Benzoyloxycinnamaldehyde Research Articles

Two Promising Anti-Cancer Compounds, 2-Hydroxycinnaldehyde and 2- Benzoyloxycinnamaldehyde: Where do we stand?

Natural bioactive compounds with anti-carcinogenic activity are gaining tremendous interest in the field of oncology. Cinnamon, an aromatic condiment commonly used in tropical regions, appeared incredibly promising as an adjuvant for cancer therapy. Indeed, its whole or active parts (e.g., bark, leaf) exhibited significant anti-carcinogenic activity, which is mainly due to two cinnamaldehyde derivatives, namely 2-hydroxycinnaldehyde (HCA) and 2- benzoyloxycinnamaldehyde (BCA). In addition to their anti-cancer activity, HCA and BCA exert immunomodulatory, anti-platelets, and anti-inflammatory activities. The highly reactive α,ßunsaturated carbonyl pharmacophore, called Michael acceptor, contributes to their therapeutic effects. The molecular mechanisms underlying their anti-tumoral and anti-metastatic effects are miscellaneous, strongly suggesting that these compounds are multi-targeting compounds. Nevertheless, unravelling the exact molecular mechanisms of HCA and BCA remains a challenging matter which is necessary for optimal controlled-drug targeting delivery, safety, and efficiency. Eventually, their poor pharmacological properties (e.g., systemic bioavailability and solubility) represent a limitation and depend both on their administration route (e.g., per os, intravenously) and the nature of the formulation (e.g., free, smart nano-). This concise review focused on the potential of HCA and BCA as adjuvants in cancer. We describe their medicinal effects as well as provide an update about their molecular mechanisms reported either in-vitro, ex-vivo, or in animal models.

P11.16 The stemness andinvasiveness of glioblastoma tumorspheres were suppressed by combined treatment with2’-hydroxycinnamaldehyde andtemozolomide

Abstract BACKGROUND Glioblastoma (GBM) is the most common and aggressive human primary brain malignancy. The key properties of GBM, stemness and invasiveness, are known to be associated with a highly unfavorable prognosis. Notably, the process of epithelial-mesenchymal transition (EMT) is closely related to the progression of GBM. On the basis of reports that 2′-hydroxycinnamaldehyde (HCA) and its derivative, 2′-benzoyloxycinnamaldehyde (BCA), suppresses EMT in several human cancer cells, we sought to evaluate the therapeutic efficacy of HCA and BCA, alone and in combination with temozolomide (TMZ), on GBM tumorspheres (TSs). MATERIAL AND METHODS Two human GBM TSs were treated with HCA, BCA, or TMZ. Therapeutic effects were evaluated by measuring ATP levels, neurosphere formation, 3D-invasion in collagen matrix, and viability. Protein expression profiles after drug treatment were evaluated by western blotting. In vivo anticancer efficacy of drugs was examined in a mouse orthotopic xenograft model. RESULTS Combined treatment of GBM TSs with HCA or BCA and TMZ significantly reduced cell viability, stemness, and invasiveness. Expression levels of stemness-, invasiveness-, and mesenchymal transition-associated markers, Zeb1, N-cadherin, and β-catenin, were also substantially decreased by the combined treatment. The combined treatment also reduced tumor growth in a mouse orthotopic xenograft model. CONCLUSION Our findings suggest that HCA and BCA, combined with TMZ, are potential therapeutic agents in the treatment of GBM.

Combined treatment with 2'-hydroxycinnamaldehyde and temozolomide suppresses glioblastoma tumorspheres by decreasing stemness and invasiveness.

Glioblastoma (GBM) is the most common and aggressive human primary brain malignancy. The key properties of GBM, stemness and invasiveness, are known to be associated with a highly unfavorable prognosis. Notably, the process of epithelial-mesenchymal transition (EMT) is closely related to the progression of GBM. On the basis of reports that 2'-hydroxycinnamaldehyde (HCA) and its derivative, 2'-benzoyloxycinnamaldehyde (BCA), suppresses EMT in several human cancer cells, we sought to evaluate the therapeutic efficacy of HCA and BCA, alone and in combination with temozolomide (TMZ), on GBM tumorspheres (TSs). Two human GBM TSs were treated with HCA, BCA, or TMZ. Therapeutic effects were evaluated by measuring ATP levels, neurosphere formation, 3D-invasion in collagen matrix, and viability. Protein expression profiles after drug treatment were evaluated by western blotting. In vivo anticancer efficacy of drugs was examined in a mouse orthotopic xenograft model. Combined treatment of GBM TSs with HCA or BCA and TMZ significantly reduced cell viability, stemness, and invasiveness. Expression levels of stemness-, invasiveness-, and mesenchymal transition-associated markers, Zeb1, N-cadherin, and β-catenin, were also substantially decreased by the combined treatment. The combined treatment also reduced tumor growth in a mouse orthotopic xenograft model. Our findings suggest that HCA and BCA, combined with TMZ, are potential therapeutic agents in the treatment of GBM.

The anticancer mechanism of 2'-hydroxycinnamaldehyde in human head and neck cancer cells.

Cinnamaldehyde has been shown to effectively induce apoptosis in a number of human cancer cells. In the present study, cinnamaldehyde derivative-induced apoptosis and its signaling pathways were assessed in p53-wild (SGT) and p53-mutant (YD-10B) human head and neck cancer cells. The cinnamaldehyde derivatives, 2'-hydroxycinnamaldehyde (HCA) and 2'-benzoyloxycinnamaldehyde (BCA), exhibited powerful anti-proliferative effects on SGT and YD-10B cells. The apoptotic effect induced by HCA or BCA was supported by caspase-3, -7, -9 and PARP activation, and confirmed by Annexin V-FITC/PI double staining. HCA induced the expression of p21 in both SGT and YD-10B cells. Furthermore, HCA induced the level of pro-apoptotic Bak1 expression while decreasing the level of anti-apoptotic Bcl-2 in both cell lines, suggesting that HCA induced the cell death pathway in a p53-independent manner. HCA also induced the expression of LC3B in SGT and YD-10B cells. Following pre-incubation with the autophagy inhibitor 3-MA, HCA-induced apoptosis was largely increased in SGT cells, while inhibited in YD-10B cells, suggesting that autophagy may actively contribute to HCA-induced apoptosis. Taken together, these observations suggest that HCA may be an effective therapeutic agent in the treatment of head and neck cancer regardless of p53 status.

DJ-1 Protects Breast Cancer Cells Against 2'-Benzoyloxycinnamaldehyde-induced Oxidative Stress Independent of Nrf2.

2'-Benzoyloxycinnamaldehyde (BCA) is a promising antitumor agent. BCA effectively inhibited proliferation of MDA-MB-435 more than in MCF-7 breast cancer cells. Our recent findings showed that DJ-1 protects MCF7 cells from BCA-induced oxidative stress via its mitochondrial translocation and inhibition of the mitochondrial perturbation (Ismail et al., 2012). In this study, we addressed the question of whether Nrf2 works downstream to DJ-1 in mediating differential antiproliferation effects in MCF-7 and MDAMB-435 breast cancer cells induced by BCA treatment. BCA upregulated the expression and induced nuclear translocalization of DJ-1 and Nrf2 in only MCF-7 cells. However, in MDA-MB-435, BCA increased only Nrf2 expression without inducing DJ-1 and/or Nrf2 protein translocalization to the nucleus. Furthermore, DJ-1 knockdown decreased DJ-1 expression in both cells without affecting Nrf2 and its downstream target γ-GCS, suggesting that DJ-1-induced cell protection and works independent of Nrf2 signaling pathway.

Dual pH-sensitive oxidative stress generating micellar nanoparticles as a novel anticancer therapeutic agent

Cancer cells are under oxidative stress due to a large production of reactive oxygen species (ROS), which involve in cell proliferation and cancer promotion and progression. On the other hand, ROS promotes cell death, depending on the rate of ROS production and the activity of antioxidant systems. Recently, “oxidation therapy” has arisen as a promising anticancer strategy, which can be achieved by inducing the generation of cytotoxic level of ROS or inhibiting the antioxidant systems in tumor cells. Here, we report oxidative stress amplifying nanoplatforms as novel anticancer therapeutics, which are able not only to suppress antioxidant but also to generate ROS simultaneously in acidic tumor microenvironments. The oxidative stress amplifying nanoplatforms are composed of dual pH-sensitive PBCAE copolymer, polymeric prodrug of BCA (benzoyloxycinnamaldehyde) and heme oxygenase-1 (HO-1) inhibiting zinc protoporphyrin (ZnPP). PBCAE was designed to incorporate ROS-generating BCA in its backbone via acid-cleavable acetal linkages and self-assemble to form micelles that encapsulate ZnPP. In vitro proof-of-concept studies revealed that ZnPP encapsulated in PBCAE micelles suppressed HO-1 to make cancer cells more vulnerable to BCA-induced ROS, leading to enhanced apoptotic cell death. In addition, ZnPP-loaded PBCAE micelles significantly suppressed the tumor growth in human cancer xenograft mouse models. We believe that oxidative stress amplifying micellar nanoparticles have a great potential as novel redox anticancer therapeutics.

EGR1-dependent PTEN upregulation by 2-benzoyloxycinnamaldehyde attenuates cell invasion and EMT in colon cancer

There has been little evidence to support EGR1 and PTEN function on the EMT of cancer cells. We tried to evaluate how these genes affect cancer cell invasion and EMT through investigating the molecular mechanism(s) of 2′-benzoyloxycinnamaldehyde (BCA). Matrigel invasion and wound healing assay, and in vivo mice model were used to evaluate the effect of BCA on colon cancer cell migration. The molecular mechanism(s) of BCA were evaluated by knock-down or overexpression of EGR1 and PTEN. BCA at 50nM increased E-cadherin and EGR1 expression without cytotoxicity. Cell migration was inhibited significantly by BCA both in vitro and in vivo. Moreover, BCA inhibits Snail and Vimentin expression, as well as β-catenin nuclear accumulation. Suppression of EGR1 by siRNA attenuated the inhibition of matrigel invasion by BCA, indicating that EGR1 is responsible for BCA effect. PTEN was upregulated by BCA treatment or EGR1 overexpression. In addition, shPTEN transfection stimulated EMT and cell invasion in vitro. Our data suggest that BCA leads to a remarkable upregulation of EGR1 expression, and that EMT and invasion is decreased via EGR1-dependent PTEN activation. These data showed a critical role of EGR1-PTEN signaling pathway in the EMT of colon cancer, as well as metastasis.

Early growth response protein 1 upregulation and nuclear translocation by 2′-benzoyloxycinnamaldehyde induces prostate cancer cell death

2′-Benzoyloxycinnamaldehyde (BCA) induces apoptosis in human cancer cells through ROS generation. BCA upregulates proapoptotic genes such as activating transcription factor 3 (ATF3), NSAID-activated gene 1 protein (NAG-1), and growth arrest and DNA-damage-inducible protein alpha (GADD45A) in prostate cancer cells. These genes are known to be induced by transcription factor early growth response protein 1 (EGR1). BCA induces significant EGR1 upregulation, while EGR1 knockdown decreases the induction of these genes with concurrent alleviation of cell death by BCA. Antioxidant glutathione pretreatment with BCA removes EGR1 expression increase, suggesting that EGR1 upregulation is dependent on oxidative stress generated by BCA. In prostate cancer cells, EGR1 localizes in the cytoplasm; however, BCA remarkably upregulates EGR1 nuclear translocalization, suggesting its possible effect as a transcriptional activator. BCA induces transient upregulation of importin-7 (IPO7) which is critical for EGR1 nuclear translocation, and IPO7 knockdown led to a significant decrease in chemosensitivity to BCA. Taken together, our findings suggest that BCA induces prostate cancer cell death via EGR1 upregulation and nuclear translocalization, followed by activation of proapoptotic target genes.

2′-Benzoyloxycinnamaldehyde inhibits nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 cells via regulation of AP-1 pathway

Cinnamaldehyde, an active compound of cinnamon, has been reported to exert various biological functions such as anti-inflammatory and anti-tumor activities. Previously, we showed that 2′-hydroxycinnamaldehyde (HCA) has an inhibitory effect on nitric oxide (NO) production through the inhibition of NF-κB signaling. In an effort to find a more effective anti-atherosclerotic agent, here we evaluated the anti-inflammatory effect of 2′-benzoyloxycinnamaldehyde (BCA) in RAW 264.7 murine macrophage cells. We showed that BCA more effectively inhibited NO production than HCA with less cytotoxicity. We also demonstrated that BCA inhibited the lipopolysaccharide (LPS)-induced expression of iNOS in a concentration-dependent manner. Signal transduction studies showed that BCA significantly inhibited the phosphorylation of SAPK/JNK and AP-1-dependent reporter gene activity. LPS-induced expression levels of JunB, c-Jun and c-Fos were also decreased by BCA treatment. Moreover, the LPS-induced DNA binding activity of AP-1 was markedly inhibited by BCA. The direct injection of BCA into mice inhibited the LPS-induced increase in plasma nitrite levels, confirming the anti-inflammatory effect of BCA in vivo. Overall, these observations suggest that BCA has the potential for use as an anti-atherosclerotic agent.

2′-Benzoyloxycinnamaldehyde-Mediated DJ-1 Upregulation Protects MCF-7 Cells from Mitochondrial Damage

2'-Benzoyloxycinnamaldehyde (BCA) is a promising antitumor agent which induces cancer cells apoptosis via reactive oxygen species (ROS) generation. BCA shows more effective antiproliferation in MDA-MB-435 than in MCF-7 breast cancer cells. DJ-1 has been known to protect cells against oxidative stress as an antioxidant because of its cysteine residues sensitive to oxidative stress. In the present study, we evaluated the mechanism of DJ-1 for cell protection from oxidative stress after BCA treatment in MCF-7 cell. BCA upregulates the expression of DJ-1 in MCF-7 cells. However, DJ-1 expression decreased continuously for 24 h after BCA treatment in MDA-MB-435 cells. DJ-1 knockdown sensitized MCF-7 cells to BCA, on the contrary, DJ-1 overexpression induced MDA-MB-435 cells less sensitive to BCA. Confocal microscopic observation showed that only in MCF-7 cells BCA increased the overlapped signal between mitochondria and DJ-1 protein. Mitochondrial membrane potential (MMP) was decreased in MDA-MB-435 cells by BCA, and DJ-1 overexpression inhibited BCA-induced MMP decrease in these cells. On the contrary, DJ-1 knockdown in MCF-7 induced MMP perturbation by BCA. These findings suggest that DJ-1 upregulation protects MCF-7 cells from BCA via inhibiting mitochondrial damage.

Differential antiproliferation effect of 2′‐benzoyloxycinnamaldehyde in K‐ras‐transformed cells via downregulation of thiol antioxidants

2'-Benzoyloxycinnamaldehyde (BCA), one of the derivatives of 2'-hydroxycinnamaldehyde (HCA) isolated from the bark of Cinnamomum cassia, induces apoptosis in human cancer cells. We found that BCA induces stronger antiproliferative effects in K-ras-transformed cells (RK3E-ras) than in isogenic non-transformed cells (RK3E). Treatment of RK3E-ras with BCA resulted in increased ROS generation and depletion of intracellular glutathione, whereas BCA-treated RK3E showed no significant increase in the ROS level with concurrent increase in intracellular glutathione (GSH). Thiol antioxidants recovered cell proliferation inhibition caused by BCA in both cell lines, while non-thiol antioxidants failed to recover cell death. BCA decreased metallothionein (MT) expression in RK3E-ras, while inducing remarkable MT expression in RK3E. The increase of intracellular GSH in RK3E is partially caused by differential induction of γ-glutamylcysteine synthetase (γ-GCS) due to BCA treatment. To evaluate the upstream pathway for differential expression of γ-GCS and MT, we analyzed early DJ-1 (PARK7) and NF-E2 p45-related factor 2 (Nrf2) changes after BCA treatment. In RK3E, DJ-1 expression considerably increased for 3 h with concurrent induction of Nrf2, whereas in RK3E-ras cells BCA decreased these protein levels. Based on these findings, it seems that the therapeutic selectivity of BCA in RK3E-ras results from decreased thiol antioxidants via decreased DJ-1 and Nrf2 expression.

2′-Hydroxycinnamaldehyde targets low-density lipoprotein receptor-related protein-1 to inhibit lipopolysaccharide-induced microglial activation

2′-Hydroxycinnamaldehyde (HCA) isolated from the stem bark of Cinnamomum cassia and its derivative 2′-benzoyloxycinnamaldehyde (BCA) were reported to have anti-angiogenic, anti-proliferative, and anti-inflammatory effects in several human cancer cells and RAW 264.7 macrophage cells. However, effects of HCA/BCA on the neuroinflammation have not been investigated. In the present study, a potential anti-neuroinflammatory effect of HCA/BCA was assessed in lipopolysaccharide (LPS)-stimulated microglial cultures and microglia/neuroblastoma cocultures. Nitric oxide production, inflammatory gene expression, and signaling pathways were investigated. HCA/BCA significantly decreased the production of nitric oxide and tumor necrosis factor-alpha (TNF-α) in microglial cells. HCA/BCA also attenuated the expression of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines such as interleukin-1β (IL-1β) and TNF-α at mRNA level via blockade of ERK, JNK, p38 MAPK, and NF-κB activation. Moreover, HCA/BCA was neuroprotective by reducing microglia-mediated neuroblastoma cell death in a microglia-neuroblastoma co-culture. Affinity chromatography and LC-MS/MS analysis identified low-density lipoprotein receptor-related protein 1 (LRP1) as a potential molecular target of HCA in microglial cells. Based on the studies using the receptor-associated protein (RAP) that blocks a ligand binding to LRP1 and the siRNA-mediated LRP1 gene silencing, we were able to conclude that HCA inhibited LPS-induced microglial activation via LRP1. Our results suggest that HCA/BCA be anti-inflammatory and neuroprotective in the CNS by targeting LRP1, and may have a therapeutic potential against neuroinflammatory diseases.

Transport and metabolism of the antitumour drug candidate 2′-benzoyloxycinnamaldehyde in Caco-2 cells

The transport and metabolism of the antitumour drug candidate 2′-benzoyloxycinnamaldehyde (BCA) was characterized in Caco-2 cells.BCA disappeared rapidly from the donor side without being transported to the receiver side during its absorptive transport across Caco-2 cells. Its metabolites 2′-hydroxycinnamaldehyde (HCA) and o-coumaric acid (OCA) were formed in both the donor and the receiver sides.HCA, in a separate study, also disappeared rapidly from the donor side, mostly being converted to its oxidative metabolite OCA during its absorptive transport across Caco-2 cells.OCA was transported rapidly in the absorptive direction across Caco-2 cells with a Papp of 25.4 ± 1.0 × 10−6 cm s−1 (mean ± standard deviation (SD), n = 3). OCA was fully recovered from both the donor and the receiver side throughout the time-course of this study.Formation of HCA from BCA was inhibited almost completely by bis(p-nitrophenyl)phosphate (BNPP), a selective inhibitor of carboxylesterases (CES), and phenylmethylsulfonyl fluoride (PMSF), a broad specificity inhibitor of esterases in Caco-2 cells, suggesting that this hydrolytic biotransformation was likely mediated predominantly by CES. Conversion of HCA to OCA was inhibited significantly by isovanillin, a selective inhibitor of aldehyde oxidase (AO). Inhibitors for xanthine oxidase (XO) and aldehyde dehydrogenase (ALDH), which are known to be involved in the oxidation of aldehydes to carboxylic acids, did not have a significant effect on the biotransformation of HCA to OCA in Caco-2 cells.In summary, the present work demonstrates that BCA is hydrolysed rapidly to HCA, followed by subsequent oxidation to OCA, in Caco-2 cells. The results provide a mechanistic understanding of the poor absorption and low bioavailability of BCA after oral administration.

2′-Benzoyloxycinnamaldehyde Inhibits Tumor Growth in H-ras12V Transgenic Mice via Downregulation of Metallothionein

Cinnamaldehydes have been reported to induce apoptosis in human carcinomas through the generation of reactive oxygen species (ROS). 2′-benzoyloxycinnamaldehyde (BCA) has been reported to inhibit tumor formation in H-ras12V transgenic mice. To see the antitumor effects of BCA, BCA was administrated intraperitoneally (50 mg/kg) to H-ras12V transgenic mice for 3 wk, and it was found that the hepatic tumor volume and the total number of tumors were decreased in BCA-treated mice as compared to control H-ras12V transgenic mice. To identify possible target genes responsible for BCA antitumor effects in H-ras12V transgenic mice, cDNA microarray analyses were performed comparing gene expression between BCA treated and control transgenic mice. We found that 42 genes were downregulated, and 40 genes were upregulated in the BCA-treated transgenic mice. The downregulated genes included several genes involved in ROS regulation and immune response (aconitase, metallothionein-1, metallothionein-2, and purine nucleoside phosphorylase). The expression of ROS-related genes, metallothionein 1 and metallothionein 2, was decreased more than twofold with BCA treatment (P < 0.001). It was confirmed by RT-PCR and immunohistochemical analyses. The inhibition of tumor formation and growth in H-ras12V transgenic mice by BCA was mediated through inhibition of the expression of the ROS scavengers metallothionein 1 and metallothionein 2.

Structure–activity analysis of 2′-modified cinnamaldehyde analogues as potential anticancer agents

The natural product 2′-hydroxycinnamaldehyde (HCA) and its analogue, 2′-benzoyloxycinnamaldehyde (BCA), have been previously shown to have antiproliferative and proapoptotic effects in vitro and inhibit tumor growth in vivo. In this study, we use structure–activity analysis to define structural features that are important for the activity of cinnamaldehyde analogues. Our results emphasize an important role for both the propenal group as well as the modification at the 2′-position. Further studies were aimed to characterize the mechanism of action of BCA. Exposure to BCA induced cell death via caspase-dependent and -independent pathways. Cell death was not due to autophagy or necrosis as a result of energy depletion or induction of reactive oxygen species. Our findings have important implications for future drug design and highlight the importance of defining molecular drug targets for this promising class of potential anticancer agents.

Plasma pharmacokinetics and metabolism of the antitumour drug candidate 2′-benzoyloxycinnamaldehyde in rats

The pharmacokinetics and metabolism of 2′-benzoyloxycinnamaldehyde (BCA) was characterized in male Sprague–Dawley rats as part of the preclinical evaluations for developing this compound as an antitumour agent.BCA was not detected in the plasma following either intravenous or oral dose, whereas its putative metabolites 2′-hydroxycinnamaldehyde (HCA) and o-coumaric acid were present at considerable levels.In separate pharmacokinetics studies, HCA exhibited a high systemic clearance and a large volume of distribution, whereas both pharmacokinetic parameters were much lower for o-coumaric acid. The terminal half-life of both metabolites was approximately 2 h.BCA was converted rapidly to HCA in rat serum, liver microsomes and cytosol in vitro; HCA was subsequently converted to o-coumaric acid in a quantitative manner only in the liver cytosol. In addition, the formation of o-coumaric acid was inhibited significantly by menadione, a specific inhibitor for aldehyde oxidase. Taken collectively, the results suggest that the rapid systemic clearance of HCA is likely due mainly to hepatic clearance occurring from aldehyde oxidase-catalysed biotransformation to o- coumaric acid.In conclusion, the present work demonstrates that the anticancer drug candidate BCA is highly likely to work as its active metabolite HCA in the body.

The Activity of 2'-Benzoyloxycinnamaldehyde Against Drug-Resistant Cancer Cell Lines

This study investigated the inhibitory effects of 2'-benzoyloxycinnamaldehyde (BCA) on cancer cells, including various drug-resistant cancer cell lines. To observe this activity, the anticancer drug-resistant cell lines were established by continuously exposing the parental cells to 5-fluorouracil (5-FU) and cyclophosphamide (CDDP), and examining the cells with the MTT assay and flow cytometric analysis. The BCA treatment produced similar growth inhibit - ory effects and apoptotic cell death on the drug-resistant cancer cells as their parental cells. The activation of the p38-mitogen activated protein kinase, an increased level of reactive oxygen species (ROS) generation and downregulation of Bcl-2 were observed in both the drug resistant and non-drug resistant cell lines. The GSH treatment effectively inhibited BCA-induced apoptosis by blocking ROS generation, suggesting that ROS is a major regulator in BCA-induced apoptotic cell death. These results suggest that BCA can be a useful drug candidate for treating drug-resistant cells.

Apoptosis induction of 2′-hydroxycinnamaldehyde as a proteasome inhibitor is associated with ER stress and mitochondrial perturbation in cancer cells

2′-Hydroxycinnamaldehyde (HCA), isolated from the stem bark of Cinnamomum cassia, and 2′-benzoyloxycinnamaldehyde (BCA), one of HCA derivatives, have antiproliferative activities on several human cancer cell lines. Our previous study suggested that reactive oxygen species (ROS) and caspase-3 are the major regulators of HCA-induced apoptosis. In the present study, we demonstrated a novel molecular target using in vitro pull-down assay by biotin-labeled HCA (biotin-HCA) in SW620 cells. We analyzed 11 differential spots of 2-dimensional gel prepared with pull-downed proteins by biotin-HCA. Among them, five spots were identified as proteasome subunits. An in vitro 26S proteasome function assay using specific fluorogenic substrates showed that HCA potently inhibits L3-like activity of the proteasome. In addition, HCA showed inhibitory action against chymotrypsin-like, trypsin-like, and PGPH-like activities. DNA microarray showed that HCA induced heat shock family and ER stress-responsive genes, which reflects the accumulation of misfolded proteins by proteasome inhibition. On western blot analysis, it was confirmed that HCA induces glucose-regulated protein, 78 kDa (GRP78) and some representative endoplasmic reticulum (ER) stress-responsive proteins. Furthermore, HCA treatment decreased mitochondrial membrane potential. The effect of HCA on cytochrome c and Bax translocation between cytosol and mitochondrial membrane was clarified using western blot analysis. These results suggest that HCA-induced apoptosis is associated with the inhibition of the proteasome activity that leads in turn to the increase of ER stress and mitochondrial perturbation.

Delayed occurrence of H- ras12V-induced hepatocellular carcinoma with long-term treatment with cinnamaldehydes

Cinnamaldehyde from the bark of Cinnamomum cassia has been reported to have antitumor activity mediated by the inhibition of farnesyl transferase. We assessed in vivo the chemo-preventive effect of cinnamaldehydes on H- ras12V-induced hepatocellular carcinoma formation. A mouse model of hepatocellular carcinoma was established by using the transgene of mutated H- ras12V under the regulation of albumin enhancer/promoter. When treated with cinnamaldehyde for 10 weeks, hepatic tumor development was delayed with 2′-benzoyloxycinnamaldehyde (BCA) compared with control hepatocellular carcinoma formation. The effect of 2′-hydroxycinnamaldehyde (HCA) was comparable. The number of lesions and the size of each lesion were significantly reduced by BCA. Cell proliferation in the lesion was detected by incorporation of 5-bromo-2′-deoxyuridine (BrdU). BCA increased the number of splenocytes, concanavalin A-stimulated splenocyte proliferation and the infiltration of lymphocytes into liver. Data suggest that the delayed hepatic tumor development observed with BCA could be mediated by a long-term immunostimulating effect on T cells.

Protein Binding Characteristics of 2′-Benzoyloxycinnamaldehyde

The protein binding characteristic of 2′-Benzoyloxycinnamaldehyde (BCA) was investigated, which has demonstrated a potent antitumor effect against several human solid tumor cell lines and in human tumor xenograft nude mice. Protein binding of BCA in human serum was 86 ± 0.91% and the predominant binding protein of BCA was fatty-acid-free human serum albumin (HSA) (81 ± 0.91%). The binding of BCA to HSA was outlined by one class, and Ka and n of BCA were 1.65 × 105 M− 1 and 0.374, respectively. Displacement studies with fluorescence probes suggested that BCA mainly binds to site I on HSA, and BCA-induced enhancement in site II binding. The limited drug–drug interaction experiments suggested that BCA influences both site I and site II drug–HSA bindings via different mechanisms; a competitive displacement and a probable allosteric conformational change in HSA, respectively.