PPemd26, an anthraquinone derivative, suppresses angiogenesis via inhibiting VEGFR2 signalling.

BACKGROUND AND PURPOSE: Angiogenesis contributes to coronary heart disease, immune disorders and numerous malignancies. VEGF-A and its receptors (VEGFRs) play a pivotal role in regulating angiogenesis. In an effort to discover more effective inhibitors of tumour angiogenesis, we have analysed the actions of a novel anthraquinone derivative, PPemd26, and explored its anti-angiogenic mechanisms. EXPERIMENTAL APPROACH: The effects of PPemd26 were evaluated in vitro using HUVEC cultures to assess proliferation, migration, invasion and tube formation. Immunoblotting was used to analyse phosphorylation of signalling kinases. Effects in vivo were assayed using Matrigel plug and xenograft mouse models. KEY RESULTS: PPemd26 significantly inhibited VEGF-A-induced proliferation, migration, invasion and tube formation of HUVECs. PPemd26 also attenuated VEGF-A-induced microvessel sprouting from rat aortic rings ex vivo and suppressed formation of new blood vessels in implanted Matrigel plugs in models of angiogenesis in vivo. In addition, PPemd26 inhibited VEGF-A-induced phosphorylation of VEGFR2 and its downstream protein kinases including Akt, focal adhesion kinase, ERK and Src. Furthermore, systemic administration of PPemd26 suppressed the growth of s.c. xenografts of human colon carcinoma in vivo. Histochemical analysis of the xenografts revealed a marked reduction in stainingfor the vascular marker CD31 and proliferation marker Ki-67. CONCLUSIONS AND IMPLICATIONS: This study provides evidence that PPemd26 suppressed tumour angiogenesis through inhibiting VEGFR2 signalling pathways, suggesting that PPemd26 is a potential drug candidate for developing anti-angiogenic agents for the treatment of cancer and angiogenesis-related diseases.

Capsaicin mediates apoptosis in human nasopharyngeal carcinoma NPC-TW 039 cells through mitochondrial depolarization and endoplasmic reticulum stress.

Capsaicin, a pungent compound found in hot chili peppers, has been reported to have antitumor activities in many human cancer cell lines, but the induction of precise apoptosis signaling pathway in human nasopharyngeal carcinoma (NPC) cells is unclear. Here, we investigated the molecular mechanisms of capsaicin-induced apoptosis in human NPC, NPC-TW 039, cells. Effects of capsaicin involved endoplasmic reticulum (ER) stress, caspase-3 activation and mitochondrial depolarization. Capsaicin-induced cytotoxic effects (cell death) through G0/G1 phase arrest and induction of apoptosis of NPC-TW 039 cells in a dose-dependent manner. Capsaicin treatment triggered ER stress by promoting the production of reactive oxygen species (ROS), increasing levels of inositol-requiring 1 enzyme (IRE1), growth arrest and DNA-damage-inducible 153 (GADD153) and glucose-regulated protein 78 (GRP78). Other effects included an increase in cytosolic Ca(2+), loss of the mitochondrial transmembrane potential (ΔΨ(m)), releases of cytochrome c and apoptosis-inducing factor (AIF), and activation of caspase-9 and -3. Furthermore, capsaicin induced increases in the ratio of Bax/Bcl-2 and abundance of apoptosis-related protein levels. These results suggest that ER stress- and mitochondria-mediated cell death is involved in capsaicin-induced apoptosis in NPC-TW 039 cells.

Comprehensive evaluation of a novel nuclear factor-kappaB inhibitor, quinoclamine, by transcriptomic analysis.

BACKGROUND AND PURPOSE: The transcription factor nuclear factor-kappaB (NF-kappaB) has been linked to the cell growth, apoptosis and cell cycle progression. NF-kappaB blockade induces apoptosis of cancer cells. Therefore, NF-kappaB is suggested as a potential therapeutic target for cancer. Here, we have evaluated the anti-cancer potential of a novel NF-kappaB inhibitor, quinoclamine (2-amino-3-chloro-1,4-naphthoquinone). EXPERIMENTAL APPROACH: In a large-scale screening test, we found that quinoclamine was a novel NF-kappaB inhibitor. The global transcriptional profiling of quinoclamine in HepG2 cells was therefore analysed by transcriptomic tools in this study. KEY RESULTS: Quinoclamine suppressed endogenous NF-kappaB activity in HepG2 cells through the inhibition of IkappaB-alpha phosphorylation and p65 translocation. Quinoclamine also inhibited induced NF-kappaB activities in lung and breast cancer cell lines. Quinoclamine-regulated genes interacted with NF-kappaB or its downstream genes by network analysis. Quinoclamine affected the expression levels of genes involved in cell cycle or apoptosis, suggesting that quinoclamine exhibited anti-cancer potential. Furthermore, quinoclamine down-regulated the expressions of UDP glucuronosyltransferase genes involved in phase II drug metabolism, suggesting that quinoclamine might interfere with drug metabolism by slowing down the excretion of drugs. CONCLUSION AND IMPLICATIONS: This study provides a comprehensive evaluation of quinoclamine by transcriptomic analysis. Our findings suggest that quinoclamine is a novel NF-kappaB inhibitor with anti-cancer potential.

Synthesis of 1-benzyl-3-(5'-hydroxymethyl-2'-furyl)indazole analogues as novel antiplatelet agents.

1-Benzyl-3-(5'-hydroxymethyl-2'-furyl)indazole (28, YC-1) was selected as the lead compound for systemic structural modification. After screening for antiplatelet activity, SARs of YC-1 analogues were established. Among these potent active derivatives, compounds 29, 30, 31, 44, and 45 functioned as potent activators of sGC and inhibitors of PDE5 with potency comparable to that of YC-1. In addition, compound 58 was found to be a selective and potent inhibitor of protease-activated receptor type 4 (PAR4)-dependent platelet activation.

Synthesis of 4-alkoxy-2-phenylquinoline derivatives as potent antiplatelet agents.

In our continuing search for novel antiplatelet agents, 4-alkoxy derivatives of 2-phenylquinoline as well as related compounds were prepared. Through biological screening, a preliminary structure antiplatelet activity relationship was established. Compounds 5-ethyl-4-methoxy-2-phenylquinoline (8), 4-ethoxy-5-ethyl-2-phenylquinoline (9), 4-ethoxycarbonylmethoxy-5-ethyl-2-phenylquinoline (10), 4-ethoxycarbonylbutoxy-5-ethyl-2-phenylquinoline (12) and 5-ethyl-4-(N-ethylcarboxido)methoxy-2-phenylquinoline (17) all demonstrated potent antiplatelet activity. Among them, compound 8 was the most potent with an IC50 value of 0.08 microM and was about 3-fold more active than indomethacin. The mechanism of antiplatelet action of 8 is possibly through its inhibition on cyclooxygenase or thromboxane synthetase.

Vector competence of Culex pipiens molestus (Diptera: Culicidae) from Taiwan for a sympatric strain of Japanese encephalitis virus.

Laboratory strains of Culex pipiens molestus Forskal and Culex tritaeniorhynchus Giles from northern Taiwan were compared for their susceptibility to the Sanhsia MQ1-2 (SH) strain of Japanese encephalitis (JE) virus isolated from Taiwan. After feeding on a sweetened blood-virus mixture, viral titers in Cx. p. molestus during the 14-d incubation period ranged from a minimum of 2.9 log10PFU (plaque forming units) per mosquito on day 3 after ingestion to a maximum of 4.65 log10PFU at day 8 and in Cx. tritaeniorhynchus from 2.6 on day 10-5.18 log10PFU per mosquito on day 13. Although virus titer in Cx. p. molestus was lower than in Cx. tritaeniorhynchus at the end of the experiment, this difference was not statistically significant. The median infective dose (ID50) for Cx. p. molestus was 2.83 log10PFU and for Cx. tritaeniorhynchus was 1.02 log10PFU per mosquito, and this difference also was not significant. There also was no significant difference between the median infective dose for transmission (TID50) per mosquito for Cx. p. molestus (5.34 log10PFU) and Cx. tritaeniorhynchus (4.59 log10PFU). We concluded that Cx. p. molestus is an effective laboratory vector of JE virus.

Hantavirus infection in Taiwan: the experience of a geographically unique area.

Hantaviruses are rodent-borne viruses, and they, mainly the Hantaan (HTN) serotype, are the causative agents of a group of febrile nephropathies known as "hemorrhagic fever with renal syndrome (HFRS). " Despite the fact that HFRS is frequently reported in China, with an annual incidence of 50,000-100,000 cases, one puzzling observation that no local case of HFRS has been confirmed in Taiwan has yet to be explained. We hypothesized that the hantavirus strain prevailing in Taiwan mainly belongs to the mild strain, the Seoul (SEO) strain, and the absence of severe disease was related to the absence of HTN. To test these hypotheses, this epidemiologic study was performed, including a seroprevalence survey and phylogenetic analysis on hantavirus isolated from the rodent population trapped in major seaports, rural, and mountainous areas of Taiwan. This study also included rodents and viruses from two isolated islands, Kinmen and Matzu, which are geographically adjacent to the east coast of mainland China. There were a total of 5,461 rodents of 16 species captured, and R. norvegicus was the most common species, with an antibody prevalence much higher in international seaports (20%) than in rural regions (approximately 5%) and intermediate in some domestic seaports. By reverse transcriptase polymerase chain reaction (RT-PCR), 33.9% of the seropositive R. norvegicus were found to have amplifiable hantavirus sequences in their lung tissues, and subsequent phylogenetic analyses indicated that almost all hantavirus in Taiwan was most closely related to the prototype SEO strain, and no HTN strain was recovered from any rodent species indigenous to Taiwan. The seroprevalence of SEO infection in R. norvegicus on Kinmen and Matzu was also different from that in southern provinces of China but closely resembled that in seaports in Taiwan, and the SEO identified was genetically linked to Taiwanese SEO strains. These results substantiate our hypotheses, and suggest that the epidemiology of hantavirus infection in Taiwan are different from that in China, where the HTN and SEO strains and HFRS concurrently prevail.

Isolation of Japanese encephalitis virus from mosquitoes collected in Northern Taiwan between 1995 and 1996.

Mosquito collections were carried out from May to October in 1995 and 1996 at Yingko and Sanhsia of Taipei County and Chunan of Miaoli County. A grand total of 13,576 mosquitoes consisting of 13 species in 407 pools were processed and inoculated into Aedes albopictus clone C6/36 cell cultures. One hundred thirty seven pools of these showed the presence of viral antigens in the infected C6/36 cell lysates which were identified by the indirect fluorescent antibody test using a monoclonal antibody against Japanese encephalitis (JE) virus. The postive pools, were divided into 97, 20, 1, 8, 1, 1, 7, and 2 pools from Culex tritaeniorhynchus Giles, Aedes albopictus Skuse, Aedes vexans nocturnus (Theobald), Armigeres subalbatus (Coquillett), Culex annulus Theobald, Culex fuscanus Wiedemann, Culex quinquefasciatus Say, and Culex sitiens Wiedemann respectively. The dominant species collected at night was Culex tritaeniorhynchus, while Aedes albopictus was the dominant species collected in daytime. Besides the pigpens, avian habitats are also a dominate source of JE virus in this study.

Susceptibility of three laboratory strains of Aedes albopictus (Diptera: Culicidae) to Japanese encephalitis virus from Taiwan.

The susceptibility of 3 laboratory strains of Aedes albopictus (Skuse) (Sanhsia [SH], Yungho [YH], Liyang [LY], and 1 strain of Culex tritaeniorhynchus Giles from northern and central Taiwan were compared for susceptibility to the MQ1-2 strain of Japanese encephalitis (JE) virus. The median infective dose (MID50) by intrathoracic inoculation was 0.23, 0.76, 1.60, and -0.03 log10 WMICLD50 (50% weanling mice intracranial lethal dose) with Ae. albopictus SH, YH, LY, and Cx. tritaeniorhynchus, respectively. After feeding on a sweetened blood-virus mixture, the oral MID50 was 2.03, 4.32, and 4.98 log10 WMICLD50 for SH, YH, and LY, respectively, and 1.02 log10 WMICLD50 for Cx. tritaeniorhynchus. The SH Ae. albopictus strain transmitted virus to normal mice after 14 d. with an average transmission rate of 45%. Based on these results, the SH strain was the most susceptible and important potential vector among 3 Ae. albopictus strains for the sympatric MQ1-2 strain of JE.

Synthesis and antiplatelet, antiinflammatory, and antiallergic activities of 2-substituted 3-chloro-1,4-naphthoquinone derivatives.

A series of 2-substituted 3-chloro-1,4-naphthoquinones was synthesized, and the antiplatelet, antiinflammatory, and antiallergic activities of these compounds were evaluated. The structure-activity relationships in this series were also examined. Most of the 2-alkyl/arylcarboxamido derivatives of 3-chloro-1,4-naphthoquinone showed potent activities with similar trends in each of the activities evaluated.

Seroepidemiology of Hantaan virus infection in Taiwan.

In order to investigate the infection rate of Hantaan virus in Taiwan, a total of 6,536 human serum samples were collected from residents, selected by stratified random sampling, from 19 townships covering four different ethnic groups: Aborigines, Fukien Taiwanese, Hakka Taiwanese, and Mainland Chinese. Serum samples were screened for Hantaan virus antibodies by indirect immunofluorescence. The prototype Hantaan virus (76/118)-infected Vero E6 cells were used as the viral antigen for the antibody detection. Among 6,536 human serum samples, 403 (6.2%) samples had Hantaan virus antibodies. The seropositive rates for males and females were 6.1% and 6.2%, respectively. A higher seropositive rate was found among Aborigines on the Orchid Islets (11.5%) and Fukien Taiwanese on the Penghu Islets (11.6%), while the lowest rate was observed among Hakka Taiwanese in the south of Taiwan (2.5%). In comparing with different ethnic groups, the highest prevalence was found among Fukien Taiwanese (8.1%) and the lowest among Mainland Chinese (4.9%). Among the different geographical areas, the highest positive rate was found in western Taiwan (7.1%) and the lowest in southern Taiwan (5.4%). Hantaan virus antibodies were also detected in 22 of 548 (4.0%) rat serum samples. The highest seropositive rate was found in rat sera collected from the Orchid Islets (21.4%). None of the rat sera collected from Hsinchu, Miaoli, Changhua, Nantu, Yunlin, Chiayi, Tainan, and Penghu Counties were positive. Hantaan virus antibodies were found in rats: Rattus rattus (20%), Bandicota indica (9.0%), Rattus norvegicus (8.3%), Bandicota nemorivaga (6.3%), Rattus losea (4.2%), and Apodemus agrarius (1.6%). Hantaan virus antibodies were not detected in rat sera collected from species of Rattus coxinga, Rattus culturatus, Mus musculus, Mus caroli, Suncus murinus, and Apodemus semotus. The results show that the Hantaan or Hantaan-related virus exists and is distributed widely in both human and rats in Taiwan.

Synthesis and antiplatelet, antiinflammatory and antiallergic activities of 2,3-disubstituted 1,4-naphthoquinones.

Modification of 2-acetamido-3-chloro-1,4-naphthoquinone, which has potent antiplatelet, antiallergic and antiinflammatory activities, led to a series of 2,3-disubstituted 1,4-naphthoquinones. Some of these compounds showed significant antiplatelet, antiallergic and antiinflammatory activities. Among them, 2-methoxy-3-chloro-1,4-naphthoquinone (15) and 2-ethoxy-3-chloro-1,4-naphthoquinone (17) exhibited potent inhibitory effects on neutrophil and mast cell degranulation. 2-Methoxy-1,4-naphthoquinone (20) and 2-ethoxy-1,4-naphthoquinone (21) exhibited potent inhibitory effect on neutrophil superoxide formation. These four compounds were thus selected for further evaluation.

Synthesis and cytotoxicity of 1,2-disubstituted naphth[2,3-d]imidazole-4,9-diones and related compounds.

As part of our continuing search for potential anticancer drug candidates that are selective against slowly growing solid tumors, we have synthesized several series of 1- and 2-substituted derivatives of the lead structure, 1-ethyl-2-methylnaphth[2,3-d]imidazole-4,9-dione (5). Their cytotoxic activity in the National Cancer Institute's in vitro cancer cell line panel is reported. In general, substitution of various alkyl, phenyl, or benzyl moieties did not improve activity, and compound 5 remains the most active naphth[2,3-d]imidazole-4,9-dione derivative. However, high levels of activity and selectivity were found with several related 2-(acylamino)-3-chloro-1,4-naphthoquinones (2f-j). Compound 2i, 2-[(2-fluorophenyl)acetamido]-3-chloro-1,4-naphthoquinone, has been selected for further in vivo testing and as an additional lead compound for further structural modification.

[Studies on the susceptibility of two northern Taiwan strains of Aedes albopictus mosquitoes to Japanese encephalitis virus].

Aedes albopictus is a dominant mosquito species in northern Taiwan. In laboratory, the vector competence of 2 geographical strains of Aedes albopictus mosquitoes to NT 113 strain of Japanese encephalitis virus was examined. The mosquito infection dose50 (MI-D50) of Sanhsia (SH) strain by intrathoracic (i.t.) inoculation was shown to be -1.1 log WMICLD50 (weanling mice intracerebrum lethal dose), while that of Yungho (YH) strain was -2.0 log WMICLD50. The infection dose for 50% mosquitoes transmission (MTID50) by i.t. inoculation was 3.5 log with SH strain but no transmission occurred with YH strain. By feeding sweetened blood-virus mixture, the MID50 with SH strain was 2.7 log though YH strain did not attain 50% infection rate. By viremic mouse blood feeding, the highest infection rate for both strains was about 30%. No evidence of virus transmission was demonstrated by oral infection.

Anopheline mosquitoes and malaria parasites in Taiwan.

Extensive taxonomic studies of the genus Anopheles were carried out in Taiwan during the first half of the 20th century. As a result, 17 species of Anopheles were identified and reported as occurring in Taiwan. However, the occurrence of two species, An. kochi and An. fluviatilis, in Taiwan is doubtful. Vector competence of the anophelines of Taiwan was also studied both in the laboratory and in the field during the same period of time, and experimental infections of 8 species of Anopheles were carried out by Anazawa in the 1920s. The results showed that all species tested were susceptible to three human plasmodia except for An. sinensis which was refractory to Plasmodium falciparum. The results of dissection of nearly 10,000 anopheline mosquitoes collected from the field by various workers suggested that An. minimus is the chief vector in Taiwan. Crithidial flagellates resembling malaria sporozoites were found in the salivary glands of 4 common species of Anopheles collected from cow-stables. These flagellates may have been misidentified as malaria sporozoites in the earlier investigations. During the first part of the operations for malaria control and eradication, anopheline mosquitoes were collected by day from 1,118 houses scattered over the island. Of the 25,656 specimens of Anopheles collected, nearly 80% were An. minimus, mostly from bedrooms. Since the highly anthropophilic and endophilic An. minimus was determined to be the chief vector of malaria in Taiwan, DDT was applied to the wall surfaces in houses for malaria control and eradication. Before spraying DDT, the rate of P. malariae infections was very high in areas where malaria is endemic. However, these infections were more quickly suppressed by DDT spraying than two other parasite species. Other details of studies on the ecology and behavior of anophelines are also presented.

[The pathogens of Taiwan mosquitoes--Coelomomyces species].

A brief mention was made of the history of world studies on the Coelomomyces fungi, the life cycle of the fungi, their importance as biological agents for mosquito control, and the known ecological information concerning the Taiwan mosquitoes parasitized by the fungi. Special accounts were made of the results of experiments infecting four mosquito species, Aedes aegypti, Ae. albopictus, Ae. triseriatus and Tripteroides aranoides with Coelomomyces stegomyiae var. chapmani, using the copepod, Phyllognathopus viguieri as an alternate host. The dead 4th instar larvae of Ae. albopictus containing sporangia were put together with healthy copepods for 10 days, then healthy 2nd instar mosquito larvae were added for a 1-day exposure. Then the larvae were transferred to clean water and subsequent examinations for signs of infection were made at 3-day intervals for a period of 21 days. The experiments demonstrated an infection rate of 59.7% and 90% respectively for Ae. albopictus and Ae. aegypti in the first trial, 18.5% and 23.3% in the 2nd trial, and none in the 3rd trial. The low infection rate in the 2nd trial and no infection in the 3rd trial were thought to be due to the extensive contamination of test water with algae. Therefore, in the 4th trial the rearing water was renewed with dechlorinated tap water and new copepods instroduced. The infection rate again rose to 41.1% and 56% respectively for the two species. Experiments with Ae. triseriatus and Tp. aranoides failed to produce signs of infection. Experimental infection of susceptible Ae. albopictus larvae with the sporangia stored at 8 degrees C showed that storage for one month produced an infection rate of 38.2%. However, storage for two months or longer produced no infection in the larvae of the same mosquito species.

A longitudinal study of the prevalence of Japanese encephalitis virus in adult and larval Culex tritaeniorhynchus mosquitoes in northern Taiwan.

Adult females and larvae of Culex tritaeniorhynchus were collected year-round for 3 1/2 years at a site near Taipei, Taiwan. One hundred sixty-four isolates of Japanese encephalitis (JE) virus were obtained from approximately 142,000 adult females and 1 isolate of the virus was obtained from approximately 382,000 larvae. Virus was recovered from adult females every year, except the first, beginning in May. The single larval isolate was from specimens collected in June. The vertical transmission of JE virus in Cx. tritaeniorhynchus as a possible inter-epidemic viral survival mechanism is examined.

Experimental vertical transmission of Japanese encephalitis virus by Culex tritaeniorhynchus and other mosquitoes.

Vertical transmission of Japanese encephalitis virus to the F1 adult stage was demonstrated in Culex tritaeniorhynchus, Cx. annulus, Cx. quinquefasciatus, and Armigeres subalbatus. Transmission to the F1 larval stage was demonstrated in Cx. pipiens, Aedes vexans, Ae. alcasidi, and A. flavus. In Cx. tritaeniorhynchus, vertical transmission rates (the percentage of parent females transmitting to progeny) varied (12-100%). Filial infection rates (the percentage of progeny infected) for a given mosquito virus combination were markedly affected by the interval of time between parental infection and oviposition, suggesting that vertical infection was not transovarial in nature but occurred at oviposition. Filial infection rates for Cx. tritaeniorhynchus also varied widely by family and, as measured in F1 larvae, rates in excess of 20% were observed in a family. Filial infection rates in Cx. tritaeniorhynchus F1 adults were about 4 times lower than those in larvae. Japanese encephalitis virus was sexually transmitted from male to female Cx. tritaeniorhynchus.

Introduced leishmaniasis on Taiwan.

Leishmaniasis is not known to be indigenous to Taiwan but a number of imported cases of visceral as well as post-kala-azar dermal leishmaniasis have been seen. Only two autochthonous cases of cutaneous-subcutaneous diseases have been documented in aborigines but no cases of visceral leishmaniasis have been reported. Although a significant number of imported cases of leishmaniasis have been seen, the disease has apparently not been established on the island.

Comparative susceptibility of mosquito species and strains to oral and parenteral infection with dengue and Japanese encephalitis viruses.

Thirty-four strains of Asian and Pacific mosquitoes belonging to 22 species of 7 genera were compared for oral and/or parenteral susceptibility to infection with 1 or more strains of each of the 4 dengue serotypes. Surprisingly, several species of common man-biting Aedes were much more susceptible to oral infection with each of the 4 dengue serotypes than was Aedes aegypti. These species included Aedes albopictus and members of the scutellaris group of the subgenus Stegomyia found on South Pacific islands. Mosquito strains and species relatively susceptible to 1 dengue serotype usually were relatively susceptible to the others also. Almost all species of Aedes tested were uniformly susceptible to parenteral infection with the dengue viruses but, with the exception of a species of Tripteroides, species of all other genera were comparatively resistant to that mode of infection. Dengue viruses usually replicated to about the same extent in orally-infected mosquitoes as they did in parenterally-infected specimens of the same species. Seventeen species of mosquitoes of 7 genera also were tested for parenteral susceptibility to infection with Japanese encephalitis virus. With the possible exception of 2 species of Anopheles, the virus replicated to about the same degree in all species tested and achieved levels considerably higher than did any of the dengue viruses in the same mosquito strain and species held under the same conditions.