A double-blind, randomized controlled trial to evaluate the safety and immunogenicity of an intranasally administered trivalent inactivated influenza vaccine with the adjuvant LTh(αK): A phase II study.

BACKGROUND: Intranasal influenza vaccines may provide protective efficacy by inducing both systemic antibodies and local secretory IgA. Live attenuated intranasal vaccines are not feasible for high-risk groups. A previously constructed inactivated vaccine with adjuvant revealed an association with neurological events in some studies. In this phase II trial, we aimed to evaluate the safety and immunogenicity of an intranasal influenza vaccine with a novel adjuvant, heat-labile enterotoxin (LT)-derived from E. coli (LTh(αK)). METHODS: This study is a multicenter, randomized controlled, double-blind, phase II trial of an intranasal influenza vaccine containing 22.5 µg of the hemagglutinin (HA) antigen of three influenza strains in combination with 2 different LTh(αK) adjuvant doses (group 1: 30 µg; group 2: 45 µg) in subjects 20-70 years old. The control vaccine was 22.5 µg of influenza HA antigen alone (group 3). The vaccine was intranasally administered on days 1 and 8. Serum anti-HA antibody and nasal secretory IgA were measured, and adverse events (AEs) were recorded prevaccination and 29 (±2) days postvaccination. RESULTS: Of 354 participants randomized in the study, 340 received two vaccine doses. AEs were mostly mild, and there was no discontinuation related to the vaccine. Only a higher frequency of diarrhea after the first dose was noted among group 2 (11.5%) than among group 3 (2.8%), and there was no significant difference after the second dose. The three groups had comparable serum anti-HA antibody immunogenicity. However, the adjuvanted vaccines induced greater mucosal IgA antibody production than the control vaccine. In a subgroup analysis, group 1 participants achieved adequate immunogenicity among both 20- to 60- and 61- to 70-year-old participants. CONCLUSION: The intranasal influenza vaccine adjuvanted with LTh(αK) is generally safe and could provide systemic and local antibody responses. Adjuvanted vaccines were significantly more immunogenic than the nonadjuvanted control vaccine in mucosal immunity. ClinicalTrials.gov Identifier: NCT03784885.

MT-4 suppresses resistant ovarian cancer growth through targeting tubulin and HSP27.

OBJECTIVE: In this study, the anticancer mechanisms of MT-4 were examined in A2780 and multidrug-resistant NCI-ADR/res human ovarian cancer cell lines. METHODS: To evaluate the activity of MT-4, we performed in vitro cell viability and cell cycle assays and in vivo xenograft assays. Immunoblotting analysis was carried out to evaluate the effect of MT-4 on ovarian cancer. Tubulin polymerization was determined using a tubulin binding assay. RESULTS: MT-4 (2-Methoxy-5-[2-(3,4,5-trimethoxy-phenyl)-ethyl]-phenol), a derivative of moscatilin, can inhibit both sensitive A2780 and multidrug-resistant NCI-ADR/res cell growth and viability. MT-4 inhibited tubulin polymerization to induce G2/M arrest followed by caspase-mediated apoptosis. Further studies indicated that MT-4 is not a substrate of P-glycoprotein (p-gp). MT-4 also caused G2/M cell cycle arrest, accompanied by the upregulation of cyclin B, p-Thr161 Cdc2/p34, polo-like kinase 1 (PLK1), Aurora kinase B, and phospho-Ser10-histone H3 protein levels. In addition, we found that p38 MAPK pathway activation was involved in MT-4-induced apoptosis. Most importantly, MT-4 also decreased heat shock protein 27 expression and reduced its interaction with caspase-3, which inured cancer cells to chemotherapy resistance. Treatment of cells with SB203580 or overexpression of dominant negative (DN)-p38 or wild-type HSP27 reduced PARP cleavage caused by MT-4. MT-4 induced apoptosis through regulation of p38 and HSP27. Our xenograft models also show the in vivo efficacy of MT-4. MT-4 inhibited both A2780 and NCI-ADR/res cell growth in vitro and in vivo. CONCLUSION: These findings indicate that MT-4 could be a potential lead compound for the treatment of multidrug-resistant ovarian cancer.

In vitro and in vivo anti-tumour effects of MPT0B014, a novel derivative aroylquinoline, and in combination with erlotinib in human non-small-cell lung cancer cells.

BACKGROUND AND PURPOSE: The purpose of the current study was to assess a novel anti-cancer drug, MPT0B014, which is not a substrate for the P-glycoprotein (P-gp) transporter, alone and in combination with erlotinib, against human non-small cell lung cancer (NSCLC). EXPERIMENTAL APPROACH: Cytotoxicity in human NSCLC cell lines was assessed by sulforhodamine B and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. Cell cycle phase distributions were estimated with FACScan flow cytometry. Protein expression was detected by Western blotting analysis. Efflux of rhodamine 123 or calcein-acetoxymethylester was used to study the P-gp profile. The A549 xenograft model in mice was used to assess in vivo anti-tumour activity. KEY RESULTS: MPT0B014 showed potent anti-proliferative activity against A549, H1299 and H226 cells. It induced G2/M arrest with down-regulation of Cdc (Tyr15) and Cdc25C, and up-regulation of cyclin B1, phospho-Cdc2 (Thr161) and Aurora A/B. P-gp-overexpressing National Cancer Institute/Adriamycin-Resistant cells were also sensitive to B014. B014-induced loss of Mcl-1 was accompanied by activation of caspases-3, -7, -8 and -9, and initiation of apoptosis. B014 in combination with erlotinib caused significant tumour inhibition in vitro and in vivo. CONCLUSIONS AND IMPLICATIONS: MPT0B014 exerted cytotoxicity against human NSCLC cell lines with little susceptibility to P-gp. Combined with the EGF receptor inhibitor, erlotinib, MPT0B014 exerted significant growth inhibition of A549 cells both in vitro and in vivo. B014 could be useful as an anti-cancer agent.

Thrombin-induced CCN2 expression in human lung fibroblasts requires the c-Src/JAK2/STAT3 pathway.

Thrombin is a multifunctional serine protease and an important fibrotic mediator that induces CCN2 expression. We previously showed that thrombin induces CCN2 expression via an ASK1-dependent JNK/AP-1 pathway in human lung fibroblasts. In this study, we further investigated the roles of c-Src, JAK2, and STAT3 in thrombin-induced CCN2 expression. Thrombin-induced CCN2 expression and CCN2-Luc activity were attenuated by a JAK inhibitor (AG490) and JAK2DN, STAT3DN, and the STAT decoy ODN. Moreover, transfection of cells with a CCN2-mtSTAT-Luc construct inhibited thrombin-induced CCN2-Luc activity. Treatment of cells with thrombin caused JAK2 phosphorylation at Tyr1007/1008 and STAT3 phosphorylation at Tyr705 in time-dependent manners. Thrombin-induced STAT3 phosphorylation was inhibited by AG490 and JAK2DN. Thrombin-induced STAT3 binding to the CCN2 promoter was analyzed by a DNA-binding affinity pull-down assay. In addition, thrombin-induced CCN2 expression and CCN2-Luc activity were inhibited by c-SrcDN and PP2 (an Src inhibitor). Transfection of cells with c-SrcDN also inhibited thrombin-induced JAK2 and STAT3 phosphorylation. Taken together, these results indicate that thrombin might activate c-Src to induce JAK2 activation, which in turn, causes STAT3 activation, and finally induces CCN2 expression in human lung fibroblasts.

Moscatilin inhibits migration and metastasis of human breast cancer MDA-MB-231 cells through inhibition of Akt and Twist signaling pathway.

Breast cancer metastasis is more resistant to chemotherapy and radiotherapy than is cancer of the visceral tissues; therefore, new treatment strategies are urgently needed. Moscatilin, derived from the orchid Dendrobrium loddigesii, has shown anticancer activity. We evaluated the mechanism by which moscatilin suppresses the migration and metastasis of human breast cancer MDA-MB-231 cells in vitro and in vivo. We demonstrated that moscatilin significantly inhibits MDA-MB-231 cell migration by using scratch assays and Boyden chambers. Transcriptional factors inducing epithelial-mesenchymal transition, such as Twist, Snail, and Akt, play important roles in cell migration and cancer metastasis. Moscatilin inhibited the mRNA and protein expression of Twist, but not that of Snail, and subsequently inhibited N-cadherin expression. However, these effects were reversed by constitutively expressing active myristoylated (myr)-Akt and Twist overexpression. Moscatilin also suppressed Akt phosphorylation. However, Akt overexpression reversed the inhibitory effects of moscatilin on phospho-Akt protein expression but not its effects on Twist. The moscatilin-mediated inhibition of cell migration was reversed by Akt and Twist overexpression, demonstrating that moscatilin blocked cell migration by inhibiting Akt and Twist. In an MDA-MB-231 metastatic animal model, moscatilin (100 mg/kg) significantly suppressed breast cancer metastasis to the lungs and reduced the number of metastatic lung nodules and lung weight without causing any toxicity. These results indicated that moscatilin inhibited MDA-MB-231 cell migration via Akt- and Twist-dependent pathways; this finding was consistent with moscatilin's antimetastatic activity in vivo. Therefore, moscatilin may be an effective compound for the prevention of human breast cancer metastasis.

Thrombin induces expression of twist and cell motility via the hypoxia-inducible factor-1α translational pathway in colorectal cancer cells.

Deep vein thrombosis associated with advanced cancer is known as Trousseau's syndrome. We hypothesized that thrombin, an activator of protease-activated receptor (PAR)-1 and PAR-4 contributes to tumor metastasis. In this study, we demonstrated that thrombin and the PAR-1 activating peptide (AP) SFLLRN, but not the PAR-4 AP GYPGKF, induced HIF-1α activities, protein expression, and cell motility in colorectal cancer cells, and these actions were significantly inhibited by the PAR-1 antagonist SCH79797. Moreover, thrombin-induced HIF-1α activity and cell motility were blocked by inhibiting important mediators of signaling transduction, including the ERK, PI3K, and mTOR pathways. These results showed that thrombin induced HIF-1α protein expression through PAR-1 and HIF-1α translational de novo protein synthesis. Twist can regulate epithelial-mesenchymal transition (EMT) and increase tumor metastasis. However, we observed that thrombin-induced HIF-1α increased Twist mRNA and its protein level was mediated by the modulation of PAR-1 activation and the HIF-1α translational pathway. In addition, Twist could increase N-cadherin but not E-cadherin to promote tumor metastasis. Overexpression of dominant-negative HIF-1α reversed thrombin-mediated Twist and Twist-induced N-cadherin expression. Moreover, siTwist inhibited Twist-induced N-cadherin and Thrombin-induced cell motility. In conclusion, our study showed that thrombin-induced HIF-1α upregulated Twist at the transcriptional level to enhance cell motility. These findings show that thrombin upregulates Twist via HIF-1α to make tumor cells malignant and also establish a link between the coagulation disorder and cancer metastasis.

Denbinobin suppresses breast cancer metastasis through the inhibition of Src-mediated signaling pathways.

Denbinobin (5-hydroxy-3,7-dimethoxy- 1,4-phenanthraquinone), a biologically active chemical isolated from Ephemerantha lonchophylla, has been demonstrated to display anti-cancer activity. Breast cancer is the leading cause of female mortality, and the high mortality is mainly attributable to metastasis. Src kinase activity is elevated in many human cancers, including breast cancer, and is often associated with aggressive disease. In the present study, we examined the anti-metastatic effects of denbinobin through decreasing Src kinase activity in human and mouse breast cancer cells. Denbinobin caused significant block of Src kinase activity in both human and mouse breast cancer cells. Moreover, phosphorylation of the signaling molecules focal adhesion kinase, Crk-associated substrate and paxillin downstream of Src was also inhibited by denbinobin. Furthermore, denbinobin inhibited the in vitro migration, invasion and in vivo metastasis of breast cancers in a mouse metastatic model. The denbinobin-treated group showed a significant reduction in tumor metastasis, orthrotopic tumor volume, and spleen enlargement compared to the control group. In addition, transfection of breast cancer cells with a plasmid coding for a constitutively active Src prevented the denbinobin-mediated phosphorylation of Src and downstream molecules and cell migration. Our findings provide evidences that denbinobin inhibits Src-mediated signaling pathways involved in controlling breast cancer migration and metastasis, suggesting that it has therapeutic potential in breast cancer treatment.

The indazole derivative YD-3 specifically inhibits thrombin-induced angiogenesis in vitro and in vivo.

Angiogenesis is a process that involves endothelial cell proliferation, migration, invasion, and tube formation, and the inhibition of these processes has implications for angiogenesis-mediated disorders. The purpose of this study was to examine the antiangiogenic efficacy of YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole], a selective thrombin inhibitor, on thrombin-induced endothelial cell proliferation and neoangiogenesis in a murine Matrigel model. First, the effect of YD-3 on angiogenesis was evaluated in vivo using the mouse Matrigel implant model. Plugs treated with 1 and 10 µM of YD-3 inhibited neovascularization induced by thrombin, protease-activated receptor (PAR) 1, and PAR-4, but not by vascular endothelial growth factor, in a concentration-dependent manner over 7 days. These results indicate that YD-3 has specific antiangiogenic activity on thrombin. YD-3 also inhibited (in a concentration-dependent manner) the ability of thrombin, PAR-1, and PAR-4, but not PAR-2, to induce the proliferation of human umbilical vascular endothelial cells, using a [³H]thymidine incorporation assay. YD-3 predominantly inhibited thrombin-induced vascular endothelial growth factor receptor 2 (Flk-1) expression, but not extracellular signal-regulated kinase 1/2 phosphorylation, using Western blot analysis. YD-3 may have benefit in elucidating pathophysiology induced by thrombin-induced angiogenesis.

Denbinobin induces apoptosis by apoptosis-inducing factor releasing and DNA damage in human colorectal cancer HCT-116 cells.

Denbinobin is a phenanthraquinone derivative present in the stems of Ephemerantha lonchophylla. We showed that denbinobin induces apoptosis in human colorectal cancer cells (HCT-116) in a concentration-dependent manner. The addition of a pan-caspase inhibitor (zVAD-fmk) did not suppress the denbinobin-induced apoptotic effect, and denbinobin-induced apoptosis was not accompanied by processing of procaspase-3, -6, -7, -9, and -8. However, denbinobin triggered the translocation of the apoptosis-inducing factor (AIF) from the mitochondria into the nucleus. Small interfering RNA targeting of AIF effectively protected HCT-116 cells against denbinobin-induced apoptosis. Denbinobin treatment also caused DNA damage, activation of the p53 tumor suppressor gene, and upregulation of numerous downstream effectors (p21WAF1/CIP1, Bax, PUMA, and NOXA). A HCT-116 xenograft model demonstrated the in vivo efficacy and low toxicity of denbinobin. Taken together, our findings suggest that denbinobin induces apoptosis of human colorectal cancer HCT-116 cells via DNA damage and an AIF-mediated pathway. These results indicate that denbinobin has potential as a novel anticancer agent.

YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole] inhibits neointima formation in balloon-injured rat carotid through suppression of expressions and activities of matrix metalloproteinases 2 and 9.

Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, and postrevascularization production of vascular smooth muscle cells may play key roles in development of arterial restenosis. We investigated the inhibitory effect of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), a benzyl indazole compound, on MMP-2 and MMP-9 activity in a balloon-injury rat carotid artery model. Injury was induced by inserting a balloon catheter through the common carotid artery; after 14 days, histopathological analysis using immunostaining and Western blotting revealed significant restenosis with neointimal formation that was associated with enhanced protein expression of MMP-2 and MMP-9. However, these effects were dose-dependently reduced by orally administered YC-1 (1-10 mg/kg). In addition, gelatin zymography demonstrated that increased MMP-2 and MMP-9 activity was diminished by YC-1 treatment. On the other hand, YC-1 inhibited hydrolysis of the fluorogenic quenching substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH(2) by recombinant MMP-2 and MMP-9 with IC(50) values = 2.07 and 8.20 muM, respectively. Reverse transcription-polymerase chain reaction analysis of MMP-2 and MMP-9 mRNA revealed that YC-1 significantly inhibited mRNA levels of MMPs. Finally, for the YC-1 treatment group, we did not observe elevation of cGMP levels using enzyme-linked immunosorbent assay, suggesting that YC-1 inhibition of neointimal formation is not through a cGMP-elevating pathway. These data show YC-1 suppression of neointimal formation is dependent on its influence on MMP-2 and MMP-9 protein, mRNA expression, and activity, but not through a cGMP-elevating effect. YC-1 shows therapeutic potential for treatment of restenosis after angioplasty.

Antitumor mechanism of evodiamine, a constituent from Chinese herb Evodiae fructus, in human multiple-drug resistant breast cancer NCI/ADR-RES cells in vitro and in vivo.

Drug resistance is one of the main obstacles to the successful treatment of cancer. The availability of agents that are highly effective against drug-resistant cancer cells is therefore essential. The present study was performed to examine the anticancer effects of evodiamine, a major constituent of the Chinese herb Evodiae fructus, in adriamycin-resistant human breast cancer NCI/ADR-RES cells. Evodiamine inhibited the proliferation of NCI/ADR-RES cells in a concentration-dependent manner with a GI50 of 0.59 +/- 0.11 microM. This agent also caused a substantial apoptosis at 1 microM. FACScan flow cytometric analysis of cell cycle progression revealed that a G2/M arrest was initiated after a 12-h exposure to the drug. Evodiamine increased tubulin polymerization as determined by the immunocytochemical and in vivo tubulin polymerization analyses. In a time- and concentration-dependent manner, evodiamine also promoted the phosphorylations of Raf-1 kinase and Bcl-2. The phosphorylation site of Raf-1 kinase was identified to be serine338. The in vivo anticancer effects of evodiamine were evaluated in Balb-c/nude mice following a tumor xenograft implantation of NCI/ADR-RES cells. The antitumor activity of evodiamine against the human multiple-drug resistant tumor xenograft was found to be superior to that of paclitaxel. Evodiamine therefore represents a highly promising chemotherapeutic agent in the treatment of human multiple-drug resistant cancer cells.