The majority of growth factor receptors are composed of extracellular, transmembrane, and cytoplasmic tyrosine kinase (TK) domains. Receptor tyrosine kinase (RTK) activation regulates many key processes including cell growth and survival. However, dysregulation of RTK has been found in a wide range of cancers, and it has been shown to correlate with the development and progression of numerous cancers. Therefore, RTK has become an attractive therapeutic target. One way to effectively block signaling from RTK is inhibition of its catalytic activity with small-molecule inhibitors. Low-molecular-weight TK inhibitors (TKIs), such as imatinib, targeting tumors with mutant c-Kit, and gefitinib, targeting non-small cell lung cancer with mutant epidermal growth factor receptor (EGFR), have received marketing approval in Japan. MET, fibroblast growth factor receptor (FGFR), and insulin-like growth factor-I receptor (IGF-IR) are frequently genetically altered in advanced cancers. TKIs of these receptors have not yet appeared on the market, but many anticancer drug candidates are currently undergoing clinical trials. Most of these TKIs were designed to compete with ATP at the ATP-binding site within the TK domain. This review will focus on small-molecule TKIs targeting MET, FGFR, and IGF-IR and discuss the merits and demerits of two types of agents, i.e., those with only one or a few targets and those directed at multiple targets. Targeting agents specifically inhibiting the target kinase were previously searched for based on the hypothesis that a narrow target window might reduce unexpected side effects, but agents with multiple targets have been recently developed to overcome tumors resistant against a single-targeting agent.
The extracellular signal-regulated kinase (ERK) pathway is a major determinant in the control of diverse cellular processes such as proliferation, survival, and motility. This pathway is often upregulated in human cancers and as such represents an attractive target for mechanism-based approaches to cancer treatment. However, specific blockade of the ERK pathway alone induces mostly cytostatic rather than proapoptotic effects, resulting in limited therapeutic efficacy. Blockade of the constitutively activated ERK pathway by an ERK kinase (MEK) inhibitor sensitizes tumor cells to apoptotic cell death induced by several cytotoxic anticancer agents including microtubule-destabilizing agents and histone deacetylase inhibitors, not only in vitro but also in tumor zenografts in vivo. Thus, low concentrations of these anticancer drugs that by themselves show little cytotoxicity effectively kill tumor cells in which the ERK pathway is constitutively activated when co-administrated with a MEK inhibitor. The combination of a cytostatic signaling pathway inhibitor (MEK inhibitors) and conventional anticancer drugs (microtubule-destabilizing agents or histone deacetylase inhibitors) provides an excellent basis for the development of safer anticancer chemotherapies with enhanced efficacy through lowering the required dose of the latter cytotoxic drugs.
Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) have crucial roles in both physiological and pathological angiogenesis. The VEGF family consists of VEGF-A (generally called VEGF), VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). These peptides show different affinities for VEGFR subtypes. VEGFR exists as three subtypes, VEGFR-1, VEGFR-2, and VEGFR-3, and is structurally related to platelet-derived growth factor receptors. All subtypes possess seven immunoglobulin-like domains in the extracellular region and a tyrosine kinase domain in the intracellular region. VEGF-A activates VEGFR-1 and VEGFR-2, whereas VEGF-B and PlGF bind to only VEFGR-1. VEGF-C and VEGF-D only bind to VEGFR-3. VEGFR-1 (fms-like tyrosine kinase-1, Flt-1) negatively regulates embryonic vasculogenesis and is involved in tumor angiogenesis via activation of monocytes and macrophages. VEGFR-2 (KDR in humans or Flk-1 in mice) is predominantly responsible for both embryonic vasculogenesis and tumor angiogenesis. In contrast, VEGFR-3 (Flt-4) regulates lymphangiogenesis. Consequently, VEGF-A and VEGFR-2 are currently the main targets for antiangiogenic therapy. Bevacizumab is a humanized monoclonal antibody against VEGF-A, and aflibercept (VEGF-Trap) is a soluble fusion protein of the extracelluar domain of VEGFR-1 and VEGFR-2 and the Fc region of immunoglobulin G (IgG). They neutralize VEGF-A, resulting in prevention of tumor angiogenesis. VEGFR tyrosine kinase inhibitors such as sunitinib and sorafenib are also effective in antiangiogenic tumor therapy by inhibiting VEGFR signaling. Anti-VEGF drugs are a promising therapy for cancer patients.
Ten years have passed since the emergence of microarray technology. Recent microarray procedures have provided reliable results on all platforms and have enabled highly reproducible gene expression measurements. Thus, nearly all technical matters regarding microarray measurements are thought to have been resolved. Treatment stratification for molecular-targeted drugs can now be achieved based on the presence of somatic mutations, gene amplification, and/or protein overexpression. However, no clinically available biomarkers have been identified for molecular-targeted drugs using microarray analysis. Microarray data as a database for the gene expressions of clinical samples may be a critical issue, especially for the development of molecular-targeted treatments. In addition, microarray analysis during early-phase clinical trials for molecular-targeted drugs is considered to provide critical information, including proof-of-concept and confirmation of the inhibition of the target molecule. Meanwhile, OncotypeDX® and MammaPrint® assays have been developed to determine the benefits of chemotherapy for breast cancer patients. These multigene-based assays are commercially available and have shown encouraging results for treatment stratification or decision-making for treatment using cytotoxic drugs in clinical settings. During the development of these assays, numerous samples and efforts were required to create a model using multi-center or inter-group investigations. Based on the success of these models, the development of further assays for determining multigene expressions is likely to increase in the future. In the present article, we introduce our data on mutant epidermal growth factor receptor (EGFR) signaling and amplification of fibroblast growth factor receptor 2 (FGFR2) using microarray analysis, and treatment stratification and clinical applications using gene expression profiles for cancer treatments are discussed.
The stems of Dendrobium thyrsiflorum RCHB.F. ex ANDRé can be processed into an important class of Traditional Chinese Medicine named “Huangcao Shihu,” which has diverse curative effects, such as nourishing yin and clearing away unhealthy heat, benefiting the stomach, and promoting the production of body fluid. The identification of the geographical origin of D. thyrsiflorum is vital for preserving its natural resource and ensuring the quality of “Huangcao Shihu.” In order to identify the origin of D. thyrsiflorum on Chinese herbal medicine market, 14 D. thyrsiflorum-specific microsatellite markers were developed in this study. Assignment tests were performed by the microsatellite marker analysis coupled with three new statistical approaches (partially Bayesian, frequency-based, and fully Bayesian methods) to determine the origin populations of 12 commercial samples of “Huangcao Shihu” collected from a medicine market in Nanjing, Jiangsu Province, China. Their genotypes were compared with those of 136 individuals belonging to five wild D. thyrsiflorum populations from China, Thailand, India, Myanmar, and Laos. Comparisons of the probabilities of 12 unknown individuals originating from each candidate population indicated that most of them appeared to originate from Myanmar and Laos. This suggests that the two countries may be the predominant sources of D. thyrsiflorum on the medicine market in Nanjing. In addition, the 14 microsatellite markers developed in this study may be an effective tool for identification of the origin of commercial available “Huangcao Shihu” and play an important role in its quality control.
Vasculogenic progenitor cells (VPCs) circulate in the blood and have the ability to differentiate into endothelial cells that make up the lining of blood vessels. Therefore, VPC transplantation is a new strategy for the treatment of ischemic diseases. Because priming/preconditioning of VPCs before transplantation enhances their regenerative potential, the present study investigated whether ent-16α,17-dihydroxy-kauran-19-oic acid (DHK) isolated from Siegesbeckia pubescens could stimulate/activate VPCs in vitro. Therefore, the effect of DHK (1—100 μM concentration) on the proliferation, migration, and tube forming of VPCs was examined in various systems, and related signaling pathways were identified. DHK treatment significantly increased the proliferation, migration, and tube formation of VPCs in a dose-dependent manner. Phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and Akt was significantly increased by DHK, but chemical inhibitors against ERK1/2 (U0126) and Akt (LY294002) significantly attenuated DHK-enhanced proliferation, migration, and tube formation of VPCs. Collectively, these results indicated that DHK shows promise as a novel VPC primer/activator.
The aim of this study is to investigate whether or not You Gui Wan (YGW), a classical herbal formula in Traditional Chinese Medicine (TCM), has an impact on rat uterine and vaginal atrophic processes induced by ovariectomy (OVX). Thirty-four OVX Sprague-Dawley (SD) rats were randomly divided into three sets, and orally administrated with YGW decoction, saline or estrogen for 11 weeks, respectively. Histomorphological changes of the uterus and vagina, and serum estradiol levels were then compared. Results showed that OVX caused a dramatic atrophy of the uterus and vagina in the rats. Estrogen replacement reversed the effect of OVX, but with a side effect of endometrial hyperplasia. YGW had no significant effect on blood estradiol concentration or uterine histology, but it significantly overturned the atrophic processes of the vaginal fold and blood vessels in the lamina propria. In order to initially explore the mechanisms underlying these effects, immunostaining of estrogen receptor (ER)-α and -β in the vagina was performed. It was shown that OVX reduced expressions of ER while YGW and estrogen replacement reversed this reduction. Our findings suggest that YGW can reverse the atrophic effect of OVX on rat vaginal plica and blood vessels in the lamina propria with little adverse effect on endometrial hyperplasia. This indicates the herbal formula as an alternative to hormone replacement therapy in the management of menopausal vaginal atrophy. Recovery of ER expressions in the vagina might be one of mechanisms underlying the effects of YGW.
The aim of the present study was to investigate whether hirsutenone affects the human ether-a-go-go related gene (hERG) K+ channels. Many drugs promote formation of the acquired form of long QT syndrome (LQTS) by blocking the hERG K+ channels. Hirsutenone, a new candidate for the treatment inflammatory skin lesions, induced a concentration-dependent decrease in hERG K+ current amplitudes. Hirsutenone significantly decreased the time constants at the onset of inactivation. However, the reductions in the time constants of steady-state inactivation and the recovery from inactivation after hirsutenone treatment were not significant. In addition, the drug had no effect on the voltage-dependent activation curve or the steady-state inactivation curve. In summary, hirsutenone potentially acts as a blocker of hERG K+ channels functioning by modifying the channel inactivation kinetics.
We investigated the effect of tacrolimus, a calcineurin inhibitor, on dextran sulfate sodium (DSS)-induced colitis. After inducing colitis in C57BL/6 mice by administering DSS solution as drinking water for 7 d, the animals were treated with tacrolimus. Severity of colonic inflammation was evaluated based on colon weight per unit length. Levels of cytokines (interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-12, and tumor necrosis factor (TNF)-α) released from isolated inflamed colons of mice treated with tacrolimus or vehicle were also measured. Treatment with tacrolimus for 14 d reduced the colon weight per unit length and suppressed the release of IFN-γ and IL-1β, but not other cytokines, in inflamed colons of colitic mice compared with vehicle-treated mice. A positive correlation was noted between colon weight per unit length and released level of IFN-γ or IL-1β. The release of IFN-γ and IL-1β was also suppressed after single dosing with tacrolimus to colitic mice. Taken together, these results suggested that tacrolimus ameliorated DSS-induced colitis by suppressing release of IFN-γ and IL-1β from inflamed colon.
Engulfment of apoptotic cells is regulated by ‘eat me’ and ‘don’t eat me' signals on the cell surface. Alterations to the ‘eat me’ signals have been well described; however, very little is known about the ‘don’t eat me' signals on the cell surface during apoptosis. In the present study, apoptosis of Jurkat cells was induced by treatment with topoisomerase II inhibitor etoposide, and then the CD31 and CD47 levels on the apoptotic cell surface and in microparticles were estimated by flow cytometry and immunoblotting methods in the presence of caspase, metalloproteinase, and Rho-associated coiled-coil containing protein kinase 1 (ROCK1) inhibitors. The CD31 and CD47 levels on the cell surface of apoptotic Jurkat cells had decreased after treatment with etoposide. These decreases in CD31 and CD47 levels on the apoptotic cell surface were almost completely suppressed by the caspase 3 inhibitor, Ac-DEVD-CHO, and partially suppressed by caspase 8 (Ac-IETD-CHO) and caspase 9 (Ac-LEHE-CHO) inhibitors but not by the metalloproteinase inhibitors GM6001 and TAPI-0. Microparticle counts in culture supernatants were higher during etoposide-induced apoptosis. The ROCK1 inhibitor, Y27632, suppressed blebbing formation and microparticle release. Moreover, flow cytometry and immunoblotting revealed CD31 and CD47 in the microparticles. These results indicate that CD31 and CD47 were released by the apoptotic Jurkat cells into the culture supernatant in microparticles, but not in soluble forms, resulting in decreased levels on the apoptotic cell surface.
The development of a simple, easy-to-use, and non-invasive vaccination system is in high demand. For transcutaneous immunization (TCI), we previously developed a hydrogel patch formulation that accelerates the penetration of an antigen (Ag) through the stratum corneum (SC) and effectively elicits Ag-specific immune responses. The present studies were performed to optimize the composition and assess the safety of the patch formulation. A hydrogel patch with a water content ratio of 5% more effectively induced an immune response compared to patches with a different composition, suggesting that the moisture content of the hydrogel patch formulation has optimal ratio for SC hydration to promote Ag penetration through the SC. TCI using a hydrogel patch induced few local and systemic adverse reactions. Based on these results, we are now advancing translational research to evaluate the safety and efficacy of our novel TCI system in humans.
The objectives of the present study were to assess pharmacokinetics, pharmacodynamics, tolerability and safety of intravenous administration of bivalirudin, a direct thrombin inhibitor, in healthy Chinese subjects. 48 subjects were equally divided into 4 groups (0.5 mg/kg, 0.75 mg/kg, 1.05 mg/kg intravenous bolus, and 0.75 mg/kg intravenous bolus followed by an infusion of 1.75 mg/kg per hour for 4 h) by a randomized, single-blind and placebo-controlled (bivalirudin groups: n=9/group; placebo groups: n=3/group) design. The safety observations showed that bivalirudin was well tolerated in the studied dose range, all adverse events were mild in severity. The half-life of bivalirudin was approximately 0.57 h (34 min), exposure increased in a dose-dependent manner. In group receiving a 0.75 mg/kg intravenous bolus followed by 1.75 mg/kg per hour infusion for 4 h, bivalirudin concentrations remained at 5000—5500 μg/l within the 4 h infusion period, which was similar to the reported data of Caucasian patients and can provide the desired anticoagulant effects. There was a strong correlation between bivalirudin concentration and anticoagulant effect. A Sigmoid model was used to fit the pharmacodynamic parameters activated clotting time (ACT), activated partial thromboplastin time (APTT) and prothrombin time (PT) and bivalirudin concentrations. The findings of this study suggest that the same dosing regimens of bivalirudin may be administered to Chinese and Caucasian patients. Ongoing and future studies in large populations may add further information.
We previously investigated the effects of an aqueous extract of maté (mate) tea, made from the leaves of Ilex paraguariensis, on the diabesity and metabolic syndrome features in a mouse model. Mate induced significant decreases in body weight (BW), body mass index, and food intake (FI). In this study, to verify the mode of action of mate on FI and consequently on BW, we examined the anorexic effects of mate on the appetite and satiety markers glucagon-like peptide 1 (GLP-1) and leptin in high-fat diet-fed ddY mice. GLP-1 is a peptide signal generated by the gastrointestinal tract, which regulates appetite and influences BW, whereas leptin is an afferent signal from the periphery to the brain in a homeostatic feedback loop that regulates adipose tissue mass, thus leading to decreased appetite and FI and increased energy expenditure. Chronic administration of mate (50, 100 mg/kg) for 3 weeks significantly reduced FI, BW, and ameliorated blood fats, liver fats, and adipose tissue. Mate induced significant increases in GLP-1 levels and leptin levels compared with the control. Acute administration of major constituents of mate showed significant increases in GLP-1 levels by dicaffeoyl quinic acids and matesaponins, and significant induction of satiety by caffeoyl quinic acids and caffeine in ddY mice. These findings suggest that mate may induce anorexic effects by direct induction of satiety and by stimulation of GLP-1 secretion and modulation of serum leptin levels.
The organotin trimethyltin (TMT) is well known to cause neuronal degeneration in the hippocampal dentate gyrus of mice. The first purpose of the present study was to examine whether the cyclooxygenase (COX) inhibitor indomethacin could ameliorate neuronal degeneration in the dentate gyrus of mice following TMT treatment in vivo. The systemic injection into mice of TMT at 2.8 mg/kg produced activation of endogenous caspase-3 and calpain, enhanced the gene expression of COX-1 and COX-2, activated microglial cells, and caused the formation of the lipid peroxidation product 4-hydroxynonenal in the hippocampus. Given at 12-h post-TMT treatment, the systemic injection of indomethacin (5 or 10 mg/kg, subcutaneously) significantly decreased the TMT-induced damage to neurons having active caspase-3 and single-stranded DNA in the dentate granule cell layer of the hippocampus. The results of the α-Fodrin degradation test revealed that the post-treatment with indomethacin was effective in attenuating TMT-induced activation of endogenous caspases and calpain in the hippocampus. In TMT-treated animals, interestingly, the post-treatment with indomethacin produced not only activation of microglial cells in the dentate gyrus but also the formation of 4-hydroxynonenal in the dentate granule cell layer. Taken together, our data suggest that COX inhibition by indomethacin ameliorated TMT-induced neuronal degeneration in the dentate gyrus by attenuating intensive oxidative stress.
Chaetoglobosin Fex (Cha Fex), a cytochalasan-based alkaloid, was isolated from marine-derived endophytic fungus Chaetomium globosum QEN-14. The knowledge of its biological function is still limited. We investigated the effects and mechanism of Cha Fex on inflammatory mediators via Toll-like receptor 4 (TLR4) signaling in macrophages. Lipopolysaccharide (LPS), TLR4 ligand, was therefore designed to active TLR4 signaling pathway, and Cha Fex significantly inhibited the LPS-induced production of tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in peritoneal macrophages and murine macrophage cell line RAW264.7. Real-time reverse transcription polymerase chain reaction (real-time RT-PCR) detection also found that Cha Fex down-regulated the mRNA expressions of these pro-inflammtory cytokines. Moreover, Cha Fex significantly attenuated the LPS-stimulated degradation of inhibitory kappa B-alpha and the subsequent translocation of the p65 subunit of nuclear factor-kappa B (NF-κB) to the nucleus. Cha Fex also reduced the phosphorylations of extracellular-signal-related kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK1/2). Furthermore, we confirmed that Cha Fex didn't affect LPS binding to the RAW264.7 cells and human monocytes, while Cha Fex was able to inhibit the increase of membrane-associated CD14 (mCD14) expression both on RAW cells and human monocytes induced by LPS to a certain degree. These results suggest that the anti-inflammatory property of Cha Fex may be attributed to NF-κB inhibition as well as the negative regulation of ERK1/2, p38, and JNK1/2 phosphorylations. On the other hand, these inhibitory effects may also be due to the blocking of mCD14 expression.
The formation of pleural effusion during pulmonary edema is an important physiological mechanism of resolution of alveolar flooding. In cases of pulmonary edema resulting from acute respiratory distress syndrome (ARDS) these effusions are exudative, having high protein load. To this end, the effect of salbutamol in the presence of protein, on the ion transport properties of the sheep parietal pleura was investigated by Ussing chamber experiments. Our results show that salbutamol increases ion transport in the presence of protein in sheep parietal pleura by stimulation of β2-adrenergic receptors since this effect was completely abolished by the specific β2-adrenergic blocker, ICI-118551. This finding may be of importance regarding the acceleration of the resolution of protein-rich pleural effusions occurring in cases of ARDS.
Gelsemium elegans BENTH and its crude extract are widely used to treat pain in China despite its apparent toxicity. The analgesic effects of gelsenicine, an active component of G. elegans, however, have not been reported. The current study examined potential analgesic effects of subcutaneously injected gelsenicine using acetic acid-induced writhing, formalin-induced nociceptive behavior, and thermal hyperalgesia caused by chronic constriction injury (CCI) in mice. Gelsenicine produced dose-dependent analgesic effects in both inflammatory and neuropathic pain models. The ED50, for either the inflammatory pain (10.4 μg/kg for writhing test, 7.4 μg/kg for formalin test) or neuropathic pain (9.8 μg/kg for thermal hyperalgesia caused by CCI model), was far below the LD50 (95% confidence interval at 100—200 μg/kg). Repeated subcutaneous injections of gelsenicine in CCI mice led to sustained attenuation of neuropathic pain after drug discontinuation. These results revealed that gelsenicine could be used safely to attenuate both inflammatory and neuropathic pain.
Ovalicin, a secondary metabolite produced by an entomopathogenic fungus Metarhizium anisopliae var. anisopliae, is currently used as an efficient biological control agent against various agricultural insect pests, but not so many biological activities have been investigated. To assess whether ovalicin has potential in ameliorating atopic dermatitis-related symptoms in mice, we first sensitized skin in the dorsal neck of Balb/c mice using compound 48/80, and scrutinized whether the compound affected the atopic dermatitis-related symptoms. The results revealed that ovalicin significantly reduces scratching behavior in a concentration-dependent fashion. Moreover, the treatment inhibits the levels of the degranulation of mast cells by 65%, and levels of histamine release by 51% at a concentration of 10 μg/ml. Together, the present data strongly suggest that ovalicin elicits potential anti-atopic activities in mice.
Human fibroblast growth factor 19 (FGF19) is an enterohepatic hormone that is involved in the regulation of hepatic metabolism of bile acids, lipids, and glucose. Farnesoid X receptor (Fxr)-null mice exhibit steatosis-like symptoms, showing higher hepatic lipid levels than with the wild-type mice. We investigated the influence of FGF19 treatment on hepatic lipogenesis in Fxr-null mice. Recombinant FGF19 treatment (400 μg/kg/d) for 3 d prevented the accumulation of lipid droplets and decreased serum alanine aminotransferase activity and hepatic lipid levels, including those of triglycerides and free fatty acids. The treatment significantly decreased the hepatic mRNA levels of acetyl-CoA carboxylase 1 (Acc1), Cd36, and sterol regulatory element-binding protein-1c (Srebp-1c) as well as those of acetyl-CoA carboxylase 2 (Acc2), stearoyl CoA desaturase 1 (Scd1), and Cyp7a1. FGF19 treatment (4 μg/kg/d) for 3 d also decreased the hepatic free fatty acid levels and mRNA levels of Acc1, Cd36, and Srebp-1c. These results indicate that FGF19-mediated signaling ameliorates disrupted hepatic lipogenesis in Fxr-null mice.
In this study, using a special diet-induced mouse model of atopic dermatitis, we tested the effect of chitosan-containing lotion (CL) on itch-related scratching associated with barrier-disrupted dry skin. HR-1 hairless mice fed a special diet exhibited apparent dry skin symptoms characterized by decreased skin hydration and increased transepidermal water loss. In the special diet-fed mice, scratching behavior was markedly enhanced for 60 min after oral administration of ethanol. When CL was applied once immediately after ethanol administration, the enhanced scratching response was significantly suppressed, but this effect was abolished within 30—40 min; when applied twice immediately and at 30 min, CL almost completely blocked scratching throughout 60 min. Comparison of CL and the chitosan-free vehicle showed that CL inhibited scratching more strongly and persistently than the vehicle, which transiently suppressed scratching only for 10 min after application. Although the decreased skin hydration was improved even by the vehicle, the increased transepidermal water loss was resolved only by CL. Skin surface temperature was much more reduced in CL-treated mice than in vehicle-treated mice. Collectively, CL has an antipruritic effect, which could be partly explained by recovery of skin barrier function and cooling of the skin.