The potential usage of caffeic acid phenethyl ester (CAPE) against chemotherapy‐induced and radiotherapy‐induced toxicity

Protection of the patients against the side effects of chemotherapy and radiotherapy regimens has attracted increasing interest of clinicians and practitioners. Caffeic acid phenethyl ester (CAPE), which is extracted from the propolis of honeybee hives as an active component, specifically inhibits nuclear factor κB at micromolar concentrations and show ability to stop 5‐lipoxygenase‐catalysed oxygenation of linoleic acid and arachidonic acid. CAPE has antiinflammatory, antiproliferative, antioxidant, cytostatic, antiviral, antibacterial, antifungal and antineoplastic properties. The purpose of this review is to summarize in vivo and in vitro usage of CAPE to prevent the chemotherapy‐induced and radiotherapy‐induced damages and side effects in experimental animals and to develop a new approach for the potential usage of CAPE in clinical trial as a protective agent during chemotherapy and radiotherapy regimens. Copyright © 2012 John Wiley & Sons, Ltd.     

Vascular effects of caffeic acid phenethyl ester (CAPE) on isolated rat thoracic aorta

This study was aimed to investigate the vascular activity of caffeic acid phenethylester (CAPE), one of the major components of honeybee propolis. Experiments were performed on rat thoracic aortic rings, mounted in an isolated organ bath and connected to an isometric force transducer. The effect of CAPE (0.1-300 microM) was evaluated on tissue pre-contracted with phenylephrine (PE, 1 microM) or with KCl (100 mM). In another set of experiments, tissue was incubated with CAPE (1-100 microM) and responses to PE (0.01-3 microM) or KCl (60 mM) were evaluated. The effect of CAPE on cytosolic Ca(2+) concentration in aortic smooth muscle cells stimulated with PE or KCl was also evaluated. CAPE (0.1-300 microM) caused a concentration-dependent relaxation (pEC(50) 4.99 +/- 0.19; Emax 100.75 +/- 1.65%; n = 4) of tissue pre-contracted with PE that was reduced by endothelium removal or by incubation with N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM). CAPE also relaxed KCl-precontracted tissue (pEC(50) 4.40 +/- 0.08; n = 4). CAPE inhibited contractile responses to PE or to KCl, and also inhibited the contractile response to PE obtained in a Ca(2+)-free medium. In addition, CAPE inhibited the increase in cytosolic Ca(2+) concentration triggered by stimulation of aortic smooth muscle cells with PE or KCl. Our results demonstrate a vascular activity for CAPE, that is only partially dependent on nitric oxide. Indeed, at high concentrations, CAPE vasorelaxant effect occurs also in absence of endothelium and it is likely due to an inhibitory effect on calcium movements through cell membranes.     

Ameliorating role of caffeic acid phenethyl ester (CAPE) against isoniazid-induced oxidative damage in red blood cells

Isoniazid (INH) still remains a first-line drug both for treatment and prophylaxis of tuberculosis, but various organs toxicity frequently develops in patients receiving this drug. We aimed to investigate possible toxic effects of INH on rat red blood cells (RBCs), and to elucidate whether Caffeic acid phenethyl ester (CAPE) prevents a possible toxic effect of INH. Experimental groups were designed as follows: control group, INH group, INH + CAPE group. Compared with the control, the INH caused a significant increase in superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels, and a decrease in glutathione peroxidase (GSH-Px) and catalase (CAT), which are recently used to monitor the development and extent of damage due to oxidative stresses. CAPE administration to INH group ameliorated above changes due to INH.     

Cytoprotective effect of caffeic acid phenethyl ester (CAPE) and catechol ring-fluorinated CAPE derivatives against menadione-induced oxidative stress in human endothelial cells

Caffeic acid phenethyl ester (CAPE), a natural polyphenolic compound with many biological activities, has been shown to be protective against ischemia-reperfusion injury. We have synthesized six new catechol ring-fluorinated CAPE derivatives and evaluated their cytotoxic and cytoprotective effects against menadione-induced cytotoxicity in human umbilical vein endothelial cells. These results provide some insights into the structural basis of CAPE cytoprotection in this assay, which does not appear to be based solely on direct antioxidant properties.     

Protective effects of caffeic acid phenethyl ester (CAPE) on amikacin-induced nephrotoxicity in rats

Amikacin (AK) has nephrotoxic side effects. AK-induced nephrotoxicity may be the consequence of oxidative stress and so anti-oxidant agents could be useful in reducing AK toxicity. Caffeic acid phenethyl ester (CAPE) was recently shown to have free radical scavenging ability and it reduces lipid peroxidation. For this purpose, the rats were distributed into three groups: (I) injected with vehicle (control); (II) injected (i.p.) with 1.2 g kg(-1) AK at a single dose; (III) injected (i.p.) with AK plus 10 micromol kg(-1) CAPE. Renal morphology was investigated by light microscopy. Tissue samples and trunk blood were also obtained to determine renal malondialdehyde (MDA), blood urea nitrogen (BUN) and creatinine (Cr) levels. MDA production was found to be higher in rats given AK than among control rats. CAPE administration before AK injection caused a significant decrease in MDA production. Morphological tubule damage in rats given AK was severe in the renal cortex, whereas in rats given AK plus CAPE, no histological changes occurred. It is concluded that CAPE could be useful for reducing the nephrotoxic effects of AK. Copyright (c) 2005 John Wiley & Sons, Ltd.     

Synthesis of a series of caffeic acid phenethyl amide (CAPA) fluorinated derivatives: Comparison of cytoprotective effects to caffeic acid phenethyl ester (CAPE)

A series of catechol ring-fluorinated derivatives of caffeic acid phenethyl amide (CAPA) were synthesized and screened for cytoprotective activity against H₂O₂ induced oxidative stress in human umbilical vein endothelial cells (HUVEC). CAPA and three fluorinated analogs were found to be significantly cytoprotective when compared to control, with no significant difference in cytoprotection between caffeic acid phenethyl ester (CAPE) and CAPA.     

The protective effects of caffeic acid phenethyl ester (CAPE) against liver damage induced by cigarette smoke inhalation in rats

The aim of this study was to investigate the histological and biochemical changes in liver of rats exposed to cigarette smoke and effects of caffeic acid phenetyl ester (CAPE) on these changes. For this purpose, 21 male Wistar rats were divided into three groups. Animals in Group I were used as control. Rats in Group II were exposed to cigarette smoke and rats in Group III were exposed to cigarette smoke and injected daily with CAPE. At the end of the 60-days experimental period, all rats were killed by decapitation and blood samples were obtained. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin levels and hepatic superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px ), malondialdehyde (MDA) contents were determined. Following routine histological procedures, liver tissue specimens were examined under a light microscope. The levels of ALT, AST, total bilirubin, SOD, GSH-Px and MDA were significantly increased in rats exposed to cigarette smoke compared with those of the controls. Light microscopic examination of liver specimens from rats exposed to cigarette smoke revealed mononuclear cell infiltration and that some of the hepatocytes had a hyperchromatic nucleus and enlarged sinusoids. The rats which were treated with CAPE along with cigarettes had partially attenuated histological changes associated with cigarette exposure. In conclusion, the damage inflicted by cigarette in the rat liver can be partially prevented by CAPE administration.     

The metabolic bioactivation of caffeic acid phenethyl ester (CAPE) mediated by tyrosinase selectively inhibits glutathione S-transferase

Glutathione S-transferase (GST) and multidrug resistance-associated proteins (MRPs) play major roles in drug resistance in melanoma. In this study, we investigated caffeic acid phenethyl ester (CAPE) as a selective GST inhibitor in the presence of tyrosinase, which is abundant in melanoma cells. Tyrosinase bioactivates CAPE to an o-quinone, which reacts with glutathione to form CAPE-SG conjugate. Our findings indicate that 90% CAPE was metabolized by tyrosinase after a 60-min incubation. LC–MS/MS analyses identified a CAPE-SG conjugate as a major metabolite. In the presence of tyrosinase, CAPE (10–25 μM) showed 70–84% GST inhibition; whereas in the absence of tyrosinase, CAPE did not inhibit GST. CAPE-SG conjugate and CAPE-quinone (25 μM) demonstrated ?85% GST inhibition via reversible and irreversible mechanisms, respectively. Comparing with CDNB and GSH, the non-substrate CAPE acted as a weak, reversible GST inhibitor at concentrations >50 μM. Furthermore, MK-571, a selective MRP inhibitor, and probenecid, a non-selective MRP inhibitor, decrease the IC₅₀ of CAPE (15 μM) by 13% and 21%, apoptotic cell death by 3% and 13%, and mitochondrial membrane potential in human SK-MEL-28 melanoma cells by 10% and 56%, respectively. Moreover, computational docking analyses suggest that CAPE binds to the GST catalytic active site. Caffeic acid, a hydrolyzed product of CAPE, showed a similar GST inhibition in the presence of tyrosinase. Although, as controls, 4-hydroxyanisole and l-tyrosine were metabolized by tyrosinase to form quinones and glutathione conjugates, they exhibited no GST inhibition in the absence and presence of tyrosinase. In conclusion, both CAPE and caffeic acid selectively inhibited GST in the presence of tyrosinase. Our results suggest that intracellularly formed quinones and glutathione conjugates of caffeic acid and CAPE may play major roles in the selective inhibition of GST in SK-MEL-28 melanoma cells. Moreover, the inhibition of MRP enhances CAPE-induced toxicity in the SK-MEL-28 melanoma cells.     

The effects of the caffeic acid phenethyl ester (CAPE) on erythrocyte membrane damage after hind limb ischaemia-reperfusion

Reactive oxygen species have been implicated in pathogenesis injury after ischaemia-reperfusion (I/R). Caffeic Acid Phenethyl Ester (CAPE), an active component of honeybee propolis extract, exhibits antioxidant and anti-inflammatory properties. The aim of this study was to investigate the effects of CAPE on erythrocyte membrane damage after hind limb I/R. Rats were divided into two groups: I/R and I/R with CAPE pre-treatment. They were anaesthetized with intramuscular ketamine 100 mg kg(-1). A 4-h I/R period was performed on the right hind limb of all animals. In the CAPE-treated group, animals received CAPE 10 microm by intraperitoneal injection 1 h before the reperfusion. At the end of the reperfusion period, a midsternotomy was performed. A 5-ml blood sample was withdrawn from the ascending aorta for biochemical assays. Serum and erythrocyte membrane MDA levels were significantly lower in the CAPE-treated group when compared to the I/R group ( p = 0.001 and p<0.001, respectively). Erythrocyte membrane Na(+)-K(+) ATPases activity in the CAPE-treated group was significantly higher than the I/R group ( p<0.001). In conclusion, CAPE seems to be effective in protecting against oxidative stress. Therefore, we suggest that in order to decrease I/R injury, pre-administration of CAPE may be a promising agent for a variety of conditions associated with I/R.     

The anticancer impacts of free and liposomal caffeic acid phenethyl ester (CAPE) on melanoma cell line (A375)

The deadliest type of skin cancer, malignant melanoma, is also the reason for the majority of skin cancer-related deaths. The objective of this article was to investigate the efficiency of free caffeic acid phenethyl ester (CAPE) and liposomal CAPE in inducing apoptosis in melanoma cells (A375) in in vitro. CAPE was loaded into liposomes made up of hydrogenated soybean phosphatidylcholine, cholesterol, and 1,2-distearoyl-sn-glycero-3 phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000], and their physicochemical properties were assessed. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was performed for comparing the cytotoxicity of free CAPE and liposomal CAPE at dosages of 10, 15, 25, 50, 75 and the highest dose of 100 μg/mL for period of 24 and 48 h on A375 cell line to calculate IC50. Apoptosis and necrosis were evaluated in A375 melanoma cancer cells using flow cytometry. Atomic force microscopy was utilized to determine the nanomechanical attributes of the membrane structure of A375 cells. To determine whether there were any effects on apoptosis, the expression of PI3K/AKT1 and BAX/BCL2 genes was analyzed using the real-time polymerase chain reaction technique. According to our results, the maximum amount of drug release from nanoliposomes was determined to be 91% and the encapsulation efficiency of CAPE in liposomes was 85.24%. Also, the release of free CAPE was assessed to be 97%. Compared with liposomal CAPE, free CAPE showed a greater effect on reducing the cancer cell survival after 24 and 48 h. Therefore, IC50 values of A375 cells treated with free and liposomal CAPE were calculated as 47.34 and 63.39 μg/mL for 24 h. After 48 h of incubation of A375 cells with free and liposomal CAPE, IC50 values were determined as 30.55 and 44.83 μg/mL, respectively. The flow cytometry analysis revealed that the apoptosis induced in A375 cancer cells was greater when treated with free CAPE than when treated with liposomal CAPE. The highest nanomechanical changes in the amount of cell adhesion forces, and elastic modulus value were seen in free CAPE. Subsequently, the greatest decrease in PI3K/AKT1 gene expression ratio occurred in free CAPE.     

Protective effect of caffeic acid phenethyl ester (CAPE) administration on cisplatin-induced oxidative damage to liver in rat

Cisplatin is one of the most active cytotoxic agents in the treatment of cancer. High doses of cisplatin have also been known to produce hepatotoxicity. Several studies suggest that supplementation with an antioxidant can influence cisplatin-induced hepatotoxicity. The present study was designed to determine the effects of cisplatin on the liver oxidant/antioxidant system, and the possible protective effects of caffeic acid phenethyl ester (CAPE) on liver toxicity induced by cisplatin. Twenty-four adult female Wistar albino rats were divided into four groups of six rats each: control, cisplatin, CAPE, and cisplatin+CAPE. Cisplatin and CAPE were injected intraperitoneally. Liver tissue was removed to study the activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), myeloperoxidase (MPO), xanthine oxidase (XO), adenosine deaminase (ADA), and the levels of malondialdehyde and nitric oxide (NO). The activities of SOD and GSH-Px increased in the cisplatin+CAPE and CAPE groups compared with the cisplatin group. CAT activity was higher in the cisplatin +CAPE group than the other three groups. XO activity was lower in the cisplatin group than the control group. MPO activity was also increased in the cisplatin group compared to the control and CAPE groups. It can be concluded that CAPE may prevent cisplatin-induced oxidative changes in liver by strengthening the antioxidant defence system by reducing reactive oxygen species and increasing antioxidant enzyme activities.     

Protective effect of caffeic acid phenethyl ester (CAPE) on lipid peroxidation and antioxidant enzymes in diabetic rat liver

The aim of this study was to examine the effect of caffeic acid phenethyl ester (CAPE) on lipid peroxidation (LPO) and the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in the liver of streptozotocin (STZ)-induced diabetic rats. Twenty-seven rats were randomly divided into three groups: group I, control non-diabetic rats (n = 9); group II, STZ-induced, untreated diabetic rats (n = 8); group III, STZ-induced, CAPE-treated diabetic rats (n = 10), which were intraperitoneally injected with CAPE (10 microM kg(-1) day(-1)) after 3 days followed by STZ treatment. The liver was excised after 8 weeks of CAPE treatment, the levels of malondialdehyde (MDA) and the activities of SOD, CAT, and GSH-Px in the hepatic tissues of all groups were analyzed. In the untreated diabetic rats, MDA markedly increased in the hepatic tissue compared with the control rats (p < 0.0001). However, MDA levels were reduced to the control level by CAPE. The activities of SOD, CAT, and GSH-Px in the untreated diabetic group were higher than that in the control group (p < 0.0001). The activities of SOD and GSH-Px in the CAPE-treated diabetic group were higher than that in the control group (respectively, p < 0.0001, p < 0.035). There were no significant differences in the activity of CAT between the rats of CAPE-treated diabetic and control groups. Rats in the CAPE-treated diabetic group had reduced activities of SOD and CAT in comparison with the rats of untreated diabetic group (p < 0.0001). There were no significant differences in the activity of GSH-Px between the rats of untreated diabetic and CAPE-treated groups. It is likely that STZ-induced diabetes caused liver damage. In addition, LPO may be one of the molecular mechanisms involved in STZ-induced diabetic damage. CAPE can reduce LPO caused by STZ-induced diabetes.     

CAPE (caffeic acid phenethyl ester) stimulates glucose uptake through AMPK (AMP-activated protein kinase) activation in skeletal muscle cells

Caffeic acid phenethyl ester (CAPE), a flavonoid-like compound, is one of the major components of honeybee propolis. In the present study, we investigated the metabolic effects of CAPE in skeletal muscle cells and found that CAPE stimulated glucose uptake in differentiated L6 rat myoblast cells and also activated AMPK (AMP-activated protein kinase). In addition, the inhibition of AMPK blocked CAPE-induced glucose uptake, and CAPE activated the Akt pathway in a PI3K (phosphoinositide 3-kinase)-dependent manner. Furthermore, CAPE enhanced both insulin-mediated Akt activation and glucose uptake. In summary, our results suggest that CAPE may have beneficial roles in glucose metabolism via stimulation of the AMPK pathway.     

Protective effects of caffeic acid phenethyl ester and thymoquinone on toluene induced liver toxicity

Toluene is an organic solvent that is toxic to humans. Caffeic acid phenethyl ester (CAPE) and thymoquinone (TQ) exhibit antioxidant and antitoxic effects. We investigated the protective effects of CAPE and TQ on toluene induced hepatotoxicity. Wistar albino rats were divided into seven groups of eight. The animals were injected intraperitoneally (i.p.) with 0.1 ml/10 g/day corn oil (control I), 0.1 ml/10 g/day corn oil + 2 ml/kg/day 10% ethanol (control II), 20 mg/kg/day TQ dissolved in 0.1 ml/10 g corn oil (TQ), 10 µmol/kg/day CAPE dissolved in 10% ethanol (CAPE), 500 mg/kg/day toluene (T), toluene and TQ together (T + TQ), or toluene and CAPE together (T + CAPE). All rats were sacrificed on day 15. Liver samples were obtained for histological analysis. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured to evaluate liver function. Liver sections from the control I and TQ groups exhibited normal histology. Sections from the T group exhibited sinusoid dilation, hemorrhage, vacuolization and necrosis. TQ and CAPE protected against toluene induced histopathological changes. AST and ALT levels were increased significantly in T group compared to both control groups. CAPE decreased significantly the toluene induced increase in AST and ALT levels, while TQ did not. CAPE and TQ exhibited some antitoxic and hepato-protective effects on toluene induced liver damage.     

Growth inhibition and modulation of antigenic phenotype in human melanoma and glioblastoma multiforme cells by caffeic acid phenethyl ester (CAPE)

The active component of the honeybee hive product propolis, caffeic acid phenethyl ester (CAPE), has been shown to display increased toxicity toward various oncogene-transformed cell lines in comparison with their untransformed counterparts (Su et al., 4: 231-242, 1991). This observation provides support for the concept that it is the transformed phenotype which is specifically sensitive to CAPE. In the present study, we have determined the effect of CAPE on the growth and antigenic phenotype of a human melanoma cell line, HO-1, and a human glioblastoma multiforme cell line, GBM-18. For comparison, we have also tested the effects of mezerein (MEZ), mycophenolic acid (MPA) and retinoic acid (RA), which can differentially modulate growth, differentiation and the antigenic phenotype in these human tumor cell lines. Growth of both cell lines was suppressed by CAPE in a dose-dependent fashion, with HO-1 cells being more sensitive than GBM-18 cells. The antiproliferative effect of CAPE was enhanced in both cell types if CAPE and MEZ were used in combination. Growth suppression was associated with morphological changes in H0-1 cells, suggesting induction of a more differentiated phenotype. CAPE also differentially modulated the expression of several antigens on the surface of the two tumor cell lines. These results suggest a potential role for CAPE as an antitumor agent, an antigenic modulating agent and possibly a differentiation inducing agent.     

Caffeic acid phenethyl ester (CAPE) protects brain against oxidative stress and inflammation induced by diabetes in rats

Diabetic patients reveal significant disorders, such as nephropathy, cardiomyopathy, and neuropathy. As oxidative stress and inflammation seem to be implicated in the pathogenesis of diabetic brain, we aimed to investigate the effects of caffeic acid phenethyl ester (CAPE) on oxidative stress and inflammation in diabetic rat brain. Diabetes was induced by a single dose of streptozotocin (45 mg kg⁻¹, i.p.) injection into rats. Two days after streptozotocin treatment 10 μM kg⁻¹ day⁻¹ CAPE was administrated and continued for 60 days. Here, we demonstrate that CAPE significantly decreased the levels of nitric oxide and malondialdehyde induced by diabetes, and the activities of catalase, glutathione peroxidase, and xanthine oxidase in the brain. However, glutathione levels were increased by CAPE. The mRNA expressions of tumor necrosis factor (TNF)-α and interferon (IFN)-γ, and inducible nitric oxide synthase (iNOS) were remarkably enhanced in brain by diabetes. CAPE treatments significantly suppressed these inflammatory cytokines (about 70% for TNF-α, 26% for IFN-γ) and NOS (completely). Anti-inflammatory cytokine IL-10 mRNA expression was not affected by either diabetes or CAPE treatments. In conclusion, diabetes induces oxidative stress and inflammation in the brain, and these may be contributory mechanisms involved in this disorder. CAPE treatment may reverse the diabetic-induced oxidative stress in rat brains. Moreover, CAPE reduces the mRNA expressions of TNF-α and IFN-γ in diabetic brain; suggesting CAPE suppresses inflammation as well as oxidative stress occurred in the brain of diabetic patients.     

Lithium-induced renal toxicity in rats: Protection by a novel antioxidant caffeic acid phenethyl ester

Lithium carbonate used in the long-term treatment of manic-depressive illness has been reported to lead to progressive renal impairment in rats and humans. Caffeic acid phenethyl ester (CAPE), a component of honeybee propolis, protects tissues from reactive oxygene species mediated oxidative stress in ischemia-reperfusion and toxic injuries. The beneficial effect CAPE on lithium-induced nephrotoxicity has not been reported yet. The purpose of this study was to examine a possible renoprotective effect of CAPE against lithium-induced nephrotoxicity in a rat model. Twenty-two adult male rats were randomly divided into three experimental groups, as follows: control group, lithium-treated group (Li), and lithium plus CAPE-treated group (Li+CAPE). Li were treated intraperitoneally (i.p.) with 25 mg/kg Li₂CO₃ solution in 0.9% NaCl twice daily for 4 weeks. CAPE was co-administered i.p. with a dose of 10 μM/kg/day for 4 weeks. Serum Li, blood urea nitrogen and plasma creatinine, urinary N-acetyl-β-D-glucosaminidase (NAG, a marker of renal tubular injury), and malondialdehyde (MDA, an index of lipid peroxidation), were used as markers of oxidative stress-induced renal impairment in Li-treated rats. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities were studied to evaluate the changes of antioxidant status in renal tissue. Serum Li levels were found high in the Li and Li+CAPE groups. In Li-administrated rats, urinary NAG and renal MDA levels were increased according to control and Li+CAPE groups (p < 0.05). CAPE caused a significant reduction in the levels of these parameters. Likewise, renal SOD, CAT and GSH-Px activities were decreased in Li-administrated animals; CAPE caused a significant increase in the activities of these antioxidant enzymes. In conclusion, CAPE treatment has a protective effect against Li-induced renal tubular damage and oxidative stress in a rat model.     

Stage-dependent modulation of limb regeneration by caffeic acid phenethyl ester (CAPE)--immunocytochemical evidence of a CAPE-evoked delay in mesenchyme formation and limb regeneration

Caffeic acid phenethyl ester (CAPE), a natural compound of bee propolis, selectively inhibits proliferation of transformed cells in several cancer models in vitro. To examine in vivo CAPE function, we used the newt regeneration blastema as a model system wherein the processes of de-differentiation and subsequent proliferation of undifferentiated cells mimic changes associated with oncogenic transformation and tumorigenesis. We have shown that a single dose of CAPE significantly increased cell proliferation at the stages of blastema growth and re-differentiation. At the de-differentiation stage, CAPE significantly stimulated proliferation of wound epidermis keratinocytes, but decreased proliferation in the blastema mesenchyme. Immunohistochemistry with a mesenchymal cell marker, vimentin, revealed a highly significant reduction of vimentin staining in the mesenchyme of CAPE-treated regenerates (p<0.001). These results, together with morphological observations indicate that, at the de-differentiation stage, CAPE stimulated wound re-epithelization, increased keratinocyte proliferation and increased thickness of the wound epidermis. However, CAPE inhibited mesenchyme formation and proliferation. The functional consequence of the CAPE inhibitory action was a delay in limb regeneration.