Mechanism Of Caffeic Acid Phenethyl Ester Research Articles

Caffeic Acid Phenethyl Ester as a DHODH Inhibitor and Its Synergistic Anticancer Properties in Combination with 5-Fluorouracil in a Breast Cancer Cell Line.

IntroductionA combination of chemotherapy agents is the best choice in breast cancer treatment to increase the patient survival rate. 5-fluorouracil (5-FU) is one of the drugs applied in combination with other drugs to control and delay development of cancer cells. Nevertheless, the occurrence of multidrug resistance and dose-limiting cytotoxicity have limited the efficacy of 5-FU treatment. Therefore, the discovery of new anti-breast cancer drugs should be pursued.ObjectiveTo study potency of a promising naturally derived compound, caffeic acid phenethyl ester (CAPE), for breast cancer treatment in single and combination with 5-FU.MethodsCytotoxicity of CAPE, 5-FU, and 5-FU+CAPE was studied by in vitro MTT experiment in MCF-7 cell line, and RT-PCR analysis was used to evaluate the change in gene expression due to the treatment. Moreover, an enzymatic assay and molecular docking analysis were applied to evaluate the possible mechanism of substance-induced apoptosis.ResultsThe study revealed that a single treatment of CAPE showed cytotoxicity with IC50 6.6 ± 1.0 µM and 6.5 ± 2.9 µM at 24 h and 48 h, respectively. Meanwhile, 5-FU showed cytostatic activity. The 5-FU + CAPE has a synergistic effect at 24 h treatment with a CI = 0.5 and an additive effect at 48 h treatment with CI = 1.0. CAPE was also found to enhances the mRNA expression of caspase-8 and BAX within 6 hours in combination with 5-FU compared to 5-FU treatment alone. Our study reveals a new mechanism of CAPE which is related to the inhibition of human dihydroorotate dehydrogenase (HsDHODH) with an IC50 of 120.7 ± 6.8 µM, by bound to the ubiquinone-binding site of the enzyme and could be responsible for inducing extrinsic and intrinsic apoptosis.ConclusionThis study demonstrated the cytotoxicity of CAPE potential to induce apoptosis of breast cancer MCF-7 cell line single and cytotoxic–cytostatic combination with 5-FU. Therefore, further studies to develop CAPE and its derivatives will be required to discover new candidates for breast cancer agents.

Caffeic acid phenethyl ester alleviated hypouricemia in hyperuricemic mice through inhibiting XOD and up-regulating OAT3

BackgroundHyperuricemia is characterized with high serum uric acids (SUAs) and directly causes suffering gout. Caffeic acid phenethyl ester (CAPE) is widely included in dietary plants and especially propolis of honey hives. Hypothesis/PurposeSince CAPE exerts a property resembling a redox shuttle, the hypothesis is that it may suppress xanthine oxidase (XOD) and alleviate hyperuricemia. The aim is to unveil the hypouricemic effect of CAPE and the underlying mechanisms. MethodsBy establishing a hyperuricemic model with potassium oxonate (PO) and hypoxanthine (HX) together, we investigated the hypouricecmic effect of CAPE. On this model, the expressions of key mRNAs and proteins, including glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1), and the activity of XOD were assayed in vivo. Also, the inhibitory effect of CAPE against XOD was assayed in vitro through enzymatic activity tests and by molecular docking. ResultsCAPE demonstrated a remarkable hypouricemic effect, which reduced the SUAs of hyperuricemic mice (401 ± 111 µmol/l) to 209 ± 56, 204 ± 65 and 154 ± 40 µmol/l (p < 0.01) at the doses of 15, 30 and 60 mg/kg respectively, depicting efficacies between 48 and 62% and approaching allopurinol's efficacy (52%). Serum parameters, body weights, inner organ coefficients, and H&E staining suggested that CAPE displayed no general toxicity and it alleviated the liver and kidney injuries caused by hyperuricemia. Mechanistically, CAPE decreased XOD activities significantly in vivo, presented an IC50 at 214.57 µM in vitro and depicted a favorable binding to XOD in molecular simulation, indicating that inhibiting XOD may be an underlying mechanism of CAPE against hyperuricemia. CAPE did decreased GLUT9 protein and down-regulated URAT1 mRNA and protein. In addition, CAPE up-regulated ATP binding cassette subfamily G member 2 (ABCG2) and organic anion transporter 3 (OAT3) mRNA and proteins in comparison with that of the hyperuricemic control. All above, CAPE may alleviate hyperuricmia through inhibiting XOD, decreasing GLUT9 and URAT1 and increasing ABCG2 and OAT3. ConclusionCAPE presented potent hypouricemic effect in hyperuricemic mice through inhibiting XOD activity and up-regulating OAT3. CAPE may be a promising treatment against hyperuricemia.

Caffeic Acid Phenethyl Ester Inhibits the Proliferation of HEp2 Cells by Regulating Stat3/Plk1 Pathway and Inducing S Phase Arrest.

Caffeic acid phenethyl ester (CAPE), an active polyphenolic component of honeybee propolis, has been demonstrated to have many medicinal properties. However, the antitumor effect and mechanism of CAPE on laryngeal carcinoma cells have not been examined. In this study, we treated HEp2 cells with various concentration of CAPE, and the results showed that CAPE can reduce the viability of HEp2 cells with IC50 values of 23.8 ± 0.7 µM for 72 h. Meanwhile, CAPE significantly inhibited activation of signal transducer and activator of transcription (Stat)3 in a concentration dependent manner in HEp2 cells and regulated the expression and transcription of Plk1. AG490, a specific Stat3 inhibitor, not only inhibited the activation and expression of Stat3, but also inhibited the expression of Plk1 in HEp2 cells, so Stat3 was probably involved in the regulation of Plk1 in HEp2 cells. In addition, treatment of CAPE leaded to a blockage of cell cycle in S phase in HEp2 cells. Therefore, CAPE inhibited the proliferation of HEp2 Cells probably by regulating Stat3/Plk1 pathway and inducing S phase arrest.

Caffeic acid phenethyl ester attenuates neuropathic pain by suppressing the p38/NF-κB signal pathway in microglia.

BackgroundManagement of neuropathic pain is still a clinical challenge. Evidence has accumulated indicating that propolis is effective in attenuating neuropathic pain; however, the mechanism is not fully understood. Our present study investigated the effects and the possible mechanism of caffeic acid phenethyl ester (CAPE), the main ingredient of propolis, in improving neuropathic pain via its inhibition on p38/NF-κB signal pathway in microglia.Materials and methodsChronic constriction injury (CCI) mice model and the microglial cell line BV-2 were used to investigate the effects and the mechanism of CAPE. Cell signaling was measured by real-time PCR, Western blotting and immunofluorescence assay.ResultsCAPE relieved neuropathic pain behaviors induced by CCI in mice. CAPE also inhibited CCI-induced activation of microglia. Furthermore, CAPE suppressed the phosphorylation of p38 mitogen-activated protein kinase, inhibited the translocation of NF-κB and decreased the expression of proinflammatory cytokines tumor necrosis factor-α, IL-1β and IL-6.ConclusionCAPE was found to be an effective and safe drug candidate for alleviating neuropathic pain by its powerful inhibition on the P38/NF-κB signal pathway.

Abstract 2591: Caffeic acid phenethyl ester (CAPE) suppresses melanoma tumor growth by inhibiting AKT/XIAP pathway

Abstract The incidence of melanoma is constantly on the rise indicating that current available therapies are not very effective. Moreover, melanoma is highly metastatic and resistant to chemotherapeutic drugs. Our previous studies have demonstrated that caffeic acid phenethyl ester (CAPE) suppress the growth of melanoma cells and induce ROS generation. However, the exact mechanism of the growth suppressive effects of CAPE was not clear. Here we determined the potential mechanism of CAPE against melanoma in vitro and in vivo. In a concentration and time-dependent study, CAPE treatment suppressed the activating phosphorylation of PI3K at Tyr-458, PDK1 at Ser-241, mTOR at Ser-2448 and AKT at Ser-473 in B16F0 and SK-MEL-28 melanoma cells. Furthermore, the expression of XIAP, survivin and BCl2 were down-regulated by CAPE treatment in both the cell lines. Significant apoptosis was observed in both the cell lines by CAPE treatment as indicated by cleavage of caspase-3 and PARP. AKT kinase activity was inhibited by CAPE in a concentration-dependent manner. Since Akt regulates XIAP, their interaction was examined by immunoprecipitation studies. Our results show that CAPE treatment decreased the interaction of AKT with XIAP. To establish the involvement of Akt in the apoptosis-inducing effects of CAPE, cells were transfected with Akt. Our results reveal that AKT over-expression attenuated the decrease in XIAP and significantly blocked CAPE-mediated apoptosis. Similarly, over-expression of XIAP further decreased CAPE-induced apoptosis. To confirm the involvement of ROS in the inhibition of AKT/XIAP pathway, cells were treated with antioxidant NAC prior to CAPE treatment. Our results indicate that NAC blocked CAPE mediated AKT/XIAP inhibition and protected the cells from apoptosis. In addition, oral administration of 10mg/kg/day CAPE substantially suppressed the growth of B16F0 tumor xenografts in C57BL/6 mice. Tumors from CAPE treated mice showed reduced phosphorylation of PI3K, AKT, mTOR and protein level of XIAP and enhanced the cleavage of caspase-3 and PARP. Taken together, our results suggest that CAPE suppresses AKT/XIAP pathway leading to apoptosis in melanoma tumor cells in vitro and in vivo. [Supported in part by R01 grant CA129038 (to S.K.S) awarded by the National Cancer Institute]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2591. doi:1538-7445.AM2012-2591

Abstract 3241: The selective inhibition of GST by CAPE enhances the cytotoxicity of doxorubicin and camptotechin in SK-MEL-28 melanoma cells

Abstract Glutathione S-transferase (GST) and multidrug resistance proteins (MRPs) play major roles in the detoxification of anti-melanoma drugs; hence diminishing their toxicity towards melanoma cells. In this study, we investigated i) the selective inhibition of GST by Caffeic Acid Phenethyl Ester (CAPE) in presence of tyrosinase, ii) the role of MRP in CAPE induced toxicity, and iii) the effect of CAPE in combination with probenecid, doxorubicin and camptotechin. The inhibition of GST was investigated using CDNB method. LC/MS/MS was used to identify a CAPE glutathione conjugate formation by tyrosinase. MTT assay, Annexin V apoptosis assay and TMRM florescence method were used in the investigation of biochemical mechanism of CAPE efficacy in human SK-MEL-28 melanoma cells. At 30 min incubation, CAPE was bioactivated 65% by tyrosinase as measured by glutathione depletion. LC/MS/MS analyses identified CAPE-glutathione conjugate m/z [MH]+ 590 as the major metabolite formed as the result of CAPE bioactivation by tyrosinase. In the presence of tyrosinase, CAPE 10-25 µM showed 47-90% GST inhibition whereas in the absence of tyrosinase, CAPE 10-25 µM led to minimal GST inhibition. Both CAPE-SG conjugate and CAPE-quinone (25 µM) demonstrated ≥90% GST inhibition via reversible and irreversible competitive mechanisms with Ki of 5 µM with respect to GSH and with Ki of 6 µM with respect to CDNB, respectively. CAPE, a non-substrate for GST, only at concentrations &amp;gt;50 µM acted as a weak reversible non-competitive and competitive GST inhibitor with Ki of 277 µM with respect to GSH and with Ki of 234 µM with respect to CDNB, respectively. Moreover, computational auto docking analyses suggest that CAPE binds to the GST catalytic active site. CAPE (15-100 µM) showed 61-86% GST inhibition in human SK-MEL-28 cell homogenate. Furthermore, probenecid, a MRP inhibitor, increased CAPE (15, 30 µM) cell toxicity by 27-29%, apoptotic cell death by 10-14%, and decreased mitochondrial membrane potential by 27-40% in SK-MEL-28 melanoma cells, suggesting that the inhibition of MRP increases CAPE induced toxicity. The combination of CAPE (5 µM) with doxorubicin (0.1 µM) demonstrated synergistic effects as the combination index was 0.53 (&amp;lt;1). Similarly, CAPE (7.5 µM) showed synergistic effects with camptotechin (0.05 µM) with a combination index of 0.75. In summary, our results suggest that CAPE was bioactivated by tyrosinase to form a CAPE-quinone and CAPE glutathione conjugate, which played a major role in the inhibition of GST. Our study also elucidates the role of MRP in the biochemical mechanism of CAPE induced toxicity in melanoma cells. For the first time, we showed that CAPE in the presence of tyrosinase acts as a selective inhibitor of GST. CAPE therapy in combination with probencid, doxorubicin and camptotechin has synergistic effects in human SK-MEL-28 melanoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3241. doi:10.1158/1538-7445.AM2011-3241

The effect of caffeic acid phenethyl ester on the functions of human monocyte-derived dendritic cells

BackgroundPropolis, an ancient herbal medicine, has been reported the beneficial effect both in asthma patients and murine model of asthma, but the mechanism was not clearly understood. In this study, the effect of caffeic acid phenethyl ester (CAPE), the most extensively studied components in propolis, on the functions of human monocyte-derived dendritic cells (MoDCs) was investigated.ResultsCAPE significantly inhibited IL-12 p40, IL-12 p70, IL-10 protein expression in mature healthy human MoDCs stimulated by lipopolysaccharides (LPS) and IL-12 p40, IL-10, IP-10 stimulated by crude mite extract. CAPE significantly inhibited IL-10 and IP-10 but not IL-12 expression in allergic patients' MoDCs stimulated by crude mite extract. In contrast, the upregulation of costimulatory molecules in mature MoDCs was not suppressed by CAPE. Further, the antigen presenting ability of DCs was not inhibited by CAPE. CAPE inhibited IκBα phosphorylation and NF-κB activation but not mitogen-activated protein kinase (MAPK) family phosphorylation in human MoDCs.ConclusionThese results indicated that CAPE inhibited cytokine and chemokine production by MoDCs which might be related to the NF-κB signaling pathway. This study provided a new insight into the mechanism of CAPE in immune response and the rationale for propolis in the treatment of asthma and other allergic disorders.