Effects Of Rhus Verniciflua Stokes Research Articles

Abstract 4123: Rhus verniciflua Stokes (RVS) inhibits PD-1 expression and induces anticancer effects via enhancing T-cell function

Abstract Rhus verniciflua Stokes (RVS) has traditionally been used for centuries as a therapeutic herb and food supplement in East Asian countries. Over the last decade, the anticancer effects of RVS have been demonstrated in various preclinical and clinical studies. RVS contains major flavonoids such as fustin, fisetin, and sulfuretin, which were known to have apoptotic or anti-proliferative activities in various cancer cell lines. In clinical retrospective studies, RVS administration exclusively suggested more favorable outcomes with prolonged overall survival in solid cancer patients. However, the effect of RVS on immuno-oncology, especially the functional properties of T cells and their phenotypes remain unclear. Dried RVS grown in Wonju (Korea) was extracted with sterile distilled water after removing the allergen (urushiol). Peripheral blood mononuclear cells (PBMCs) from breast cancer patients with HLA-A*02:01 were isolated using a vacutainer tube and cultured for T cell expansion in the presence of interleukin (IL)-2 for 14 days. Cells were enriched above 90% CD3+ T cells after 14 days of expansion. Red fluorescent protein (RFP)-expressing HLA-A*02:01-matched breast cancer cell lines (MCF7 and MDA-MB-231), were used to determine the anticancer activity of T cells in the presence or absence of RVS. For in vitro killing assays, MCF7 and MDA-MB-231 cell lines expressing RFP were co-cultured with T cells with or without RVS treatment for 48 h, and cell viability was measured by the MTT assay. The phenotypic characteristics of the T cells were profiled by staining with various T cell surface markers, including CD3, CD4, CD8, PD-1, and CTLA4. RVS toxicity in the target cells (MCF7 and MDA-MB-231) was not observed at RVS concentrations up to 250 ug/mL. The anticancer activity of T cells against breast cancer cells was significantly increased by adding both 10 ug/mL and 100ug/mL of RVS. T cells co-cultured with MCF7 and MDA-MB-231 cells in the presence of 100 ug/mL of RVS showed 20.6% increases in cytotoxicity in MCF7 cells and 36.2% in MDA-MB-231 cells compared to no RVS treatment. Interestingly, relative reductions in programmed death-1 (PD-1) were found in T cells co-cultured with target cells by adding RVS, even though there was no significant difference in the other markers observed. Our findings showed that RVS could improve the function of T cells against cancer cells in the tumor microenvironment, as interpreted by reduced PD-1 expression in T cells after RVS treatment. Therefore, the components of RVS are candidates for restoring T cells exhausted against cancer. Active compounds should be identified for clinical efficacy, and further studies are necessary for combination strategies with conventional cancer treatments, such as chemotherapy and radiotherapy. Citation Format: Seoyoung Kim, Young-Kwan Lee, Hyoun Jong Moon, Sunki Lim, Yujeong Gho, Wang Jun Lee, Sanghun Lee, Excelsisbio Inc.. Rhus verniciflua Stokes (RVS) inhibits PD-1 expression and induces anticancer effects via enhancing T-cell function. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4123.

Genotoxicity of Water Extract from Bark-Removed Rhus verniciflua Stokes

Rhus verniciflua Stokes (RVS) has been traditionally used as an herbal remedy to support the digestive functions in traditional Korean medicine. Additionally, the pharmacological effects of RVS, including antioxidative, antimicrobial and anticancer activities, have been well-reported. The genotoxicity of RVS, however, is elusive; thus, we evaluated the genotoxicity of RVS without bark (RVX) for safe application as a resource of functional food or a medical drug. To evaluate the genotoxicity of RVX, we used a bacterial reverse mutation test, chromosomal aberration test and comet assay, according to the “Organization for Economic Co-operation and Development” (OECD) guidelines. Briefly, for the reverse mutation test, samples (5000, 1667, 556, 185, 62 and 0 μg/plate of RVX or the positive control) were treated with a precultured strain (TA98, TA100, TA1535, TA1537 or WP2µvrA) with or without the S9 mix, in which RVX partially induced a reverse mutation in four bacterial strains. From the chromosomal aberration test and comet assay, the RVX samples (556, 185, 62, 20 and 0 μg/mL of RVX or the positive control) were treated in a Chinese hamster ovary cell line (CHO-K1 cells) in the conditions of the S9 mix absent or S9 mix present and in Chang liver cells and C2C12 myoblasts, respectively. No chromosomal aberrations in CHO-K1 or DNA damage in Chang liver cells and C2C12 myoblasts was observed. In conclusion, our results suggest the non-genotoxicity of RVX, which would be helpful as a reference for the safe application of bark-removed Rhus verniciflua Stokes as functional raw materials in the food, cosmetics or pharmaceutical fields.

Extract of Rhus verniciflua stokes protects against renal ischemia-reperfusion injury by enhancing Nrf2-mediated induction of antioxidant enzymes.

Ischemia-reperfusion injury (IRI) may cause acute kidney disease (AKD) by mediating the oxidative stress-induced apoptosis of parenchymal cells. The extract of Rhus verniciflua Stokes (RVS) is used as a traditional herbal medicine as it exhibits anti-oxidant, anti-apoptotic and anti-inflammatory properties. Therefore, the current study investigated the therapeutic effect and the underlying mechanism of RVS on IRI-induced AKD in vivo and in vitro. The current study assessed the effects of RVS on a mouse model of renal IRI and in hypoxic human renal tubular epithelial HK-2 cells. The results demonstrated that the IRI-induced elevation of blood urea nitrogen, serum creatinine and lactate dehydrogenase was significantly attenuated by the intraoral administration of RVS (20 mg/kg/day) for 14 days prior to surgery. It was demonstrated that IRI surgery induced histological damage and cellular apoptosis in renal parenchyma, which were attenuated by pretreatment with RVS. Furthermore, in HK-2 cells incubated with 300 µM CoCl2 to induce chemical hypoxia, it was demonstrated that RVS treatment significantly inhibited cell death and the production of reactive oxygen species (ROS). Furthermore, RVS treatment upregulated the levels of endogenous antioxidant enzymes, including heme oxygenase-1 and catalase, as well as their upstream regulator nuclear factor erythroid 2-related factor 2, in HK-2 cells. Taken together, these results suggested that the intraoral administration of RVS induces a therapeutic effect on IRI-induced AKD. These effects are at least partly due to the attenuation of ROS production via upregulation of the antioxidant defense system in renal tubular cells.

Extract of Rhus verniciflua Stokes enhances Th1 response and NK cell activity

Rhus verniciflua Stokes (RVS) is a common poison ivy that causes allergic dermatitis. However, it has been used in Korean Medicine as a therapeutic herb, traditionally for the treatment of abdominal masses. Recent studies have demonstrated evidence of anti-tumoral effects by RVS. However, the effect of RVS on tumor-targeting immune cells such as NK cells and helper T cells remains to be verified. The purposes of this study are as follows: (1) determine whether RVS increases NK cell activity and (2) determine whether RVS increases the expression of cytokines, such as IFN-γ, TNF-α, IL-4, IL-5, and IL-10, from Th1 and Th2 cells. NK cells and CD4 T-cells from the spleens of BALB/c mice were highly purified using magnetic beads. CD4 T-cells were polarized to Th1-cells by rIL-12 and anti-IL-4 antibody or to Th2-cells by rIL-4 and anti-IL-12 antibody. RVS directly increased NK cell activity in a calcein-AM release assay and augmented IFN-γ and TNF-α secretion from Th1-cells in an ELISA assay. Thus, these results suggest that RVS may stimulate therapeutic immune responses against cancer via NK cells and Th1 cells.

건칠의 항주름 효과와 미백효과 연구

Objectives : Recently, the demands for the effective and safe depigmentating and anti-aging agents of the skin have increased due to the medical, pharmaceutical and cosmetic reasons. The aim of this study is to search new materials from the traditional herbal medicines which inhibit the aging process of skin in vitro.Methods : Human dermis cell (HS68) was used to test the effect of Rhus verniciflua Stokes (RVS). 80% ethanol or water extracts were screened for their inhibitory activities against elastase. Elastase inhibition effect was tested by microplate reader instrument. And MMP-1 suppression effect of RVS was tested by western blot. These cells were investigated the viability by MTS assay. And also the inhibition effect of tyrosinase by RVS was tested.Results : RVS (final concentrstion 1 ㎎/㎖ appeared over 30% of inhibition of elastase activity. So we are investigated anti wrinkle effects of Rhus verniciflua Stokes look through MMP-1 inhibition activity, also Extracts of RVS showed higher anti-tyrosinase activity than arbutin as final concentration 1 mg/ml. These results suggest that herbal medicines could be strong potential sources of inhibition of anti-aging and whitening effects for the skin.Colclusions : RVS was the best suppressor candidate of elastase activity among other control oriental drugs. It was found that RVS did not have toxicity to cells. We found proper concentration of RVS to treat in HS68 culture and investigated not only wrinkle care effect but also whitening effect of RVS.

Rhus verniciflua Stokes (RVS) and butein induce apoptosis of paclitaxel-resistant SKOV-3/PAX ovarian cancer cells through inhibition of AKT phosphorylation.

BackgroundRhus verniciflua Stokes (RVS) belongs to the Anacardiaceae family and traditionally used for cancer treatment. RVS and butein, a major compound of RVS, were known to induce apoptosis via AKT inhibition in cancer cells. Thus, in this study, we investigated the effect of RVS and its derivative compounds (fisetin, quercetin, butein) on cell death in SKOV-3/PAX cells.MethodsThe 80 % ethanol extract of RVS and its derivative compounds (fisetin, quercetin, butein) were prepared. The cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Apoptotic cells were detected by staining with propidium iodide (PI) and Annexin V-fluorescein isothiocyanate/7-aminoactinomycin D (Annexin V-FITC/7-AAD). The expression level of intracellular signaling related-proteins in apoptosis and growth were measured by western blot assay.ResultsWe found that RVS and butein suppressed the growth of SKOV-3/PAX cells in a dose-dependent manner. We also found that RVS and butein produced the cleavage of caspase-9, -8, -3, and PARP. Similarly, sub-G1 phase and Annexin V-FITC positive cells were increased by RVS and butein. Moreover, RVS and butein significantly reduced AKT phosphorylation in SKOV-3/PAX cells. PI3K inhibitor LY294002 caused PARP cleavage supporting our finding.ConclusionOur data clearly indicate that RVS and butein induce apoptosis of SKOV-3/PAX cells through inhibition of AKT activation. RVS and butein could be useful compounds for the treatment for paclitaxel resistant-ovarian cancer.

Heartwood extract of Rhus verniciflua Stokes and its active constituent fisetin attenuate vasoconstriction through calcium-dependent mechanism in rat aorta.

Rhus verniciflua Stokes (RVS) exert cardiovascular protective activity by promoting blood circulation, but its active ingredients and underlying mechanism have yet to be identified. This study investigated the vascular effects of RVS, focusing on vasoconstriction and smooth muscle Ca(2+) signaling. RVS heartwood extract attenuated contraction of aortic rings induced by the vasoconstrictors serotonin and phenylephrine, and inhibited the Ca(2+) signaling evoked by serotonin in vascular smooth muscle cells. Subsequent activity-guided fractionation identified fisetin as an active constituent exerting a Ca(2+) inhibitory effect. Fisetin could inhibit major Ca(2+) mobilization pathways including extracellular Ca(2+) influx mediated by the L-type voltage-gated Ca(2+) channel, Ca(2+) release from the intracellular store and store-operated Ca(2+) entry. In accordance with Ca(2+) inhibitory effect, fisetin attenuated vasoconstriction by serotonin and phenylephrine. These results suggest that the anticontractile effect, which is presumably mediated by inhibition of Ca(2+) signaling, may contribute to the improvement of blood circulation by RVS.

Rhus vernicifluaStokes attenuates cholestatic liver cirrhosis-induced interstitial fibrosis via Smad3 down-regulation and Smad7 up-regulation

Cholestatic liver cirrhosis (CLC) eventually proceeds to end-stage liver failure by mediating overwhelming deposition of collagen, which is produced by activated interstitial myofibroblasts. Although the beneficial effects of Rhus verniciflua Stokes (RVS) on various diseases are well-known, its therapeutic effect and possible underlying mechanism on interstitial fibrosis associated with CLC are not elucidated. This study was designed to assess the protective effects of RVS and its possible underlying mechanisms in rat models of CLC established by bile duct ligation (BDL). We demonstrated that BDL markedly elevated the serological parameters such as aspartate aminotransferase, alanine transaminase, total bilirubin, and direct bilirubin, all of which were significantly attenuated by the daily uptake of RVS (2 mg/kg/day) for 28 days (14 days before and after operation) via intragastric route. We observed that BDL drastically induced the deterioration of liver histoarchitecture and excessive deposition of extracellular matrix (ECM), both of which were significantly attenuated by RVS. In addition, we revealed that RVS inhibited BDL-induced proliferation and activation of interstitial myofibroblasts, a highly suggestive cell type for ECM production, as shown by immunohistochemical and semi-quantitative detection of α-smooth muscle actin and vimentin. Finally, we demonstrated that the anti-fibrotic effect of RVS was associated with the inactivation of Smad3, the key downstream target of a major fibrogenic cytokine, i.e., transforming growth factor β (TGF-β). Simultaneously, we also found that RVS reciprocally increased the expression of Smad7, a negative regulatory protein of the TGF-β/Smad3 pathway. Taken together, these results suggested that RVS has a therapeutic effect on CLC, and these effects are, at least partly, due to the inhibition of liver fibrosis by the downregulation of Smad3 and upregulation of Smad7.

The effect of Rhus verniciflua Stokes (RVS) for anti-aging and whitening of skin

The purpose of this study is to investigate the effect of new materials from the traditional herbal medicines which induce anti-aging and anti-melanogenesis process of skin in vitro. Among 22 traditional herbal medicines which were tested in this study, Rhus vernciflua stokes (RVS) showed prominent effects for anti-aging and whitening of skin compared to other herbal extracts. In our study, RVS showed the highest elastase inhibitory effect and a higher anti-tyrosinase activity. Pretreatment with RVS significantly inhibited the expressions of UVB-induced MMP-1 protein and mRNA in a dose-dependent manner, and also inhibited the UV-induced activation of extracellular signal regulated kinase (ERK). In whitening, it was found that RVS significantly inhibits melanin biosynthesis in a dose-dependent manner. Moreover, α-MSH-induced increases of microphthalmia-associated transcription factor (MITF) and tyrosinase were significantly inhibited in B16F1 melanoma cells treated with RVS. Therefore, these data suggest that RVS extracts could be a potential agent for the prevention and treatment of skin photoaging and hyperpigmentation.

Anti-inflammatory and anti-proliferative effects of Rhus verniciflua Stokes in RAW264.7 cells

Inflammatory response is a major defense mechanism against pathogens and chemical or mechanical injury. Rhus verniciflua Stokes (RVS) has traditionally been used as an ingredient in East Asian medicine for the treatment of gastritis, stomach cancer and atherosclerosis. The aim of the current study was to analyze the effect of RVS on LPS‑induced inflammatory responses in the RAW264.7 mouse macrophage cell line. RAW264.7 cells were treated with various concentrations of RVS and LPS at specific time points. WST assay, trypan blue assay and quantification of activated cells revealed that RVS suppressed cell proliferation in a dose‑dependent manner. RVS induced G1 cell cycle arrest, suppressed iNOS and COX‑2 mRNA expression induced by LPS and decreased intracellular ROS levels induced by LPS. In addition, RVS induced PARP and caspase‑3 cleavage suggesting that RVS causes cell death. Results of the present study indicate that RVS may be advantageous in treating inflammatory disease.

Detoxified Extract of Rhus verniciflua Stokes Inhibits Rotenone-Induced Apoptosis in Human Dopaminergic Cells, SH-SY5Y

Rhus verniciflua Stokes (RVS), traditionally used as a food supplement and in traditional herbal medicine for centuries in Korea, is known to possess various pharmacological properties. Environmental neurotoxins such as rotenone, a specific inhibitor of complex I provide models of Parkinson's disease (PD) both in vivo and in vitro. In this study, we investigated the neuroprotective effect of RVS against rotenone-induced toxicity in human dopaminergic cells, SH-SY5Y. Cells exposed to rotenone for 24 h-induced cellular injury and apoptotic cell death. Pretreatment of cells with RVS provided significant protection to SH-SY5Y cells. Further, RVS offered remarkable protection against rotenone-induced oxidative stress and markedly inhibited mitochondrial membrane potential (MMP) disruption. RVS also attenuated the up-regulation of Bax, Caspase-9 and Caspase-3 and down-regulation of Bcl-2. Moreover, pretreatment with RVS prevented the decrease in tyrosine hydroxylase (TH) levels in SH-SY5Y cells. Interestingly, RVS conferred profound protection to human dopaminergic cells by preventing the downregulation of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). These results suggest that RVS may protect dopaminergic neurons against rotenone-induced apoptosis by multiple functions and contribute to neuroprotection in neurodegenerative diseases, such as PD.

A Detoxified Extract ofRhus vernicifluaStokes Upregulated the Expression of BDNF and GDNF in the Rat Brain and the Human Dopaminergic Cell Line SH-SY5Y

Rhus verniciflua Stokes (RVS) has traditionally been used as a food supplement and a traditional herbal medicine for centuries in Korea. This study attempted to evaluate the effects of RVS on the expression of Brain derived neurotrophic factor (BDNF) and Glial cell line-derived neurotrophic factor (GDNF) in SH-SY5Y cells and the rat brain. The results indicated that RVS is a potent inducer of Neurotrophic factor (NTF) production both in vitro and in vivo. Treatment with 10 µg/ml and 10 mg/kg RVS for 4 h of SH-SY5Y cells and rats yielded significant increases in BDNF and GDNF protein levels. We also detected BDNF and GDNF immunoreactive neurons in the rat brain. Both BDNF and GDNF-immunohistochemical staining was markedly enhanced in the animals treated with RVS. These results suggest that RVS serves as an ideal adjuvant in regard to regulating NTF expression, and can contribute to neuroprotection in neurodegenerative diseases.

Early antiallergic inflammatory effects of Rhus verniciflua stokes on human mast cells

Rhus verniciflua Stokes (RVS) is a traditional medicine used in Korea, Japan and China to treat various diseases including catharsis, diaphoretic gastritis and stomach cancer. However, the effects of RVS on allergic inflammatory diseases are unknown to date. This study showed the antiallergic inflammatory effects of RVS on human mast cells (HMC-1) which were stimulated by phorbol myristate acetate (PMA) and calcium ionophore A23187. RVS inhibited the expressions of TNF-alpha, IL-6 and IL-8 that were stimulated by treatment with both PMA and A23187. Among the mitogen-activated protein kinases (MAPKs), extracts of RVS suppressed the phosphorylation of ERK and p38, whereas RVS increased the phosphorylation of JNK in HMC-1. Consistent with the regulation of MAPKs, it was found that RVS inhibited the nuclear translocation of nuclear factor (NF)-kappaB via inhibition of the phosphorylation of IkappaB-alpha, which are important processes in controlling inflammatory responses. Taken together, these results suggest that RVS modulates the expressions of signal molecules related to allergic inflammatory responses mainly through the ERK signaling pathway, suggesting that RVS could be used as a treatment for mast cell-derived allergic inflammatory diseases.

Rhus verniciflua Stokes prevents cisplatin-induced cytotoxicity and reactive oxygen species production in MDCK-I renal cells and intact mice

Cisplatin-induced oxidative stress can cause liver and kidney damage, thus limiting therapeutic efficacy. Thus, in the present study, since Rhus verniciflua Stokes (RVS) containing flavonoids has antioxidant effects, we investigated whether it can protect cisplatin-induced toxicity in vitro and in vivo, The in vitro effects of RVS on the cell viability and reactive oxygen species (ROS) production were investigated using cisplatin-treated Madin-Darby Canine kidney (MDCK)-I renal cells. Its in vivo effects were also studied in BALB/c mice inoculated with CT-26 colon adenocarcinoma cells and treated with cisplatin with or without RVS. Liver and renal functions were assessed together with indices of tissue oxidation. RVS prevented cisplatin-induced cytotoxicity and ROS release against MDCK-I cells. RVS alone exerted modest antitumor activity against CT-26 cells. When used concurrently with cisplatin, RVS prevented the increases in serum blood urea nitrogen (BUN), creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and NO, while reducing liver and kidney tissue MDA content, and increasing catalase, glutathione (GSH), and superoxide dismutase (SOD) activities. Moreover, the antitumor efficacy of cisplatin was not altered by concurrent administration of RVS. These findings demonstrate that RVS prevents cisplatin-induced toxicity in vitro and in vivo via an antioxidant activity without hurting its antitumor effectiveness, suggesting that RVS can be usefully applied to the neoplastic patients as a combined chemopreventive agent with cisplatin.

Evaluation of Genotoxicity of Water and Ethanol Extracts from Rhus verniciflua Stokes (RVS).

Rhus verniciflua Stokes (RVS), one of traditional medicinal plants in Asia, was found to have pharmacological activities such as antioxidative and antiapoptotic effects, raising the possibility for the development of a novel class of anti-cancer drugs. Thus, potential genotoxic effects of RVS in three short-term mutagenicity assays were investigated, which included the Ames assay, in vitro Chromosomal aberration test, and the in vivo Micronucleus assay. In Ames test, the addition of RVS water extracts at doses from 313 up to 5000 mg/plate induced an increase more than 2-fold over vehicle control in the number of revertant colonies in TA98 and TA1537 strains for detecting the frame-shift mutagens. The similar increase in reversion frequency was observed after the addition of RVS ethanol extracts. To assess clastogenic effect, in vitro chromosomal aberration test and in vivo micronucleus assay were performed using Chinese hamster lung cells and male ICR mice, respectively. Both water and ethanol extracts from RVS induced significant increases in the number of metaphases with structural aberrations mostly at concentrations showing the cell survival less than 60% as assessed by in vitro CA test. Also, there was a weak but statistically significant increase in number of micronucleated polychromatic erythrocytes (MNPCEs) in mice treated with water extract at 2000 mg/kg while ethanol extracts of RVS at doses of up to 2000 mg/kg did not induce any statistically significant changes in the incidence of MNPCEs. Therefore, our results lead to conclusion that RVS acts as a genotoxic material based on the available in vitro and in vivo results.

Rhus verniciflua Stokes Extract: Radical Scavenging Activities and Protective Effects on H2O2-Induced Cytotoxicity in Macrophage RAW 264.7 Cell Lines

The goal of this study was to determine the effects of Rhus verniciflua stokes (RVS) on the radical scavenging activities and H2O2-induced cytotoxicity in macrophage 264.7 cells. An 80% ethanol extract of RVS was purified by ion exchange chromatography (IEC). Purified RVS extract contained a high amount of phenolics (55.5 g/100 g of extract) and flavonoids (5.9 g/100 g of extract) and its antioxidant activities, such as DPPH, superoxide anion and hydroxyl radical scavenging activities, was higher than the unpurified RVS extract. Purified RVS extract significantly reduced intracellular ROS formation caused by H2O2. Purified RVS extract also prevented the cell death of macrophage RAW 264.7 cells induced by H2O2. Interestingly, co-treated purified RVS extract effectively reduced thiobarbituric acid reactive substance (TBARS) formation, and attenuated catalase depletion at concentration of 100 microg/ml (p<0.05). Study using PDA-HPLC analyses have found that the purified RVS extract contains six major low molecular compounds as p-coumaric acid, fustin, kaempferol-3-O-glucoside, sulfuretin, butein and kaempferol. These results indicate that purified RVS extract, tested in this study, could contribute to the antioxidant activities and inhibition of intracellular ROS level.