Analgesic effects of systemic fentanyl on cancer pain are mediated by not only central but also peripheral opioid receptors in mice.

Fentanyl is an opioid commonly prescribed for cancer pain. Using melanoma-bearing mice, we investigated whether peripheral action would contribute to fentanyl analgesia in cancer pain. Intravenous injection of fentanyl inhibited mechanical nociception in healthy mice, which was markedly inhibited by the opioid antagonist naloxone, but not naloxone methiodide, a peripherally acting opioid antagonist. Melanoma-bearing mice showed mechanical allodynia and spontaneous licking, a pain-related behavior, which were suppressed by intravenous and local injections of fentanyl. Both naloxone and naloxone methiodide inhibited the analgesic effect of intravenous fentanyl to the same degree. Electrophysiological analysis showed that melanoma growth increased the spontaneous and mechanical stimuli-evoked activity of the tibial nerve, which were inhibited by intravenous fentanyl. There was a greater expression of µ- opioid receptors in skin with a melanoma mass than in the contralateral normal skin. In addition, we found µ-opioid receptors in cultured melanoma cells. There was no difference between the number of µ-opioid receptors in the dorsal root ganglia and spinal cord of the melanoma-bearing and contralateral skin side. These results suggest that the analgesic effect of systemic fentanyl is produced via central and peripheral µ- opioid receptors in cancer pain, and cancer cells are a key site of peripheral action.

COP9 signalosome subunit 5 regulates cancer metastasis by deubiquitinating SNAIL.

Cancer metastasis is a major cause of mortality in cancer patients. The transcription factor SNAIL plays an important role in cancer metastasis and progression, and its expression is tightly regulated by the ubiquitin-proteasome system through the balance between ubiquitin ligases and deubiquitinating enzymes. While several ubiquitin ligases of SNAIL have been identified, it is not yet clear regarding deubiquitinating enzyme. In this study, we identified COP9 signalosome subunit 5 (COPS5) as a deubiquitinating enzyme of SNAIL by using siRNA library screening. COPS5 downregulation significantly reduced the expression of SNAIL and impaired the metastatic potential of lung cancer cells both in vitro and in vivo. Importantly, we demonstrated that COPS5 binds to SNAIL and stabilizes its expression by deubiquitination. Furthermore, we observed the positive correlation between COPS5 and SNAIL expression in the clinical tissue samples of lung adenocarcinomas by using tissue microarray analysis. These findings provide strong evidence that COPS5 can be a new therapeutic target for cancer metastasis as a deubiquitinating enzyme of SNAIL.

Retrospective screening of microarray data to identify candidate IFN-inducible genes in a HTLV-1 transformed model.

HuT-102 cells are considered one of the most representable human T-lymphotropic virus 1 (HTLV-1)-infected cell lines for studying adult T-cell lymphoma (ATL). In our previous studies, genome-wide screening was performed using the GeneChip system with Human Genome Array U133 Plus 2.0 for transforming growth factor-ß-activated kinase 1 (TAK1)-, interferon regulatory factor 3 (IRF3)- and IRF4-regulated genes to demonstrate the effects of interferon-inducible genes in HuT-102 cells. Our previous findings demonstrated that TAK1 induced interferon inducible genes via an IRF3-dependent pathway and that IRF4 has a counteracting effect. As our previous data was performed by manual selection of common interferon-related genes mentioned in the literature, there has been some obscure genes that have not been considered. In an attempt to maximize the outcome of those microarrays, the present study reanalyzed the data collected in previous studies through a set of computational rules implemented using 'R' software, to identify important candidate genes that have been missed in the previous two studies. The final list obtained consisted of ten genes that are highly recommend as potential candidate for therapies targeting the HTLV-1 infected cancer cells. Those genes are ATM, CFTR, MUC4, PARP14, QK1, UBR2, CLEC7A (Dectin-1), L3MBTL, SEC24D and TMEM140. Notably, PARP14 has gained increased attention as a promising target in cancer cells.

NK Cells Control Tumor-Promoting Function of Neutrophils in Mice.

Although natural killer (NK) cells are recognized as direct antitumor effectors, the ability of NK cells to control cancer-associated inflammation, which facilitates tumor progression, remains unknown. In this study, we demonstrate that NK cells control tumor-promoting inflammation through functional modification of neutrophils. NK cells control the tumor-promoting function of neutrophils through an IFNγ-dependent mechanism. Tumor progression in an NK cell-depleted host is diminished when the IL17A-neutrophil axis is absent. In NK cell-depleted mice, neutrophils acquire a tumor-promoting phenotype, characterized by upregulation of VEGF-A expression, which promotes tumor growth and angiogenesis. A VEGFR inhibitor which preferentially suppressed tumor growth in NK cell-depleted mice was dependent on neutrophils. Furthermore, the systemic neutropenia caused by an antimetabolite treatment showed an anticancer effect only in mice lacking NK cells. Thus, NK cells likely control the tumor-promoting and angiogenic function of neutrophils. Cancer Immunol Res; 6(3); 348-57. ©2018 AACR.

Coptidis Rhizoma induces intrinsic apoptosis through BAX and BAK activation in human melanoma.

Malignant melanoma has exhibited a rising incidence in recent years worldwide. Although various molecular targeted drugs are being researched and developed for melanoma patients, their efficacy appears to be unsatisfactory. Over the past few years, several reports have demonstrated that Coptidis Rhizoma water extracts (CR) or its major active chemical component, berberine, has anticancer activities in various types of cancer, including melanoma. However, their underlying mechanisms have not been well understood. In the present study, we determined that CR suppressed melanoma cell viability, which was mainly mediated through apoptosis. In addition, the expression levels of anti-apoptotic proteins, BCL2A1, MCL1 and BCL-w, were strongly suppressed by CR treatment. Furthermore, multi-domain pro-apoptotic proteins BAX and BAK were activated by CR treatment and were also required for the CR-induced apoptosis. Collectively, CR or some formulations containing CR, may be effective safe treatment strategies for human melanoma.

Ethanolic extract of Thevetia peruviana flowers enhances TNF-α and TRAIL-induced apoptosis of human cervical cancer cells via intrinsic and extrinsic pathways.

Tumor necrosis factor-α (TNF-α) and TNF-related apoptosis-inducing ligand (TRAIL) are promising candidates for cancer treatment due to their ability to induce apoptosis through death receptor stimulation. However, their usage may be limited due to the resistance of cancer cells to TNF-α- and TRAIL-induced apoptosis. Currently, there is interest in screening for natural products that can sensitize cancer cells to TNF-α- and TRAIL-induced apoptosis for their use in combination with TNF-α or TRAIL. It was previously reported that the bark extract of Thevetia peruviana showed a reversal effect on TRAIL-resistance in human gastric adenocarcinoma cell lines. In the present study, the effects of the ethanolic extract of T. peruviana flowers on TNF-α- and TRAIL-induced apoptosis of human cervical cancer HeLa cells were investigated in vitro by determining cell viability and apoptosis using a WST-1 cell proliferation assay and immunoblot analysis, respectively. The ethanolic extract of T. peruviana flowers promoted TNF-α and TRAIL-mediated cell death through the activation of the caspase cascade, poly(ADP-ribose) polymerase and BH3-interacting domain death agonist cleavage. Combined treatment using the extract plus TNF-α resulted in downregulation of anti-apoptotic protein, including myeloid cell leukemia sequence-1, B-cell lymphoma-extra large (Bcl-XL), X-linked inhibitor of apoptosis protein and survivin, while the combined treatment with TRAIL downregulated Bcl-XL. Thus, the ethanolic extract of T. peruviana flowers has potential in sensitizing the TNF-α- and TRAIL-induced apoptosis of HeLa cells via the intrinsic and extrinsic pathways.

Chemosensitizing Effect of Saikosaponin B on B16F10 Melanoma Cells.

Cancer cell resistance to chemotherapy is one of the obstacles for better cancer treatment, and inflammatory signaling pathways, such as NF-κB signaling pathway, have been recognized to be involved in such chemoresistance. In this study, we aim to identify a new approach for overcoming cancer chemoresistance by using natural compounds. As a result of screening by using Murine B16F10 melanoma cell line constitutively expressing NF-κB luciferase reporter gene, we identified Saikosaponin B2 as an effective inhibitor for etoposide-induced NF-κB activation in B16F10NFkB cells. Saikosaponin B2 sensitized etoposide-induced cell death in B16F10 melanoma cells through the induction of apoptosis. Along with apoptosis induction, we observed an induction of γ-H2AX expression, which is a molecular signature for DNA damage, upon the combination treatment of etoposide and Saikosaponin B2. Among Saikosaponin family compounds, we found that Saikosaponin B1, but not Saikosaponin A, sensitized etoposide-induced cytotoxicity implicating the structural requirement of Saikosaponin B for such chemosensitization. By testing the combination of Saikosaponin B1 and B2 with 9 clinical anticancer drugs, Saikosaponin B showed a certain preference in the combination with those tested anticancer drugs. Collectively, we conclude Saikosaponin B can be an attractive adjuvant for enhancing the clinical effect of cancer chemotherapy.

Highly oxygenated antiausterity agents from the leaves of Uvaria dac.

From the chloroform extract of the leaves of Uvaria dac, four new highly-oxygenated cyclohexene derivatives named uvaridacols I-L (1-4) were isolated together with nine previously reported compounds (5-13). Their structures were determined based on the extensive NMR spectroscopic data and circular dichroism spectroscopic analysis. Among the new compounds, uvaridacol L (4) displayed strong preferential cytotoxicity in the nutrient deprived medium against five different tested pancreatic cancer cell lines, PANC-1 (PC50, 20.1µM), PSN-1 (PC50, 9.7µM), MIA PaCa-2 (PC50, 29.1µM), Capan-1 (73.0µM) and KLM-1 (25.9µM).

Nonthmicin, a Polyether Polyketide Bearing a Halogen-Modified Tetronate with Neuroprotective and Antiinvasive Activity from Actinomadura sp.

Nonthmicin (1), a new polyether polyketide bearing a chlorinated tetronic acid, was isolated from the culture extract of a soil-derived Actinomadura strain. The structure of 1 was elucidated by interpretation of NMR and MS spectroscopic data, and the absolute configuration of 1 was proposed on the basis of the crystal structure of its dechloro congener ecteinamycin (2) also isolated from the same strain. Tetronic acids modified by halogenation have never been reported from natural products. Compounds 1 and 2 were found to have neuroprotective activity and antimetastatic properties at submicromolar concentrations in addition to antibacterial activity.

AKT-STAT3 Pathway as a Downstream Target of EGFR Signaling to Regulate PD-L1 Expression on NSCLC cells.

While cancer development and progression can be controlled by cytotoxic T cells, it is also known that tumor-specific CD8+T cells become functionally impaired by acquiring a group of inhibitory receptors known as immune checkpoints. Amongst those, programmed death-1 (PD-1) is one of the most recognized negative regulators of T cell function. In non-small lung cancers (NSCLCs), the aberrant activation of epidermal growth factor receptor (EGFR) is known to induce PD-L1 expression and further the treatment with gefitinib, a tyrosine kinase inhibitor (TKI) for EGFR, decrease the expression of PD-L1 on NSCLC. Given the acquired resistance to gefitinib treatment frequently observed by developing secondary-site mutations limiting its efficacy, it is important to understand the downstream mechanism of activated-EGFR signaling for regulating PD-L1 in NSCLC. In this study, we demonstrated that AKT-STAT3 pathway could be a potential target for regulating the surface expression of PD-L1 on NSCLCs with aberrant EGFR activity and, further, the inhibition of AKT or STAT3 activity could down-regulate the expression of PD-L1 even in gefitinib-resistant NSCLCs. These results highlight an importance of AKT-STAT3 pathway as a promising target for potentiating anti-tumor immune responses by regulating PD-L1 expression on cancer cells with aberrant EGFR activity.

IL-17A-producing CD30(+) Vδ1 T cells drive inflammation-induced cancer progression.

Although it has been suspected that inflammation is associated with increased tumor metastasis, the exact type of immune response required to initiate cancer progression and metastasis remains unknown. In this study, by using an in vivo tumor progression model in which low tumorigenic cancer cells acquire malignant metastatic phenotype after exposure to inflammation, we found that IL-17A is a critical cue for escalating cancer cell malignancy. We further demonstrated that the length of exposure to an inflammatory microenvironment could be associated with acquiring greater tumorigenicity and that IL-17A was critical for amplifying such local inflammation, as observed in the production of IL-1ß and neutrophil infiltration following the cross-talk between cancer and host stromal cells. We further determined that γδT cells expressing Vδ1 semi-invariant TCR initiate cancer-promoting inflammation by producing IL-17A in an MyD88/IL-23-dependent manner. Finally, we identified CD30 as a key molecule in the inflammatory function of Vδ1T cells and the blockade of this pathway targeted this cancer immune-escalation process. Collectively, these results reveal the importance of IL-17A-producing CD30(+) Vδ1T cells in triggering inflammation and orchestrating a microenvironment leading to cancer progression.

P38 pathway as a key downstream signal of connective tissue growth factor to regulate metastatic potential in non-small-cell lung cancer.

Although the secretory matricellular protein connective tissue growth factor (CTGF) has been reported to be related to lung cancer metastasis, the precise mechanism by which CTGF regulates lung cancer metastasis has not been elucidated. In the present study, we show the molecular link between CTGF secretion and the p38 pathway in the invasive and metastatic potential of non-small-cell lung cancer (NSCLC). Among three different human NSCLC cell lines (PC-14, A549, and PC-9), their in vitro invasiveness was inversely correlated with the level of CTGF secretion. By supplementing or reducing CTGF secretion in NSCLC culture, dysregulation of the invasive and metastatic potential of NSCLC cell lines was largely compensated. By focusing on the protein kinases that are known to be regulated by CTGF, we found that the p38 pathway is a key downstream signal of CTGF to regulate the metastatic potential of NSCLC. Importantly, a negative correlation between CTGF and phosphorylation status of p38 was identified in The Cancer Genome Atlas lung adenocarcinoma dataset. In the context of the clinical importance of our findings, we showed that p38 inhibitor, SB203580, reduced the metastatic potential of NSCLC secreting low levels of CTGF. Collectively, our present findings indicate that the CTGF/p38 axis is a novel therapeutic target of NSCLC metastasis, particularly NSCLC secreting low levels of CTGF.

Targeting the ataxia telangiectasia mutated pathway for effective therapy against hirsutine-resistant breast cancer cells.

The present authors have recently demonstrated that hirsutine, one of the major alkaloids in Uncaria species, promotes cell apoptosis by inducing DNA damage and suppresses metastasis of breast cancer cells. Despite its potent anti-cancer activity, certain types of human breast cancer cells exhibit resistance to hirsutine. To maximize the clinical utility of hirsutine therapy against breast cancer, it is critical to explore the underlying mechanism that protects hirsutine-resistant breast cancer cell lines. To identify potential targets for overcoming hirsutine-resistance, the present study investigated a library of kinase inhibitors in combination with hirsutine treatment in the hirsutine-resistant human breast carcinoma MCF-7 cell line. Amongst the 96 compounds tested, inhibitors of the ataxia telangiectasia mutated (ATM) pathway sensitized MCF-7 cells to hirsutine-induced cell death along with a sustained DNA damage response. This sensitization of MCF-7 cells to the hirsutine-induced DNA damage response by interfering with the ATM pathway did not require p53. Instead, radical oxygen species generation was significantly increased in hirsute and ATM inhibitor-treated MCF-7 cells. In conclusion, the present findings suggest the importance of the ATM pathway for optimizing the anti-cancer effect of hirsutine in breast cancer cells.

Inhibition of p38 mitogen-activated protein kinase potentiates the apoptotic effect of berberine/tumor necrosis factor-related apoptosis-inducing ligand combination therapy.

It was previously reported that berberine (BBR) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) exhibited a synergistic apoptotic effect on triple negative breast cancer (TNBC) cells. In addition, the BBR/TRAIL combination treatment sensitized TRAIL-resistant TNBC cells to TRAIL. The aim of the present study was to investigate a novel pathway for enhancing the apoptotic effect of BBR/TRAIL through mitogen-activated protein kinases (MAPKs). Selective inhibitors and small interfering RNAs were utilized to understand the role of p38 MAPK in this pathway. The results demonstrated that p38 MAPK was activated in response to the combination therapy in TRAIL-resistant TNBC cells. In addition, it was revealed that the inhibition of p38 enhanced apoptosis in epidermal growth factor receptor (EGFR)-overexpressing MDA-MB-468 TNBC cells and EGFR-mutant PC-9 non-small-cell lung carcinoma cells, which was associated with the downregulation of EGFR serine phosphorylation. Viability assays for these two cell lines also confirmed the significant reduction of cell viability following p38 inhibition in BBR/TRAIL-treated cells. In conclusion, the present study provided novel evidence for the role of p38 in suppressing BBR/TRAIL-mediated apoptosis and its association with EGFR, which may explain the mechanism of treatment resistance in certain types of cancer.

Immune adjuvant effect of Juzentaihoto, a Japanese traditional herbal medicine, on tumor vaccine therapy in a mouse model.

Japanese traditional herbal medicine (Kampo) have been used to improve the general physical condition after surgery and to mitigate the side effects of radiation and chemotherapy in tumor patients. Juzentaihoto (JTT) consists of ten medical herbs, and is also called Shi-Quan-Da-Bu-Tang in Chinese herbal medicine. Among Kampo medicines, JTT has especially gained attention as a biological response modifier. Currently, clinical trials of various tumor vaccine therapies are being performed world-wide. However, tumor antigens that are inoculated as vaccines do not have high immunogenicity; thus, it is difficult to obtain an effective therapeutic effect. Thus, it is necessary to develop a tumor vaccine adjuvant that is more potent and very safe. In the present study, we examined the efficacy of JTT as an oral adjuvant when given together with tumor vaccines. As a result, JTT enhanced the phagocytic ability of OVA antigen and the presentation ability of OVA antigen in dendritic cells in vitro. Furthermore, tumor growth was markedly decreased, and the survival period was significantly prolonged in mice inoculated with mouse lymphoma, which is expressed with tumor model antigen. In conclusion, these findings suggest that JTT can be used with tumor vaccines as an immune adjuvant.

Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2.

Crosstalk between inflammatory signalling pathways and receptor tyrosine kinases has been revealed as an indicator of cancer malignant progression. In the present study, we focus on EphA2 receptor tyrosine kinase, which is overexpressed in many human cancers. It has been reported that ligand-independent phosphorylation of EphA2 at Ser-897 is induced by Akt. We show that inflammatory cytokines promote RSK-, not Akt-, dependent phosphorylation of EphA2 at Ser-897. In addition, the RSK-EphA2 signalling pathway controls cell migration and invasion of metastatic breast cancer cells. Moreover, Ser-897-phosphorylated EphA2 co-localizes with phosphorylated active form of RSK in various human tumour specimens, and this double positivity is related to poor survival in lung cancer patients, especially those with a smoking history. Taken together, these results indicate that the phosphorylation of EphA2 at Ser-897 is controlled by RSK and the RSK-EphA2 axis might contribute to cell motility and promote tumour malignant progression.

Multi-pathway cellular analysis on crude natural drugs/herbs from Japanese Kampo formulations.

Kampo formulations comprise a number of crude natural drugs/herbs as constituents. The crude drugs/herbs have been traditionally classified by their traditional classifications or efficacies in Kampo medicines; however, it has been difficult to establish the scientific link between experimental evidence and traditional classifications in Kampo medicine. To clarify such traditional conceptions, we tested 112 crude drugs/herbs that are major components of Kampo formulations, in the multi-pathway analysis of 10 well-studied transcriptional activities including CREB, ERSF, HIF-1α, IRFs, MYC, NF-κB, p53, SMAD, SOX2, and TCF/LEF in A549 human lung cancer cells. By clustering the results of multi-pathway analysis with the Spearman rank-correlation coefficient and Ward linkage, three distinct traditional categories were significantly enriched in the major groupings, which are heat-clearing and dampness-drying herbs, acrid and warm exterior-resolving herbs, and acrid and cool exterior-resolving herbs. These results indicate that these crude drugs/herbs have similar effects on intracellular signaling and further imply that the traditional classifications of those enriched crude drugs/herbs can be supported by such experimental evidence. Collectively, our new in vitro multi-pathway analysis may be useful to clarify the mechanism of action of crude drugs/herbs and Kampo formulations.

Mammary tissue microenvironment determines T cell-dependent breast cancer-associated inflammation.

Although the importance of the host tissue microenvironment in cancer progression and metastasis has been established, the spatiotemporal process establishing a cancer metastasis-prone tissue microenvironment remains unknown. In this study, we aim to understand the immunological character of a metastasis-prone microenvironment in a murine 4T1 breast tumor model, by using the activation of nuclear factor-κb (NF-κB) in cancer cells as a sensor of inflammatory status and by monitoring its activity by bioluminescence imaging. By using a 4T1 breast cancer cell line stably expressing an NF-κB/Luc2 reporter gene (4T1 NF-κB cells), we observed significantly increased bioluminescence approximately 7 days after metastasis-prone orthotopic mammary fat-pad inoculation but not ectopic s.c. inoculation of 4T1 NF-κB cells. Such in vivo NF-κB activation within the fat-pad 4T1 tumor was diminished in immune-deficient SCID or nude mice, or T cell-depleted mice, suggesting the requirement of host T cell-mediated immune responses. Given the fat-pad 4T1 tumor expressed higher inflammatory mediators in a T cell-dependent mechanism compared to the s.c. tumor, our results imply the importance of the surrounding tissue microenvironment for inflaming tumors by collaborating with T cells to instigate metastatic spread of 4T1 breast cancer cells.

Role of chemokine CX3CL1 in progression of multiple myeloma via CX3CR1 in bone microenvironments.

Several chemokines/chemokine receptors such as CXCL12, CCL3, CXCR4 and CCR1 attract multiple myelomas to specific microenvironments. In the present study, we investigated whether the CX3CL1/CX3CR1 axis is involved in the interaction of the multiple myeloma cells with their microenvironment. The expression of CX3CR1 (also known as fractalkine) was detected in three of the seven human myeloma cell lines. CX3CL1-induced phosphorylation of Akt and ERK1/2 was detected in the CX3CR1-positive cell lines, but not in the CX3CR1-negative cell lines. In addition, CX3CL1-induced cell adhesion to fibronectin and vascular cell adhesion molecule-1 (VCAM-1) in the human myeloma RPMI-8226 cell line. We also investigated whether a relationship existed between myeloma cells and osteoclasts that may function via the CX3CL1/CX3CR1 axis. Conditioned medium from CX3CL1-stimulated RPMI-8226 cells drastically increased the osteoclast differentiation. Collectively, the results from the present study support the concept of the CX3CL1-mediated activation of the progression of the multiple myeloma via CX3CR1. Thus, CX3CR1 may represent a potential therapeutic target for the treatment of multiple myeloma in a bone microenvironment.

Anti-inflammatory effect of cinnamaldehyde in Helicobacter pylori induced gastric inflammation.

Cinnamomum cassia is widely employed for gastrointestinal complaints such as dyspepsia, flatulence, diarrhea, and vomiting. Studies report cinnamaldehyde (CM) as a major active constituent of cinnamon. The aim of this study was to evaluate the anti-inflammatory mechanism of CM on Helicobacter (H.) pylori-infected gastric epithelial cells in order to validate cinnamon traditional use in gastrointestinal (GI)-related disorders. AGS/MKN-45 cells and H. pylori (193C) were employed for co-culture experiments. Anti-H. pylori cytotoxic and anti-adhesion activity of CM were determined. Enzyme linked immunosorbent assay, real time polymerase chain reaction analysis and immunoblotting were used to measure the effect on interleukin-8 (IL-8) secretion/expression. The effect on activation of nuclear factor kappa B (NF-κB) was determined by immunoblot analysis. The non-cytotoxic CM (≤125 µM) was also non-bactericidal at the given time, suggesting the effect in H. pylori/cell co-culture system was not due to alteration in H. pylori viability or the toxicity to the cells. Also, CM did not show any anti-adhesion effect against H. pylori/cell co-culture. However, pre-incubation of the cells with CM significantly inhibited the IL-8 secretion/expression from H. pylori-infected cells (p<0.01). In addition, CM suppressed H. pylori-induced NF-κB activation and prevented degradation of inhibitor (I)-κB This study provides evidence that the anti-inflammatory effect of C. cassia on H. pylori-infected gastric cells is due to blockage of the NF-κB pathway by cinnamaldehyde. This agent can be considered as a potential candidate for in vivo and clinical studies against various H. pylori related gastric pathogenic processes.