A Sulfur Containing Melanogenesis Substrate, N-Pr-4-S-CAP as a Potential Source for Selective Chemoimmunotherapy of Malignant Melanoma.

N-propionyl-4-S-cysteaminylphenol (N-Pr-4-S-CAP) is a substrate for tyrosinase, which is a melanin biosynthesis enzyme and has been shown to be selectively incorporated into melanoma cells. It was found to cause selective cytotoxicity against melanocytes and melanoma cells after selective incorporation, resulting in the induction of anti-melanoma immunity. However, the underlying mechanisms for the induction of anti-melanoma immunity remain unclear. This study aimed to elucidate the cellular mechanism for the induction of anti-melanoma immunity and clarify whether N-Pr-4-S-CAP administration could be a new immunotherapeutic approach against melanoma, including local recurrence and distant metastasis. A T cell depletion assay was used for the identification of the effector cells responsible for N-Pr-4-S-CAP-mediated anti-melanoma immunity. A cross-presentation assay was carried out by using N-Pr-4-S-CAP-treated B16-OVA melanoma-loaded bone marrow-derived dendritic cells (BMDCs) and OVA-specific T cells. Administration of N-Pr-4-S-CAP induced CD8+ T cell-dependent anti-melanoma immunity and inhibited the growth of challenged B16F1 melanoma cells, indicating that the administration of N-Pr-4-S-CAP can be a prophylactic therapy against recurrence and metastasis of melanoma. Moreover, intratumoral injection of N-Pr-4-S-CAP in combination with BMDCs augmented the tumor growth inhibition when compared with administration of N-Pr-4-S-CAP alone. BMDCs cross-presented a melanoma-specific antigen to CD8+ T cells through N-Pr-4-S-CAP-mediated melanoma cell death. Combination therapy using N-Pr-4-S-CAP and BMDCs elicited a superior anti-melanoma effect. These results suggest that the administration of N-Pr-4-S-CAP could be a new strategy for the prevention of local recurrence and distant metastasis of melanoma.

Effects of Arm-cranking Training with Electrical Muscle Stimulation on Vessel Function.

This study aimed to determine whether arm-cranking training with electrical muscle stimulation (EMS) results in a greater improvement in vessel function than performing the same exercise without EMS. First, nine healthy young men performed two 20-min arm-cranking trials at 50% V˙O2max with and without EMS applied to the lower limbs. The flow-mediated vasodilation (FMD) of the right brachial artery was measured using a high-resolution ultrasound device. Both FMD and normalized FMD were increased significantly after the arm-cranking with EMS trial, and significant differences were observed between the two trials. Second, 16 healthy adult men were randomly assigned to either the arm-cranking exercise training (A) group or arm-cranking training with EMS (A+EMS) group. The subjects were engaged in 20 min of arm-cranking at 50% V˙O2max twice a week for 8 weeks with/without EMS applied to the lower limbs. The FMD increased significantly after A+EMS training session and the FMD in A+EMS group was significantly higher than that in the A group. These results indicate that acute/chronic endurance arm-cranking with EMS applied to the lower limbs improves the brachial artery endothelial function more markedly than the same exercise without EMS.

Immunomodulation of Melanoma by Chemo-Thermo-Immunotherapy Using Conjugates of Melanogenesis Substrate NPrCAP and Magnetite Nanoparticles: A Review.

A major advance in drug discovery and targeted therapy directed at cancer cells may be achieved by the exploitation and immunomodulation of their unique biological properties. This review summarizes our efforts to develop novel chemo-thermo-immunotherapy (CTI therapy) by conjugating a melanogenesis substrate, N-propionyl cysteaminylphenol (NPrCAP: amine analog of tyrosine), with magnetite nanoparticles (MNP). In our approach, NPrCAP provides a unique drug delivery system (DDS) because of its selective incorporation into melanoma cells. It also functions as a melanoma-targeted therapeutic drug because of its production of highly reactive free radicals (melanoma-targeted chemotherapy). Moreover, the utilization of MNP is a platform to develop thermo-immunotherapy because of heat shock protein (HSP) expression upon heat generation in MNP by exposure to an alternating magnetic field (AMF). This comprehensive review covers experimental in vivo and in vitro mouse melanoma models and preliminary clinical trials with a limited number of advanced melanoma patients. We also discuss the future directions of CTI therapy.

Heat shock protein 47 confers chemoresistance on pancreatic cancer cells by interacting with calreticulin and IRE1α.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most chemoresistant cancers. An understanding of the molecular mechanism by which PDAC cells have a high chemoresistant potential is important for improvement of the poor prognosis of patients with PDAC. Here we show for the first time that disruption of heat shock protein 47 (HSP47) enhances the efficacy of the therapeutic agent gemcitabine for PDAC cells and that the efficacy is suppressed by reconstituting HSP47 expression. HSP47 interacts with calreticulin (CALR) and the unfolded protein response transducer IRE1α in PDAC cells. Ablation of HSP47 promotes both the interaction of CALR with sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase 2 and interaction of IRE1α with inositol 1,4,5-triphosphate receptor, which generates a condition in which an increase in intracellular Ca2+ level is prone to be induced by oxidative stimuli. Disruption of HSP47 enhances NADPH oxidase-induced generation of intracellular reactive oxygen species (ROS) and subsequent increase in intracellular Ca2+ level in PDAC cells after treatment with gemcitabine, resulting in the death of PDAC cells by activation of the Ca2+ /caspases axis. Ablation of HSP47 promotes gemcitabine-induced suppression of tumor growth in PDAC cell-bearing mice. Overall, these results indicated that HSP47 confers chemoresistance on PDAC cells and suggested that disruption of HSP47 may improve the efficacy of chemotherapy for patients with PDAC.

Detection of a major QTL related to smut disease resistance inherited from a Japanese wild sugarcane using GRAS-Di technology.

Smut disease of sugarcane causes considerable yield losses and the use of resistant varieties is the best control practice. Our group identified a Japanese wild sugarcane with highly smut disease resistance named 'Iriomote8'. In this study, we conducted QTL analysis for smut disease resistance using a mapping population derived from a resistant variety 'Yaenoushie', in which resistance is inherited from 'Iriomote8'. We identified 4813 non-redundant markers using GRAS-Di technology and developed a linkage map of mapping parents. We evaluated smut disease resistance of the mapping population by the inoculation test. Consequently, a large number of clones did not show the disease symptoms and the distribution of smut disease incidence tended to be "L shaped". Composite interval mapping detected an identical QTL for indices of smut disease incidence with a markedly high LOD score (26.6~45.6) at the end of linkage group 8 of 'Yaenoushie'. This QTL explained approximately 50% of the cases of smut disease incidence. In the mapping population, there were no correlations between the indices of smut disease incidence and other agronomic traits. In conclusion, this QTL could be used for marker-assisted selection to significantly improve smut disease resistance without negative effects on other agronomic traits.

ERO1α is a novel endogenous marker of hypoxia in human cancer cell lines.

BACKGROUND: Hypoxia is an important factor that contributes to tumour aggressiveness and correlates with poor prognosis and resistance to conventional therapy. Therefore, identifying hypoxic environments within tumours is extremely useful for understanding cancer biology and developing novel therapeutic strategies. Several studies have suggested that carbonic anhydrase 9 (CA9) is a reliable biomarker of hypoxia and a potential therapeutic target, while pimonidazole has been identified as an exogenous hypoxia marker. However, other studies have suggested that CA9 expression is not directly induced by hypoxia and it is not expressed in all types of tumours. Thus, in this study, we focused on endoplasmic reticulum disulphide oxidase 1α (ERO1α), a protein that localises in the endoplasmic reticulum and is involved in the formation of disulphide bonds in proteins, to determine whether it could serve as a potential tumour-hypoxia biomarker. METHODS: Using quantitative real-time polymerase chain reaction, we analysed the mRNA expression of ERO1α and CA9 in different normal and cancer cell lines. We also determined the protein expression levels of ERO1α and CA9 in these cell lines by western blotting. We then investigated the hypoxia-inducible ERO1α and CA9 expression and localisation in HCT116 and HeLa cells, which express low (CA9-low) and high (CA9-high) levels of CA9, respectively. A comparative analysis was performed using pimonidazole, an exogenous hypoxic marker, as a positive control. The expression and localisation of ERO1α and CA9 in tumour spheres during hypoxia were analysed by a tumour sphere formation assay. Finally, we used a mouse model to investigate the localisation of ERO1α and CA9 in tumour xenografts using several cell lines. RESULTS: We found that ERO1α expression increased under chronic hypoxia. Our results show that ERO1α was hypoxia-induced in all the tested cancer cell lines. Furthermore, in the comparative analysis using CA9 and pimonidazole, ERO1α had a similar localisation to pimonidazole in both CA9-low and CA9-high cell lines. CONCLUSION: ERO1α can serve as a novel endogenous chronic hypoxia marker that is more reliable than CA9 and can be used as a diagnostic biomarker and therapeutic target for cancer.

Endoplasmic reticulum oxidase 1α is critical for collagen secretion from and membrane type 1-matrix metalloproteinase levels in hepatic stellate cells.

Upon liver injury, excessive deposition of collagen from activated hepatic stellate cells (HSCs) is a leading cause of liver fibrosis. An understanding of the mechanism by which collagen biosynthesis is regulated in HSCs will provide important clues for practical anti-fibrotic therapy. Endoplasmic reticulum oxidase 1α (ERO1α) functions as an oxidative enzyme of protein disulfide isomerase, which forms intramolecular disulfide bonds of membrane and secreted proteins. However, the role of ERO1α in HSCs remains unclear. Here, we show that ERO1α is expressed and mainly localized in the endoplasmic reticulum in human HSCs. When HSCs were transfected with ERO1α siRNA or an ERO1α shRNA-expressing plasmid, expression of ERO1α was completely silenced. Silencing of ERO1α expression in HSCs markedly suppressed their proliferation but did not induce apoptosis, which was accompanied by impaired secretion of collagen type 1. Silencing of ERO1α expression induced impaired disulfide bond formation and inhibited autophagy via activation of the Akt/mammalian target of rapamycin signaling pathway, resulting in intracellular accumulation of collagen type 1 in HSCs. Furthermore, silencing of ERO1α expression also promoted proteasome-dependent degradation of membrane type 1-matrix metalloproteinase (MT1-MMP), which stimulates cell proliferation through cleavage of secreted collagens. The inhibition of HSC proliferation was reversed by treatment with MT1-MMP-cleaved collagen type 1. The results suggest that ERO1α plays a crucial role in HSC proliferation via posttranslational modification of collagen and MT1-MMP and, therefore, may be a suitable therapeutic target for managing liver fibrosis.

Anthocyanin and proanthocyanidin contents, antioxidant activity, and in situ degradability of black and red rice grains.

OBJECTIVE: An experiment was conducted to assess the antioxidant contents and activities of colored rice grains and to evaluate their nutritive characteristics in terms of chemical composition and in situ ruminal degradation. METHODS: Ten cultivars of colored rice grains (Oryza sativa L.) collected from several areas of Japan were studied, and control rice without pigment, maize, barley, and wheat grains were used as control grains. Their chemical compositions, pigment, polyphenol contents, total antioxidant capacity (TAC), and degradation characteristics were determined. RESULTS: The starch contents of the colored rice grains were in the range of 73.5% to 79.6%, similar to that of the control rice grain. The black and red rice grains contained anthocyanin (maximum: 5,045.6 µg/g) and proanthocyanidin (maximum: 3,060.6 µg/g) at high concentrations as their principal pigments, respectively. There were significantly (p<0.05) positive relationships among the pigment contents, polyphenol content, and TAC values in the colored and control rice grains, indicating that the increase in pigment contents also contributed to the increased polyphenol content and TAC values in the colored rice grains. The dry matter and starch degradation characteristics, as represented by c (fractional degradation rate of slowly degradable fraction) and by the effective degradability, of the colored rice grains and the control rice grain were ranked as follows among commonly used grains: wheat>barley ≥rice>maize. The colored rice grains also included the most-digestible starch, since their potential degradable fraction and actual degradability at 48 h incubation were almost 100%. CONCLUSION: Colored rice grains have high potential to be used as antioxidant sources in addition to starch sources in ruminants.

Microenvironmental stresses induce HLA-E/Qa-1 surface expression and thereby reduce CD8(+) T-cell recognition of stressed cells.

Hypoxia and glucose deprivation are often observed in the microenvironment surrounding solid tumors in vivo. However, how they interfere with MHC class I antigen processing and CD8(+) T-cell responses remains unclear. In this study, we analyzed the production of antigenic peptides presented by classical MHC class I in mice, and showed that it is quantitatively decreased in the cells exposed to either hypoxia or glucose deprivation. In addition, we unexpectedly found increased surface expression of HLA-E in human and Qa-1 in mouse tumor cells exposed to combined oxygen and glucose deprivation. The induced Qa-1 on the stressed tumor model interacted with an inhibitory NKG2/CD94 receptor on activated CD8(+) T cells and attenuated their specific response to the antigen. Our results thus suggest that microenvironmental stresses modulate not only classical but also nonclassical MHC class I presentation, and confer the stressed cells the capability to escape from the CD8(+) T-cell recognition.

Hypoxia augments MHC class I antigen presentation via facilitation of ERO1-α-mediated oxidative folding in murine tumor cells.

To establish an effective cancer immunotherapy, it is crucial that cancer cells present a cancer-specific antigen in a hypoxic area, a hallmark of the tumor microenvironment. Here, we show the impact of hypoxia on MHC class I antigen presentation in vitro and in vivo in murine tumors. Activation of antigen-specific CTLs by tumor cells that had been pre-incubated under a condition of hypoxia was enhanced compared with that by tumor cells pre-incubated under a condition of normoxia. Cell surface expression of MHC class I-peptide complex on the tumor cells was increased under a condition of hypoxia, thereby leading to higher susceptibility to specific CTLs. We show that the hypoxia-inducible ER-resident oxidase ERO1-α plays an important role in the hypoxia-induced augmentation of MHC class I-peptide complex expression. ERO1-α facilitated oxidative folding of MHC class I heavy chains, thereby resulting in the augmentation of cell surface expression of MHC class I-peptide complex under hypoxic conditions. These results suggest that since the expression of MHC class I-peptide complex is augmented in a hypoxic tumor microenvironment, strategies for inhibiting the function of regulatory T cells and myeloid-derived suppressor cells and/or immunotherapy with immune checkpoint inhibitors are promising for improving cancer immunotherapy.

Cancer-associated oxidoreductase ERO1-α drives the production of tumor-promoting myeloid-derived suppressor cells via oxidative protein folding.

Endoplasmic reticulum disulfide oxidase ERO1-α plays a role in the formation of disulfide bonds in collaboration with protein disulfide isomerase. Disulfide bond formation is required for the proper conformation and function of secreted and cell surface proteins. We found that ERO1-α was overexpressed in a variety of tumor types; therefore, we examined its role in tumor growth. In BALB/c mice, knockdown of ERO1-α within 4T1 mouse mammary gland cancer (KD) cells caused retardation of in vivo tumor growth compared with tumor growth of scrambled control (SCR) cells. In contrast, when ERO1-α-overexpressed 4T1 (OE) cells were compared with mock control cells, OE cells showed augmented tumor growth. However, differences in tumor growth were not observed among four groups of nude mice, suggesting that expression of ERO1-α diminished antitumor immunity. We observed dense peritumoral granulocytic infiltrates in tumors of wild-type 4T1 and SCR cells but not KD cells, and these cells were identified as polymorphonuclear myeloid-derived suppressor cells (MDSCs). In addition, production of G-CSF and CXCL1/2, which have intramolecular disulfide bonds, from KD cells was significantly decreased compared with that from SCR cells. In contrast, OE cells produced a larger amount of these molecules than did mock cells. These changes were regulated at the posttranscriptional level. These results suggest that overexpression of ERO1-α in the tumor inhibits the T cell response by recruiting polymorphonuclear MDSCs via regulation of MDSC-prone cytokines and chemokines.

Cancer-Associated Oxidase ERO1-α Regulates the Expression of MHC Class I Molecule via Oxidative Folding.

ERO1-α is an oxidizing enzyme that exists in the endoplasmic reticulum and is induced under hypoxia. It reoxidizes the reduced form of protein disulfide isomerase that has oxidized target proteins. We found that ERO1-α is overexpressed in a variety of tumor types. MHC class I H chain (HC) has two disulfide bonds in the α2 and α3 domains. MHC class I HC folding is linked to the assembly of MHC class I molecules because only fully disulfide-bonded class I HCs efficiently assemble with ß2-microglobulin. In this study, we show that ERO1-α associates with protein disulfide isomerase, calnexin, and immature MHC class I before being incorporated into the TAP-1-associated peptide-loading complex. Importantly, ERO1-α regulates the redox state as well as cell surface expression of MHC class I, leading to alteration of susceptibility by CD8(+) T cells. Similarly, the ERO1-α expression within cancer cells was associated with the expression level of MHC class I in colon cancer tissues. Thus, the cancer-associated ERO1-α regulates the expression of the MHC class I molecule via oxidative folding.

Vitamin A and insulin are required for the maintenance of hepatic stellate cell quiescence.

Transdifferentiation of vitamin A-storing hepatic stellate cells (HSCs) to vitamin A-depleted myofibroblastic cells leads to liver fibrosis. Vitamin A regulates lipid accumulation and gene transcription, suggesting that vitamin A is involved in the maintenance of HSC quiescence under a physiological condition. However, the precise mechanism remains elusive because there is no appropriate in vitro culture system for quiescent HSCs. Here, we show that treatment of quiescent HSCs with vitamin A partially maintained the accumulation of lipid droplets and expression of quiescent HSC markers (glial fibrillary acidic protein, peroxisome proliferator-activator receptor-γ and CCAAT/enhancer-binding protein-α) and also the expression of myofibroblastic markers (α-smooth muscle actin, heat shock protein 47 and collagen type I). On the other hand, combined treatment with vitamin A and insulin sustained the characteristic of HSC quiescence and completely suppressed the expression of myofibroblastic markers through activation of the JAK2/STAT5 signaling pathway and increased expression of sterol regulatory element binding protein-1. These treated HSCs transdifferentiated to myofibroblastic cells under a culture condition with fetal bovine serum. The results suggest an important role of vitamin A and insulin in the maintenance of HSC quiescence under a physiological condition.

Cancer-associated oxidoreductase ERO1-α drives the production of VEGF via oxidative protein folding and regulating the mRNA level.

BACKGROUND: Endoplasmic reticulum disulfide oxidase 1-α (ERO1-α) is an oxidase that exists in the endoplasmic reticulum and has a role in the formation of disulfide bonds of secreted proteins and cell-surface proteins. Recently, we reported that ERO1-α is present in high levels in various types of tumours, and that ERO1-α is a novel factor of poor prognosis. However, how ERO1-α affects a tumour in vivo and why patients who have a tumour with a high expression level of ERO1-α have a poor prognosis are still unknown. Therefore, to clarify the mechanism, we investigated the effect of ERO1-α on a tumour from the point of view of angiogenesis. METHODS: The effect of ERO1-α on tumour growth and angiogenesis was analysed by using non-obese diabetic-severe combined immunodeficient mice. The production of vascular endothelial growth factor (VEGF) in MDA-MB-231 cells with ERO1-α- overexpression or with ERO1-α knockdown was measured. The role of ERO1-α on VEGF expression was investigated. In triple-negative breast cancer cases, the relationship between expression of ERO1-α and angiogenesis was analysed. RESULTS: We found that the expression of ERO1-α promoted tumour growth in a mouse study and angiogenesis. The effects of ERO1-α on angiogenesis were mediated via oxidative protein folding of VEGF and enhancement of VEGF mRNA expression by using MDA-MB-231. In triple-negative breast cancer cases, the expression of ERO1-α related to the number of the blood vessel. Furthermore, we found that ERO1-α was a poor prognosis factor in triple-negative breast cancer. CONCLUSIONS: Our study has established a novel link between expression of ERO1-α and secretion of VEGF, providing new evidence for the effectiveness of ERO1-α-targeted therapy in patients with ERO1-α-expressed cancer.

Heat shock protein 90 associates with Toll-like receptors 7/9 and mediates self-nucleic acid recognition in SLE.

Systemic lupus erythematosus (SLE) is a prototype systemic autoimmune disease, and disease activity is associated with serum IFN-α level. Plasmacytoid dendritic cells (pDCs) sense microbial as well as self-nucleic acids by TLRs 7 and 9 and produce a large amount of IFN-α. Here, we show that heat shock protein 90 (Hsp90) associates with and delivers TLR7/9 from the ER to early endosomes for ligand recognition. Inhibition of Hsp90 by various approaches including the use of Hsp90 inhibitor, a geldanamycin derivative, suppressed the Hsp90 association with TLR7/9, which resulted in inhibition of IFN-α production, leading to improvement of SLE symptoms in mice. Notably, we observed that serum Hsp90 is clearly increased in patients with active SLE compared with that in patients with inactive disease. Furthermore, we demonstrated that serum Hsp90 detected in SLE patients binds to self-DNA and/or anti-DNA Ab, thus leading to stimulation of pDCs to produce IFN-α. Our data demonstrate that Hsp90 plays a crucial role in the pathogenesis of SLE and that an Hsp90 inhibitor will therefore provide a new therapeutic approach to SLE and other nucleic acid-related autoimmune diseases.

A novel nuclear DnaJ protein, DNAJC8, can suppress the formation of spinocerebellar ataxia 3 polyglutamine aggregation in a J-domain independent manner.

Polyglutamine (polyQ) diseases comprise neurodegenerative disorders caused by expression of expanded polyQ-containing proteins. The cytotoxicity of the expanded polyQ-containing proteins is closely associated with aggregate formation. In this study, we report that a novel J-protein, DNAJ (HSP40) Homolog, Subfamily C, Member 8 (DNAJC8), suppresses the aggregation of polyQ-containing protein in a cellular model of spinocerebellar ataxia type 3 (SCA3), which is also known as Machado-Joseph disease. Overexpression of DNAJC8 in SH-SY5Y neuroblastoma cells significantly reduced the polyQ aggregation and apoptosis, and DNAJC8 was co-localized with the polyQ aggregation in the cell nucleus. Deletion mutants of DNAJC8 revealed that the C-terminal domain of DNAJC8 was essential for the suppression of polyQ aggregation, whereas the J-domain was dispensable. Furthermore, 22-mer oligopeptide derived from C-termilal domain could suppress the polyQ aggregation. These results indicate that DNAJC8 can suppress the polyQ aggregation via a distinct mechanism independent of HSP70-based chaperone machinery and have a unique protective role against the aggregation of expanded polyQ-containing proteins such as pathogenic ataxin-3 proteins.

Activated hepatic stellate cells are dependent on self-collagen, cleaved by membrane type 1 matrix metalloproteinase for their growth.

Stellate cells are distributed throughout organs, where, upon chronic damage, they become activated and proliferate to secrete collagen, which results in organ fibrosis. An intriguing property of hepatic stellate cells (HSCs) is that they undergo apoptosis when collagen is resolved by stopping tissue damage or by treatment, even though the mechanisms are unknown. Here we disclose the fact that HSCs, normal diploid cells, acquired dependence on collagen for their growth during the transition from quiescent to active states. The intramolecular RGD motifs of collagen were exposed by cleavage with their own membrane type 1 matrix metalloproteinase (MT1-MMP). The following evidence supports this conclusion. When rat activated HSCs (aHSCs) were transduced with siRNA against the collagen-specific chaperone gp46 to inhibit collagen secretion, the cells underwent autophagy followed by apoptosis. Concomitantly, the growth of aHSCs was suppressed, whereas that of quiescent HSCs was not. These in vitro results are compatible with the in vivo observation that apoptosis of aHSCs was induced in cirrhotic livers of rats treated with siRNAgp46. siRNA against MT1-MMP and addition of tissue inhibitor of metalloproteinase 2 (TIMP-2), which mainly inhibits MT1-MMP, also significantly suppressed the growth of aHSCs in vitro. The RGD inhibitors echistatin and GRGDS peptide and siRNA against the RGD receptor αVß1 resulted in the inhibition of aHSCs growth. Transduction of siRNAs against gp46, αVß1, and MT1-MMP to aHSCs inhibited the survival signal of PI3K/AKT/IκB. These results could provide novel antifibrosis strategies.

Heat shock protein 90 targets a chaperoned peptide to the static early endosome for efficient cross-presentation by human dendritic cells.

The presentation of an exogenous antigen in a major histocompatibility complex class-I- restricted fashion to CD8(+) T cells is called cross-presentation. Heat shock proteins (HSPs) such as Hsp70, gp96, and Hsp90 have been shown to elicit efficient CTL responses by cross-presentation through an as-yet entirely unknown mechanism. Hsp90 is the most abundant cytosolic HSP and is known to act as a molecular chaperone. We have shown that a tumor antigen peptide complexed with Hsp90 could be cross-presented by dendritic cells (DCs) through an endosomal pathway in a murine system. However, it has not been determined whether human DCs also cross-present an Hsp90-peptide complex and induce peptide-specific CTLs. In this study, we found that an Hsp90-cancer antigen peptide complex was efficiently cross-presented by human monocyte-derived DCs and induced peptide-specific CTLs. Furthermore, we observed that the internalized Hsp90-peptide complex was strictly sorted to the Rab5(+), EEA1(+) static early endosome and the Hsp90-chaperoned peptide was processed and bound to MHC class I molecules through an endosome-recycling pathway. Our data indicate that targeting of the antigen to a "static" early endosome by Hsp90 is essential for efficient cross-presentation.

EpsinR, a target for pyrenocine B, role in endogenous MHC-II-restricted antigen presentation.

While the presentation mechanism of antigenic peptides derived from exogenous proteins by MHC class II molecules is well understood, relatively little is known about the presentation mechanism of endogenous MHC class II-restricted antigens. We therefore screened a chemical library of 200 compounds derived from natural products to identify inhibitors of the presentation of endogenous MHC class II-restricted antigens. We found that pyrenocine B, a compound derived from the fungus Pyrenochaeta terrestris, inhibits presentation of endogenous MHC class II-restricted minor histocompatibility antigen IL-4 inducible gene 1 (IL4I1) by primary dendritic cells (DCs). Phage display screening and surface plasmon resonance (SPR) analysis were used to investigate the mechanism of suppressive action by pyrenocine B. EpsinR, a target molecule for pyrenocine B, mediates endosomal trafficking through binding of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Lentiviral-mediated short hairpin (sh) RNA downregulation of EpsinR expression in DCs resulted in a decrease in the responsiveness of CD4+ T cells. Our data thus suggest that EpsinR plays a role in antigen presentation, which provides insight into the mechanism of presentation pathway of endogenous MHC class II-restricted antigen.

Heat shock protein DNAJB8 is a novel target for immunotherapy of colon cancer-initiating cells.

The aim of the present study was to establish cancer stem-like cell/cancer-initiating cell (CSC/CIC)-targeting immunotherapy. The CSC/CIC are thought to be essential for tumor maintenance, recurrence and distant metastasis. Therefore they are reasonable targets for cancer therapy. In the present study, we found that a heat shock protein (HSP) 40 family member, DnaJ (Hsp40) homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in CSC/CIC derived from colorectal cancer (CRC) cells rather than in non-CSC/CIC. Overexpression of DNAJB8 enhanced the expression of stem cell markers and tumorigenicity, indicating that DNAJB8 has a role in CRC CSC/CIC. A DNAJB8-specific cytotoxic T lymphocyte (CTL) response could be induced by a DNAJB8-derived antigenic peptide. A CTL clone specific for DNAJB8 peptide showed higher killing activity to CRC CSC/CIC compared with non-CSC/CIC, and CTL adoptive transfer into CRC CSC/CIC showed an antitumor effect in vivo. Taken together, the results indicate that DNAJB8 is expressed and has role in CRC CSC/CIC and that DNAJB8 is a novel target of CRC CSC/CIC-targeting immunotherapy.