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Differentiation therapy with all-trans retinoic acid (ATRA) is well established for acute promyelocytic leukemia (APL). However, the narrow application and tolerance development of ATRA remain to be improved. A number of kinase inhibitors have been reported to induce cell differentiation. In this study, we investigated several combinations of these kinase inhibitors. Recently, we revealed that the Akt inhibitor triciribine (TCN) efficiently induces differentiation of NB4 APL cells and acute myeloid leukemia (AML) M2-derived HL-60 cells through activation of the ERK/MAPK pathway. In the present study, we found that the p38 MAPK inhibitor PD169316 had profoundly enhanced the TCN effect for differentiation of NB4 and HL-60 cells. Morphologically, the combination of these two agents efficiently reduced the nuclear-to-cytoplasmic ratio and induced the expression of myelomonocytic markers (CD11b, CD11c) and some ectopic markers (erythroid glycophorin A, lymphoid CD7 and CD20), as determined by PCR and flow cytometry analyses. Western blotting analysis revealed that these agents efficiently induced phosphorylation of ERK. To clarify the molecular mechanisms involved in the TCN and PD169316-induced differentiation, we performed microarray analyses using NB4 cells. Pathway analysis using DAVID software indicated that “viral protein interaction with cytokine and cytokine receptor” and “cytokine-cytokine receptor interaction” were enriched with high significance. Real-time PCR analysis demonstrated that genes for components of these pathways, including chemokines like CCL1 , CCL2 , CCL3 , CCL5 , and CXCL8 as well as cytokines and receptors like CSF1 , IL-10 , IL-10RA , IL-10RB , IL-1β , and TNFSF10 , were upregulated in NB4 and HL-60 cells during TCN and PD169316-induced differentiation.

Differentiation therapy using all-trans retinoic acid (ATRA) for acute promyelocytic leukemia (APL) is well established. However, because the narrow application and tolerance development of ATRA need to be improved, we searched for another efficient myeloid differentiation inducer. Kinase activation is involved in leukemia biology and differentiation block. To identify novel myeloid differentiation inducers, we used a Kinase Inhibitor Screening Library. Using a nitroblue tetrazolium dye reduction assay and real-time quantitative PCR using NB4 APL cells, we revealed that, PD169316, SB203580, SB202190 (p38 MAPK inhibitor), and triciribine (TCN) (Akt inhibitor) potently increased the expression of CD11b. We focused on TCN because it was reported to be well tolerated by patients with advanced hematological malignancies. Nuclear/cytoplasmic (N/C) ratio was significantly decreased, and myelomonocytic markers ( CD11b and CD11c ) were potently induced by TCN in both NB4 and acute myeloid leukemia (AML) M2 derived HL-60 cells. Western blot analysis using NB4 cells demonstrated that TCN promoted ERK1/2 phosphorylation, whereas p38 MAPK phosphorylation was not affected, suggesting that activation of the ERK pathway is involved in TCN-induced differentiation. We further examined that whether ATRA may affect phosphorylation of ERK and p38, and found that there was no obvious effect, suggesting that ATRA induced differentiation is different from TCN effect. To reveal the molecular mechanisms involved in TCN-induced differentiation, we performed microarray analysis. Pathway analysis using DAVID software indicated that “hematopoietic cell lineage” and “cytokine-cytokine receptor interaction” pathways were enriched with high significance. Real-time PCR analysis demonstrated that components of these pathways including IL1β, CD3D, IL5RA, ITGA6, CD44, ITGA2B, CD37, CD9, CSF2RA, and IL3RA, were upregulated by TCN-induced differentiation. Collectively, we identified TCN as a novel myeloid cell differentiation inducer, and trials of TCN for APL and non-APL leukemia are worthy of exploration in the future.

Immunotherapy with immune‐checkpoint therapy has recently been used to treat oral squamous cell carcinomas (OSCCs). However, improvements in current immunotherapy are expected because response rates are limited. Transforming growth factor‐β (TGF‐β) creates an immunosuppressive tumor microenvironment (TME) by inducing the production of regulatory T‐cells (Tregs) and cancer‐associated fibroblasts and inhibiting the function of cytotoxic T‐lymphocytes (CTLs) and natural killer cells. TGF‐β may be an important target in the development of novel cancer immunotherapies. In this study, we investigated the suppressive effect of TGF‐β on CTL function in vitro using OSCC cell lines and their specific CTLs. Moreover, TGFB1 mRNA expression and T‐cell infiltration in 25 OSCC tissues were examined by in situ hybridization and multifluorescence immunohistochemistry. We found that TGF‐β suppressed the function of antigen‐specific CTLs in the priming and effector phases in vitro. Additionally, TGF‐β inhibitor effectively restored the CTL function, and TGFB1 mRNA was primarily expressed in the tumor invasive front. Interestingly, we found a significant negative correlation between TGFB1 mRNA expression and the CD8+ T‐cell/Treg ratio and between TGFB1 mRNA expression and the Ki‐67 expression in CD8+ T‐cells, indicating that TGF‐β also suppressed the function of CTLs in situ. Our findings suggest that the regulation of TGF‐β function restores the immunosuppressive TME to active status and is important for developing new immunotherapeutic strategies, such as a combination of immune‐checkpoint inhibitors and TGF‐β inhibitors, for OSCCs. We found that, TGF‐β suppressed the function of antigen‐specific CTLs in the priming and effector phases in vitro. And, we found a significant negative correlation between TGFB1 mRNA expression and the CD8+ T‐cell/Treg ratio. Inhibition of TGF‐β restores the immunosuppressive TME to active status by a mechanism different from that of immune‐checkpoint inhibitors, suggesting that the combination of both may lead to a new therapeutic strategy for OSCCs.

Regulatory T (Treg) cells are important negative regulators of immune homeostasis, but in cancers they tone down the anti-tumor immune response. They are distinguished by high expression levels of the chemokine receptor CCR4, hence their targeting by the anti-CCR4 monoclonal antibody mogamulizumab holds therapeutic promise. Here we show that despite a significant reduction in peripheral effector Treg cells, clinical responses are minimal in a cohort of patients with advanced CCR4-negative solid cancer in a phase Ib study (NCT01929486). Comprehensive immune-monitoring reveals that the abundance of CCR4-expressing central memory CD8 + T cells that are known to play roles in the antitumor immune response is reduced. In long survivors, characterised by lower CCR4 expression in their central memory CD8 + T cells possessed and/or NK cells with an exhausted phenotype, cell numbers are eventually maintained. Our study thus shows that mogamulizumab doses that are currently administered to patients in clinical studies may not differentiate between targeting effector Treg cells and central memory CD8 + T cells, and dosage refinement might be necessary to avoid depletion of effector components during immune therapy. Elimination of regulatory T cells via the anti-CCR4 monoclonal antibody, mogamulizumab, is expected to augment anti-tumour immune response. Authors show here that although regulatory T cell targeting is successful, clinical improvement remains minimal in patients with solid tumours due to concomitant and unintended depletion of central memory CD8 + T cells.

The renin–angiotensin system (RAS) and the kallikrein– kinin system (KKS) closely interact with each other [1, 2] (Fig. 1). In the RAS, angiotensinogen cleavage by renin generates angiotensin I, which is thereafter cleaved by angiotensin-converting enzyme (ACE) to form angiotensin II. Angiotensin II then binds to the angiotensin II type 1 (AT1) receptor, resulting in the induction of vasoconstriction, inflammation, apoptosis, hypertrophy, cell proliferation, fibrosis, and oxidative stress [3]. On the other hand, angiotensin-(1-7), which is generated by the cleavage of angiotensin II by ACE2, by the cleavage of angiotensin I by neprilysin (NEP), or by the cleavage of angiotensin-(1-9) by ACE/NEP, binds to the Mas receptor and induces vasodilatation, antiinflammation, antiremodeling, antihypertrophy, antiproliferation, antiapoptosis, and antioxidation effects [3, 4]. Recently, oral administration of angiotensin-(1-7) has been reported to prevent obesity and hepatic inflammation in rats fed a high-fat diet [5].

Immune‐checkpoint inhibitors improve the survival of head and neck squamous cell carcinoma (HNSCC) patients. Although recent studies have demonstrated that the tumor immune microenvironment (TIME) has critical roles in immunotherapy, the precise mechanisms involved are unclear. Therefore, further investigations of TIME are required for the improvement of immunotherapy. The frequency of effector regulatory T‐cells (eTregs) and the expression of immune‐checkpoint molecules (ICM) on eTregs and conventional T‐cells (Tconvs) both in peripheral blood lymphocytes (PBL) and tumor‐infiltrating lymphocytes (TIL) from HNSCC patients were analyzed by flow cytometry and their distributions were evaluated by multi‐color immunofluorescence microscopy. High frequency eTreg infiltration into HNSCC tissues was observed and high expressions of CD25, FOXP3, stimulatory‐ICM (4‐1BB, ICOS, OX40 and GITR) and inhibitory‐ICM (programmed cell death‐1 [PD‐1] and cytotoxic T‐lymphocyte‐associated protein‐4 [CTLA‐4]) were found on invasive eTregs. In contrast, the expression of stimulatory‐ICM on Tconvs was low and the expression of inhibitory‐ICM was high. In addition, ICM‐ligands (programmed cell death‐1 [PD‐L1], galectin‐9 and CEACAM‐1) were frequently expressed on cancer cells. PD‐L1 and galectin‐9 were also expressed on macrophages. PD‐1+ T‐cells interacted with PD‐L1+ cancer cells or PD‐L1+ macrophages. This suggested that in TIL, eTregs are highly activated, but Tconvs are exhausted or inactivated by eTregs and immune‐checkpoint systems, and ICM and eTregs are strongly involved in the creation of an immunosuppressive environment in HNSCC tissues. These suggested eTreg targeting drugs are expected to be a combination partner with immune‐checkpoint inhibitors that will improve immunotherapy of HNSCC. Frequencies of eTregs populations (CD45RA‐FOXP3hi) in CD4+ T‐cells of PBL and TIL from HNSCC patients were compared. High frequency of eTreg infiltration into HNSCC tissues was revealed. In addition, MFI of CD25 and FOXP3 in eTreg of TIL were higher than in PBL, which indicates that eTreg in TIL are not only highly infiltrated into the tissues but also are highly activated.

Aims/Introduction Early diagnosis of diabetes‐associated cardiac autonomic neuropathy using the coefficient of variation of R‐R intervals (CVRR) may improve outcomes for individuals with diabetes. The present study examined the associations of decreased CVRR at rest and during deep breathing (DB) with other autonomic nerve function parameters. Materials and Methods The electronic records of 141 inpatients with diabetes (22–65 years) admitted to our hospital between March 2015 and March 2019 were analyzed retrospectively. After assessment by exclusion criteria, 51 inpatients were included. All inpatients were assessed for peripheral and autonomic nerve function, clinical characteristics, and physical abilities. Results Inpatients with decreased CVRR at rest (n = 9 (17.6%)) and during DB (n = 12 (23.5%)) had a longer duration of known diabetes, a higher prevalence of diabetic retinopathy, lower body mass index (BMI), skeletal mass index (SMI), and knee extension strength, and a higher proportion of impaired standing balance. Decreased CVRR at rest was associated with a greater fall in diastolic BP from supine to standing, higher resting HR, longer QTc, longer time of voiding, and sensory symptoms. Conclusions Decreased CVRR at rest and during deep breathing was associated with lower BMI, SMI, and knee strength and a higher proportion of impaired standing balance among non‐elderly inpatients with diabetes. Decreased CVRR at rest appeared more strongly associated with a greater orthostatic BP decline, higher resting heart rate, longer QTc, lower urinary tract dysfunction, and sensory symptoms than a decreased CVRR during deep breathing. To explore the associations of a decreased coefficient of variation of R‐R intervals (CVRR) at rest and during deep breathing (DB) with other autonomic nerve function parameters. Fifty‐one non‐elderly inpatients aged 22–65 years were assessed for peripheral and autonomic nerve function, clinical characteristics, and physical abilities. Decreased CVRR at rest appeared more strongly associated with a greater diastolic BP fall from supine to standing, higher resting heart rate, longer QTc, lower urinary tract dysfunction, and sensory symptoms than decreased CVRR during deep breathing.

Docetaxel (DOC) is one of the most effective chemotherapeutic agents against castration-resistant prostate cancer (CRPC). Despite an impressive initial clinical response, the majority of patients eventually develop resistance to DOC. In tumor metabolism, where tumors preferentially utilize anaerobic metabolism, lactate dehydrogenase (LDH) serves an important role. LDH controls the conversion of pyruvate to lactate, with LDH-A, one of the predominant isoforms of LDH, controlling this metabolic process. In the present study, the role of LDH-A in drug resistance of human prostate cancer (PC) was examined by analyzing 4 PC cell lines, including castration-providing strains PC3, DU145, LNCaP and LN-CSS (which is a hormone refractory cell line established from LNCaP). Sodium oxamate (SO) was used as a specific LDH-A inhibitor. Changes in the expression level of LDH-A were analyzed by western blotting. Cell growth and survival were evaluated with a WST-1 assay. Cell cycle progression and apoptotic inducibility were evaluated by flow cytometry using propidium iodide and Annexin V staining. LDH expression was strongly associated with DOC sensitivity in PC cells. SO inhibited growth of PC cells, which was considered to be caused by the inhibition of LDH-A expression. Synergistic cytotoxicity was observed by combining DOC and SO in LN-CSS cells, but not in LNCaP cells. This combination treatment induced additive cytotoxic effects in PC-3 and DU145 cells, caused cell cycle arrest in G2-M phase and increased the number of cells in the sub-G1 phase of cell cycle in LN-CSS cells. SO promoted DOC induced apoptosis in LN-CSS cells, which was partially caused by the inhibition of DOC-induced increase in LDH-A expression. The results strongly indicated that LDH-A serves an important role in DOC resistance in advanced PC cells and inhibition of LDH-A expression promotes susceptibility to DOC, particularly in CRPC cells. The present study may provide valuable information for developing targeted therapies for CRPC in the future.

Indoleamine 2,3‐dioxygenase 1 (IDO) is an enzyme catabolizing tryptophan (Trp) into the kynurenine (Kyn) pathway. The purpose of the present study was to determine the clinical significance of Trp catabolism in newly diagnosed Hodgkin lymphoma (HL) patients. We quantified serum Trp and Kyn in 52 HL patients, and analyzed their associations with different clinical parameters including serum soluble CD30 concentration. The IDO expression was evaluated in the patients’ affected lymph nodes. The cohort comprised 22 male and 30 female patients (age range, 15‐81 years; median, 45 years), with a 5‐year overall survival (OS) of 88.6%. The OS was significantly shorter for patients with a high Kyn/Trp ratio (OS at 5 years, 60.0% vs 92.2%), for those with stage IV disease, and for those with lymphocytopenia (<600/mm3 and/or <8% white blood cell count). The latter two parameters are components of the international prognostic score for advanced HL. In contrast, there were no significant differences in OS according to age, serum albumin, hemoglobin, sex, white blood cell count, or serum soluble CD30 (≥ or <285.6 ng/mL). Multivariate analysis using the three variables stage, lymphocytopenia, and serum Kyn/Trp ratio showed that only the latter significantly affected OS. Indoleamine 2,3‐dioxygenase 1 was produced by macrophages/dendritic cells, but not by HL tumor cells, and IDO levels determined by immunohistochemistry had a significant positive correlation with the serum Kyn/Trp ratio. In conclusion, quantification of serum Kyn and Trp is useful for predicting prognosis of individual HL patients. A high serum Kyn/Trp ratio was a significant detrimental prognostic factor in Hodgkin Lymphoma. Measurement of serum Kyn and Trp concentrations will be useful for predicting prognosis of the individual Hodgkin Lymphoma patient. Hodgkin Lymphoma, especially in patients with a high serum Kyn/Trp ratio, is an appropriate disease for testing these novel cancer immunotherapies targeting IDO.

The prognosis of multiple myeloma (MM) has drastically improved owing to the development of new drugs, such as proteasome inhibitors and immunomodulatory drugs. Nevertheless, MM is an extremely challenging disease, and many patients are still refractory to the existing therapies, thus requiring new treatment alternatives. Venetoclax is a selective, orally bioavailable inhibitor of BCL-2 that shows efficacy in MM not only as a single agent but also in combination therapy, especially for MM patients with translocation t(11;14). However, many patients are refractory to this drug. Here, we treated the MM cell lines KMS12PE and KMS27 with a combination treatment of venetoclax targeting BCL-2 and daratumumab targeting CD38 to evaluate the synergistic cytotoxicity of these drugs in vitro. MM cell lines were co-cultured with natural killer (NK) cells at an effector:target ratio of 0.3:1 in the presence of serial concentrations of daratumumab and venetoclax, and the resulting apoptotic MM cells were detected by flow cytometry using annexin V. These results indicated that the antibody-dependent cell-mediated NK cytotoxicity was enhanced in KMS12PE and KMS27 cells harboring t(11;14) with a high BCL-2 expression, suggesting that the combination treatment of venetoclax and daratumumab should be especially effective in patients with these characteristics.