Ethanolic Extract of Propolis and CAPE as Cardioprotective Agents against LPS and IFN-α Stressed Cardiovascular Injury.

The inflammatory process is triggered by several factors such as toxins, pathogens, and damaged cells, promoting inflammation in various systems, including the cardiovascular system, leading to heart failure. The link between periodontitis as a chronic inflammatory disease and cardiovascular disease is confirmed. Propolis and its major component, caffeic acid phenethyl ester (CAPE), exhibit protective mechanisms and anti-inflammatory effects on the cardiovascular system. The objective of the conducted study was to assess the anti-inflammatory effects of the Polish ethanolic extract of propolis (EEP) and its major component-CAPE-in interferon-alpha (IFN-α), lipopolysaccharide (LPS), LPS + IFN-α-induced human gingival fibroblasts (HGF-1). EEP and CAPE were used at 10-100 µg/mL. A multiplex assay was used for interleukin and adhesive molecule detection. Our results demonstrate that EEP, at a concentration of 25 µg/mL, decreases pro-inflammatory cytokine IL-6 in LPS-induced HGF-1. At the same concentration, EEP increases the level of anti-inflammatory cytokine IL-10 in LPS + IFN-α-induced HGF-1. In the case of CAPE, IL-6 in LPS and LPS + IFN-α induced HGF-1 was decreased in all concentrations. However, in the case of IL-10, CAPE causes the highest increase at 50 µg/mL in IFN-α induced HGF-1. Regarding the impact of EEP on adhesion molecules, there was a noticeable reduction of E-selectin by EEP at 25, 50, and100 µg/mL in IFN-α -induced HGF-1. In a range of 10-100 µg/mL, EEP decreased endothelin-1 (ET-1) during all stimulations. CAPE statistically significantly decreases the level of ET-1 at 25-100 µg/mL in IFN-α and LPS + IFN-α. In the case of intercellular adhesion molecule-1 (ICAM-1), EEP and CAPE downregulated its expression in a non-statistically significant manner. Based on the obtained results, EEP and CAPE may generate beneficial cardiovascular effects by influencing selected factors. EEP and CAPE exert an impact on cytokines in a dose-dependent manner.

Caffeic acid phenethyl ester inhibits neuro-inflammation and oxidative stress following spinal cord injury by mitigating mitochondrial dysfunction via the SIRT1/PGC1α/DRP1 signaling pathway.

BACKGROUND: The treatment of spinal cord injury (SCI) has always been a significant research focus of clinical neuroscience, with inhibition of microglia-mediated neuro-inflammation as well as oxidative stress key to successful SCI patient treatment. Caffeic acid phenethyl ester (CAPE), a compound extracted from propolis, has both anti-inflammatory and anti-oxidative effects, but its SCI therapeutic effects have rarely been reported. METHODS: We constructed a mouse spinal cord contusion model and administered CAPE intraperitoneally for 7 consecutive days after injury, and methylprednisolone (MP) was used as a positive control. Hematoxylin-eosin, Nissl, and Luxol Fast Blue staining were used to assess the effect of CAPE on the structures of nervous tissue after SCI. Basso Mouse Scale scores and footprint analysis were used to explore the effect of CAPE on the recovery of motor function by SCI mice. Western blot analysis and immunofluorescence staining assessed levels of inflammatory mediators and oxidative stress-related proteins both in vivo and in vitro after CAPE treatment. Further, reactive oxygen species (ROS) within the cytoplasm were detected using an ROS kit. Changes in mitochondrial membrane potential after CAPE treatment were detected with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide. Mechanistically, western blot analysis and immunofluorescence staining were used to examine the effect of CAPE on the SIRT1/PGC1α/DRP1 signaling pathway. RESULTS: CAPE-treated SCI mice showed less neuronal tissue loss, more neuronal survival, and reduced demyelination. Interestingly, SCI mice treated with CAPE showed better recovery of motor function. CAPE treatment reduced the expression of inflammatory and oxidative mediators, including iNOS, COX-2, TNF-α, IL-1ß, 1L-6, NOX-2, and NOX-4, as well as the positive control MP both in vitro and in vivo. In addition, molecular docking experiments showed that CAPE had a high affinity for SIRT1, and that CAPE treatment significantly activated SIRT1 and PGC1α, with down-regulation of DRP1. Further, CAPE treatment significantly reduced the level of ROS in cellular cytoplasm and increased the mitochondrial membrane potential, which improved normal mitochondrial function. After administering the SIRT1 inhibitor nicotinamide, the effect of CAPE on neuro-inflammation and oxidative stress was reversed.On the contrary, SIRT1 agonist SRT2183 further enhanced the anti-inflammatory and antioxidant effects of CAPE, indicating that the anti-inflammatory and anti-oxidative stress effects of CAPE after SCI were dependent on SIRT1. CONCLUSION: CAPE inhibits microglia-mediated neuro-inflammation and oxidative stress and supports mitochondrial function by regulating the SIRT1/PGC1α/DRP1 signaling pathway after SCI. These effects demonstrate that CAPE reduces nerve tissue damage. Therefore, CAPE is a potential drug for the treatment of SCI through production of anti-inflammatory and anti-oxidative stress effects.

Caffeic acid phenethyl ester mediates apoptosis in serum-starved HT29 colon cancer cells through modulation of heat shock proteins and MAPK pathways.

Colorectal cancer (CRC) is among the most prevalent gastrointestinal cancers of epithelial origin worldwide, with over 2 million cases detected every year. Emerging evidence suggests a significant increase in the levels of inflammatory and stress-related markers in patients with CRC, indicating that oxidative stress and lipid peroxidation may influence signalling cascades involved in the progression of the disease. However, the precise molecular and cellular basis underlying CRC and their modulations during bioactive compound exposure have not yet been deciphered. This study examines the effect of caffeic acid phenethyl ester (CAPE), a natural bioactive compound, in HT29 CRC cells grown under serum-supplemented and serum-deprived conditions. We found that CAPE inhibited cell cycle progression in the G2/M phase and induced apoptosis. Migration assay confirmed that CAPE repressed cancer invasiveness. Protein localisation by immunofluorescence microscopy and protein expression by western blot analysis reveal increased expressions of key inflammatory signalling mediators such as p38α, Jun N-terminal kinase and extracellular signal-regulated kinase (ERK) proteins. Molecular docking data demonstrates that CAPE shows a higher docking score of -5.35 versus -4.59 to known p38 inhibitor SB203580 as well as a docking score of -4.17 versus -3.86 to known ERK1/2 inhibitor AZD0364. Co-immunoprecipitation data reveals that CAPE treatment effectively downregulates heat shock protein (HSP) expression in both sera-supplemented and limited conditions through its interaction with mitogen-activated protein kinase 14 (MAPK14). These results suggest that stress induction via serum starvation in HT29 CRC cells leads to the induction of apoptosis and co-ordinated activation of MAPK-HSP pathways. Molecular docking studies support that CAPE could serve as an effective inhibitor to target p38 and MAPK compared to their currently known inhibitors.

Myconoside and Calceolarioside E Restrain UV-Induced Skin Photoaging by Activating NRF2-Mediated Defense Mechanisms.

Chronic and excessive ultraviolet (UVA/UVB) irradiation exposure is known as a major contributor to premature skin aging, which leads to excessive reactive oxygen species generation, disturbed extracellular matrix homeostasis, DNA damage, and chronic inflammation. Sunscreen products are the major preventive option against UVR-induced photodamage, mostly counteracting the acute skin effects and only mildly counteracting accelerated aging. Therefore, novel anti-photoaging and photopreventive compounds are a subject of increased scientific interest. Our previous investigations revealed that the endemic plant Haberlea rhodopensis Friv. (HRE) activates the antioxidant defense through an NRF2-mediated mechanism in neutrophiles. In the present study, we aimed to investigate the photoprotective potential of HRE and two of its specialized compounds-the phenylethanoid glycosides myconoside (MYC) and calceolarioside E (CAL)-in UVA/UVB-stimulated human keratinocytes in an in vitro model of photoaging. The obtained data demonstrated that the application of HRE, MYC, and CAL significantly reduced intracellular ROS formation in UVR-exposed HaCaT cells. The NRF2/PGC-1α and TGF-1ß/Smad/Wnt signaling pathways were pointed out as having a critical role in the observed CAL- and MYC-induced photoprotective effect. Collectively, CAL is worth further evaluation as a potent natural NRF2 activator and a promising photoprotective agent that leads to the prevention of UVA/UVB-induced premature skin aging.

Caffeic Acid Phenethyl Ester (CAPE), a natural polyphenol to increase the therapeutic window for lung adenocarcinomas.

BACKGROUND AND PURPOSE: Lung cancers are highly resistant to radiotherapy, necessitating the use of high doses, which leads to radiation toxicities such as radiation pneumonitis and fibrosis. Caffeic Acid Phenethyl Ester (CAPE) has been suggested to have anti-proliferative and pro-apoptotic effects in tumour cells, while radioprotective anti-inflammatory and anti-oxidant effects in the normal tissue. We investigated the radiosensitizing and radioprotective effects of CAPE in lung cancer cell lines and normal tissue in vitro and ex vivo, respectively. MATERIALS AND METHODS: The cytotoxic and radiosensitizing effects of CAPE in lung cancer were investigated using viability and clonogenic survival assays. The radioprotective effects of CAPE were assessed in vitro and ex vivo using precision cut lung slices (PCLS). Potential underlying molecular mechanisms of CAPE focusing on cell cycle, cell metabolism, mitochondrial function and pro-inflammatory markers were investigated. RESULTS: Treatment with CAPE decreased cell viability in a dose-dependent manner (IC50 57.6 ± 16.6 µM). Clonogenic survival assays showed significant radiosensitization by CAPE in lung adenocarcinoma lines (p < 0.05), while no differences were found in non-adenocarcinoma lines (p ≥ 0.13). Cell cycle analysis showed an increased S-phase (p < 0.05) after incubation with CAPE in the majority of cell lines. Metabolic profiling showed that CAPE shifted cellular respiration towards glycolysis (p < 0.01), together with mitochondrial membrane depolarization (p < 0.01). CAPE induced a decrease in NF-κB activity in adenocarcinomas and decreased pro-inflammatory gene expression in PCLS. CONCLUSION: The combination of CAPE and radiotherapy may be a potentially effective approach to increase the therapeutic window in lung cancer patients.

Phenolic acids from Prunella vulgaris alleviate cardiac remodeling following myocardial infarction partially by suppressing NLRP3 activation.

Acute myocardial infarction (MI) is one of the leading causes of mortality around the world. Prunella vulgaris (Xia-Ku-Cao in Chinese) is used in traditional Chinese medicine practice for the treatment of cardiovascular diseases. However, its active ingredients and mechanisms of action on cardiac remodeling following MI remain unknown. In this study, we investigated the cardioprotective effect of P. vulgaris on MI rat models. MI rats were treated with aqueous extract of P. vulgaris or phenolic acids from P. vulgaris, including caffeic acid, ursolic acid or rosmarinic acid, 1 day after surgery and continued for the following 28 days. Then the cardioprotective effect, such as cardiac function, inflammatory status, and fibrosis areas were evaluated. RNA-sequencing (RNA-seq) analysis, real-time polymerase chain reaction (PCR), western blotting, and ELISA were used to explore the underlying mechanism. In addition, ultra-high performance liquid chromatography/mass spectrometer analysis was used to identify the chemicals from P. vulgaris. THP-1NLRP3-GFP cells were used to confirm the inhibitory effect of P. vulgaris and phenolic acids on the expression and activity of NLRP3. We found that P. vulgaris significantly improved cardiac function and reduced infarct size. Meanwhile, P. vulgaris protected cardiomyocyte against apoptosis, evidenced by increasing the expression of anti-apoptosis protein Bcl-2 in the heart and decreasing lactate dehydrogenase (LDH) levels in serum. Results from RNA-seq revealed that the therapeutic effect of P. vulgaris might relate to NLRP3-mediated inflammatory response. Results from real-time PCR and western blotting confirmed that P. vulgaris suppressed NLRP3 expression in MI heart. We also found that P. vulgaris suppressed NLRP3 expression and the secretion of HMGB1, IL-1ß, and IL-18 in THP-1NLRP3-GFP cells. Further studies indicated that the active components of P. vulgaris were three phenolic acids, those were caffeic acid, ursolic acid, and rosmarinic acid. These phenolic acids inhibited LPS-induced NLRP3 expression and activity in THP-1 cells, and improved cardiac function, suppressed inflammatory aggregation and fibrosis in MI rat models. In conclusion, our study demonstrated that P. vulgaris and phenolic acids from P. vulgaris, including caffeic acid, ursolic acid, and rosmarinic acid, could improve cardiac function and protect cardiomyocytes from ischemia injury during MI. The mechanism was partially related to inhibiting NLRP3 activation.

Caffeic acid phenethyl ester attenuates indomethacin-induced gastric ulcer in rats.

Gastric ulcer is one of the most frequent gastrointestinal ailments worldwide. Indomethacin, one of the most potent NSAIDs, suffers undesirable ulcerogenic activity. Caffeic acid phenethyl ester (CAPE) has known health benefits. The current study examined the potential of CAPE to combat indomethacin-induced gastric ulcers in rats. Animals were randomized into 5 groups: control, Indomethacin (50 mg/kg) mg/kg), Indomethacin + CAPE (5 mg/kg/day), Indomethacin + CAPE (10 mg/kg), and Indomethacin + Omeprazole (30 mg/kg). CAPE prevented the rise in ulcer index, attenuated histopathological changes and preserved gastric mucin concentration. CAPE efficiently significantly prevented accumulation of malondialdehude (MDA) and prevented exhaustion of the enzymatic activities of catalase (CAT) and superoxide dismutase (SOD). Further, CAPE prevented the rise in the expression of tumor necrosis factor-α (TNF-α), cyclo-oxygenase-2 (COX-2) and nuclear factor kapp-B (NFκB). This was associated with down-regulation of Bax and up-regulation of Bcl-2 mRNA. Finally, CAPE prevented induced indomethacin-induced decrease in heat shock protein 70 (HSP70) in gastric tissues. In conclusion, CAPE possesses the ability to prevent indomethacin-induced gastric ulcer in rats. This involves, at least partially, antioxidation, anti-inflammation, anti-apoptosis and enhancement of HSP70 expression.

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.

Caffeic acid phenethyl ester: Unveiling its potential as a potent apoptosis inducer for combating hypopharyngeal squamous cell carcinoma.

Hypopharyngeal squamous cell carcinoma (HSCC) is a relatively rare form of head and neck cancer that is notorious for its poor prognosis and low overall survival rate. This highlights the need for new therapeutic options for this malignancy. The objective of the present study was to examine the ability of caffeic acid phenethyl ester (CAPE), which is an active compound found in propolis, to combat HSCC tumor growth. CAPE exerted its tumor­suppressive activity in HSCC cell lines through the induction of apoptosis. Mechanistically, the CAPE­mediated apoptotic process was attributed to the perturbation of the mitochondrial membrane potential and the activation of caspase­9. CAPE also modulated survivin and X­linked inhibitor of apoptosis, which are potent members of the inhibitors of apoptosis protein family, either through transcriptional or post­translational regulation, leading to HSCC cell line death. Therefore, the findings of the present study suggested that CAPE is an effective treatment alternative for HSCC via the stimulation of mitochondria­dependent apoptosis.

Ethyl caffeate combined with fluconazole exhibits efficacy against azole-resistant oropharyngeal candidiasis via the EFGR/JNK/c-JUN signaling pathway.

Ethyl caffeate (EC) is a phenylpropanoid compound derived from Elephantopus scaber. In our previous work, EC was investigated to have a strong synergistic antifungal effect against azole-resistant strains of Candida albicans when combined with fluconazole (FLU). However, the protective effect and mechanism of EC + FLU on oropharyngeal candidiasis (OPC) caused by drug-resistant strains of C. albicans have not been investigated. This study aimed to investigate the protective effect and mechanism of EC combined with FLU against C. albicans-resistant strains that lead to OPC. An OPC mouse model revealed that EC + FLU treatment reduced fungal load and massive hyphal invasion of tongue tissues, and ameliorated the integrity of the tongue mucosa. Periodic acid-Schiff staining results showed more structural integrity of the tongue tissues and reduced inflammatory cell infiltration after EC + FLU treatment. Phosphorylation of EGFR (epidermal growth factor receptor) and other proteins in the EFGR/JNK (c-Jun N-terminal kinase)/c-JUN (transcription factor Jun) signaling pathway was significantly downregulated by EC + FLU. EGFR and S100A9 mRNA expression were also reduced. The above results were verified in FaDu cells. ELISA results showed that the concentration of inflammatory factors in the cell supernatant was significantly reduced after EC combined with FLU treatment. Molecular docking revealed that EC exhibited high binding energy to EGFR. In conclusion, EC enhances the susceptibility of azole-resistant C. albicans to FLU, and the underlying mechanism is related to the inhibition of the EGFR/JNK/c-JUN signaling pathway. This result suggests that EC has potential to be developed as an antifungal sensitizer to treat OPC caused by azole-resistant C. albicans.

Caffeic acid phenethyl ester inhibits MDA-MB-231 cell proliferation in inflammatory microenvironment by suppressing glycolysis and lipid metabolism.

Caffeic acid phenethyl ester (CAPE) is one of the main active ingredients of propolis with good antitumor activities. However, the potential effects of CAPE on the glycolysis and lipid metabolism of tumor cells are unclear. Here, the anti-tumor effects of CAPE on MDA-MB-231 cells in an inflammatory microenvironment stimulated with lipopolysaccharide (LPS) were studied by estimating the inflammatory mediators and the key factors of glycolysis and lipid metabolism. The CAPE treatment obviously inhibited proliferation, migration, invasion, and angiogenesis, and the mitochondrial membrane potential was decreased in the LPS-stimulated MDA-MB-231 cells. Compared with the LPS group, pro-inflammatory mediators, including toll-like receptor 4 (TLR4), tumor necrosis factor alpha (TNF-α), NF-kappa-B inhibitor alpha (IκBα), interleukin (IL)-1ß, and IL-6, as well as interleukin-1 receptor-associated kinase 4 (IRAK4), declined after the CAPE treatment. Additionally, CAPE significantly down-regulated the levels of glucose transporter 1 (GLUT1), glucose transporter 3 (GLUT3), and the key enzymes of glycolysis-hexokinase 2 (HK2), phosphofructokinase (PFK), pyruvate kinase muscle isozyme M2 (PKM2), and lactate dehydrogenase A (LDHA). Moreover, CAPE treatment decreased the levels of key lipid metabolism proteins, including acetyl coenzyme A carboxylase (ACC), fatty acid synthase (FASN), and free fatty acid (FFA)-transported-related protein CD36. After adding the glycolysis inhibitor 2-deoxy-D-glucose (2-DG), the inhibitory effects of CAPE on cell viability and migration were not significant when compared with the LPS group. In summary, the antitumor activity of CAPE in vitro was mainly via the modulation of the inflammatory mediators and the inhibition of key proteins and enzymes in glucose and lipid metabolism.

Caffeic Acid Phenethyl Ester Reduces the Adverse Effects of Nicotine on the Endometrium.

Background: Tobacco smoke contains various toxins that negatively affect the human reproductive system. Caffeic acid phenethyl ester (CAPE), a potent antioxidant, has protective effects on the reproductive system against oxygen-free radicals, methotrexate, and pesticides. Herein, the effect of CAPE on some key markers of endometrial receptivity has been evaluated. Methods: A cross-sectional study was conducted during 2018-2019 in the Department of Clinical Biochemistry, School of Medicine, Fasa University of Medical Sciences (Fasa, Iran). Primary endometrial cells were divided into five groups, namely control, nicotine, CAPE, vehicle, and nicotine+CAPE. Real-time polymerase chain reaction (PCR) and methylation-specific PCR were performed to evaluate gene expressions and methylation, respectively. Appropriate doses of CAPE and nicotine were determined using the MTT assay. Data were analyzed using SPSS software (version 16.0) with a one-way analysis of variance. P<0.01 was considered statistically significant. The fold change was calculated using the 2-∆ΔCT method. Results: Treatment of cells with nicotine significantly reduced the expression of C-X-C motif chemokine ligand 12 (CXCL12), fibroblast growth factor 2 (FGF2), and vascular endothelial growth factor A (VEGF-A) genes (P<0.0001). However, the expression levels increased significantly when treated with nicotine+CAPE (P<0.0001). Despite the reduced CXCL12 gene expression in cells treated with nicotine, CXCL12 was unmethylated in all study groups, indicating that the methylation status of the CXCL12 gene was not affected by nicotine or CAPE. Conclusion: CAPE can be a suitable agent to protect female smokers from the harmful effects of nicotine. This manuscript is available as a preprint on the Research Gate website.

The effect of caffeic acid on tendon healing in rats with an Achilles tendon injury model.

OBJECTIVES: This study aims to examine the effect of caffeic acid on tendon healing histopathologically and biomechanically in rats with an Achilles tendon injury model. MATERIALS AND METHODS: Twenty male Wistar-albino rats were used in this study. The rats were divided into two groups as the experimental group and control group. All rats underwent a bilateral achillotomy injury model and then surgical repair. Postoperatively, for four weeks, the experimental group was given intraperitoneal caffeic acid (100 mg/kg/day suspended in saline), while the control group was given only intraperitoneal saline. At the end of four weeks, after sacrificing each rat, right Achilles tendons were subjected to biomechanical analysis and the Achilles tendons were subjected to histopathological analysis. Bonar and Movin scores were used for histopathological analysis. In biomechanical analysis, tensile test was applied to Achilles tendons until rupture. For each tendon, failure load, displacement, cross-sectional area, maximum energy, total energy, length, stiffness, ultimate stress and strain parameters were recorded. RESULTS: According to Bonar and Movin scoring, the experimental group had lower scoring values than the control group (p=0.002 and p=0.002, respectively). Bonar scoring parameters were analyzed separately. Vascularity, collagen, and ground substance scores were lower in the experimental group compared to the control group (p=0.001, p=0.003, and p=0.047, respectively). No significant difference was found for tenocyte (p=0.064). In biomechanical analysis, failure load, displacement, ultimate stress, strain, and stiffness values were found to be higher in the experimental group compared to the control group (p=0.049, p=0.005, p=0.028, p=0.021, and p=0.049, respectively). CONCLUSION: The caffeic acid contributed positively to tendon healing histopathologically and biomechanically in rats with an Achilles tendon injury model.

Caffeic Acid Phenethyl Ester (CAPE) Synergistically Enhances Paclitaxel Activity in Ovarian Cancer Cells.

Caffeic acid phenethyl ester (CAPE) belongs to the phenols found in propolis. It has already shown strong antiproliferative, cytotoxic and pro-apoptotic activities against head and neck cancers and against breast, colorectal, lung and leukemia cancer cells. Ovarian cancer is one of the most dangerous gynecological cancers. Its treatment involves intensive chemotherapy with platinum salts and paclitaxel (PTX). The purpose of this study was to evaluate whether the combined use of CAPE and paclitaxel increases the effectiveness of chemotherapeutic agents. The experiment was performed on three ovarian cancer lines: OV7, HTB78, and CRL1572. The effect of the tested compounds was assessed using H-E staining, a wound-healing test, MTT and the cell death detection ELISAPLUS test. The experiment proved that very low doses of PTX (10 nM) showed a cytotoxic effect against all the cell lines tested. Also, the selected doses of CAPE had a cytotoxic effect on the tested ovarian cancer cells. An increase in the cytotoxic effect was observed in the OV7 line after the simultaneous administration of 10 nM PTX and 100 µM CAPE. The increase in the cytotoxicity was dependent on the CAPE dosage (50 vs. 100 µM) and on the duration of the experiment. In the other cell lines tested, the cytotoxic effect of PTX did not increase after the CAPE administration. The administration of PTX together with CAPE increased the percentage of apoptotic cells in the tested ovarian cancer cell lines. Moreover, the simultaneous administration of PTX and CAPE enhanced the anti-migration activity of the chemotherapeutic used in this study.

Feasibility of caffeic acid as a crosslinking agent in modifying acellular extracellular matrices.

Acellular extracellular matrices (aECM) are commonly utilized, both experimentally and clinically, in the regenerative medicine field. However, some disadvantages such as rapid degradation, poor mechanical properties, chronic inflammatory reactions and low antioxidant activity have limited their further application. In this study the feasibility of caffeic acid as a crosslinking agent in fixing small intestinal submucosa (SIS) was evaluated. The ninhydrin assay, swelling ratio and FTIR spectra indicated that caffeic acid can efficiently react with free amino groups to crosslink SIS and the highest crosslinking index reached 21.60 ± 1.37%. Moreover, the shrinkage temperature of SIS remarkably increased from 59 °C to about 80 °C and the degradation rate of CA-SIS was all lower than 6%, demonstrating their improved biostability and hydrothermal stability. Importantly, the antioxidant activity of CA-SIS ranged from 55% to 90%, statistically higher than that of native SIS (37.33 ± 2.94%). Additionally the cytotoxicity test presented that the cytotoxicity grade of CA-SIS was 1 or 0, whilst large numbers of living HUVECs were attached to the surface of the material and exhibited high cell viability. These results indicated their excellent cytocompatibility. The data of subcutaneous implant displayed that the number of inflammatory cells in 2%- and 2.5%CA-SIS groups remained at a low level (below 100 cells/field) while that of the native SIS group continued increasing, finally reaching 142.33 ± 30.92 cells/field. In conclusion, caffeic acid is a promising candidate for modifying aECM and may play a vital role in the design and fabrication of tissue engineering scaffolds.

Intralipid and caffeic acid phenethyl ester reverse the neurotoxic effects of organophosphate poisoning in rats.

Background: Organophosphate (Op)-containing herbicides continue to be widely used in the world. Although its usage and intoxication are widespread, the studies on organophosphate-induced neurotoxicity and treatment protocols are very few in the literature. Aims: This study aimed to investigate any potential effects of caffeic acid phenyl ester with/without intralipid on neurotoxicity produced by acute intoxication of glyphosate isopropylamine in an experimental rat model. Materials And Methods: Forty-nine wistar albino rats were randomly allotted into seven experimental groups: I, control; II, intralipid (IL); III, caffeic acid phenyl esther (CAPE); IV, glyphosate isopropylamine (GI); V, GI + IL; VI, GI + CAPE; and VII, GI + IL + CAPE. Total antioxidant and oxidant status levels were gauged, and the oxidative stress index was calculated in the serum samples. On the other hand, the tissues were analyzed with hematoxylin-eosin (HE) staining protocol and counted up by immunohistochemical method. Statistical evaluations were conducted using SPSS 11.5 for Windows (SPSS, Chicago, IL, USA). Results: Compared to the control, IL, and GI + IL + CAPE groups, the GI group significantly decreased the total antioxidant levels in brain tissues. In a supportive nature, a significant increase in the oxidative site index (OSI) in the GI group compared to other groups. Especially standing out point of these findings is the significant difference between the GI + IL + CAPE and the GI group. Parallelly, histopathological analysis extended severe neurotoxicity in the GI group. Neurotoxic status was reduced significantly in the GI + CAPE + IL group. The histopathologic examinations confirmed biochemical results. The results also revealed that CAPE and IL, probably their antioxidant effects, have a rehabilitative effect on neurotoxicity caused by GI. Conclusion: Therefore, CAPE and IL may function as potential cleansing and scavenger agents for supportive therapy regarding tissue damage or facilitate the therapeutic effects of the routine treatment of the patient with GI poisoning.

Caffeic acid phenethyl ester suppresses metastasis of breast cancer cells by inactivating FGFR1 via MD2.

BACKGROUND: Tumor metastasis is the main cause of death for breast cancer patients. Caffeic acid phenethyl ester (CAPE) has strong anti-tumor effects with very low toxicity and may be a potential candidate drug. However, the anti-metastatic effect and molecular mechanism of CAPE on breast cancer need more research. METHODS: MCF-7 and MDA-MB-231 breast cancer cells were used here. Wound healing and Transwell assay were used for migration and invasion detection. Western blot and RT-qPCR were carried out for the epithelial-to-myofibroblast transformation (EMT) process investigation. Western blot and immunofluorescence were performed for fibroblast growth factor receptor1 (FGFR1) phosphorylation and nuclear transfer detection. Co-immunoprecipitation was used for the FGFR1/myeloid differentiation protein2 (MD2) complex investigation. RESULTS: Our results suggested that CAPE blocks the migration, invasion, and EMT process of breast cancer cells. Mechanistically, CAPE inhibits FGFR1 phosphorylation and nuclear transfer while overexpression of FGFR1 reduces the anti-metastasis effect of CAPE. Further, we found that FGFR1 is bound to MD2, and silencing MD2 inhibits FGFR1 phosphorylation and nuclear transfer as well as cell migration and invasion. CONCLUSION: This study illustrated that CAPE restrained FGFR1 activation and nuclear transfer through MD2/FGFR1 complex inhibition and showed good inhibitory effects on the metastasis of breast cancer cells.

Chemoproteomics and Phosphoproteomics Profiling Reveals Salvianolic Acid A as a Covalent Inhibitor of mTORC1.

Salvianolic acid A (SAA), a major active ingredient of Salvia miltiorrhiza Bunge (Danshen), displays strong antiproliferative activity against cancer cells. However, their protein targets remain unknown. Here, we deconvoluted the protein targets of SAA using chemoproteomics and phosphoproteomics. By using alkynylated SAA as a probe, we discovered that SAA is a covalent ligand that can modify cellular proteins via its electrophilic α,ß-unsaturated ester moiety. The subsequent chemoproteomics profiling revealed that 46 proteins were covalently modified by SAA, including Raptor, a subunit of mTORC1 for recruiting substrates for mTORC1. Although gene ontology enrichment analysis of these proteins suggested that SAA displays a promiscuous protein interaction, phosphoproteomics profiling revealed that the SAA modulated phosphoproteins were mainly enriched in the signaling pathways of PI3K-Akt-mTOR, which is closely related to cell growth and proliferation. This was confirmed by the biochemical assay with purified mTORC1, a Western blot assay with phospho-specific antibodies, and a cellular thermal shift assay. Our work discovered that SAA is a covalent ligand for protein modification and mTORC1 is one of its targets. Moreover, our work demonstrated that the integrative profiling of chemoproteomics and phosphoproteomics can be a powerful tool for target deconvolution for bioactive natural products.

Caffeic Acid Phenethyl Ester and Caffeamide Derivatives Suppress Oral Squamous Cell Carcinoma Cells.

Caffeic acid phenethyl ester (CAPE) contains antibiotic and anticancer activities. Therefore, we aimed to investigate the anticancer properties and mechanisms of CAPE and caffeamide derivatives in the oral squamous cell carcinoma cell (OSCC) lines SAS and OECM-1. The anti-OSCC effects of CAPE and the caffeamide derivatives (26G, 36C, 36H, 36K, and 36M) were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. Cell cycle and total reactive oxygen species (ROS) production were analyzed using flow cytometry. The relative protein expression of malignant phenotypes was determined via Western blot analysis. The results showed that 26G and 36M were more cytotoxic than the other compounds in SAS cells. After 26G or 36M treatment for 48 h, cell cycle S phase or G2/M phase arrest was induced, and cellular ROS increased at 24 h, and then decreased at 48 h in both cell lines. The expression levels of cell cycle regulatory and anti-ROS proteins were downregulated. In addition, 26G or 36M treatment inhibited malignant phenotypes through mTOR-ULK1-P62-LC3 autophagic signaling activated by ROS generation. These results showed that 26G and 36M induce cancer cell death by activating autophagy signaling, which is correlated with altered cellular oxidative stress.

Caffeic acid phenethyl ester analogues as selective inhibitors of 12-lipoxygenase product biosynthesis in human platelets.

The inflammatory response is an essential process for the host defence against pathogens. Lipid mediators are important in coordinating the pro-inflammatory and pro-resolution phases of the inflammatory process. However, unregulated production of these mediators has been associated with chronic inflammatory diseases such as arthritis, asthma, cardiovascular diseases, and several types of cancer. Therefore, it is not surprising that enzymes implicated in the production of these lipid mediators have been targeted for potential therapeutic approaches. Amongst these inflammatory molecules, the 12-hydroxyeicosatetraenoic acid (12(S)-HETE) is abundantly produced in several diseases and is primarily biosynthesized via the platelet's 12-lipoxygenase (12-LO) pathway. To this day, very few compounds selectively inhibit the 12-LO pathway, and most importantly, none are currently used in the clinical settings. In this study, we investigated a series of polyphenol analogues of natural polyphenols that inhibit the 12-LO pathway in human platelets without affecting other normal functions of the cell. Using an ex vivo approach, we found one compound that selectively inhibited the 12-LO pathway, with IC50 values as low as 0.11 µM, with minimal inhibition of other lipoxygenase or cyclooxygenase pathways. More importantly, our data show that none of the compounds tested induced significant off-target effects on either the platelet's activation or its viability. In the continuous search for specific and better inhibitors targeting the regulation of inflammation, we characterized two novel inhibitors of the 12-LO pathway that could be promising for subsequent in vivo studies.