Koichiro Kawaguchi

The cancer-promoting gene fatty acid-binding protein 5 (FABP5) is epigenetically regulated during human prostate carcinogenesis

FABPs (fatty-acid-binding proteins) are a family of low-molecular-mass intracellular lipid-binding proteins consisting of ten isoforms. FABPs are involved in binding and storing hydrophobic ligands such as long-chain fatty acids, as well as transporting these ligands to the appropriate compartments in the cell. FABP5 is overexpressed in multiple types of tumours. Furthermore, up-regulation of FABP5 is strongly associated with poor survival in triple-negative breast cancer. However, the mechanisms underlying the specific up-regulation of the FABP5 gene in these cancers remain poorly characterized. In the present study, we determined that FABP5 has a typical CpG island around its promoter region. The DNA methylation status of the CpG island in the FABP5 promoter of benign prostate cells (PNT2), prostate cancer cells (PC-3, DU-145, 22Rv1 and LNCaP) and human normal or tumour tissue was assessed by bisulfite sequencing analysis, and then confirmed by COBRA (combined bisulfite restriction analysis) and qAMP (quantitative analysis of DNA methylation using real-time PCR). These results demonstrated that overexpression of FABP5 in prostate cancer cells can be attributed to hypomethylation of the CpG island in its promoter region, along with up-regulation of the direct trans-acting factors Sp1 (specificity protein 1) and c-Myc. Together, these mechanisms result in the transcriptional activation of FABP5 expression during human prostate carcinogenesis. Importantly, silencing of Sp1, c-Myc or FABP5 expression led to a significant decrease in cell proliferation, indicating that up-regulation of FABP5 expression by Sp1 and c-Myc is critical for the proliferation of prostate cancer cells.

High expression of Fatty Acid-Binding Protein 5 promotes cell growth and metastatic potential of colorectal cancer cells

Fatty acid‐binding proteins (FABPs) are responsible for binding and storing hydrophobic ligands such as long‐chain fatty acids, and for transporting these ligands to the appropriate compartments within the cell. The present study demonstrates that the FABP5 gene is upregulated in colorectal cancer cells compared to normal colon cells in a manner that correlates with disease stage and that FABP5 significantly promotes colorectal cancer cell growth and metastatic potential. Thus, FABP5 might be a promising prognostic or therapeutic biomarker candidate in human colorectal cancer.

SYNTHESIS OF PROCYANIDINS C2 AND C1 USING LEWIS ACID MEDIATED EQUIMOLAR CONDENSATION

Synthesis of procyanidin C2 and C1 was achieved via a stereoselective intermolecular condensation of equimolar amount of dimeric catechin or epicatechin nucleophile and monomeric catechin or epicatechin electrophile using Lewis acid. In the case of synthesis of procyanidin C2, AgBF4 and AgOTf afforded condensed product in excellent yield. As to the synthesis of procyanidin C1, Yb(OTf)3 was effective for equimolar condensation. INTRODUCTION Proanthocyanidins are known as condensed or non-hydrolysable tannins. These tannins are widely found in the vegetables, fruits and the bark of trees. Proanthocyanidins have been reported to exhibit strong free-radical scavenging and antioxidative activities. Many biological activities such as antitumor, antiviral, anti-inflammatory, and inhibition of DNA polymerase were reported. Thus proanthocyanidins are increasingly recognized as possessing health beneficial effects for humans. Tannin extracts from the nature give various types of polyphenols. Because their identification as well as purification is extremely difficult, further investigation of biological activities, i. e. mechanism of action remains unsolved. In these days, in order to obtain procyanidin oligomers in pure state, synthetic efforts have been devoted. As to the synthesis of catechin and/or epicatechin trimers, several synthetic studies were reported. The typical synthetic methods are as follows. The first example is pioneering work of the synthesis of procyanidins reported by Saito et al. This method requires to use nucleophilic partner in large excess (3.0 to 4.5 eq.) for condensation to prevent further oligomerization. The disadvantage of this procedure is that excess nucleophilic partner needs to be removed after condensation. The next one is use of C-8 bromide derivative to prevent forming further oligomeration. Quite recent example is using C-8 bromide and C-8 metallated nucleophile for the selective synthesis of C-4 and C-8 bound catechin trimers. So far, little attention has been paid to the screening of Lewis acids for equimolar condensation to construct the skeleton of procyanidin trimers. We have already reported a stereoselective synthesis of catechin trimer derivative using equimolar condensation. However, synthesis of epicatechin trimer using equimolar condensation has not been reported yet. In this article, we wish to report total synthesis of procyanidin C2 (1) and the recent progress of the synthesis of procyanidin C1 (2) via equimolar condensation between dimeric catechin or epicatechin nucleophile and monomeric catechin or epicatechin electrophile (Figure 1). Figure 1. The structures of procyanidin C2 (1) and C1 (2). RESULTS AND DISCUSSION We have investigated various Lewis acids including Ag, Cu, and Yb for equimolar condensation between dimeric catechin nucleophile 3 and monomeric catechin electrophile 4. Among these Lewis acids, we found that silver Lewis acids such as AgBF4 and AgOTf gave condensed product in excellent yield Scheme 1). Scheme 1. Equimolar condensation between dimeric catechin nucleophile 3 and monomeric catechin electrophile 4. The condensed product 5 was obtained in high yield, we next pursued to establish total synthesis of procyanidin C2 (1). Hydrolysis of the acetate of 5 using NaOMe under reflux did not furnish 6 although this reaction worked well in the case of procyaidin B1-B4. Thus diol 5 was once acetylated using Ac2O to give triacetate and subsequent reduction with DIBALH afforded 6 in 45% yield through 2 steps. However, the yield of transformation from 5 to 6 was not high enough, we tried the Kozikowski’s method using n-Bu4NOH to afford 6 in 66% yield. The H and C NMR spectral data of 6 were identical with those of the reported values. The 3,4-cis trimer could not be detected. This result indicated that electrophile 4 reacted with nucleophile 3 at C-4 position in a stereoselective manner to afford 3,4-trans trimer 5. The benzyl ether 6 was deprotected to yield procyanidin C2 (1) by hydrogenolysis over Pearlman’s catalyst. As to the purification of 1, when the crude products were directly evaporated, partially insoluble materials remained. We met with some difficulty to purify 1 after debenzylation. We tried separation by ODS cartridge column chromatography and preparative HPLC, however, we could not obtain 1 in a satisfying yield. Kozikowski and colleagues reported that lyophilization is useful for purification of these type compounds. Thus we used this method to afford procyanidin C2 (1) in good yield. We confirmed that lyophilized procyanidin C2 (1) was pure by HPLC analysis. The optical rotation value and mass spectrum data of synthetic 1 were in good accordance with those of the reported values. With regard to the H and C NMR spectral data of 1, we could not find them in any reports to the best of our knowledge. In this article, we wish to supply these data in experimental section and supporting information for future reference. The H NMR spectral datum of peracetate 7 was in good agreement with that of the reported values (Scheme 2). 5 6 procyanidin C2 (1) 7 n-Bu4NOH H2, Pd(OH)2/C 66% O BnO

Epicatechin oligomers longer than trimers have anti-cancer activities, but not the catechin counterparts

Since procyanidins (oligomeric catechin or epicatechin) were reported to exhibit health benefits, much attention has been paid to the synthesis of these compounds, especially those that are longer than trimers. In the present study, syntheses of cinnamtannin A3 (epicatechin pentamer), A4 (epicatechin hexamer), catechin tetramer, pentamer, arecatannin A2 (epicatechin-epicatechin-epicatechin-catechin) and A3 (epicatechin-epicatechin-epicatechin-epicatechin-catechin) were achieved. The key reaction was a Lewis acid mediated equimolar condensation. The antitumor effects of these synthesized compounds against a human prostate cancer cell line (PC-3) were investigated. Among the tested compounds, cinnamtannin A3, A4 and arecatannin A3, which possess epicatechin oligomers longer than tetramers as the basic scaffold, showed significant activities for suppression of cell growth, invasion and FABP5 (fatty acid-binding protein 5) gene expression. Effects on cell cycle distribution showed that cell cycle arrest in the G2 phase was induced. Furthermore, these epicatechin oligomers suppressed significantly the expression of the cancer-promoting gene, FABP5, which is related to cell proliferation and metastasis in various cancer cells. Interestingly, the suppressive activities were associated with the degree of oligomerization of epicatechin. Thus, synthetic studies clearly demonstrate that epicatechin oligomers longer than trimers have significant anti-tumorigenic activities, but not the catechin counterparts.

A novel fatty acid-binding protein 5-estrogen-related receptor α signaling pathway promotes cell growth and energy metabolism in prostate cancer cells

Epidermal or cutaneous fatty acid-binding protein is an intracellular lipid-binding protein, also known as FABP5, and its expression level is closely related to cancer cell proliferation and metastatic activities in various types of carcinoma. However, the molecular mechanisms of FABP5 in cancer cell proliferation and its other functions have remained unclear. In the present study, we have clearly revealed that FABP5 activated expression of metabolic genes (ATP5B, LCHAD, ACO2, FH and MFN2) via a novel signaling pathway in an ERRα (estrogen-related receptor α)-dependent manner in prostate cancer cell lines. To clarify the novel function of FABP5, we examined the activation mechanisms of the ERRα target genes via FABP5. A direct protein-protein interaction between FABP5 and ERRα was demonstrated by immunoprecipitation and GST pull-down assays. We have clearly revealed that FABP5 interacted directly with transcriptional complex containing ERRα and its co-activator PGC-1β to increase expression of the ERRα target genes. In addition, we have shown that FABP5 knockdown induced high energy stress leading to induction of apoptosis and cell cycle arrest via AMPK-FOXO3A signaling pathway in prostate cancer cells, suggesting that FABP5 plays an important role in cellular energy status directing metabolic adaptation to support cellular proliferation and survival.

Fatty acid-binding protein 5 (FABP5) promotes lipolysis of lipid droplets, de novo fatty acid (FA) synthesis and activation of nuclear factor-kappa B (NF-κB) signaling in cancer cells

Fatty acid-binding proteins (FABPs) are involved in binding and storing hydrophobic ligands such as long-chain fatty acids, as well as transporting them to the appropriate compartments in the cell. Epidermal fatty acid-binding protein (FABP5) is an intracellular lipid-binding protein that is abundantly expressed in adipocytes and macrophages. Previous studies have revealed that the FABP5 expression level is closely related to malignancy in various types of cancer. However, its precise functions in the metabolisms of cancer cells remain unclear. Here, we revealed that FABP5 knockdown significantly induced downregulation of the genes expression, such as hormone-sensitive lipase (HSL), monoacylglycerol lipase (MAGL), elongation of long-chain fatty acid member 6 (Elovl6), and acyl-CoA synthetase long-chain family member 1 (ACSL1), which are involved in altered lipid metabolism, lipolysis, and de novo FA synthesis in highly aggressive prostate and breast cancer cells. Moreover, we demonstrated that FABP5 induced inflammation and cytokine production through the nuclear factor-kappa B signaling pathway activated by reactive oxygen species and protein kinase C in PC-3 and MDA-MB-231 cells. Thus, FABP5 might regulate lipid quality and/or quantity to promote aggressiveness such as cell growth, invasiveness, survival, and inflammation in prostate and breast cancer cells. In the present study, we have revealed for the first time that high expression of FABP5 plays a critical role in alterations of lipid metabolism, leading to cancer development and metastasis in highly aggressive prostate and breast cancer cells.