Application of project-based service-learning courses in medical education: trials of curriculum designs during the pandemic.

BACKGROUND: Due to COVID-19, face-to-face service activities in service-learning courses have become unfeasible. To address this challenge, this study aims to integrate project-based learning into medical education's service-learning curriculum. This study also seeks to evaluate the effectiveness of this instructional approach and identify factors that influence its success. METHODS: A total of 135 first-year medical students enrolled in a mandatory 1-credit service-learning course were recruited. The course involved various service activities aligned with the needs of the local community. The students were organized into 12 groups, each working on different service-learning projects, such as raising health awareness or educating the public about specific diseases. Following the completion of the course, a questionnaire was distributed among the students to gather feedback on the course, and 122 (valid responses were collected, representing a response rate of 90.3%). RESULTS: The results indicated that the project-based service-learning course significantly improved students' "interpersonal communication skills," their ability to "learn and grow from work," and their sense of "professionalism" (all p ≤ 0.037). Among the various aspects of service learning, the highest agreement was observed for "executing the project," followed by "group discussions and project formulation," "overall course review," "review of project outcomes," "outcome presentations," "teaching proposal writing and project brainstorming," "sharing of service-learning experiences by teachers," and "sharing of service-learning experiences by teaching assistants." Students also found certain factors to be beneficial in enhancing the learning effectiveness of service-learning courses, including "prize money for service-learning outcomes," "funding for service-learning activities," and "consultations from medical personnel" (all p ≤ 0.01). However, "course credit" and "photography software" did not show significant effects (both p > 0.05). The most preferred resources or activities for future service-learning courses were "course credit" and "face-to-face service-learning activities." CONCLUSIONS: The project-based learning method improved the learning effectiveness in service-learning courses. Students perceived that the number of course credits reflects the course value and plays a pivotal role in enhancing the learning effectiveness in service-learning courses. During the epidemic, students still expect to have face-to-face service activities in service-learning courses. Therefore, without the impact of the epidemic, service learning courses should return to face-to-face service activities.

2-Phenyl-4-quinolone (YT-1) induces G2/M phase arrest and an intrinsic apoptotic mechanism in human leukemia cells.

The present study aimed to investigate the biological effects of the new compound 2­phenyl­4­quinolone (YT­1) on human leukemia cells. Cell viability was determined by propidium iodide (PI) exclusion method followed by flow cytometry. Our results showed that YT­1 inhibited the cell viability and resulted in morphologic changes to the U937, HL­60 and K562 cells, respectively. Among them, U937 cells were the most sensitive cell line. On the contrary, YT­1 had no cytotoxic effects on human fetal skin fibroblast WS1 cells. Flow cytometric analysis indicated that YT­1 induced G2/M phase arrest and apoptosis (sub­G1 population) in U937 cells. The presence of apoptotic bodies evidenced by DAPI staining and DNA fragmentation detected by agarose gel electrophoresis further supported the induction of apoptosis in the YT­1­treated U937 cells. Annexin V/PI staining of U937 cells confirmed that the early apoptotic event occurred after YT­1 exposure. YT­1 disrupted the mitochondrial membrane potential (ΔΨm) in a time­dependent manner. YT­1 increased the protein levels of Bax and Bak but decreased Bcl­2 and Bid protein levels in U937 cells in a time­dependent manner. In addition, YT­1 stimulated the expression of cytochrome c and proteolytic activation of caspase­3 and caspase­9 after exposure to YT­1 in U937 cells. In summary, YT­1 suppressed the viability of U937 leukemia cells through the intrinsic apoptosis pathway. YT­1 is a potential chemotherapeutic candidate for the treatment of leukemia.

ITR­284 modulates cell differentiation in human chronic myelogenous leukemia K562 cells.

ITR­284 is a carboxamide analog that can inhibit proliferation in human promyelocytic leukemia HL-60 cells. To understand the effects and molecular mechanisms of ITR­284 in human erythromyeloblastoid leukemia, we treated K562 cells with different concentrations of ITR­284 (0, 2, 4, 6, 8 and 10 nM) and all-trans retinoic acid (ATRA) (0, 0.1, 0.5, 1, 5 and 10 µM) for 24 h. The IC50 of ITR­284 was ~10 nM in K562 cells treated for 24 h as determined by MTT assay. May-Grünwald-Giemsa staining and nitro blue tetrazolium (NBT) assays were used to determine cell morphology changes and differentiation after ITR­284 and ATRA treatment. In addition, mRNA expression levels of hematopoietic factors, including GATA­1, NF-E2 and GATA­2, were elevated, while expression levels of BCR­ABL were downregulated in K562 cells after 24 h of treatment with ITR­284 as determined by quantitative reverse transcription polymerase chain reaction. In addition, western blot analyses showed that FOXM1, GLI 1 and c-MYC protein levels were decreased by ITR­284. Taken together, our data show that ITR­284 induced K562 cell differentiation, which led to decreased tumorigenesis. Our findings suggest that ITR­284 could be a potential candidate for treating chronic myelogenous leukemia.

YC-1 induces G0/G1 phase arrest and mitochondria-dependent apoptosis in cisplatin-resistant human oral cancer CAR cells.

Oral cancer is a serious and fatal disease. Cisplatin is the first line of chemotherapeutic agent for oral cancer therapy. However, the development of drug resistance and severe side effects cause tremendous problems clinically. In this study, we investigated the pharmacologic mechanisms of YC-1 on cisplatin-resistant human oral cancer cell line, CAR. Our results indicated that YC-1 induced a concentration-dependent and time-dependent decrease in viability of CAR cells analyzed by MTT assay. Real-time image analysis of CAR cells by IncuCyte™ Kinetic Live Cell Imaging System demonstrated that YC-1 inhibited cell proliferation and reduced cell confluence in a time-dependent manner. Results from flow cytometric analysis revealed that YC-1 promoted G0/G1 phase arrest and provoked apoptosis in CAR cells. The effects of cell cycle arrest by YC-1 were further supported by up-regulation of p21 and down-regulation of cyclin A, D, E and CDK2 protein levels. TUNEL staining showed that YC-1 caused DNA fragmentation, a late stage feature of apoptosis. In addition, YC-1 increased the activities of caspase-9 and caspase-3, disrupted the mitochondrial membrane potential (AYm) and stimulated ROS production in CAR cells. The protein levels of cytochrome c, Bax and Bak were elevated while Bcl-2 protein expression was attenuated in YC-1-treated CAR cells. In summary, YC-1 suppressed the viability of cisplatin-resistant CAR cells through inhibiting cell proliferation, arresting cell cycle at G0/G1 phase and triggering mitochondria-mediated apoptosis. Our results provide evidences to support the potentially therapeutic application of YC-1 on fighting against drug resistant oral cancer in the future.

Cortactin is a prognostic marker for oral squamous cell carcinoma and its overexpression is involved in oral carcinogenesis.

EMS1 (chromosome eleven, band q13, mammary tumor and squamous cell carcinoma-associated gene 1) gene amplification and the concomitant cortactin overexpression have been reported to associate with poor prognosis and tumor metastasis. In this study, we examined cortactin expression by immunohistochemistry in human oral tumors and murine tongue tumors which were induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO). The immunostaining results show over- to moderate expression of cortactin in 85% (104/122) of oral squamous cell carcinoma (OSCC) tissues and in all 15 leukoplakia tissues examined. Further, statistical analysis indicates that cortactin overexpression appears to be a predictor for shorter survival and poorer prognosis in OSCC patients. In an animal model, cortactin is shown to upregulate in infiltrating squamous cell carcinoma, papilloma, and epithelia with squamous hyperplasia, indicating that cortactin induction is an early event during oral carcinogenesis. It is suggested that cortactin expression is mediated in the progression of pre-malignancy to papilloma, based on earlier cortactin induction in pre-malignancy preceding cyclin D1 in papilloma. In conclusion, cortactin overexpression is frequently observed in human OSCC and mouse tongue tumors. Thus, cortactin may have an important role in the development of oral tumors in human and mice. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 799-812, 2017.

CCY-1a-E2 induces G2/M phase arrest and apoptotic cell death in HL-60 leukemia cells through cyclin-dependent kinase 1 signaling and the mitochondria-dependent caspase pathway.

Our previous study demonstrated that 2-[(3-methoxybenzyl)oxy]benzaldehyde (CCY-1a-E2) is a potent compound that acts against multiple human leukemia cell lines. CCY-1a-E2 was also shown to have efficacious anti­leukemic activity in vivo. However, the molecular mechanism of action of CCY­1a­E2 attributed to its anticancer effect remains poorly understood. In the present study, CCY­1a­E2 suppressed cell viability in multiple leukemia cell lines (HL­60, K562, KG­1 and KG­1a) via inhibition of cell proliferation, cell cycle arrest and induction of apoptosis. CCY­1a­E2 exhibited a marked toxic effect on HL­60 cells and displayed low cytotoxicity in normal human peripheral blood mononuclear cells (PBMCs). Results from flow cytometric analysis indicated that CCY­1a­E2 promoted G2/M phase arrest and promoted apoptosis in the HL­60 cells. CCY­1a­E2 treatment upregulated cyclin B, cyclin­dependent kinase 1 (CDK1), cell division cycle 25C (cdc25C) and p21 protein expression. CCY­1a­E2 caused apoptotic cell death and DNA fragmentation as determined by 4',6­diamidino­2­phenylindole (DAPI) staining and DNA gel electrophoresis. Elevated activities of caspase­8, ­9 and ­3 were observed during CCY­1a­E2­induced cell apoptosis; their specific inhibitors were found to block CCY­1a­E2­induced apoptosis, respectively. Moreover, CCY­1a­E2 time­dependently disrupted the mitochondrial membrane potential (ΔΨm), and it enhanced the protein levels of Fas/CD95, cytochrome c, Bax, cleaved PARP, as well as attenuated Bcl­2 expression in the HL­60 cells. Our results provide direct evidence that supports the future potential therapeutic application of CCY-1a-E2 in leukemia.

Purification of Cu/Zn superoxide dismutase from Piper betle leaf and its characterization in the oral cavity.

The aim of this study was to purify protein(s) from Piper betle leaf for identification and further characterization. A functionally unknown protein was purified to apparent homogeneity with a molecular mass of 15.7 kDa and identified as Cu/Zn superoxide dismutase (SOD). The purified SOD appeared to be monomeric and converted to its dimeric form with increased enzymatic activity in betel nut oral extract. This irreversible conversion was mainly induced by slaked lime, resulting from the increase in pH of the oral cavity. Oral extract from chewing areca nut alone also induced SOD dimerization due to the presence of arginine. The enhanced activity of the SOD dimer was responsible for the continuous production of hydrogen peroxide in the oral cavity. Thus, SOD may contribute to oral carcinogenesis through the continuous formation of hydrogen peroxide in the oral cavity, in spite of its protective role against cancer in vivo.

Peroxidase as the major protein constituent in areca nut and identification of its natural substrates.

Numerous reports illustrate the diverse effects of chewing the areca nut, most of which are harmful and have been shown to be associated with oral cancer. Nearly all of the studies are focused on the extract and/or low molecular weight ingredients in the areca nut. The purpose of this report is to identify the major protein component in the areca nut. After ammonium sulfate fractionation, the concentrated areca nut extract is subjected to DEAE-cellulose chromatography. A colored protein is eluted at low NaCl concentration and the apparently homogeneous eluent represents the major protein component compared to the areca nut extract. The colored protein shares partial sequence identity with the royal palm tree peroxidase and its peroxidase activity is confirmed using an established assay. In the study, the natural substrates of areca nut peroxidase are identified as catechin, epicatechin, and procyanidin B1. The two former substrates are similarly oxidized to form a 576 Da product with concomitant removal of four hydrogen atoms. Interestingly, oxidation of procyanidin B1 occurs only in the presence of catechin or epicatechin and an additional product with an 864 Da molecular mass. In addition, procyanidin B1 is identified as a peroxidase substrate for the first time.

Inhibition of formyl peptide-stimulated phospholipase D activation by Fal-002-2 via blockade of the Arf6, RhoA and protein kinase C signaling pathways in rat neutrophils.

Three recently developed selective phospholipase D (PLD) inhibitors N-(2-(4-(2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)ethyl)-2-naphthamide (VU0155056), (S)-N-(1-(4-(5-chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)piperidin-1-yl)propan-2-yl)-2-naphthamide (VU0155069), and N-(2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4,5]decan-8-yl)ethyl)quinoline-3-carboxamide (VU0285655-1) inhibited O2 (•-) generation in formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils. A novel 2-phenyl-4-quinolone compound 6-chloro-2-(2-chlorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (Fal-002-2), which inhibited O2 (•-) generation, also reduced the fMLP- but not phorbol ester-stimulated PLD activity (IC50 16.0 ± 5.0 µM). Fal-002-2 attenuated the interaction of PLD1 with ADP-ribosylation factor (Arf) 6, Ras homology (Rho) A and protein kinase C (PKC) isoforms (α, ßI, and ßII), and also inhibited the membrane recruitment of Arf6 and RhoA in fMLP-stimulated neutrophils, but not in GTPγS-stimulated cell-free system. The cellular levels of GTP-bound Arf6 and GTP-bound RhoA were reduced by Fal-002-2. Fal-002-2 also attenuated the membrane recruitment of Rho-associated protein kinase 1, phosphorylation of myosin light chain 2 at Thr18/Ser19 and PLD1 at Thr147, and the interaction of Arf6 with both arfaptin 1 and phosphatidylinositol 4-phosphate 5-kinase 1A. The association between RhoA and Vav, the interaction of Vav with both Lyn and Lck, the membrane recruitment of Vav, and the phosphorylation of Vav at Tyr174, but not Src family at Tyr416, were all attenuated by Fal-002-2 in fMLP-stimulated neutrophils. These results indicate that Fal-002-2 is not a direct PLD inhibitor, but the inhibition of fMLP-stimulated PLD activity by Fal-002-2, which partly accounts for its suppression of O2 (•-) generation, is attributable to the blockade of both Arf6 and RhoA activation and attenuation of the interaction of Arf6, RhoA and PKC isoforms with PLD1 in rat neutrophils.

In vivo evaluation of the synthesized novel 2-benzyloxybenzaldehyde analog CCY-1a-E2 for the treatment of leukemia in the BALB/c mouse WEHI-3 allograft model.

Our previous study demonstrated that the 2-benzyloxybenzaldehyde analog CCY-1a-E2 is a potent compound against HL-60 human leukemia cell lines. To investigate the potential therapeutic application of CCY-1a-E2 for leukemia, we analyzed the antileukemic effects and safety of CCY-1a-E2 in the BALB/c mouse WEHI-3 allograft model. Our results showed that CCY-1a-E2 decreased the percentage of viable cells in a concentration-dependent manner. The IC(50) of CCY-1a-E2 was 5 µM for the 24-h treatment of WEHI-3 cells. We examined the antileukemic activity of CCY-1a-E2 in the BALB/c mouse WEHI-3 allograft model. The CCY-1a-E2 (100 mg/kg) group was not found to have significantly decreased body weight compared with the control group, while the leukemia group was found to have significantly decreased body weight compared with the control mice. The CCY-1a-E2 (100 mg/kg) group showed no difference in spleen and liver weight, but significantly decreased levels of CD11b and CD45 compared with the leukemia group. In safety evaluation analysis, CCY-1a-E2 had no adverse effects on renal, hepatic and hematological parameters. Based on these observations, CCY-1a-E2 has efficacious antileukemic activity in the BALB/c mouse WEHI-3 allograft model.

Inhibition of formyl peptide-stimulated superoxide anion generation by Fal-002-2 occurs mainly through the blockade of the p21-activated kinase and protein kinase C signaling pathways in ratneutrophils.

In formyl-Met-Leu-Phe (fMLP)-stimulated rat neutrophils, a synthetic compound, 6-chloro-2-(2-chlorophenyl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (Fal-002-2), inhibited superoxide anion (O2(•-)) generation with an IC50 value of about 11µM, which was not mediated by scavenging the generated O2(•-) or by a cytotoxic effect on neutrophils. Fal-002-2 effectively attenuated the phosphorylation of Ser residues in p47(phox) and the association between p47(phox) and p22(phox) in fMLP-stimulated neutrophils. The interaction of p47(phox) with protein kinase C (PKC) isoforms (α, ßI, ßII, δ and ζ) was attenuated by Fal-002-2 with a similar IC50 value to that required for inhibition of O2(•-) generation, whereas Fal-002-2 had no prominent effect on PKC isoform membrane translocation and did not affect the kinase activity. Moreover, Fal-002-2 had no effect on the phosphorylation of Akt and downstream glycogen synthase kinase-3ß, only slightly affected the intracellular free Ca(2+) concentration, phosphorylation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase (MAPK), but effectively attenuated the downstream MAPK-activated protein kinase-2 phosphorylation. The interaction of p21-activated kinase (PAK) 1with p47(phox), phosphorylation of PAK1 (Thr423/Ser144) and the membrane recruitment of PAK1 were effectively inhibited by Fal-002-2. Fal-002-2 also blocked the activation of Rac1 and Cdc42 in a concentration range that effectively inhibited PAK activation. Taken together, these results suggest that Fal-002-2 inhibits fMLP-stimulated O2(•-) generation in neutrophils mainly through the blockade of PKC and PAK signaling pathways and partly through p38 MAPK signaling.

p38 Mitogen-activated protein kinase and extracellular signal-regulated kinase signaling pathways are not essential regulators of formyl peptide-stimulated p47(phox) activation in neutrophils.

Three structurally unrelated p38 mitogen-activated protein kinase (MAPK) inhibitors, (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole (SB203580), 1-5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl] urea (BIRB 796) and 5-(2,6-dichlorophenyl)-2-[2,4-difluorophenyl]thio]-6H-pyrimido[1,6-b]pyridazin-6-one (VX 745) showed approximately 40% inhibition of formyl-Met-Leu-Phe (fMLP)-stimulated neutrophil superoxide anion (O2(•-)) generation at concentrations that greatly diminished p38 MAPK activity. However, a significant inhibition of p47(phox) activation occurred at concentrations much higher than the corresponding IC50 values of these inhibitors in blocking p38 MAPK activity. 4-Ethyl-2(p-methoxyphenyl)-5-(4'-pyridyl)-IH-imidazole (SB202474), an inactive analogue of SB203580, at a concentration (30µM) which significantly attenuated p38 MAPK activity, had no effect on p47(phox) activation, whereas it inhibited O2(•-) generation with an IC50 value of approximately 16µM. Moreover, both SB203580 and BIRB 796 had no effect on protein kinase B (PKB)/Akt Ser473 phosphorylation and S100A9 protein membrane translocation at concentrations that effectively blocked p38 MAPK activity. Pretreatment of cells with two structurally unrelated MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors, 2-(2-amino-3-methoxy-phenyl)-chromen-4-one (PD 98059) and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), at concentrations that effectively blocked MEK activity, attenuated p47(phox) phosphorylation but did not affect the recruitment of p47(phox) to p22(phox) or O2(•-) generation. Both p47(phox) activation and O2(•-) generation were attenuated by a protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X) in the concentration range that effectively blocked PKC activity. Taken together, these results suggest that the ERK-mediated Ser phosphorylation of p47(phox) is not implicated in the assembly of NADPH oxidase or O2(•-) generation, and that O2(•-) generation is partly attributable to p38 MAPK signaling through mechanisms other than p47(phox) activation, Akt activation and S100A9 membrane recruitment in fMLP-stimulated neutrophils.

AKT serine/threonine protein kinase modulates baicalin-triggered autophagy in human bladder cancer T24 cells.

Baicalin is one of the major compounds in the traditional Chinese medicinal herb from Scutellaria baicalensis Georgi. We investigated the molecular mechanisms of cell autophagy induced by baicalin in human bladder cancer T24 cells. Baicalin inhibited cell survival as shown by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. Baicalin did not induce apoptotic cell death in T24 cells by TUNEL and caspase-3 activity assay. Baicalin induced the acidic vesicular organelle cell autophagy marker, manifested by acridine orange (AO) and monodansylcadaverine (MDC) staining and cleavage of microtubule-associated protein 1 light chain 3 (LC3). The protein expression levels of the Atg 5, Atg 7, Atg 12, Beclin-1 and LC3-II were upregulated in T24 cells after baicalin treatment. Inhibition of autophagy by 3-methyl-adenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) reduced the cleavage of LC3 in T24 cells after baicalin treatment. Furthermore, protein expression levels of phospho-AKT (Ser473) and enzyme activity of AKT were downregulated in T24 cells after baicalin treatment. In conclusion, baicalin triggered cell autophagy through the AKT signaling pathway in T24 cells.

Early induction of cytokines/cytokine receptors and Cox2, and activation of NF-κB in 4-nitroquinoline 1-oxide-induced murine oral cancer model.

The purpose of this study was to identify the genes induced early in murine oral carcinogenesis. Murine tongue tumors induced by the carcinogen, 4-nitroquinoline 1-oxide (4-NQO), and paired non-tumor tissues were subjected to microarray analysis. Hierarchical clustering of upregulated genes in the tumor tissues revealed an association of induced genes with inflammation. Cytokines/cytokine receptors induced early were subsequently identified, clearly indicating their involvement in oral carcinogenesis. Hierarchical clustering also showed that cytokine-mediated inflammation was possibly linked with Mapk6. Cox2 exhibited the greatest extent (9-18 fold) of induction in the microarray data, and its early induction was observed in a 2h painting experiment by RT-PCR. MetaCore analysis showed that overexpressed Cox2 may interact with p53 and transcriptionally inhibit expression of several downstream genes. A painting experiment in transgenic mice also demonstrated that NF-κB activates early independently of Cox2 induction. MetaCore analysis revealed the most striking metabolic alterations in tumor tissues, especially in lipid metabolism resulting from the reduction of Pparα and Rxrg. Reduced expression of Mapk12 was noted, and MetaCore analysis established its relationship with decreased efficiency of Pparα phosphorylation. In conclusion, in addition to cytokines/cytokine receptors, the early induction of Cox2 and NF-κB activation is involved in murine oral carcinogenesis.

The signaling mechanisms mediating the inhibitory effect of TCH-1116 on formyl peptide-stimulated superoxide anion generation in neutrophils.

In fMLP (formyl-Met-Leu-Phe)-stimulated rat neutrophils, a mixture of regioisomers benzo[a]furo[2,3-c]phenazine-10-carboxylic acid and benzo[a]furo[2,3-c]phenazine-11-carboxylic acid (TCH-1116) inhibited O(2)(-) (superoxide anion) generation, which was not mediated by scavenging the generated O(2)(-) or by a cytotoxic effect on neutrophils. TCH-1116 had no effect on the arachidonic acid-induced NADPH oxidase activation in a cell-free system, whereas it effectively attenuated the phosphorylation of Ser residues in p47(phox) and the association between p47(phox) and p22(phox) in fMLP-stimulated neutrophils. The interaction of p47(phox) with PKC (protein kinase C) isoforms (α, ßI, ßII, δ and ζ) was attenuated by TCH-1116, whereas TCH-1116 did not affect the PKC isoforms membrane translocation, phosphorylation (Ser660) and kinase activity. TCH-1116 effectively attenuated the association between PKB/Akt (protein kinase B) and p47(phox), Akt phosphorylation (Thr308/Ser473) and kinase activities of Akt and human recombinant PDK (3-phosphoinositide-dependent kinase) 1, whereas it had no effect on recruitment of Akt, phospho-PDK1 (Ser241) and p110γ to membrane. Moreover, the interaction of p21-activated kinase (PAK) 1 with p47(phox) and the phosphorylation of PAK1 (Thr423 but not Ser144) were inhibited by TCH-1116, but without affecting the membrane recruitment of PAK1. The cellular cyclic AMP level was not changed by TCH-1116. Taken together, these results suggest that TCH-1116 inhibits fMLP-stimulated O(2)(-) generation in rat neutrophils through the blockade of PKC, Akt and PAK signaling pathways.

Co-treating with arecoline and 4-nitroquinoline 1-oxide to establish a mouse model mimicking oral tumorigenesis.

The aim of this study was to establish an effective mouse model of oral cancer and to use this model to identify potential markers of oral tumor progression. C57BL/6JNarl mice were treated with arecoline, 4-nitroquinoline 1-oxide (4-NQO), or both arecoline and 4-NQO in high and low doses for 8 weeks to induce oral tumor. The induced oral lesions were observed for 20 weeks to assess the efficiency of cancer induction and survival rate of the mice. In addition, two target proteins that are frequently overexpressed during tongue cancer tumorigenesis, alphaB-crystallin and Hsp27, were examined by immunohistochemical analysis. In mice exposed to 4-NQO (200 microg/mL) and arecoline (500 microg/mL), the tongue lesions showed evidence of hyperplasia, papilloma, dysplasia, and carcinoma, and the lesions were pathologically similar to those lesions in human oral cancer. The tongue tumor incidence rate was 100% in mice exposed to concomitant 4-NQO (200 microg/mL) and arecoline (500 microg/mL) treatment, 57% in mice exposed to 4-NQO only, and 0% in mice exposed to arecoline only. Immunohistochemical analysis demonstrated that, consistent with human studies, alphaB-crystallin and Hsp27 were upregulated in murine oral tumors. In conclusion, we have established a powerful animal model that enables the study of the promoting effects of arecoline on tongue tumorigenesis. Data subsequently attained from this mouse model support a role for alphaB-crystallin and Hsp27 as clinical markers for tumor progression.

Inhibition of lipopolysaccharide-stimulated NO production by a novel synthetic compound CYL-4d in RAW 264.7 macrophages involving the blockade of MEK4/JNK/AP-1 pathway.

In the present study, a novel synthetic compound 4-(2-(cyclohex-2-enylidene)hydrazinyl)quinolin-2(1H)-one (CYL-4d) was found to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production without affecting cell viability or enzyme activity of expressed inducible NO synthase (iNOS) in RAW 264.7 macrophages. CYL-4d exhibited parallel inhibition of LPS-induced expression of iNOS protein, iNOS mRNA and iNOS promoter activity in the same concentration range. LPS-induced activator protein-1 (AP-1) DNA binding, AP-1-dependent reporter gene activity and c-Jun nuclear translocation were all markedly inhibited by CYL-4d with similar efficacy, whereas CYL-4d produced a weak inhibition of nuclear factor-kappaB (NF-kappaB) DNA binding, NF-kappaB-dependent reporter gene activity and p65 nuclear translocation without affecting inhibitory factor-kappa B alpha (I kappa B alpha) degradation. CYL-4d had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and its upstream activator MAPK kinase (MEK) 3, whereas it significantly attenuated the phosphorylation of c-Jun, c-Jun NH(2)-terminal kinase (JNK) and its upstream activator MEK4 in a parallel concentration-dependent manner. Other Toll-like receptors (TLRs) ligands (peptidoglycans, double-stranded RNA, and oligonucleotide containing unmethylated CpG motifs)-induced iNOS protein expression were also inhibited by CYL-4d. Furthermore, the NO production from BV-2 microglial cells as well as rat alveolar macrophages in response to LPS was diminished by CYL-4d. These results indicate that the blockade of NO production by CYL-4d in LPS-stimulated RAW 264.7 cells is attributed mainly to interference in the MEK4-JNK-AP-1 signaling pathway. CYL-4d inhibition of NO production is not restricted to TLR4 activation and immortalized macrophage-like cells.

Inhibition of lipopolysaccharide-stimulated NO production by crotafuran B in RAW 264.7 macrophages involves the blockade of NF-kappaB activation through the increase in IkappaBalpha synthesis.

Crotafuran B, a natural pterocarpanoid isolated from Crotalaria pallida, inhibited the lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (IC50 16.4+/-0.7 microM) and inducible nitric oxide synthase (iNOS) protein and mRNA expression (IC50 11.5+/-0.6 microM and 11.8+/-2.2 microM, respectively), but not via its cytotoxicity or the inhibition of iNOS enzyme activity, in RAW 264.7 macrophages. Crotafuran B also reduced the iNOS promoter activity (IC50 13.4+/-0.1 microM) in piNOS-LUC-transfected cells. Crotafuran B treatment inhibited the p65 nuclear translocation and the nuclear factor-kappaB (NF-kappaB) DNA binding activity in LPS-activated macrophages. Crotafuran B also reduced the NF-kappaB transcriptional activity in pNF-kappaB-LUC-transfected cells. Crotafuran B had no effect on the LPS-induced phosphorylation of inhibitory kappaBalpha (IkappaBalpha), but enhanced the cellular level of IkappaBalpha that rebounded to the basal levels and increased the IkappaBalpha mRNA expression. These results indicate that the crotafuran B inhibition of NO production involves a decrease in the iNOS gene expression via the inhibition of NF-kappaB activation through the increase in IkappaBalpha synthesis.

Synthesis and anticancer activity of benzyloxybenzaldehyde derivatives against HL-60 cells.

A series of benzyloxybenzaldehyde derivatives were prepared and tested against the HL-60 cell line for anticancer activity. Preliminary structure-activity relationships were established. It was discovered that 2-(benzyloxy)benzaldehyde (17), 2-(benzyloxy)-4-methoxybenzaldehyde (26), 2-(benzyloxy)-5-methoxybenzaldehyde (27), 2-(benzyloxy)-5-chlorobenzaldehyde (28), 2-[(3-methoxybenzyl)oxy]benzaldehyde (29), 2-[(2-chlorobenzyl)oxy]benzaldehyde (30), and 2-[(4-chlorobenzyl)oxy]benzaldehyde (31) exhibited significant activity at 1-10 microM. Among them, compound 29 was the most potent one. The morphological assessment and DNA fragmentation analysis indicated that these compounds arrested cell cycle progression at G2/M phase and induced cell apoptosis. They resulted in the loss of mitochondrial membrane potential after 12h of treatment.

Selective induction of G2/M arrest and apoptosis in HL-60 by a potent anticancer agent, HMJ-38.

We previously reported that HMJ-38 was the most potent 2-phenyl-4-quinozolinone derivative in inhibiting tubulin polymerization and showed significant cytotoxicity against several human tumor cell lines. In this work, we studied its cytotoxic effect on HL-60 leukemia cells and the underlying mechanisms. We first investigated the effects of HMJ-38 on viability, cell cycle and induction of apoptosis in HL-60 and normal human peripheral blood mononuclear cells (PBMC). After 24-hour treatment with HMJ-38, a dose- and time-dependent decrease in the viability of HL-60 cells was observed and the approximate IC50 was 4.48 microM. The cytotoxic effect of HMJ-38 on PBMC was less significant than that on HL-60 cells, either with 24 or 48 hours of treatment. Cell cycle analysis showed that HMJ-38 induced significant G2/M arrest and apoptosis in HL-60 cells. The HMJ-38-induced G2/M arrest occurred before the onset of apoptosis. Within 24 hours of treatment, HMJ-38 influenced the CDK/cyclin B activity by increasing Chk1, Wee1 and p21 and decreasing Cdc25C protein levels. The HMJ-38-induced apoptosis was further confirmed by morphological assessment and DNA fragmentation assay. Induction of apoptosis in HMJ-38-treated HL-60 cells was accompanied by an apparent increase of cytosolic cytochrome c, down-regulation of Bcl-2, up-regulation of Bax and cleavage of pro-caspase-9, -3 and poly(ADP)ribosylpolymerase (PARP). The results of the significant reduction of caspase activities and apoptosis by caspase inhibitors indicated that the HMJ-38-induced apoptosis was mainly mediated by activation of caspases-9 and -3. HMJ-38 also activated ERK in HL-60 cells. Pre-incubating cells with ERK inhibitors (U0126 and PD98059) attenuated the HMJ-38-induced ERK activation and apoptosis. Nevertheless, cells remained arrested in G2/M. These results suggest that HMJ-38 is a potent anticancer drug and it shows a remarkable action on cell cycle before commitment for apoptosis is reached.