Design,synthesis and pharmacological evaluation of caffeic acid phenethyl ester acylation as multifunctional neuroprotective agents against oxidative stress injury

4-Acylated or 3,4-diacylated caffeic acid phenethyl ester (CAPE) was prepared as prodrug to improve its stability and lipid solubility. Their neuroprotective activities were assessed by H2O2 model and 6-OHDA model. The results showed that target compounds displayed positive abilities to protect PC12 nerve cells from oxidative stress injury, superior to that of CAPE. Additionally, target compounds showed high blood-brain barrier permeability.     

Studies on the structure-activity relationship of caffeate derivatives as neuroprotective agents

In the present study, novel ester derivatives of CAPE were designed and synthesized as neuroprotective agents. The anti-inflammatory and antioxidant activities of these compounds were evaluated at the cellular level, while the blood-brain barrier (BBB) permeability was predicted by parallel artificial membrane permeability assay (PAMPA). The results revealed that phenolic hydroxyl groups and double bonds in the structure of CAPE had important effects on neuroprotective activities. Accordingly,a preliminary structure-activity relationship was summarized in this paper. In addition, we observed a significant improvement on BBB permeability. These results provided important references for the structural modification and optimization of CAPE in the future.     

Caffeic acid phenethyl ester (CAPE) prevents transformation of human cells by arsenite (As) and suppresses growth of As-transformed cells

Recent evidence suggests that inflammatory cytokines and growth factors contribute to arsenite (As)-induced human carcinogenesis. We investigated the expression of inflammatory cytokine mRNAs during the transformation process induced by chronic As exposure in non-tumorigenic human osteogenic sarcoma (N-HOS) cells using gene arrays, and results were confirmed by RT-PCR and protein arrays. Caffeic acid phenethyl ester (CAPE), a naturally occurring immunomodulating agent, was used to evaluate the role of inflammatory factors in the process of As-mediated N-HOS cell transformation and in As-transformed HOS (AsT-HOS) cells. We found that an 8-week continuous exposure of N-HOS to 0.3 microM arsenite resulted in HOS cell transformation. That exposure also caused substantial decreases in inflammatory cytokine mRNAs, such as interleukin (IL) IL-1alpha, IL-2, IL-8, IL-18, MCP-1, TGF-beta2, and TNF-alpha, while it increased c-jun mRNA in a time-dependent manner. Co-incubation of N-HOS with As and CAPE (0.5-2.5 microM) prevented As-mediated declines in cytokine mRNAs in the co-treated cells, as well as their transformation to anchorage independence, while it caused decreases in c-jun mRNA. CAPE (up to 10 microM) had no effect on growth of N-HOS cells. However, CAPE (1-10 microM) treatment of AsT-HOS cells inhibited cell growth, induced cell cycle G2/M arrest, and triggered apoptosis, accompanied by changes in cytokine gene expression, as well as decreases in cyclin B1 and cdc2 abundance. Resveratrol (RV) and (-)(.) epigallocatechin gallate (EGCG), preventive agents present in grapes and green tea, respectively, induced similar changes in AsT-HOS cell growth but required much higher doses than CAPE to cause 50% growth arrest (<2.5 microM CAPE versus 25 microM RV or 50 microM EGCG). Overall, our findings suggest that inflammatory cytokines play an important role in the suppressive effects of CAPE on As-induced cell transformation and in the selective cytotoxicity of CAPE to As-transformed HOS cells.     

The antioxidant caffeic acid phenethyl ester induces apoptosis associated with selective scavenging of hydrogen peroxide in human leukemic HL-60 cells

Caffeic acid phenethyl ester (CAPE), an active component of propolis, has many biological and pharmacological activities including antioxidation and tumor cell cytotoxicity. We examined the type of cell death in human leukemic HL-60 cells after CAPE treatment in order to elucidate the relationship between CAPE-induced alterations of the redox state and apoptosis. CAPE treatment (6 microg/ml) resulted in marked growth inhibition up to 70.3+/-4.0% at day 2. This inhibition was partially blocked by pretreatment with N-acetyl-L-cycteine (NAC). Agarose gel electrophoresis showed evident DNA fragmentation after CAPE treatment. CAPE induced a significant decrease in mitochondrial transmembrane potential to about half of the untreated level after 6 h and a rapid depletion of intracellular glutathione (GSH) down to 41.7+/-6.0% after 1 h. Pretreatment of HL-60 cells with NAC reversed the GSH depletion and partially rescued cells from CAPE-induced apoptosis. With regard to intracellular reactive oxygen species, CAPE caused a fast and profound scavenging of H202 (19% of untreated cells after a 2-h treatment) but not of superoxide anion. These results suggest that apoptosis induced by CAPE is associated with mitochondrial dysfunction, GSH depletion and selective scavenging of H2O2 in human leukemic HL-60 cells.     

Caffeic acid phenethyl ester (CAPE) protects rat skeletal muscle against ischemia-reperfusion-induced oxidative stress

Oxygen-derived free radicals have been implicated in the pathogenesis of skeletal muscle injury after ischemia-reperfusion. Caffeic acid phenethyl ester, an active component of propolis extract, exhibits antioxidant properties. The aim of this study was to assess the effects of caffeic acid phenethyl ester (CAPE) and alpha-tocopherol (vit E) on ischemia/reperfusion (I/R) injury in a rat hind limb ischemia/reperfusion model. For this purpose, ischemia was induced in anesthetized rats by unilateral (right) femoral artery clipping for 2 h followed by 2 h of reperfusion. Four groups were studied: sham, I/R, I/R+CAPE and I/R+vit E. Drugs were administered intraperitoneally after 1 h of ischemia and I/R rats received saline vehicle. After 2 h of reperfusion, venous blood was sampled and the right gastrocnemius muscle was harvested. Plasma and tissue were assayed for malondialdehyde (MDA), superoxide dismutase (SOD) and nitric oxide (NO) metabolites. Tissue was also assayed for catalase (CAT) activity. Both tissue and plasma NO levels, MDA levels, SOD activities was significantly increased in I/R groups compared to control groups. The two treated groups showed decreased MDA and NO in both muscle and plasma compared to the I/R group. No differences were noted in muscle tissue SOD in three I/R groups, but SOD activity were increased in the plasma of I/R+CAPE and I/R+vit E groups compared with I/R group. Whereas tissue CAT activity was not changed among groups. Our results indicate that CAPE has antioxidant properties similar to those of vit E in this model and may attenuate the harmful effects of hind limb I/R in skeletal muscle.     

Protective role of caffeic acid phenethyl ester (cape) on gentamicin-induced acute renal toxicity in rats

The toxicity of gentamicin (GEN) in the kidney seems to relate to the generation of reactive oxygen species (ROS). Caffeic acid phenethyl ester (CAPE) has been demonstrated to have antioxidant, free radical scavenger and anti-inflammatory effects. It has been proposed that antioxidant maintain the concentration of reduced glutathione (GSH) may restore the cellular defense mechanisms and block lipid peroxidation thus protect against the toxicity of wide variety of nephrotoxic chemicals. We investigated the effects of CAPE on GEN-induced changes in renal malondialdehyde (MDA), a lipid peroxidation product, nitric oxide (NO) generation, superoxide dismutase (SOD), catalase (CAT) activities, GSH content, blood urea nitrogen (BUN) and serum creatinine (Cr) levels. Morphological changes in the kidney were also examined. A total of 32 rats were equally divided into four groups which were: (1) control, (2) injected with intraperitoneally (i.p.) GEN, (3) injected with i.p. GEN+CAPE and (4) injected with i.p. CAPE. GEN administration to control rats increased renal MDA and NO generation but decreased SOD and CAT activities, and GSH content. CAPE administration with GEN injections caused significantly decreased MDA, NO generation and increased SOD, CAT activities and GSH content when compared with GEN alone. Serum level of BUN and Cr significantly increased as a result of nephrotoxicity. CAPE also, significantly decreased serum BUN and Cr levels. Morphological changes in the kidney due to GEN, including tubular necrosis, were evaluated qualitatively. In addition, CAPE reduced the degree of kidney tissue damage induced by GEN. Both biochemical findings and histopathological evidence showed that administration of CAPE reduced the GEN-induced kidney damage. Our results indicated that CAPE acts in the kidney as a potent scavenger of free radicals to prevent the toxic effects of GEN both at the biochemical and histological level. Thus, CAPE could be effectively combined with GEN treatment.     

Immunomodulatory effect of caffeic acid phenethyl ester in Balb/c mice

Caffeic acid phenethyl ester (CAPE), an the active component of propolis, is known to have anticarcinogenic, antiviral and various biological activities; however, the effect of CAPE on the immunomodulatory activity in vivo remains unknown. We have investigated the effect of CAPE on the immune system in female Balb/c mice. CAPE (0, 5, 10, 20 mg/kg) was given to mice orally for 14 days. Immunomodulatory activity was evaluated by assessment of body and organ weight, lymphocyte blastogenesis, plaque-forming cell (PFC) assay, lymphocyte subpopulation by flow cytometry and cytokine production. Even though the change of body weight was not observed in CAPE-administered group, thymus weight and/or cellularity of thymus and spleen are decreased at the all dose groups of CAPE (5, 10, 20 mg/kg). On the other hand, CAPE had no effect on B lymphocyte proliferation induced by lipopolysaccharide (LPS) but increased T lymphocyte blastogenesis induced by concanavalin A (Con A) at the dose of 20 mg/kg. In the case of lymphocyte subpopulation, the population of T and B cells was not changed but CD4(+) T cell subsets are significantly increased in exposure to CAPE. The antibody responses to T lymphocyte dependent antigen, sheep red blood cell and keyhole limpet hemocyanin (KLH) were increased more than 10 mg/kg in CAPE-treated group. Likewise, the cytokine, IL-2, IL-4 and IFN-gamma were significantly increased at the dose of 20 mg/kg CAPE group. These results suggest that CAPE could have immunomodulatory effects in vivo.     

Caffeic acid phenethyl ester (CAPE) analogues: potent nitric oxide inhibitors from the Netherlands propolis

The MeOH and water extracts of the Netherlands propolis were tested for their inhibitory activity toward nitric oxide (NO) production in lipopolysaccharide (LPS)-activated murine macrophage-like J774.1 cells. Both of the extract possessed significant NO inhibitory activity with IC(50) values of 23.8 and 51.5 microg/ml, respectively. Then 13 phenolic compounds obtained from the MeOH extract showing stronger NO inhibition were examined on their NO inhibitory activities. Caffeic acid phenethyl ester (CAPE) analogues, i.e., benzyl caffeate, CAPE and cinnamyl caffeate, possessed most potent NO inhibitory activities with IC(50) values of 13.8, 7.64 and 9.53 microM, respectively, which were two- to four-fold stronger than the positive control N(G)-monomethyl-L-arginine (L-NMMA; IC(50), 32.9 microM). Further study on the synthetic analogues of CAPE revealed that both of 3-phenylpropyl caffeate (18; IC(50), 7.34 microM) and 4-phenylbutyl caffeate (19; IC(50), 6.77 microM) possessed stronger NO inhibitory activity than CAPE (10) and that elongation of alkyl side chain of alcoholic parts of caffeic acid esters enhance the NO inhibitory activity. In addition, it was found that CAPE analogues having longer carbon chain (>C(5)) in alcoholic part showed toxic effects toward J774.1 cells. This NO inhibitory effect may directly correlate with antiinflammatory properties of the Netherlands propolis.     

Caffeic acid phenethyl ester protects nigral dopaminergic neurons via dual mechanisms involving haem oxygenase-1 and brain-derived neurotrophic factor

BACKGROUND AND PURPOSE

Caffeic acid phenethyl ester (CAPE) is a component of honey bee propolis that can induce expression of haem oxygenase-1 (HO-1). Because HO-1 induction has been suggested to protect dopaminergic neurons in the substantia nigra, we examined the effect of CAPE in experimental models of dopaminergic neurodegeneration.

EXPERIMENTAL APPROACH

Neuroprotective effect of CAPE was investigated in rat organotypic midbrain slice cultures and in vivo, using a mouse model of dopaminergic neurodegeneration induced by intranigral injection of LPS and intrastriatal injection of 6-hydroxydopamine.

KEY RESULTS

CAPE protected dopaminergic neurons in slice cultures from IFN-γ/LPS-induced injury. The effect of CAPE was inhibited by zinc protoporphyrin IX, an HO-1 inhibitor, and by neutralizing antibody against brain-derived neurotrophic factor (BDNF). A p38 MAPK inhibitor SB203580 prevented activation of NF-E2-related factor 2, attenuated increased expression of HO-1 and BDNF, and blocked the neuroprotective actions of CAPE. In the LPS-injected mouse model, daily intraperitoneal administration of CAPE protected dopaminergic neurons, up-regulated HO-1 and BDNF, and reduced the increase of activated microglia/macrophages. Neuroprotective effects of CAPE against LPS-induced injury was prevented by zinc protoporphyrin IX or anti-BDNF antibody. CAPE protected dopaminergic neurons and alleviated methamphetamine-induced rotational behaviour also in 6-hydroxydopamine hemiparkinsonian mice.

CONCLUSION AND IMPLICATIONS

CAPE is a novel type of neuroprotective agent whose actions are mediated by both HO-1 and BDNF. These findings may provide novel clues to develop neuroprotective agents for treatment of neurodegenerative disorders.     

Evaluation of polyphenolic acid esters as potential antioxidants

Six polyphenolic acid esters were synthesized and their antioxidative properties were evaluated in three model systems [2,2'-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH)-induced lipid peroxidation system, and the dye-bleaching assay of peroxynitrite radical]. Among these compounds, we found that compounds 4 [3,4-dihydroxy-benzoic acid-(2-phenoxyethyl ester)], and 5 [3,4-dihydroxy-cinnamic acid-(2-phenoxyethyl ester)] provided comparable activity to caffeic acid phenethyl ester (CAPE) in the DPPH model. Compound 3 [2,5-dihydroxy-benzoic acid-(2-phenoxyethyl ester)], was found to be more active than CAPE in the AAPH system, it also displayed about 2-fold greater activity than CAPE in the peroxynitrite radical model. These results suggest that these phenolic acid ester derivatives, with their potent anti-oxidant activities, may have useful applications as antioxidants.     

Protective effect of caffeic acid phenethyl ester (CAPE) on myocardial ischemia-reperfusion-induced apoptotic cell death

Occlusion of coronary artery causes cardiomyocyte dysfunction. Reperfusion relieves ischemia by providing cells with metabolites and oxygen, thereby preventing extensive tissue damage. Although reperfusion salvages the myocardium, it also initiates a series of events including myocardial apoptosis and necrosis. The common inducers of apoptosis include reactive oxygen species (ROS). Caffeic acid phenethyl ester (CAPE) is known as an antioxidative, anti-inflammatory effects, may protect myocardial ischemia-reperfusion (MI/R)-induced apoptosis. We have previously reported that CAPE reduced MI/R-induced necrosis. Therefore, this study was focused to investigate protective effect of CAPE on the distinct form of cell death; apoptosis in an in vivo rat model. To produce MI/R, a branch of the descending left coronary artery was occluded for 30 min followed by 2 h reperfusion. ECG changes, blood pressure (BP), and heart rate (HR) were measured before occlusion and continued both occlusion and reperfusion. CAPE (50 micromol/kg) was given 10 min before ischemia via jugular vein. Extensive formation of DNA strand breaks, the typical biochemical feature of apoptosis, was detected with the use of the terminal deoxynucleotidyl transferase (TdT)-mediated d UTP-biotin nick and labeling (TUNEL) method. Also, cysteine aspartate specific proteinase (caspase)-3 and caspase-9 activities a universal effector of apoptosis, were determined. Trunk blood was extracted to determine the serum contents related to oxidant-antioxidant status. In hemodynamic parameters, there was no significant difference in HR or BP values among any group. CAPE administration had no a significant effect on hemodynamic parameters during ischemia or reperfusion. Control group revealed extensive TUNEL-positive cardiomyocytes especially in free wall of left ventricule, interventiculare septum and nearly apex zone. Intensity of TUNEL-positive cardiomyocytes reduced as a result of CAPE treatment compared to control group in the same sections. Result of the caspase activities was found to correlate with TUNEL evaluation. CAPE also, ameliorated antioxidant status. We propose that CAPE acts in the heart as a potent scavenger of free radicals to prevent the apoptotic effect of I/R. Further studies are needed to elucidate the mechanisms of apoptotic death machinery.     

Caffeic acid phenethyl ester,a propolis polyphenolic,attenuates potentially cadmium-induced testicular dysfunction in mice

Abstract

Cadmium (Cd) as environmental pollutant can induce severe damage, particularly to the testis. This study investigated the effects of Caffeic acid phenethyl ester (CAPE) on testicular dysfunction induced by Cd. Adult mice were intraperitoneally injected with cadmium chloride (CdCl₂) with different doses of CAPE pretreatment. After CdCl₂ injection, body/testis weight ratio decreased, Cd levels accumulated and zinc levels decreased in testis. Furthermore, Cd intoxication caused a significant increase of oxidative stress levels, antioxidant enzymes activities, and glutathione levels. Interestingly, significant improvements were observed after the administration of CAPE. Our results demonstrated the protective effect of CAPE, linking Cd testicular dysfunction to oxidative stress.     

Caffeic acid phenethyl ester (CAPE) prevents methotrexate-induced hepatorenal oxidative injury in rats

Objectives This study aimed to investigate the antioxidant and anti‐inflammatory effects of caffeic acid phenethyl ester (CAPE) on the methotrexate (MTX)‐induced hepatorenal oxidative damage in rats. Methods Following a single dose of methotrexate (20 mg/kg), either vehicle (MTX group) or CAPE (10 µmol/kg, MTX + CAPE group) was administered for five days. In other rats, vehicle (control group) or CAPE was injected for five days, following a single dose of saline injection. After decapitation of the rats, trunk blood was obtained, and the liver and kidney tissues were removed for histological examination and for the measurement of malondialdehyde (MDA) and glutathione (GSH) levels and myeloperoxidase (MPO) and sodium potassium‐adenosine triphosphatase (Na⁺/K⁺‐ATPase) activity. TNF‐α and IL‐1β levels were measured in the blood. Key findings Methotrexate administration increased the tissue MDA levels, MPO activity and decreased GSH levels and Na⁺/K⁺‐ATPase activity, while these alterations were reversed in the CAPE‐treated MTX group. Elevated TNF‐α and IL‐1β levels were also reduced with CAPE treatment. Conclusions The results of this study revealed that CAPE, through its anti‐inflammatory and antioxidant actions, alleviates methotrexate‐induced oxidative damage, which suggests that CAPE may be of therapeutic benefit when used with methotrexate.     

Protective effect of caffeic acid phenethyl ester (CAPE) on fluoride-induced oxidative stress and apoptosis in rat endometrium

High fluoride intake may affect biological systems by increasing free radicals, which may enhance lipid peroxidation levels of the tissues, thus leading to oxidative damage. Caffeic acid phenethyl ester (CAPE), a component of honeybee propolis, protects tissues from reactive oxygen species mediated oxidative stress in ischemia-reperfusion and toxic injuries. Several studies suggest that supplementation with anti-oxidant can influence fluoride induced tissue damage. The aims of this study was to investigate the possible role of malondialdehyde (MDA) levels and activity of superoxide dismutase (SOD) and catalase (CAT), in the pathogenesis of fluoride-induced endometrial damage and to demonstrate the effect of CAPE, the potent antioxidant, in decreasing the toxicity. Twenty-four adult female rats were randomly divided into three experimental groups, as follows: control group, fluoride-treated group (F), and fluoride plus CAPE-treated group (F+CAPE). Fluoride was given orally as 30mg/L NaF solution in spring water daily for 45 days. CAPE was co-administered intraperitoneally (i.p.) with a dose of 10μM/(kgday) for 46 days. Extensive formation of DNA strand breaks, the typical biochemical feature of apoptosis, was detected with the use of the terminal deoxynucleotidyl transferase (TdT)-mediated d UTP-biotin nick and labeling (TUNEL) method. The activities of antioxidant enzymes such as SOD and CAT as well as the concentration of MDA, as an indicator of lipid peroxidation, were measured to evaluate oxidative stress in homogenates of the endometrium. Fluoride administration increased MDA levels (p<0.05), decreased SOD (p<0.05) and CAT (p<0.05) activities. CAPE co-administration with fluoride treatments caused significantly decreased MDA levels (p<0.05), increased SOD (p<0.05) and CAT (p<0.05) activities in endometrial tissue when compared with F alone. Diffuse apoptosis in glandular epithelium and stromal cells was found by TUNEL method in endometrial tissues of rats treated with fluoride. The severity of these lesions was reduced by administration of CAPE. In conclusion, our study demonstrated that MDA may play an important role in the pathogenesis of fluoride-induced oxidative endometrial damage. CAPE may have protective aspects in this process by its antioxidant and anti-inflammatory effect.     

Involvement of tumor suppressor protein p53 and p38 MAPK in caffeic acid phenethyl ester-induced apoptosis of C6 glioma cells

Caffeic acid phenethyl ester (CAPE), an active component of propolis, has many biological and pharmacological activities including antioxidant, anti-inflammation, antiviral action, and anticancer effect. Our previous studies showed that CAPE exhibited significant cytotoxicity in oral cancer cells. Herein we further investigated the cytotoxicity potential of CAPE and the mechanism of its action in C6 glioma cells. The data exhibited that C6 glioma cells underwent internucleosomal DNA fragmentation 24 hr after the treatment of CAPE (50 microM). The proportion of C6 glioma cells with hypodiploid nuclei was increased to 24% at 36 hr after the exposure. Further results showed that CAPE induced the release of cytochrome c from mitochondria into cytosol, and the activation of CPP32. CAPE application also enhanced the expression of p53, Bax, and Bak. Finally, the potential signaling components underlying CAPE induction of apoptosis were elucidated. We found that CAPE activated extracellular signal-regulated kinase (ERKs) and p38 mitogen-activated protein kinase (p38 MAPK) in C6 glioma cells. More importantly, p38 kinase formed a complex with p53 after the treatment of CAPE for 0.5 hr. The expression of p53, phospho-serine 15 of p53, and Bax, and inactivate form of CPP32 was suppressed by a pretreatment of a specific p38 MAPK inhibitor, SB203580. The resultant data suggest that p38 MAPK mediated the CAPE-induced p53-dependent apoptosis in C6 glioma cells.     

Effect of caffeic acid phenethyl ester on gastric acid secretion in vitro

Caffeic acid phenethyl ester (CAPE), one of the major components of propolis (honeybee resin), has demonstrated a wide spectrum of activities including suppression of eicosanoids by inhibition of cyclooxygenase-1 and cyclooxygenase-2 enzyme activities. The aim of this study was to investigate the effect of CAPE on basal and secretagogues-stimulated gastric acid secretion in vitro. In the isolated, lumen-perfused, stomach preparation of mouse, CAPE (10-100 microM) did not affect the basal gastric acid secretion nor the secretion stimulated by histamine, pentagastrin, isobutyl methylxanthine and high levels of K+. By contrast, CAPE increased the gastric acid secretion induced by the muscarinic receptor agonist, 5-methylfurmethide (5-MEF). CAPE also inhibited the acetylcholinesterase activity in an in vitro colorimetric assay. Eserine (10 microM), a well known acetylcholinesterase inhibitor, also increased 5-MEF-stimulated acid secretion. Our results show that CAPE increases gastric acid secretion stimulated by an acetylcholine agonist receptor likely through inhibition of acetylcholinesterase activity.     

A preliminary study of the levels of testis oxidative stress parameters after MK-801-induced experimental psychosis model: protective effects of CAPE

We evaluated the effects of caffeic acid phenethyl ester (CAPE) on antioxidant enzyme levels and histopathologic changes in dizocilpine (MK-801) induced schizophrenic rat testis. A total of 30 adult male Wistar-Albino rats were divided into three groups. Group-I was used as control. Rats in the Group-II were intraperitoneally injected with MK-801, whereas those in Group-III were intraperitoneally injected with CAPE in addition to MK-801. The testes were collected for biochemical and histopathological examinations. Antioxidant enzyme activities, malondialdehyde, protein carbonyl and nitric oxide levels in testicular tissues were analyzed with spectrophotometric methods. Induction of schizophrenia resulted in a significant oxidative stress by increasing the levels of antioxidant enzymes. Tissue malondialdehyde and protein carbonyl levels were also increased. Treatment with CAPE led to significant decrease in oxidative injury. Administration of CAPE reduced the detrimental histopathologic changes caused by MK-801. The results showed that experimentally induced schizophrenia caused oxidative stress in testes of rats and treatment with CAPE reduced these harmful effects.     

Absorption properties and effects of caffeic acid phenethyl ester and its p-nitro-derivative on P-glycoprotein in Caco-2 cells and rats

Context: Caffeic acid phenethyl ester (CAPE), isolated from honeybee propolis, has pharmacological applications. A synthesized CAPE derivative, p-nitro-caffeic acid phenethyl ester (CAPE-NO₂), showed similar activities with CAPE. The pharmacological activities of CAPE and CAPE-NO₂ are related to their absorption properties.

Objective: To understand the pharmacokinetic profiles of CAPE and CAPE-NO₂ in rats and investigate the absorption mechanisms and effects on P-glycoprotein in Caco-2 cells.

Materials and methods: The pharmacokinetic profiles of CAPE and CAPE-NO₂ were obtained after oral administration (10?mg/kg) to rats. Transport studies of CAPE and CAPE-NO₂ (5, 10, 20?μM) were performed in Caco-2 cell model. P-gp activities were assayed by rhodamine 123 cellular retention. Expression of P-gp was determined after the cells were administrated with CAPE and CAPE-NO₂ (5, 20?μM) for 48 and 72?h.

Results: The AUC_(0?t) of CAPE-NO₂ (3239.9?±?352?ng?×?h/mL) was two-time greater than CAPE (1659.6?±?152?ng?×?h/mL) in rats. The P_app values of CAPE and CAPE-NO₂ were (4.86?±?0.90)?×?10^?6?cm/s and (12.34?±?1.6)?×?10^?6?cm/s, respectively. The accumulation of rhodamine 123 was increased by 1.3- to 1.9-fold and 1.4- to 2.3-fold in CAPE and CAPE-NO₂ groups after 1?h administration, respectively. However, CAPE and CAPE-NO₂ increased the P-gp levels by 2.1- and 1.7-fold, respectively.

Conclusion: The absorption of CAPE-NO₂ can be enhanced in rats and Caco-2 cells compared with CAPE. The two compounds are potential inhibitors of P-gp. The increased P-gp levels generated by CAPE and CAPE-NO₂ played a role as a defense mechanism by limiting intracellular xenobiotic levels.     

Caffeic Acid Phenethyl Ester (CAPE) derived from propolis, a honeybee product, inhibits growth of breast cancer stem cells

Cancer stem cells (CSC) are chemoresistant and implicated in tumor recurrence, metastasis and high patient mortality; thus substances impairing CSC activity, could be invaluable as novel cancer therapeutics. We previously showed that CAPE (caffeic acid phenethyl ester), a component of propolis, a honeybee product, inhibits growth of MDA-MB-231 (MDA-231) cells, mdr gene expression, NF-κB, EGFR, and VEGF. We hypothesized that CAPE also acts by interfering with CSC-mediated effects. We isolated breast CSC (bCSC) from MDA-231 cells, a model of human triple-negative breast cancer, and mouse xenografts. bCSC grow as mammospheres (MMS) and when dissociated into single cells, form MMS again, a sign of self-renewal. bCSC exhibited the characteristic CD44(+)/CD24(-/low) phenotype and generated progenitors in the presence of serum, a CSC trait responsible for regenerating tumor mass. CAPE caused dose-dependent bCSC self-renewal inhibition and progenitor formation. Clonal growth on soft agar was inhibited dose-dependently, but apoptosis was not induced as determined by Annexin-V/PI assay. Instead, bCSC were noted to significantly progress from a quiescent cell cycle state in G0/G1 (82%), S phase (12%) to a cycling state with an increase in S phase (41%) and subsequent decrease in G0/G1 (54%). Treatment of bCSC with CAPE (4.5-days) decreased CD44 levels by 95%, while another cell population containing 10-100-fold lower CD44 content concurrently increased. Results suggest that CAPE causes pronounced changes in bCSC characteristics manifested by inhibition of self renewal, progenitor formation, clonal growth in soft agar, and concurrent significant decrease in CD44 content, all signs of decreased malignancy potential.     

Inhibition of angiogenesis by propolis

Propolis, obtained from honeybee hives, has been used in Oriental folk medicine as an anti-inflammatory, anti-carcinogenic, and immunomodulatory agent. There is considerable evidence suggesting that angiogenesis and chronic inflammation are codependent. Blockage of angiogenesis results in an anti-inflammatory effect. Ethanol (EEP) and ether extracts of propolis (REP), and caffeic acid phenethyl ester (CAPE), an active component of propolis, were examined for their anti-angiogenic activities using the chick embryo chorioallantoic membrane (CAM), and the calf pulmonary arterial endothelial (CPAE) cell proliferation, assays. The presence of EEP, REP and CAPE inhibited angiogenesis in the CAM assay and the proliferation of CPAE cells. The results suggest that anti-angiogenic activities of EEP, REP and CAPE are also responsible for their anti-inflammatory effect.