Syu-ichi Kanno

Inhibitory effect of the alkyl side chain of caffeic acid analogues on lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages

Caffeic acid esters, one of the components of propolis, are known to show a variety of biological effects such as anti-tumor, anti-oxidant, and anti-inflammatory activities. Although, the anti-inflammatory activities of caffeic acid esters have been studied by analyzing their structure, the detailed mechanisms of their activities remain unclear. Thus, in this study, we examined the function of the ester functional group and the alkyl side chain (alcoholic part) and transformed caffeic acid to several derivatives. The inhibitory effect of these derivatives on NO production in murine macrophage RAW264.7 cells was dependent on the length and size of the alkyl moiety, and undecyl caffeate was the most potent inhibitor of NO production. In addition, individual experiments using undecanol, caffeic acid, undecanol plus caffeic acid, and undecyl caffeate showed that the connection between caffeic acid and the alkyl chain is critical for activity. Amide and ketone derivatives showed that not only the ester functional group but also the amide and ketone functional groups exhibit an inhibitory effect on NO production.

Structures and Biological Evaluations of Agelasines Isolated from the Okinawan Marine Sponge Agelas nakamurai.

Three new N-methyladenine-containing diterpenes, 2-oxoagelasines A (1) and F (2) and 10-hydro-9-hydroxyagelasine F (3), were isolated from the Okinawan marine sponge Agelas nakamurai Hoshino together with eight known agelasine derivatives, 2-oxoagelasine B (4), agelasines A (5), B (6), D (7), E (8), F (9), and G (10), and ageline B (11). The structures of 1-3 were assigned on the basis of their spectroscopic data and their comparison with those of the literature. Compounds 3 and 5-11 inhibited the growth of Mycobacterium smegmatis with inhibition zones of 10, 14, 15, 18, 14, 20, 12, and 12 mm at 20 μg/disc, respectively. All compounds were inactive (IC50 > 10 μM) against Huh-7 (hepatoma) and EJ-1 (bladder carcinoma) human cancer cell lines. Three 2-oxo derivatives (1, 2, and 4) exhibited markedly reduced biological activity against M. smegmatis. Moreover, compound 10 inhibited protein tyrosine phosphatase 1B (PTP1B) activity with an IC50 value of 15 μM.

Acacetin induces apoptosis in human T cell leukemia Jurkat cells via activation of a caspase cascade

Flavonoids are naturally occurring antioxidants, with several flavonoids shown to have chemopreventive effects on cancer. We investigated the effects of the flavonoid acacetin on human T cell leukemia Jurkat cells. Acacetin inhibited the proliferation of Jurkat cells by inducing apoptosis in a concentration- and time-dependent manner. Acacetin-induced cell death was characterized by changes in nuclear and cell morphology. Treatment of Jurkat cells with acacetin also induced caspase-3, -8 and -9 activities in a time-dependent manner. Acacetin-induced apoptosis was blocked by a broad-spectrum caspase inhibitor, a caspase-3 inhibitor and a caspase-8 inhibitor, but not by a caspase-9 inhibitor. In addition, acacetin promoted the expression of FAF1, phosphor-FADD, Apaf-1 and cytochrome c. Acacetin-induced apoptosis was also accompanied by upregulation of Bax, and downregulation of Bcl-2. Taken together, these results suggest that acacetin may induce apoptosis in T cell leukemia cells, possibly by activating the Fas-mediated pathway. These findings may help in designing cancer therapeutic and chemopreventive agents.

Albumin modulates docosahexaenoic acid-induced cytotoxicity in human hepatocellular carcinoma cell lines

Fish oil-containing diets rich in cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) provide protection against tumorigenesis. The mechanisms of the cytotoxic effects of DHA include the production of reactive oxygen species (ROS). Albumin has antioxidant property and binds fatty acids, it may protect the cells against the DHA-induced cytotoxicity. In this study, we compared the susceptibility of three human hepatocellular carcinoma (HCC) cell lines (HepG2, Hep3B, Huh7) to the cytotoxic effects of DHA, and examined the changes in the susceptibility following albumin overexpression using transfection vectors or albumin downregulation using small interfering RNA (siRNA). HepG2 cells were the most susceptible to DHA-induced cytotoxicity and increased oxidative activities by DHA compared to Hep3B and Huh7 cells. The cytotoxic effects of DHA were concentration-dependently abrogated by typical antioxidants, a radical scavenger, an iron chelator and incubation with exogenous albumin. Overexpression of albumin in HepG2 cells markedly attenuated DHA-induced oxidative activities and cytotoxicity. Furthermore, knockdown of albumin in both Hep3B and Huh7 cells significantly enhanced the effects of DHA. The results of our in vitro experiments indicate that the cytotoxic effects of DHA on HCC cell lines are modulated by albumin.

Papuamine and haliclonadiamine, obtained from an Indonesian sponge Haliclona sp., inhibited cell proliferation of human cancer cell lines

The extract of an Indonesian marine sponge Haliclona sp. showed potent cytotoxicity against human solid cancer cell lines, MCF-7 (breast), LNCap (prostate), Caco-2 (colon) and HCT-15 (colon) cells. Study on nuclear morphological changes and flow cytometric analysis suggested that the component(s) in the extract would induce an apoptosis to these cancer cells. Bioassay-guided isolation yielded two pentacyclic alkaloids, papuamine (1) and haliclonadiamine (2), which inhibited cell proliferation of six human cancer cell lines with IC50 values of 0.93–1.50 and 1.00–4.44 µM, respectively. Compounds 1 and 2 accumulated lymphoma U937 cells at sub-G1 phase and induced a condensation of chromatin and fragmentation of nucleus.

Absolute structures and bioactivities of euryspongins and eurydiene obtained from the marine sponge Euryspongia sp. collected at Iriomote Island.

Three unique sesquiterpenes, named euryspongins A-C (1-3), have been isolated from the marine sponge Euryspongia sp. The absolute configuration of 1 was assigned as (4R,6R,9S) by comparing its experimental Electronic Circular Dichroism (ECD) spectrum with the calculated ECD spectra of both enantiomers, and the absolute configurations of 2, 3 and artifact 4 were suggested on the basis of that of 1 by assuming common biogenesis of 1-3. These absolute configurations were opposite to those depicted in the previous communication. Further separation of the remaining fractions lead to the isolation of a new C11-polyketide, named as eurydiene (5), together with a known C11-polyketide, nakitriol (6). The structure of 5 was assigned on the basis of its spectroscopic data as a bicyclic alcohol with a diene side chain. Dehydroeuryspongin A (4) inhibited protein tyrosine phosphatase 1B (PTP1B), an important target enzyme for the treatment of type II diabetes and obesity, with an IC50 value of 3.58μM. Moreover, compound 4 did not inhibit the proliferation of human hepatoma Huh-7 cells at 100μM. One of the locations in which PTP1B has been detected is hepatocytes. Compounds 1-3, 5, and 6 were not active against PTP1B. The growth of human colon (HCT-15) and T-cell lymphoma (Jurkat) cells was not disturbed by compounds 1-6.

Papuamine causes autophagy following the reduction of cell survival through mitochondrial damage and JNK activation in MCF-7 human breast cancer cells

We previously reported that extracts of an Indonesian marine sponge Haliclona sp. showed potent cytotoxicity and the induction of apoptosis against human solid cancer cell lines. In this study, we examine the cytotoxic mechanism of the major chemical compound, papuamine, on MCF-7 human breast cancer cells. Papuamine at 5 μM did not show significant cytotoxic effects after incubation for 24 h, but autophagosome vesicular formation was apparent. At 10 μM of papuamine, significant reduction in cell survival was observed at 12 h, and increases in autophagy at this concentration were time-dependent and apparent before the appearance of cytotoxic effects. Both the release of cytochrome c to the cytosol and increase in Bax in the mitochondrial fraction were found to be concentration-dependent. Moreover, mitochondrial membrane potential shows concentration- and time-dependent decreases with exposure to papuamine. The release of cytochrome c has been shown to be accompanied by an increase in JNK activation. 3-Methyladenine (MA), a classical autophagy inhibitor showed increased JNK activation by exposure to papuamine. In conclusion, our results indicate that papuamine causes earlier onset autophagy and delayed reduction of cell survival through mitochondrial damage and JNK activation in MCF-7 cells.

Pifithrin-alpha has a p53-independent cytoprotective effect on docosahexaenoic acid-induced cytotoxicity in human hepatocellular carcinoma HepG2 cells

Pifithrin-alpha (PFT) is an inhibitor of p53 and is known to protect against a variety of p53-mediated genotoxic agents. In this report, we examined the inhibitory effects of PFT against docosahexaenoic acid (DHA)-induced cytotoxicity in the human hepatocellular carcinoma (HCC) cell line HepG2. PFT significantly abrogated DHA-induced cytotoxicity in wild-type HepG2 cells (normal expression of p53) and after p53-knockdown by siRNA, as well as in Hep3B (p53 null) and Huh7 (p53 mutant) cells. DHA-induced cytotoxicity is mediated by induction of oxidative stress, and PFT inhibited this event, but it does not exert antioxidant effects. PFT significantly suppressed the release of cytochrome c from mitochondria to cytosol, as well as changes in the mitochondrial membrane potential (ΔΨM) by DHA. Therefore, protection of mitochondria by PFT is crucial for its inhibition of DHA-induced cytotoxicity. Although it has been reported that PFT is able to block p53 function, our data suggest that PFT also has a p53-independent inhibition mechanism. This work provided insights into the mechanisms of PFT action on DHA-induced cytotoxicity in HCC.

Involvement of p21waf1/cip1 expression in the cytotoxicity of the potent histone deacetylase inhibitor spiruchostatin B towards susceptible NALM-6 human B cell leukemia cells

Spiruchostatin B (SP-B) is a potent histone deacetylase (HDAC) inhibitor that has potential for the chemotherapy of leukemia. The aim of this study was to study the susceptibility of human leukemia cell lines to SP-B. We found that NALM-6 human B cell leukemia cells are the most susceptible to SP-B. There was a low correlation between the expression of HDAC1 mRNA and HDI susceptibility of leukemia cells. NALM-6 has higher endogenous p21waf1/cip1 mRNA expression than other leukemia cells. SP-B-induced cytotoxicity was mediated by induction of histone acetylation via inhibition of HDACs, and this effect of SP-B was associated with apoptosis, which was mediated by caspase activation in NALM-6 cells. SP-B time-dependently increased the size of the sub-G1 (apoptotic) peak, and this effect correlated with SP-B induction of cellular apoptotic features such as changes in nuclear morphology. SP-B significantly increased p21waf1/cip1 expression prior to induction of apoptosis. In conclusion, NALM-6 cells, which have a higher expression of p21waf1/cip1 mRNA than other leukemia cell lines, were susceptible to SP-B-induced cytotoxicity that resulted in induction of apoptosis. Our findings may be useful when establishing a therapeutic strategy based on SP-B.

Lack of a rewarding effect and a locomotor-enhancing effect of the selective μ-opioid receptor agonist amidino-TAPA

Rationale and objectivesPsychological dependence is one of the worst side effects of morphine. It limits the clinical availability of morphine and non-patient morphine users suffer from addiction. An analgesic, which is more potent than morphine but without the liability of psychological dependence, has long been sought in the clinic. We have recently developed a new μ-opioid receptor agonist, Nα-amidino-Tyr-D-Arg-Phe-β-Ala (amidino-TAPA), as a potent analgesic with an antinociceptive profile that is distinct from morphine, including the release of endogenous κ-opioid peptides. The activation of κ-opioid receptors has been suggested to suppress the development of psychological dependence by μ-opioid receptor agonists. In the present study, the psychological dependence liability and the related locomotor-enhancing effect of amidino-TAPA were evaluated.ResultsAmidino-TAPA injected subcutaneously produced an extremely potent and longer lasting antinociception than morphine in ddY mice, prodynorphin-knockout mice, and wild-type C57BL/6J mice. Unlike subcutaneously injected morphine, which had potent locomotor-enhancing and rewarding effects at antinociceptive doses in ddY mice, amidino-TAPA injected subcutaneously did not induce significant locomotor-enhancing and rewarding effects at antinociceptive or even higher doses in ddY mice. In wild-type C57BL/6J mice, amidino-TAPA showed the same pharmacological profile (potent antinociception, lack of locomotor-enhancing and rewarding effects) as in ddY mice. However, amidino-TAPA produced potent locomotor-enhancing and rewarding effects at antinociceptive doses in prodynorphin-knockout mice.ConclusionsThe present results suggest that amidino-TAPA is a potent analgesic without the liability of psychological dependence because it releases endogenous κ-opioid peptides.