Examination of aminophenol-containing compounds designed as antiproliferative agents and potential atypical retinoids.

Retinoic acid (RA, 1), an oxidized form of vitamin A, binds to retinoic acid receptors (RAR) and retinoid X receptors (RXR) to regulate gene expression and has important functions such as cell proliferation and differentiation. Synthetic ligands regarding RAR and RXR have been devised for the treatment of various diseases, particularly promyelocytic leukemia, but their side effects have led to the development of new, less toxic therapeutic agents. Fenretinide (4-HPR, 2), an aminophenol derivative of RA, exhibits potent antiproliferative activity without binding to RAR/RXR, but its clinical trial was discontinued due to side effects of impaired dark adaptation. Assuming that the cyclohexene ring of 4-HPR is the cause of the side effects, methylaminophenol was discovered through structure-activity relationship research, and p-dodecylaminophenol (p-DDAP, 3), which has no side effects or toxicity and is effective against a wide range of cancers, was developed. Therefore, we thought that introducing the motif carboxylic acid found in retinoids, could potentially enhance the anti-proliferative effects. Introducing chain terminal carboxylic functionality into potent p-alkylaminophenols significantly attenuated antiproliferative potencies, while a similar structural modification of weakly potent p-acylaminophenols enhanced growth inhibitory potencies. However, conversion of the carboxylic acid moieties to their methyl esters completely abolished the cell growth inhibitory effects of both series. Insertion of a carboxylic acid moiety, which is important for binding to RA receptors, abolishes the action of p-alkylaminophenols, but enhances the action of p-acylaminophenols. This suggests that the amido functionality may be important for the growth inhibitory effects of the carboxylic acids.

Effects of Antibacterial Agents on Cancerous Cell Proliferation.

Myelosuppression, a side effect of anticancer drugs, makes people more susceptible to infectious diseases by compromising the immune system. When a cancer patient develops a contagious disease, treatment with an anticancer drug is suspended or postponed to treat the infectious disease. If there was a drug that suppresses the growth of cancer cells among antibacterial agents, it would be possible to treat both infectious diseases and cancer. Therefore, this study investigated the effect of antibacterial agents on cancer cell development. Vancomycin (VAN) had little effect on cell proliferation against the breast cancer cell, MCF-7, prostate cancer cell, PC-3, and gallbladder cancer cell, NOZ C-1. Alternatively, Teicoplanin (TEIC) and Daptomycin (DAP) promoted the growth of some cancer cells. In contrast, Linezolid (LZD) suppressed the proliferation of MCF-7, PC-3, and NOZ C-1 cells. Therefore, we found a drug that affects the growth of cancer cells among antibacterial agents. Next, when we examined the effects of the combined use of existing anticancer and antibacterial agents, we found VAN did not affect the growth suppression by anticancer agents. However, TEIC and DAP attenuated the growth suppression of anticancer agents. In contrast, LZD additively enhanced the growth suppression by Docetaxel in PC-3 cells. Furthermore, we showed that LZD inhibits cancer cell growth by mechanisms that involve phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway suppression. Therefore, LZD might simultaneously treat cancer and infectious diseases.

Increased Vancomycin Clearance in Patients with Solid Malignancies.

Vancomycin (VAN) is an anti-microbial agent used to treat a number of bacterial infections, which has a high incidence of nephrotoxicity. We examined the pharmacokinetics of VAN retrospectively based on trough concentrations at large scale and identified pharmacokinetic differences between Japanese patients having solid malignancy and non-malignancy patients. Data were analyzed from 162 solid malignancy patients and 261 non-malignancy patients, including the patient's background, VAN dose, and pharmacokinetics of VAN. We failed to detect differences in values for VAN clearance or shorter elimination half-lives between these two groups. In contrast, multiple regression analysis under adjusting for confounding factors by propensity score, showed that VAN clearance significantly increased in relation to solid malignancies in each stage. We conclude that VAN clearance in solid malignancy patients is increased and that the blood concentration of VAN becomes lower than expected. These results suggest that early monitoring of VAN levels in solid malignancy patients might be essential for maintaining desired effects without side-effects.

Effects of ß-carotene on oxazolone-induced atopic dermatitis in hairless mice.

Skin acts as a barrier, which protects internal tissues and promotes moisture retention. Atopic dermatitis (AD) is an inflammatory skin disease associated with a variety of genetic and environmental factors that involve helper T cells. ß-Carotene (provitamin A) exhibits antioxidant activity and activates the immune system. However, it is not clear whether inflammation in AD skin is improved by posttreatment with ß-carotene. In the current study, we investigated the effects of ß-carotene on the skin of hairless mice with oxazolone-induced inflammation/oedema (Ox-AD mice). We found that skin inflammation was significantly reduced by oral administration of ß-carotene. In addition, treatment with ß-carotene suppressed protein levels of TNF-α, IL-1ß and MCP-1, as well as mRNA expression associated with IL-1ß, IL-6, IL-4 and Par-2 in skin tissues. Furthermore, the mRNA and protein levels of filaggrin, a structural protein in the epidermal stratum corneum, were elevated by ß-carotene administration as compared with Ox-AD mice. ß-Carotene significantly reduced the activity of proMMP-9, but not proMMP-2. These results suggest that in Ox-AD mice, ß-carotene improves skin inflammation by suppressing the expression of inflammatory factors, promoting filaggrin expression and reducing MMP-9 activity. ß-Carotene is a potent anti-inflammatory agent that improves the barrier functions of AD skin.

Growth Inhibition of Human Breast and Prostate Cancer Cells by Cinnamic Acid Derivatives and Their Mechanism of Action.

Cancer is the leading cause of death and there is a particularly pressing need to develop effective treatments for breast and prostate cancer. In the current study, we show the inhibitory effects of cinnamic acid derivatives, including caffeic acid phenethyl ester (CAPE, 1), on the growth of breast and prostate cancer cells. Among the compounds examined, 3,4,5-trihydroxycinnamic acid decyl ester (6) showed the most potent inhibition of cancer cell growth by the induction of apoptosis. Compound 6 could be a new anti-cancer agent for use against breast and prostate cancer.

Augmented Renal Clearance of Vancomycin in Hematologic Malignancy Patients.

The pharmacokinetics of vancomycin (VAN) was retrospectively examined based on trough concentrations at large scale to identify pharmacokinetic differences between Japanese hematologic malignancy and non-malignancy patients. Data from 261 hematologic malignancy patients and 261 non-malignancy patients, including the patient's background, VAN dose, and pharmacokinetics of VAN estimated by an empirical Bayesian method, were collected and analyzed. Our results showed significantly higher values for VAN clearance and shorter elimination half-lives in patients with hematologic malignancies than non-malignancy patients. In addition, multiple regression analysis under adjusting for confounding factors by propensity score, showed that VAN clearance significantly increased in relation to hematologic malignancies. In conclusion, since in hematologic cancer patients VAN clearance is increased, the blood concentration of VAN becomes lower than expected and this may contribute to the survival of resistant bacteria when VAN is administered at low doses. These results suggest that early monitoring of VAN levels in hematologic cancer patients might be recommended to maintain desired effects without side-effects.

Transcriptional regulation of acetoacetyl-CoA synthetase by Sp1 in neuroblastoma cells.

Acetoacetyl-CoA synthetase (AACS) is the enzyme responsible for cholesterol and fatty acid synthesis in the cytosol. We have previously shown that AACS has an important role in normal neuronal development and that knockdown of SREBP-2, which orchestrates cholesterol synthesis, resulted in the downregulation of AACS mRNA levels. In this study, we investigated the transcriptional mechanism of AACS in Neuro-2a, neuroblastoma cells. Luciferase assay showed that the minimal core promoter of the mouse AACS gene is located in a region with 110 bps upstream from the transcription start site. Mutagenesis studies showed that the Sp1 binding site was crucial for AACS promoter activity. ChIP assay and DNA affinity precipitation assay showed that Sp1 binds to the Sp1 binding site on the promoter region of AACS. Moreover, overexpression of Sp1 increased AACS mRNA levels. Knockdown of AACS resulted in a decrease in histone deacetylase 9, associated with gene silencing. These results suggest that Sp1 regulates gene expression of AACS in Neuro-2a cells and ketone body utilization affects the balance of histone acetylation.

Retinoylation (covalent modification by retinoic acid) of Rho-GDIß in the human myeloid leukemia cell line HL60 and its functional significance.

Retinoic acid (RA) has a variety of biological effects in mammalian cells and tissues. It is well known that RA induces differentiation of human acute promyelocytic leukemia (APL) HL60 cells, fresh human APL cells, and clinical remission in patients with APL. Retinoylation (acylation of proteins by RA) is a possible pathway for RA action. However, an understanding of the role that retinoylation plays in the actions of RA is lacking. In the current study, several retinoylated proteins were detected in RA-treated HL60 fractions following Mono Q anion exchange chromatography and analysis using two-dimensional polyacrylamide gel electrophoresis. One of the retinoylated proteins was identified as Rho-GDIß (28kDa) by TOF-MS and co-migration with Rho-GDIß (28kDa). Truncated Rho-GDIß (23kDa, N∆19), a product of cleavage by caspase-3, was not retinoylated. RA covalently bound to the Thr2 residue in Rho-GDIß (5kDa), which is the second product resulting from the cleavage of Rho-GDIß (28kDa) by caspase-3. RA treatment increased the level of Rho-GDIß (28kDa) and decreased the level of Rho-GDIß (23kDa). RA did not induce caspase-3 activity or Rho-GDIß mRNA expression. It is likely that retinoylation of Rho-GDIß increases its metabolic stability.

Protein kinase A activation by retinoic acid in the nuclei of HL60 cells.

BACKGROUND: Activation of protein kinase A (PKA) occurs during retinoic acid (RA)-induced granulocytic differentiation of human promyelocytic leukemia HL60 cells. It is known that the RIIα regulatory subunit of PKA, is modified by RA (retinoylated) in the early stages of differentiation. We have investigated the effects of RA on PKA during cell differentiation in order to understand the potential significance of this process in the retinoylation of RIIα subunits. METHODS: Immunoblotting, immunoprecipitation, confocal microscopy, PCR, and PKA activity assays were employed for characterizing the effects of RA on PKA. RESULTS: We found that RA induces intracellular mobility of RIIα and the activation of PKA in HL60 cells. Increases in RIIα levels were observed in RA-treated HL60 cells. RA treatment altered intracellular localization of the PKA subunits, RIIα and Cα, and increased their protein levels in the nuclei as detected by both immunoblotting and immunostaining analyses. Coincident with the increase in nuclear Cα, RA-treated HL60 cells showed increases in both the protein phosphorylation activity of PKA and the levels of phosphorylated proteins in nuclear fractions as compared to control cells. In addition, RIIα protein was stabilized in RA-treated HL60 cells as compared to control cells. CONCLUSIONS: These results suggest that RA stabilizes RIIα protein and activates PKA in the nucleus, with a resultant increase in the phosphorylation of nuclear proteins. GENERAL SIGNIFICANCE: Our evidence suggests that retinoylation of PKA might contribute to its stabilization and activation and that this could potentially participate in RA's ability to induce granulocytic differentiation of HL60 cells.

Anticancer efficacy of p-dodecylaminophenol against high-risk and refractory neuroblastoma cells in vitro and in vivo.

Neuroblastoma is an aggressive and drug-resistant refractory cancer. The human high-risk neuroblastoma cell line, SK-N-AS (non-amplified N-myc) is derived from stromal cells and it is resistant to treatment with retinoic acid (1, RA), which is a chemotherapeutic agent used to induce neuronal cellular differentiation of neuroblastomas. We have developed p-dodecylaminophenol (3, p-DDAP), based on N-(4-hydroxyphenyl)retinamide (2, 4-HPR), a synthetic amide of 1, since 1 and 2 are associated with the side-effect of nyctalopia. In order to evaluate the effects of 3 on high-risk neuroblastomas, we employed SK-N-AS cells as well asa second high-risk human neuroblastoma cell line, IMR-32, which is derived from neuronal cells (amplified N-myc, drug sensitive). Compound 3 suppressed cell growth of SK-N-AS and IMR-32 cells more effectively than 1, 2, p-decylaminophenol (4, p-DAP), N-(4-hydroxyphenyl)dodecananamide (5, 4-HPDD) or N-(4-hydroxyphenyl)decananamide (6, 4-HPD). In SK-N-AS cells, 3 induced G0/G1 arrest and apoptosis to a greater extent than 1 and 2. In IMR-32 cells, 3 induced apoptosis to a similar extent as 1 and 2, potentially by inhibiting N-myc expression. In addition, i.p. administration of 3 suppressed tumor growth in SK-N-AS-implanted mice in vivo. Since 3 showed no effects on blood retinol concentrations, in contrast to reductions following the administration of 2, it exhibited excellent anticancer efficacy against high-risk neuroblastoma SK-N-AS and IMR-32 expressing distinct levels of N-myc. Compound 3 may have potential for clinical use in the treatment of refractory neuroblastoma with reduced side effects.

Effects of Pre- and Post-Administration of Vitamin A on the Growth of Refractory Cancers in Xenograft Mice.

Vitamin A is an essential nutrient that is obtained from the daily diet. The major forms of vitamin A in the body consist of retinol, retinal, retinoic acid (RA), and retinyl esters. Retinal is fundamental for vision and RA is used in clinical therapy of human acute promyelocytic leukemia. The actions of retinol and retinyl palmitate (RP) are not known well. Recently, we found that retinol is a potent anti-proliferative agent against human refractory cancers, including gallbladder cancer, being more effective than RA, while RP was inactive. In the current study, we determined serum retinol concentrations in xenograft mice bearing tumors derived from four refractory cancer cell lines. We also examined the effects of vitamin A on proliferation of human gallbladder cancer cells in vivo. Serum retinol concentrations were significantly lower in xenograft mice with tumors derived from various refractory cancer cell lines as compared with control mice. The growth of tumors was inhibited with increasing serum retinol concentrations obtained post-administration of RP. In addition, pre-administration of RP increased serum retinol concentrations and suppressed tumor growth. These results indicate that administration of RP can maintain retinol concentrations in the body and that this might suppress cancer cell growth and attachment. The regulation of vitamin A concentration in the body, which is critical biomarker of health, could be beneficial for cancer prevention and therapy.

Growth Inhibition of Refractory Human Gallbladder Cancer Cells by Retinol, and Its Mechanism of Action.

Among the constituents of the essential nutrient vitamin A, retinol is a potent suppressor of refractory cancer cell growth linked to tumor progression, showing greater efficacy than retinoic acid (RA). However, the mechanisms of retinol action on human refractory cancer are not known well. In the current study, we examined the actions of retinol on proliferation of human gallbladder cancer NOZ C-1 cells. Retinol and RA inhibited the proliferation of human NOZ C-1 cells in dose-dependent manner, while RA was less potent than retinol. Cell incorporation of RA was approximately two-fold higher than retinol and was not correlated with anti-proliferative activity. Retinol did not affect caspase-3 activity or mRNA expression of Bax and Bcl-2, which are associated with apoptosis. In addition, protein expression of phosphorylated extracellular signal-regulated kinase (p-ERK)/ERK and p-Akt/Akt were not significantly changed by retinol treatment. In contrast, retinol treatment significantly increased the mRNA expression of endoplasmic reticulum (ER) stress factors (heme oxygenase 1 (HMOX1), CCAAT/enhancer-binding protein homologous protein (CHOP), 78 kDa glucose-regulated protein (GRP78), and DnaJ (Hsp40) homolog, subfamily B, member 9 (DNAJB9)). Furthermore, the number of cells in the G0/G1 phase was increased, while the number of cells in the S phase were decreased by retinol treatment. Retinol increased expression of the autophagy-associated protein, LC3-II. These results indicate that retinol is a potent suppressor of gallbladder cancer cell growth by mechanisms that involve ER stress, which results in autophagy and cell cycle delay. This suggests that retinol might be useful for anticancer prevention and therapy in the clinic.

High-fat diet-induced obesity stimulates ketone body utilization in osteoclasts of the mouse bone.

Previous studies have shown that high-fat diet (HFD)-induced obesity increases the acetoacetyl-CoA synthetase (AACS) gene expression in lipogenic tissue. To investigate the effect of obesity on the AACS gene in other tissues, we examined the alteration of AACS mRNA levels in HFD-fed mice. In situ hybridization revealed that AACS was observed in several regions of the embryo, including the backbone region (especially in the somite), and in the epiphysis of the adult femur. AACS mRNA expression in the adult femur was higher in HFD-fed mice than in normal-diet fed mice, but this increase was not observed in high sucrose diet (HSD)-induced obese mice. In addition, HFD-specific increases were observed in the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and interleukin (IL)-6 genes. Moreover, we detected higher AACS mRNA expression in the differentiated osteoclast cells (RAW 264), and found that AACS mRNA expression was significantly up-regulated by IL-6 treatment only in osteoclasts. These results indicate the novel function of the ketone body in bone metabolism. Because the abnormal activation of osteoclasts by IL-6 induces bone resorption, our data suggest that AACS and ketone bodies are important factors in the relationship between obesity and osteoporosis.

Inhibitory Effects of Retinol Are Greater than Retinoic Acid on the Growth and Adhesion of Human Refractory Cancer Cells.

Vitamin A constituents include retinal, which plays a role in vision, and retinoic acid (RA), which has been used in the therapy of human acute promyelocytic leukemia. However, the effects on cancer of retinol (Rol) and its ester, retinyl palmitate (RP) are not known well. In the current study, we examined the effects of these agents on proliferation and adhesion of various cancer cells. Rol exhibited dose-dependent inhibition of the proliferation of human refractory and prostate cancer cells, while RA and RP showed little or no effect. In contrast, RA inhibited the growth of human breast cancer cells to a greater extent than Rol at low concentrations, but not at high concentrations. Rol suppressed adhesion of refractory and prostate cancer cells to a greater extent than RA, while it suppressed adhesion of breast cancer cells as well as RA and of JHP-1 cells less effectively than RA. These results indicate that Rol is a potent suppressor of cancer cell growth and adhesion, which are both linked to metastasis and tumor progression. Rol might be useful for the clinical treatment of cancer.

25-Gb/s broadband silicon modulator with 0.31-V·cm VπL based on forward-biased PIN diodes embedded with passive equalizer.

We investigated the broadband operations of a silicon Mach-Zehnder modulator (MZM) based on a forward-biased-PIN diode. The phase shifter was integrated with a passive-circuit equalizer to compensate for the narrowband characteristics of the diodes, which consists of a simple resistance of doped silicon and a parallel-plate metal capacitance. The device structure was simple and fabricated using standard CMOS processes. The measured results for a 50-Ω driver indicated there was a small VπL of 0.31 V·cm and a flat frequency response for a 3-dB bandwidth (f(3dB)) of 17 GHz, which agree well with the designed values. A 25-Gb/s large-signal operation was obtained using binary signals without pre-emphasis. The modulator showed a linear modulation property to the applied voltage, due to the metal capacitance of the equalizer.

Inhibitory effects of hydroxylated cinnamoyl esters on lipid absorption and accumulation.

Obesity is a risk factor associated with several lifestyle-related diseases, for example, diabetes, high blood pressure, hyperlipidemia and cancer. Caffeic acid 2-phenylethyl ester (CAPE, 1), a naturally-occurring compound found in various plants and propolis, which exhibits anti-inflammatory, immunomodulatory and cytotoxic activities and inhibits 3T3-L1 differentiation to adipocytes. As part of our efforts to moderate lifestyle-related diseases, we synthesized analogs of 1 and studied their effects on pancreatic lipase activities, lipid absorption, and 3T3-L1 differentiation. We found that catechols 1-4 show inhibitory activities against pancreatic lipase in a dose-dependent manner in vitro. Compounds 1-3 proved to be more potent inhibitors of pancreatic lipase than 5, 6, 8, and 9, which have one hydroxyl group, respectively. Compound 7 has three aromatic hydroxyl groups and restrains greater lipase inhibitory activity than the other compounds. In addition, 7 and 3 significantly suppress a rise in blood triglyceride (TG) levels in mice given corn oil orally. Furthermore, 2 and 3 are more potent at preventing 3T3-L1 differentiation (lipid accumulation) than 1, while 7 is more potent than 3, 8, and 9 in these assays. Compounds 2, 3, and 7 inhibit lipid absorption and accumulation, with new compound 7 being the most potent. These results indicate that 7 may have potential benefits as a health agent with anti-obesity properties.

Inhibitory effects of p-alkylaminophenol on melanogenesis.

Melanin protects the skin against ultraviolet (UV) rays. It is produced in excess by UV radiation, which causes skin disorders and pigmentation. Retinoic acid (RA) decreases the levels of epidermal melanin by suppressing the expression of melanogenic enzymes including tyrosinase, which is the rate-limiting enzyme in melanin synthesis. However, RA shows inflammatory effects on the skin. In an effort to develop potent inhibitors of melanin synthesis, new aminophenol derivatives were synthesized based on structure-activity relationship studies of N-(4-hydroxyphenyl)retinamide (1), a derivative of RA. We investigated the inhibitory effects of a series of aminophenols on melanogenesis using B16 melanoma cells. p-Decylaminophenol (3) was the most potent agent examined, showing significant inhibition of B16 tyrosinase activities at concentrations less than what was required to achieve a similar level of inhibition by the well-known tyrosinase inhibitor, kojic acid. Compound 3 decreased melanin content and inhibited protein and mRNA expression for the tyrosinase-related protein-1 (TRP-1). It also inhibited the microphthalmia-associated transcription factor (MITF), a master transcription factor in melanogenesis. Compound 3 suppressed MEK/ERK signal pathways involved in the activation and expression of MITF. The data indicate that 3 inhibits TRP-1 expression by decreasing MITF expression through suppressing MEK/ERK signal pathways. This results in the reduction of melanin in B16 cells. Compound 3 might be an alternative to RA as a potent inhibitor of melanogenesis.

Effect of p-aminophenols on tyrosinase activity.

Tyrosinase is involved in the synthesis of melanin in the skin and hair as well as neuromelanin in the brain. This rate limiting enzyme catalyzes two critical steps (reactions) in melanogenesis; the hydroxylation of tyrosine to form DOPA and the subsequent oxidation of DOPA into dopaquinone. Several new aminophenol derivatives have been synthesized based on structure-activity relationship studies of N-(4-hydroxyphenyl)retinamide (1), a derivative of retinoic acid. In order to find new tyrosinase inhibitors, we investigated the effects of these p-aminophenols, including p-decylaminophenol (3), on the activity of mushroom tyrosinase. Compound 3 was the most potent agent, showing significant inhibition as compared with control. The inhibitory effects of 3 on tyrosinase activities were greater than seen with kojic acid, a well-known potent inhibitor of tyrosinase activity, which also causes adverse effects, including rash and dermatitis. A Lineweaver-Burk kinetic analysis of inhibition showed that 3 suppresses tyrosinase activity in a non-competitive fashion for both substrates, tyrosine and DOPA. These results suggest that 3 might be a useful alternative to kojic acid as a tyrosinase inhibitor.

50-Gb/s ring-resonator-based silicon modulator.

We achieved 50-Gb/s operation of a ring-resonator-based silicon modulator for the first time. The pin-diode phase shifter, which consists of a side-wall-grating waveguide, was loaded into the ring resonator. The forward-biased operation mode was applied, which exhibited a V(π)L as small as 0.28 V · cm at 25 GHz. The driving voltage and optical insertion loss at 50-Gb/s were 1.96 V(pp) and 5.2 dB, respectively.

Inhibitory effects of p-dodecylaminophenol on the invasiveness of human fibrosarcoma cell line HT1080.

Cancer is a major cause of death, and the development of new anticancer drugs is urgently needed. Invasion and metastasis are the primary causes of death due to cancer rather than growth of the primary tumor. In the current study, we examined the anti-invasive effects of p-dodecylaminophenol (1), which was developed based on N-(4-hydroxyphenyl)retinamide (2), a synthetic amide of all-trans-retinoic acid (3). In HT1080 cells 1 inhibited growth, induced apoptosis and arrested the cell cycle in S phase in a dose-dependent manner. In addition, 1 significantly suppressed cell invasion, and the activity and mRNA expression of matrix metalloproteinase-9 (MMP-9). Furthermore, the expression of the reversion-inducing cysteine-rich protein with Kazal motifs (RECK), which is a negative regulator of MMP-9, was increased by treatment with 1. These results suggest that 1 could be an effective anti-cancer agent that suppresses cell growth through apoptosis induction and cell cycle arrest, which also inhibits cell invasion by decreasing MMP-9 expression due to an increase in RECK. Compound 1 might be useful clinically as a new and potent anticancer agent that could overcome adverse side effects of the retinoids.