQ=1ng/ml), accuracy (both intra- and interday analytical recovery within 100±15%) and precision (both intra- and interday RSD < 15%). The matrix effect was insignificant. The pharmacokinetics of pinostilbene was subsequently profiled in Sprague–Dawley rats. Upon intravenous administration (5 or 10mg/kg), pinostilbene displayed rapid clearance (Cl=129±42 or 107±31ml/min/kg) and extremely short mean transit time (MTT=6.24±0.41 or 8.52±1.38min). After oral dosing (50mg/kg), the bioavailability of pinostilbene was limited but highly erratic (F=1.87±2.67%). Pharmacokinetic comparison among pinostilbene, resveratrol and some resveratrol analogues suggested that stilbenes with meta-hydroxyl group(s) may be associated with metabolic instability and subsequently suffer from rapid clearance and low oral bioavailability. The information obtained from this study will facilitate further exploration on pinostilbene as well as other resveratrol analogues.

TRPA1 channels as targets for resveratrol and related stilbenoids.

A series of twenty resveratrol analogues was synthesized and tested on TRPA1 and TRPV1 channels. None was able to significantly modulate TRPV1 channels. Conversely, most of them exhibited remarkably higher TRPA1 modulating activity than resveratrol. Optimal potency was observed with ortho monoxygenated stilbenes 6 and 17.

Abstract A39: 3,4',5- Trismethoxybenzophenone inhibits the growth of human hepatocarcinoma cells via cell-cycle arrest at the G2/M phase

Abstract 3,4',5-trismethoxybenzophenone (TMBP) is an analogue of the natural dietary polyphenolic compound resveratrol (3,5,4'-trihydro-trans-stilbene, abbreviated as RVT). Natural RVT belongs to the phytoalexin class of phytochemicals that are antibiotics produced by plants in response to stimuli such as injury, ultraviolet irradiation, stress, and fungal attack. RVT and its analogues have also been shown to prevent hepatic steatosis. In this study we focused on TMBP which is a methylated derivative of RVT. Studies have shown that TMBP exhibits added biological effects and a better pharmacokinetic profile compared to RVT due to the presence of methoxy groups. The human hepatoma cell line (FCA4 cells) that harbors a subgenomic selectable HCV replicon was used for this study. The Huh7 cell line lacking the replicon served as the corresponding control. MTS assay was used to determine the cytotoxicity of TMBP at various concentrations. Wound healing assay was performed to determine the effect of the analogue on cell migration. Flow cytometry and magnetic levitation were used to measure the effect of compound on cell cycle and 3D spheroid formation. Our data showed that treating liver cancer cells with TMBP resulted in a significant dose and time dependent growth inhibition combined with G2/M-phase cell cycle arrest at 5 μM with (IC50) at 7.5 μM. TMBP at 5μM inhibited the formation of 3D spheroids, and also repressed the migration of cancer cells. Current therapies available for treating hepatocellular carcinoma (HCC) have a high incidence of recurrence and postoperative death in patients. Exploring different strategies with high pharmaceutical function and low toxicity is of great urgency for treating HCC. Our studies show that we have identified a promising analogue for treating HCC. Citation Format: Christopher J. Patton, Pritika Khadka, Hari Kotturi. 3,4',5- Trismethoxybenzophenone inhibits the growth of human hepatocarcinoma cells via cell-cycle arrest at the G2/M phase. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A39.

The in vitro propagation of Helichrysum umbraculigerum

Stilbenoids are a specific class of phenolic compounds, some of which show medicinal potential. Novel stilbenoids have previously been isolated from the South African plant Helichrysum umbraculigerum [1]. These prenylated resveratrol analogues are well known for their applications, which have mainly been discovered in research conducted on Cannabis sativa [2]. The applications include possible anti-inflammatory, antimicrobial and anticancer effects. H. umbraculigerum is known to be used by South African traditional healers. Exploitation of medicinal properties will require large numbers of plants, but H. umbraculigerum has proved difficult to cultivate. The aim of this study was to optimise an in vitro propagation protocol for H. umbraculigerum. Leaf cuttings were sterilised using 70% ethanol followed by 2% sodium hypochlorite containing 1 drop of Tween-20®. They were then washed in distilled water and placed onto Murashige and Skoog (MS) media. Uncontaminated leaf cuttings were placed onto MS media supplemented with different combinations of plant growth regulators. Shoots were produced most efficiently on MS media containing a specific concentration of thidiazuron (TDZ). These shoots were subsequently moved into the light, after which they started producing roots. Plantlets were successfully acclimatised using Jiffy-7C® pellets. The in vitro propagation protocol developed will allow for the production of large amounts of plant material for future research conducted on H. umbraculigerum.

Pterostilbene exerts an anti-inflammatory effect via regulating endoplasmic reticulum stress in endothelial cells

Pterostilbene (PT), an analog of resveratrol, exerts a potent anti-inflammatory effect. However, the protective effects of PT against inflammation in endothelial cells have not been elucidated. Previous studies have confirmed that endoplasmic reticulum stress (ERS) plays an important role in regulating the pathological process of endothelial cell inflammation. In this study, we explored the effect of PT on the tumor necrosis factor-α (TNF-α)-induced inflammatory response in human umbilical vein endothelial cells (HUVECs) and elaborated the role of ERS in this process. TNF-α treatment significantly upregulated the levels of inflammation-related molecules in cell culture media, increased the adhesion of monocytes to HUVECs, and enhanced the expression of the MMP9 and ICAM proteins in HUVECs. Additionally, TNF-α potently increased ERS-related protein levels, such as GRP78 and p-eIF2α. However, PT treatment reversed the increased production of inflammatory cytokines and the adhesion of monocytes to HUVECs, as well as reduced the TNF-α-induced effects exerted by ERS-related molecules. Furthermore, thapsigargin (THA), an ERS inducer, attenuated the protective effect of PT against TNF-α-induced inflammation and ERS in HUVECs. Additionally, the downregulation of ERS signaling using siRNA targeting eIF2α and IRE1 not only inhibited ERS-related molecules but also simulated the therapeutic effects of PT on TNF-α-induced inflammation. In summary, PT treatment potently attenuates inflammation in vascular endothelial cells, which at least partly depends on the reduction of ERS.

(Z)3,4,5,4'-trans-tetramethoxystilbene, a new analogue of resveratrol, inhibits gefitinb-resistant non-small cell lung cancer via selectively elevating intracellular calcium level.

Calcium is a second messenger which is required for regulation of many cellular processes. However, excessive elevation or prolonged activation of calcium signaling would lead to cell death. As such, selectively regulating calcium signaling could be an alternative approach for anti-cancer therapy. Recently, we have identified an effective analogue of resveratrol, (Z)3,4,5,4′-trans-tetramethoxystilbene (TMS) which selectively elevated the intracellular calcium level in gefitinib-resistant (G-R) non-small-cell lung cancer (NSCLC) cells. TMS exhibited significant inhibitory effect on G-R NSCLC cells, but not other NSCLC cells and normal lung epithelial cells. The phosphorylation and activation of EGFR were inhibited by TMS in G-R cells. TMS induced caspase-independent apoptosis and autophagy by directly binding to SERCA and causing endoplasmic reticulum (ER) stress and AMPK activation. Proteomics analysis also further confirmed that mTOR pathway, which is the downstream of AMPK, was significantly suppressed by TMS. JNK, the cross-linker of ER stress and mTOR pathway was significantly activated by TMS. In addition, the inhibition of JNK activation can partially block the effect of TMS. Taken together, TMS showed promising anti-cancer activity by mediating calcium signaling pathway and inducing apoptosis as well as autophagy in G-R NSCLC cells, providing strategy in designing multi-targeting drug for treating G-R patients.