Intravenous administration of human mesenchymal stem cells derived from adipose tissue and umbilical cord improves neuropathic pain via suppression of neuronal damage and anti-inflammatory actions in rats.

Mesenchymal stem cells (MSCs), which are isolated from adipose tissue (AD-MSCs), umbilical cord (UC-MSCs), or bone marrow, have therapeutic potential including anti-inflammatory and immunomodulatory activities. It was recently reported that MSCs are also effective as a therapeutic treatment for neuropathic pain, although the underlying mechanisms have yet to be resolved. Therefore, in this study, we investigated the effects of human AD- and UC-MSCs on neuropathic pain and its mechanisms using rat models of partial sciatic nerve ligation (PSNL). AD- or UC-MSCs were intravenously administered 4 days after PSNL. Antinociceptive effects were then evaluated using the von Frey and weight-bearing tests. We found that, 3-9 days after the administration of AD- or UC-MSCs to PSNL-exposed rats, both the mechanical threshold and differences in weight-bearing of the right and left hind paws were significantly improved. To reveal the potential underlying antinociceptive mechanisms of MSCs, the levels of activation transcription factor 3- and ionized calcium-binding adapter molecule 1-positive cells were measured by immunohistochemical analysis. AD- and UC-MSCs significantly decreased the levels of these proteins that were induced by PSNL in the dorsal root ganglia. Additionally, UC-MSC significantly improved the PSNL-induced decrease in the myelin basic protein level in the sciatic nerve, indicating that UC-MSC reversed demyelination of the sciatic nerve produced by PSNL. These data suggest that AD- and UC-MSCs may help in the recovery of neuropathic pain via the different regulation; AD-MSCs exhibited their effects via suppressed neuronal damage and anti-inflammatory actions, while UC-MSCs exhibited their effects via suppressed neuronal damage, anti-inflammatory actions and remyelination.

HepG2-NIAS cells, a new subline of HepG2 cells that can enhance not only CYP3A4 activity but also expression of drug transporters and form bile canaliculus-like networks by the oxygenation culture via a collagen vitrigel membrane.

We reported the enhanced liver-specific function and structure of HepG2 cells by the oxygenation culture via a collagen vitrigel membrane (CVM). The cells were conditioned in our laboratory for a long period, so their characteristics may change from the original HepG2 cells registered in RIKEN cell bank (RCB) with the number of 1648 (HepG2-RCB1648 cells). We named the conditioned HepG2-RCB1648 cells in our laboratory as HepG2-NIAS cells. Here, we clarified the features of HepG2 cells with three different culture histories by analyzing their morphology and viability, CYP3A4 activity, the potential to form bile canaliculus-like structures, and the expression of drug transporters. On plastic, HepG2-NIAS cells grew as a monolayer without the formation of large aggregates involving dead cells that were observed in HepG2-RCB1648 cells and HepG2-RCB1886 cells. In the oxygenation culture via a CVM, the CYP3A4 activity of HepG2-NIAS cells increased to almost half level in direct comparison to that of differentiated HepaRG cells cultured on a collagen-coated plate; however, that of HepG2-RCB1648 cells and HepG2-RCB1886 cells was almost not detected. HepG2-NIAS cells formed bile canaliculus-like networks in which fluorescein was accumulated after the exposure of fluorescein diacetate, although HepG2-RCB1648 cells and HepG2-RCB1886 cells did not possess the potential. Also, immunohistological observations revealed that HepG2-NIAS cells remarkably enhanced the expression of drug transporters, NTCP, OATP1B1, OATP1B3, BSEP, MDR1, MRP2, and BCRP. These results suggest that HepG2-NIAS cells are a new subline of HepG2 cells useful for drug development studies. HepG2-NIAS cells were registered in RCB with the number of 4679.

Metabolome Shift in Both Metastatic Breast Cancer Cells and Astrocytes Which May Contribute to the Tumor Microenvironment.

The role of astrocytes in the periphery of metastatic brain tumors is unclear. Since astrocytes regulate central nervous metabolism, we hypothesized that changes in astrocytes induced by contact with cancer cells would appear in the metabolome of both cells and contribute to malignant transformation. Coculture of astrocytes with breast cancer cell supernatants altered glutamate (Glu)-centered arginine-proline metabolism. Similarly, the metabolome of cancer cells was also altered by astrocyte culture supernatants, and the changes were further amplified in astrocytes exposed to Glu. Inhibition of Glu uptake in astrocytes reduces the variability in cancer cells. Principal component analysis of the cancer cells revealed that all these changes were in the first principal component (PC1) axis, where the responsible metabolites were involved in the metabolism of the arginine-proline, pyrimidine, and pentose phosphate pathways. The contribution of these changes to the tumor microenvironment needs to be further pursued.

The Japanese Herbal Medicine Hangeshashinto Induces Oral Keratinocyte Migration by Mediating the Expression of CXCL12 Through the Activation of Extracellular Signal-Regulated Kinase.

Several clinical studies have reported that Japanese herbal medicine Hangeshashinto (HST) has beneficial effects on chemotherapy-induced oral ulcerative mucositis (OUM). Our previous research demonstrated that HST improves chemotherapy-induced OUM through human oral keratinocyte (HOK) migration, which was suppressed by mitogen-activated protein kinase (MAPK) and C-X-C chemokine receptor 4 (CXCR4) inhibitors. However, the association between these molecules and HOK migration was unclear. Here, we examined the effects of HST on the expression of CXCR4/CXCR7 and C-X-C motif chemokine ligands 11 and 12 (CXCL11/CXCL12) in HOKs. Our results indicated that HST upregulated CXCL12, but not CXCR4, CXCR7, nor CXCL11 in HOKs. HST-induced expression of CXCL12 was significantly suppressed by an inhibitor of extracellular signal-regulated kinase (ERK), but not of p38 and c-Jun N-terminal kinase (JNK). In addition, HST induced phosphorylation of ERK in HOKs. These findings suggest that HST enhances HOK migration by upregulating CXCL12 via ERK.

Author Correction: The Japanese herbal medicine Hangeshashinto enhances oral keratinocyte migration to facilitate healing of chemotherapy-induced oral ulcerative mucositis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Novel microvascular endothelial model utilizing a collagen vitrigel membrane and its advantages for predicting histamine-induced microvascular hyperpermeability.

INTRODUCTION: The evaluation of microvascular permeability is crucial for drug development. Nonetheless, there are few reliable test methods in vitro due to the lack of vascular endothelial models suitable for quantitative analyses. The purpose of this study is to construct a novel microvascular endothelial model with the high endothelial barrier function and sensitivity to physiological stimuli utilizing a collagen vitrigel membrane (CVM) composed of high-density collagen fibrils. METHODS: Human microvascular endothelial cells (HMVECs) were cultured for 6 days in a CVM chamber with or without human dermal fibroblasts (HDFs) cocultured on the reverse surface of the CVM. The endothelial barrier function was evaluated by measurement of transendothelial electrical resistance (TEER) and macromolecular permeation. RESULTS: The TEER value of a monolayer of HMVECs cultured on the CVM was 15-20 Ωï½¥cm2 during the culture period while it reached over 60 Ωï½¥cm2 by coculturing with HDFs. The TEER value was decreased from 5.7 to 3.4 Ωï½¥cm2 by 100 µM histamine in the monolayer model and from 50 to 32 Ωï½¥cm2 by 1 nM histamine in the coculture model, respectively. Interestingly, the permeability coefficient of the compound with a molecular weight of not 376 and 40,000 but 4000 was selectively increased in the histamine-treated coculture model. DISCUSSION: HMVECs cocultured with HDFs via a CVM formed the tight endothelial barrier and showed high responsivity to histamine. The model might be useful for exploring molecules that modulate microvascular permeability and pass through the microvascular endothelial barrier.

A novel method for evaluating activity of transient receptor potential channels using a cellular dielectric spectroscopy.

Cellular dielectric spectroscopy (CDS) is a novel technology enabling pharmacological evaluation of multiple receptor types with a label-free cell-based assay. We evaluated activities of a family of ligand-gated channels, transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels by an electrical impedance-based biosensor (CellKey™ system) using CDS. Measures of both potency (EC50) and efficacy (Emax) of these agonists with CellKey™ were almost identical to those made using the traditional Ca2+ influx assay in TRPV1- or TRPA1-expressing cells, suggesting that CellKey™ is a simpler and easier means of evaluating TRP activities.

The Japanese herbal medicine Hangeshashinto enhances oral keratinocyte migration to facilitate healing of chemotherapy-induced oral ulcerative mucositis.

Chemotherapy often induces oral ulcerative mucositis (OUM) in patients with cancer, characterized by severe painful inflammation. Mouth-washing with the Japanese herbal medicine hangeshashinto (HST) ameliorates chemotherapy-induced OUM in patients with colorectal cancer. Previously, we demonstrated that HST decreased interleukin 1ß-induced prostaglandin E2 production in human oral keratinocytes (HOKs) and OUM-induced mechanical or spontaneous pain in rats. However, HST effects on tissue repair functions in HOKs remain unclear. Here, we examined the effects of HST on scratch-induced wound healing in vitro and in vivo. In vitro, HST enhanced wound healing mainly through scratch-induced HOK migration. Screening of the seven constituent medicinal herbs and their major components revealed that Scutellaria root, processed ginger, and Glycyrrhiza components mainly induced the scratch-induced HOK migration. Pharmacokinetic analyses indicated that the active ingredient concentrations in rat plasma following oral HST administration were below the effective doses for HOK migration, suggesting direct effects of HST in OUM. Mitogen-activated protein kinase and C-X-C chemokine receptor 4 inhibitors significantly suppressed HST-induced HOK migration. Moreover, HST enhanced tissue repair in our OUM rat model. Thus, HST likely enhanced OUM tissue repair through oral keratinocyte migration upon MAPK and CXCR4 activation and may be useful in patients with cancer-associated OUM.

Possible biased analgesic of hydromorphone through the G protein-over ß-arrestin-mediated pathway: cAMP, CellKey™, and receptor internalization analyses.

Morphine, fentanyl, and oxycodone are widely used as analgesics, and recently hydromorphone has been approved in Japan. Although all of these are selective for µ-opioid receptors (MORs) and have similar structures, their analgesic potencies and adverse effects (AEs) are diverse. Recent molecular analyses of MOR signaling revealed that the G protein-mediated signaling pathway causes analgesic effects and the ß-arrestin-mediated signaling pathway is responsible for AEs. We used several cell-based analyses that selectively measure cellular responses activated by either G protein- or ß-arrestin-mediated pathways. GloSensor™ cAMP, CellKey™, and receptor internalization assays were performed with four different types of cells stably expressing differentially labelled MOR. EC50 values measured by cAMP and CellKey™ assays had potencies in the order fentanyl ≤ hydromorphone < morphine ≤ oxycodone, all also exhibiting full agonist responses. However, in the internalization assay, only fentanyl elicited a full agonist response. Hydromorphone had the strongest potency next to fentanyl; however, contribution of the ß-arrestin-mediated pathway was small, suggesting that its effect could be biased toward the G protein-mediated pathway. Based on these properties, hydromorphone could be chosen as an effective analgesic.

A novel strategy for treatment of cancer cachexia targeting xanthine oxidase in the brain.

Cancer cachexia is a systemic wasting syndrome characterized by anorexia and loss of body weight. The xanthine oxidase (XO) inhibitor febuxostat is one of the promising candidates for cancer cachexia treatment. However, cachexic symptoms were not alleviated by oral administration of febuxostat in our cancer cachexia model. Metabolomic analysis with brains of our cachexic model showed that purine metabolism was activated and XO activity was increased, and thus suggested that febuxostat would not reach the brain. Accordingly, targeting XO in the brain, which controls appetite, may be an effective strategy for treatment of cancer cachexia.

Trichostatin A modulates cellular metabolism in renal cell carcinoma to enhance sunitinib sensitivity.

Although sunitinib is the first-line drug for progressive renal cell carcinoma (RCC), most patients experience its tolerance. One possible way of overcoming drug resistance is combination therapy. Epigenetic modifier is one of the candidate drug group. A recent evidence suggests that cell metabolism is regulated by epigenetic mechanisms. Epigenetic abnormalities lead to changes in metabolism and may contribute to drug resistance and progression of RCC. Consequently, we investigated whether trichostatin A (TSA), a potent histone-deacetylase (HDAC) inhibitor, alters sunitinib-induced cytotoxicity and metabolism in RCC cells at epigenetic regulatory concentrations. Combined metabolome and transcriptome analysis suggested that TSA impacts on energy productive metabolic pathways, such as those involving TCA cycle and nucleotide metabolism especially for increase of hyperphosphorylated form. Combination of sunitinib and TSA increased cell death with PARP cleavage, an early marker of mitochondrial apoptosis, whereas receptor tyrosine kinase signaling, which is the target of sunitinib, was not altered by TSA. Finally, the established sunitinib resistant-RCC cell (786-O Res) was also exposed to sunitinib and TSA combination, resulting in significant growth inhibition. In summary, it was suggested that TSA reduces sunitinib resistance by triggering intracellular metabolome shifts regarding energy metabolism, that is the first recognized mechanism as an HDAC inhibitor.

Leukemia inhibitory factor via the Toll-like receptor 5 signaling pathway involves aggravation of cachexia induced by human gastric cancer-derived 85As2 cells in rats.

Cancer cachexia is highly prevalent in gastric cancer patients and characterized by decreased food consumption and body weight. We previously created a rat model of cancer cachexia using MKN45cl85 and 85As2 cells derived from human gastric cancer. The 85As2 cells induced cachexia more potently compared to MKN45cl85 cells. To clarify the mechanism underlying the difference in the cachexia-inducing ability of these cells, we conducted DNA microarray analysis, focusing on cell proliferation and the production of leukemia inhibitory factor (LIF), a cachexia-inducing factor. The plasma human LIF levels of 85As2-induced cachexic rats increased as symptoms worsened, whereas the plasma levels of MKNcl85 were low. 85As2 cells displayed more genetic changes compared to MKN45cl85 cells, which were related to Toll-like receptor (TLR) 4/5 signaling. Stimulation of both cells with TLR4 (lipopolysaccharide) or TLR5 (flagellin) agonists did not affect proliferation. However, in 82As2 cells, LIF production was significantly increased by stimulation with TLR5, which was suppressed by an inhibitor of interleukin-1 receptor-associated kinase-1/4, which are important factors in the TLR5 signaling pathway. The increase in LIF production resulting from activation of the TLR5 signaling pathway may contribute to the cachexia-inducing ability of 85As2 cells.

Multifunctional Actions of Ninjinyoeito, a Japanese Kampo Medicine: Accumulated Scientific Evidence Based on Experiments With Cells and Animal Models, and Clinical Studies.

Herbal medicines are currently employed for the treatment of several types of diseases, and also employed for the improvement of Quality of Life (QOL) of patients over the world, in particular, in Asian countries. In Japan, a Japanese herbal medicine namely kampo medicine has been prescribed for the improvement of QOL of patients. Ninjinyoeito (NYT), composed of 12 herbal plants, is one of kampo medicines and used for helping recovery of diseases and improving several symptoms that suffer patients such as anemia, anorexia and fatigue. Recent scientific research approaches to kampo medicines with cells and animal models enable to prove that NYT has multiple functions for improvement of symptoms. Also, clinical studies using NYT support such actions to be widely used for the improvement of symptoms that reduce the QOL of patients.

Modulation of synaptic inputs in magnocellular neurones in a rat model of cancer cachexia.

In cancer cachexia, abnormal metabolism and neuroendocrine dysfunction cause anorexia, tissue damage and atrophy, which can in turn alter body fluid balance. Arginine vasopressin, which regulates fluid homeostasis, is secreted by magnocellular neurosecretory cells (MNCs) of the hypothalamic supraoptic nucleus. Arginine vasopressin secretion by MNCs is regulated by both excitatory and inhibitory synaptic activity, alterations in plasma osmolarity and various peptides, including angiotensin II. In the present study, we used whole-cell patch-clamp recordings of brain slices to determine whether hyperosmotic stimulation and/or angiotensin II potentiate excitatory synaptic input in a rat model of cancer cachexia, similar to their effects in normal (control) rats. Hyperosmotic (15 and 60 mmol L-1   mannitol) stimulation and angiotensin II (0.1 µmol L-1 ) increased the frequency, but not the amplitude, of miniature excitatory postsynaptic currents in normal rats; in model rats, both effects were significantly attenuated. These results suggest that cancer cachexia alters supraoptic MNC sensitivity to osmotic and angiotensin II stimulation.

Conflicting Roles of Connexin43 in Tumor Invasion and Growth in the Central Nervous System.

The tumor microenvironment is known to have increased levels of cytokines and metabolites, such as glutamate, due to their release from the surrounding cells. A normal cell around the tumor that responds to the inflammatory environment is likely to be subsequently altered. We discuss how these abnormalities will support tumor survival via the actions of gap junctions (GJs) and hemichannels (HCs) which are composed of hexamer of connexin43 (Cx43) protein. In particular, we discuss how GJ intercellular communication (GJIC) in glioma cells, the primary brain tumor, is a regulatory factor and its attenuation leads to tumor invasion. In contrast, the astrocytes, which are normal cells around the glioma, are "hijacked" by tumor cells, either by receiving the transmission of malignant substances from the cancer cells via GJIC, or perhaps via astrocytic HC activity through the paracrine signaling which enable the delivery of these substances to the distal astrocytes. This astrocytic signaling would promote tumor expansion in the brain. In addition, brain metastasis from peripheral tissues has also been known to be facilitated by GJs formed between cerebral vascular endothelial cells and cancer cells. Astrocytes and microglia are generally thought to eliminate cancer cells at the blood-brain barrier. In contrast, some reports suggest they facilitate tumor progression as tumor cells take advantage of the normal functions of astrocytes that support the survival of the neurons by exchanging nutrients and metabolites. In summary, GJIC is essential for the normal physiological function of growth and allowing the diffusion of physiological substances. Therefore, whether GJIC is cancer promoting or suppressing may be dependent on what permeates through GJs, when it is active, and to which cells. The nature of GJs, which has been ambiguous in brain tumor progression, needs to be revisited and understood together with new findings on Cx proteins and HC activities.

Sodium butyrate enhances the growth inhibitory effect of sunitinib in human renal cell carcinoma cells.

Sunitinib (SU) is a small molecule that inhibits the receptor tyrosine kinase (RTK) signaling pathway, and has been clinically used to treat advanced renal cell carcinoma (RCC). However, SU is not always effective as RCC is a highly chemoresistant type of cancer. One of the factors that confer chemoresistance to RCC is a hypoxic condition. Lack of oxygen activates hypoxia-inducible factor (HIF) protein, which is followed by the upregulation of growth factors, including vascular endothelial growth factor and activation of the RTK signaling pathway. In this context, histone deacetylase inhibitors (HDACIs) are considered prominent combined agents for SU as they downregulate the expression of HIFs. Therefore, the present study aimed to investigate the effectiveness of combined treatment with SU and sodium butyrate (NaBu), an HDACI. Long-term exposure to these agents exerted a stronger growth inhibitory effect in RCC cell lines compared with single treatment groups. Furthermore, combined treatment suppressed HIF-2α protein, which was induced under hypoxic conditions. In addition, this combination sustained the activity of the RTK signaling pathway to the level of intact cells, although a single treatment with SU or NaBu was demonstrated to increase this activity. Overall, it is suggested that the combination of SU and NaBu is effective for overcoming drug resistance in RCC.

Connexin 43 enhances Bax activation via JNK activation in sunitinib-induced apoptosis in mesothelioma cells.

The constituent protein of gap junctions, connexin (Cx), interacts with various proteins via its C-terminus region, including kinases, cell-adhesion proteins, and a pro-apoptotic protein, Bax. This molecular interaction may affect expression and functioning of the interacting proteins and modulate the cellular physiology. In our previous work, Cx43 was found to interact directly with Bax and in the presence of sunitinib, lead to the Bax-mediated apoptosis in mesothelioma cells. In this study, we investigated the mechanism of how Cx43 promotes Bax-mediated apoptosis using the same cell line. Treatment with sunitinib increased the expression of the active conformation of the Bax protein, which was predominantly localized at the mitochondria, only in Cx43-transfected cells. Bax oligomerization and decrease in the mitochondrial membrane potential were also observed. The involvement of c-Jun N-terminal kinase (JNK) in the interaction of Cx43 and Bax was further examined. Treatment with sunitinib increased the expression of phosphorylated (active) form of JNK only in the Cx43-transfected cells. Phosphorylated JNK and active Bax were co-localized, and the co-localization was suppressed by the knockdown of Cx43. Moreover, JNK inhibition clearly suppressed Bax activation. In conclusion, we identified a novel Cx43-JNK-Bax axis regulating the process of apoptosis for the first time.

Effect of enhanced expression of connexin 43 on sunitinib-induced cytotoxicity in mesothelioma cells.

Connexin (Cx) makes up a type of intercellular channel called gap junction (GJ). GJ plays a regulatory role in cellular physiology. The Cx expression level is often decreased in cancer cells compared to that in healthy ones, and the restoration of its expression has been shown to exert antiproliferative effects. This work aims to evaluate the effect of the restoration of connexin 43 (Cx43) (the most ubiquitous Cx subtype) expression on sunitinib (SU)-induced cytotoxicity in malignant mesothelioma (MM) cells. Increased Cx43 expression in an MM cell line (H28) improved the ability of SU to inhibit receptor tyrosine kinase (RTK) signaling. Moreover, higher Cx43 expression promoted SU-induced apoptosis. The cell viability test revealed that Cx43 enhanced the cytotoxic effect of SU in a GJ-independent manner. The effect of Cx43 on a proapoptotic factor, Bax, was then investigated. The interaction between Cx43 and Bax was confirmed by immunoprecipitation. Furthermore, higher Cx43 expression increased the production of a cleaved (active) form of Bax during SU-induced apoptosis with no alteration in total Bax expression. These findings indicate that Cx43 most likely increases sensitivity to SU in H28 through direct interaction with Bax. In conclusion, we found that Cx43 overcame the chemoresistance of MM cells.

Elacridar enhances the cytotoxic effects of sunitinib and prevents multidrug resistance in renal carcinoma cells.

Intrinsic drug resistance occurs in many renal carcinomas and is associated with increased expression of multidrug resistant proteins, which inhibits intracellular drug accumulation. Multidrug resistant protein 1, also known as P-glycoprotein, is a membrane drug efflux pump belonging to the ATP-binding cassette (ABC) transporter superfamily. ABC Sub-family B Member 2 (ABCG2) is widely distributed and is involved in the multidrug resistant phenotype. Sunitinib is a tyrosine kinase inhibitor used to treat kidney cancer that disrupts signaling pathways responsible for abnormal cancer cell proliferation and tumor angiogenesis. Multiple drug resistance is important in tyrosine kinase inhibitor-induced resistance. We hypothesized that inhibition of multidrug resistant transporters by elacridar (dual inhibitor of P-glycoprotein and ABCG 2) might overcome sunitinib resistance in experimental renal cell carcinoma. Human renal carcinoma cell lines 786-O, ACHN, and Caki-1 were treated with sunitinib or elacridar alone, or in combination. We showed that elacridar significantly enhanced sunitinib cytotoxicity in 786-O cells. P-glycoprotein activity, confirmed by P-glycoprotein function assay, was found to be inhibited by elacridar. ABCG2 expression was low in all renal carcinoma cell lines, and was suppressed only by combination treatment in 786-O cells. ABCG2 function was inhibited by sunitinib alone or combination with elacridar but not elacridar alone. These findings suggest that sunitinib resistance involves multidrug resistance transporters, and in combination with elacridar, can be reversed in renal carcinoma cells by P-glycoprotein inhibition.