The emergence of drug resistance is a major obstacle to the successful pharmacological treatment of cancer. Tumor heterogeneity is one of the key factors underlying drug resistance. Cancer cell heterogeneity in tumors is caused by genetic mutation and by the existence of cancer stem cells (CSCs). CSCs are defined as a subpopulation of highly tumorigenic cancer cells with self-renewal activity. It has been reported that various types of cancer involve CSCs, and that CSCs are generally resistant to anticancer drugs. Therefore, CSC-targeting agents could allow for more effective pharmacological treatment of cancer. Using a comprehensive gene expression study and functional genomic approach, we are trying to identify CSC-specific survival factors, as well as candidate compounds that interfere with CSC-selective survival signaling. These CSC-targeting drugs could be promising new therapeutic agents which would suppress the emergence of drug-resistant cells and enhance the effect of antitumor agents.
Aberrant activation of Wnt/β-catenin canonical signaling is observed in multiple malignant tumors, and is recognized as an attractive therapeutic target for molecular targeted drugs. This signaling pathway is also involved in maintaining pluripotency in adult stem cells. Therefore, lowering potential stem cell toxicity is a key factor for the development of a Wnt/β-catenin signaling inhibitor. Here, we show Wnt/β-catenin pathway inhibitors with low toxicity, identified through phenotype-based screening using zebrafish embryos. Artificial activation of the Wnt/β-catenin pathway in fertilized eggs, which are often considered the “ultimate stem cells”, results in an “eyeless” phenotype in zebrafish embryos. Screening for compounds that rescue this “eyeless” phenotype and have no effects on normal embryogenesis could help us identify Wnt/β-catenin pathway inhibitors with minimal stem cell toxicities, at least at a concentration that suppresses aberrant signaling. Chemical suppressors of the “eyeless” phenotype include novel and known compounds with different modes of action. Some of these compounds diminish the activation of crosstalk between other signaling pathways and the Wnt/β-catenin pathway. These inhibitors reduced tumor growth in ApcMin/+ mice and did not show apparent toxicities. Thus, our screening for chemical suppressors of the “eyeless” phenotype allowed us to successfully identify inhibitors for the Wnt/β-catenin pathway with low toxicity.
The extracellular matrix (ECM) provides a variety of biological signals for cell regulation. It is apparent that some of these signals are derived from functional sites, which are concealed in the higher structure of ECM protein molecules. Previously, we found that fibronectin, a ubiquitous cell adhesive ECM protein, harbors a cryptic functional site termed FNIII14, which can be exposed through the processing of fibronectin by inflammatory proteinases, including matrix metalloproteinase-2 (MMP-2). FNIII14, once exposed, induces a conformational change in beta1-integrins necessary for their functional inactivation, resulting in weakened cell adhesion to the ECM. Interestingly, eukaryotic peptide elongation factor 1A (eEF1A) was recently identified as a membrane receptor mediating this anti-adhesive effect of FNIII14. Here, we show that exposure of FNIII14 from the fibronectin matrix, and its interaction with the membrane receptor eEF1A, contributes to the migration and invasion of cancer cells. Furthermore, an in vivo experiment using a mouse xenograft model shows that FNIII14 could be a promising target for preventing lung metastasis of melanoma.
Receptor tyrosine kinases (RTKs) are known to be key regulators of cancer cell proliferation, migration, invasion and metastatic spread. Ligand-binding to the extracellular domain triggers canonical activation of the intracellular tyrosine kinase domain. In contrast, it has become evident that RTKs are also regulated by non-canonical tyrosine kinase-independent mechanisms via phosphorylation of their serine/threonine residues. In this review, I mainly introduce our recent findings on the non-canonical regulation of epidermal growth factor receptor (EGFR), ErbB2 and erythropoietin-producing hepatocellular receptor A2 (EphA2), and discuss the roles of non-canonical activation of RTKs in cancer progression and resistance to targeted cancer agents. Further characterization of non-canonical regulation will contribute to the development of new target cancer therapies.
Multidrug resistance (MDR) in cancer is a major problem in clinical settings: MDR correlates with a patient's poor prognosis and decreased quality of life. Recently, MDR was found to be involved in various signal pathways, so the inhibition of signal molecules by molecular targeting drugs may help overcome MDR. In addition, the acquisition of MDR is shown to be associated with the overexpression of drug efflux pumps such as P-glycoprotein (MDR1), which in turn affects the regulation of the expression of cell survival factors, B-cell leukemia protein 2 (Bcl-2) family proteins, etc. We analyzed the mechanisms of MDR in hematopoietic malignancies, and showed that the activation of signaling molecules regulated the expression of drug efflux pumps and cell survival factors, thus suggesting that molecular targeting drugs are potentially useful as anti-MDR agents. In this review, I focus on recent advancements in understanding the mechanisms of MDR with respect to hematopoietic malignancies: (1) exploration of molecular targets for overcoming MDR in anti-cancer drug-resistant cell lines, (2) the mechanism of drug resistance through the cytokine autocrine loop, and (3) cell-cell interaction with bone marrow stromal cells, along with the application of molecular targeting drugs for overcoming MDR.
Drug resistance is a critical problem inhibiting the effective use of targeted molecular cancer therapies. Investigators have revealed a variety of resistance mechanisms, including alterations in drug targets, activation of pro-survival pathways, and the ineffective induction of cell death. The key alterations driving this resistance are likely condition-dependent, and a detailed analysis would be required to characterize these diverse alterations under a variety of conditions in order to facilitate practical precision medicine for treating individual cancer patients. We have been investigating the molecular mechanisms of anti-cancer drug resistance, and analyzed our original resistant cells against anti-mitotic kinase inhibitors. This study suggests that novel mechanisms reduce cytokinetic dysregulation caused by those inhibitors, and anti-apoptotic activities are associated with resistant phenotypes. These observations suggest that the activation of various bypass mechanisms may allow cancer cells to avoid the selective antiproliferative effect of molecularly targeted drugs, and such bypass activation mechanism would thus be a critical target for designing combination chemotherapy to overcome non-genetic drug resistance.
Kampo medicine is an original traditional medicine in Japan. Currently, 148 ethical Kampo formulations (Kampo prescription drugs) are registered in the National Health Insurance Price List. Kampo medicines can be prescribed under the national insurance system, which shows that they are part of conventional medicine in Japan. Japan has a unified drug approval system that does not distinguish between Western and Kampo medicines, and both are subject to the same regulations. The application for the market approval of ethical Kampo medicines is based on the general notification for drugs, i.e., “Handling of Ethical Combination Drugs” in “Precautions Necessary When Applying for Drug Marketing Approval” (Yakushokushinsa Notification No. 1121-12 of November 21, 2014). Furthermore, applications for the market approval of ethical Kampo medicines should follow the Kampo-specific notification of “Handling of Ethical Kampo Medicines” (Yakushin Notification No. 804 of June 25, 1980). Data from comparative studies with standard decoctions must be submitted with approval applications according to Yakushin 2 Notification No. 120 of May 31, 1985. The safety, efficacy, and quality of Kampo medicines are comprehensively assured by the Japanese Pharmacopoeia, Good Manufacturing Practice, Good Agricultural and Collection Practices, marketing approval certificate, approval standard, and pharmacovigilance. I believe that the basic framework for the market approval of ethical Kampo medicines has been established as described above. The key factors for the practical application of superior manufacturing technology and research achievements and the promotion of drug development are the specific guidelines for the approval of drugs of herbal origin.
Ephedra Herb (Japanese name: mao) is defined as the terrestrial stem of Ephedra species. It is one of the most important crude drugs in Kampo prescriptions such as maoto, kakkonto, and shoseiryuto. The traditional biological properties of Ephedra Herb are mainly attributable to ephedrine alkaloids. Recently, our research group has found that Ephedra Herb extract also shows alkaloid-independent pharmacological action; therefore, we focused on polyphenols, other active ingredients of Ephedra Herb extract. Reports on the constituents of the extract other than ephedrine alkaloids are limited, although condensed tannins of procyanidin A-type have been found in some Ephedra species. To determine the components associated with the antitumor activity of Ephedra Herb extract, we investigated the phenolic compounds in the extract and characterized 10 compounds, including the new compound herbacetin 7-O-neohesperidoside, together with catechins and flavonoids with an apigenin or a herbacetin skeleton, and phenylpropanoids such as syringin. Of the 10 compounds, isovitexin 2″-O-rhamnoside was obtained in the greatest yield. Here, we describe the elucidation of part of the polyphenolic fraction from Ephedra Herb extract.
Ephedra Herb is a crude drug for the treatment of headache, bronchial asthma, nasal inflammation, and the common cold. Although it has been considered that ephedrine alkaloids (EAs) are the principal active ingredients of Ephedra Herb, EAs are known to induce palpitations, hypertension, insomnia, and dysuria as major side effects. Therefore, the administration of EAs-containing drugs to patients with cardiovascular-related diseases is strongly contraindicated. Previously, we isolated herbacetin 7-O-neohesperidoside from Ephedra Herb. In addition, we found that herbacetin, a flavonoid aglycone in Ephedra Herb, had antiproliferative and analgesic effects. Therefore, the prospect of preparing safer natural medicines without the adverse effects associated with EAs was appealing. In this symposium review, to achieve the aim of producting a clinically useful Ephedra Herb extract with none of the adverse effects associated with EAs, I present an efficient preparation method of EAs-free Ephedra Herb extract, together with its chemical composition, antiproliferative effects, and a putative marker for quality control.
Ephedra Herb is defined in the 17th edition of the Japanese Pharmacopoeia as the terrestrial stem of Ephedra sinica STAPF., Ephedra intermedia SCHRENK et C.A. MEYER, or Ephedra equisetina BUNGE (Ephedraceae) which contains more than 0.7% ephedrine alkaloids (ephedrine and pseudoephedrine). The primary effects and adverse effects of Ephedra Herb are traditionally believed to be mediated by ephedrine alkaloids. We recently reported that Ephedra Herb extract (EHE) exhibits antimetastatic and antitumor effects by suppressing the hepatocyte growth factor-c-Met signaling pathway through the inhibition of c-Met tyrosine kinase activity. We confirmed that the non-alkaloidal fraction of EHE had c-Met-inhibitory activity. Moreover, we discovered herbacetin glycosides in EHE and demonstrated that herbacetin, the aglycone of the glycosides, shows c-Met-inhibitory activity and analgesic action. These findings suggest that some pharmacological actions of EHE may be produced by its non-alkaloidal fraction, which does not cause the adverse effects of ephedrine alkaloids. Therefore, we prepared ephedrine alkaloids-free EHE (EFE) by removing ephedrine alkaloids from EHE using ion-exchange column chromatography. EFE had c-Met-inhibitory action, analgesic effects, and antiinfluenza activity similar to EHE but had no toxicity. Now, we are evaluating the safety of EFE in healthy volunteers and its efficacy in patients to obtain licensing approval for its therapeutic use in the future.
Ephedra Herb is classified “pungent, slightly bitter, and warm” in tastes and natures, and is used to provide warmth to the body, dispel coldness, remove dampness, and reduce pain. Similar herbs are “pungent and hot” chili peppers, “pungent and hot” evodia fruit,” “pungent and warm” ginger, “pungent and hot” processed ginger, “pungent and hot” Zanthoxylum fruit, etc. These herbs are prescribed to provide heat to the outer or inner body. Some pungent components such as capsaicin, evodiamine, gingerol, and shogaol are known to be activators of transient receptor potential vanilloid 1 (TRPV1). TRPV1, a pain receptor, is activated in response to irritant chemicals such as capsaicin and high heat (>43℃) and strongly acidic conditions (pH<6). The typical TRPV1 activator capsaicin has various effects such as improvement of peripheral circulation, enhancement of thermogenesis, and pain relief. These effects are commonly observed for the “pungent and hot/warm” herbs, suggesting that TRPV1 stimulation plays an important part in their pharmacological action. In this study, we demonstrated that Ephedra Herb extract (EHE) shows strong TRPV1 activation, although ephedrine didn't show such effects. Both EHE and ephedrine alkaloids-free EHE (EFE) expressed similar analgesic action following oral administration, suggesting the presence of active components other than ephedrine alkaloids. Furthermore, EFE did not show side effects such as loss of sleep and irregular heartbeat in mice. Caution needs to be exercised while prescribing Ephedra Herb because it contains ephedrine. The application of EFE in Kampo medicine might be a better alternative in some cases.
We aimed to test the clinical usefulness of ephedrine alkaloids-free Ephedra Herb extract (EFE), which has been proven to be effective and safe in animal studies. First, we performed a safety trial with 7 healthy volunteers, and increased white blood cell counts were observed in 2. However, it was unknown whether this observation was a result of EFE administration. Then, we implemented a controlled, double-blind, randomized crossover trial as the second safety trial for EFE. The trial was conducted under strict conditions, and the participants were managed in the hospital and monitored using an increased number of endpoints. Twelve healthy volunteers were randomly allocated to group 1, who were administered EFE first, or group 2, who were administered Ephedra Herb first. In both groups, the drug was administered for 6 d. After a 4-week washout period with no drug administration, each group was administered the alternate drug for 6 d. In the third stage, a dose-response trial is planned to test the effectiveness and safety of EFE on chronic joint pain. Patients with chronic knee joint pain will be randomly allocated to three groups, and each group will be administered EFE prepared from different amounts of Ephedra Herb. Until this stage, the studies will be conducted as investigator-initiated clinical trials. In the fourth stage, industry-sponsored clinical trials will be planned. This multicenter trial will involve 300 patients with joint pain, neuralgia, and/or muscle pain.
An increasingly large number of pharmacological and physiological works on fatty acids have shown that the functional properties of fatty acids are regulated by the amount of individual fatty acid intake and the distribution of fatty acids among organs. Recently, it has been determined that G-protein-coupled receptor 40/free fatty acid receptor 1 (GPR40/FFA1) is activated by long-chain fatty acids, such as docosahexaenoic acid (DHA). GPR40/FFA1 is mainly expressed in the β cell of the pancreas, spinal cord and brain. It is reported that this receptor has a functional role in controlling blood glucose levels via the modulation of insulin secretion. However, its physiological function in the brain remains unknown. Our previous studies have shown that GPR40/FFA1 is expressed in pro-opiomelanocortin (POMC)-positive neurons of the arcuate nucleus, serotonergic neurons in the nucleus raphe magnus, and in noradrenergic neurons in the locus coeruleus. Furthermore, the intracerebroventricular injection of DHA or GW9508, which is a selective GPR40/FFA1 agonist, attenuates formalin-induced inflammatory pain behavior through increasing β-endorphin release in the hypothalamus. It also suppresses complete Freund's adjuvant-induced mechanical allodynia and thermal hyperalgesia. Our findings suggest that brain free long-chain fatty acids-GPR40/FFA1 signaling might have an important role in the modulation of endogenous pain control systems. In this review, I discuss the current status and our recent study regarding a new pain regulatory system via the brain long chain fatty acid receptor GPR40/FFA1 signal.
The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, and the nucleoprotein (NP) interact with the genomic RNA of influenza viruses and form ribonucleoproteins. Especially, the PB2 subunit binds to the host RNA cap [7-methylguanosine triphosphate (m7GTP)] and supports the endonuclease activity of PA to “snatch” the cap from host pre-mRNAs. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is necessary for interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m7GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m7GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of 2 or 3 amino acid residues of the VRG site to alanine significantly reduced PB2's binding ability to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication. I will also discuss some novel functions of NP in this review.
Recently, the investigation of bioactive constituents from natural products has been performed extensively around the world, yet many plants remain to be studied, even now. The author is especially interested in saponins, which are included in various natural products and Kampo medicines. Based on these study aims, we isolated the chemical constituents of Dianthus japonicus and Clethra barbinervis. The aerial parts of Dianthus japonicus were isolated into nine new (1-9) and seven known oleanane-type triterpene saponins. The structural elucidation of their chemical constituents was examined by several spectroscopic methods. Two new C-glycosyl flavones, two glycosidic derivative of anthranilic acid amides and a maltol glucoside were also isolated from this plant. The isolated compounds were evaluated according to cytotoxic inhibition toward A549 cell lines. Compounds 7-9 showed moderate inhibitory activity. The isolation of a MeOH extract of Clethra barbinervis leaves led to three new triterpene glucosides, i.e. an ursane (ryobunin A, 22), a seco-ursane (ryobunin B, 23) and an oleanane-type glucoside (ryobunin C, 24), along with four known compounds.
Radiotherapy (RT) and chemoradiotherapy (CRT) is widely accepted as the standard treatment for head and neck cancer (HNC). Oral mucositis (OM) often develops as an adverse reaction in HNC patients that receive RT or CRT involving S-1. However, little is known about the risk factors for OM in HNC patients. We retrospectively evaluated patients' pre-treatment clinical data in order to identify the risk factors for severe OM in HNC patients that are treated with RT or CRT involving S-1. We analyzed the cases of 129 patients who received RT or CRT involving S-1 for HNC. The endpoint of the survey was the occurrence of severe OM (≥grade 2). Risk factors that were significantly related to severe OM were identified using logistic regression analysis. The patients' mean age was 69.3±10.1 years, and 118 (92%) of the patients were male. The primary tumor was located in the oropharynx in 21.7% of cases. Severe OM occurred in 85.0% of cases. In the univariate analysis, the following variables were found to be associated with severe OM: age, the type of radiotherapy, disease stage, and chemotherapy. In the multivariate analysis, the location of the primary tumor and chemotherapy were identified as significant risk factors that contributed independently to the risk of severe OM (p<0.05). Our analysis suggests that cancer of the oropharynx and CRT are important risk factors for severe OM in HNC patients that undergo RT or CRT involving S-1.
To enable community pharmacists to provide empathic patient counseling, we developed and validated a training program based on cognitive reframing, which is one of the cognitive behavioral therapies. We divided 24 community pharmacists into two groups, providing training to the intervention group. The duration of the training program was two hours per session, with a total of eight hours. We conducted a survey of the intervention group to evaluate their training experience. In addition, we performed two role-play scenarios on patient counseling using simulated patients, evaluating the patient counseling alliance scores and the degrees of the psychological distance between the patients and pharmacists. The degree of satisfaction correlated with four training items, including “explanation by comics”. When pharmacists felt that the cognitive behavioral therapy approach was successful, no significant differences were found in the patient counseling alliance grades. However, the psychological distance between the patients and pharmacists was smaller. We were able to infer that a cognitive behavioral therapy approach could decrease the psychological distance between patients and pharmacists, thereby enabling empathic patient counseling.
Cancer patients often develop mental conditions, including anxiety and depressive disorder. And, patients with chemotherapy often exhibit mental changes including anxiety and depressive disorder. Furthermore, it is well known that the frequency of delirium is increased in cancer patients receiving terminal care. Thus, the psychiatric and cognitive functions of cancer survivors are significantly influenced by their quality of life. In this study, we performed a retrospective analysis to identify the risk factors for psychiatric disorders in lung cancer survivors. Most lung cancer survivors that were diagnosed with psychiatric disorders had stage 4 disease. In addition, it was found that an increase in disease stage and high doses of opioids are associated with an increased risk of psychiatric disorders in lung cancer survivors. These findings suggest that it is necessary to consider the mental changes experienced by lung cancer patients during disease progression.