IL-7-primed bystander CD8 tumor-infiltrating lymphocytes optimize the antitumor efficacy of T cell engager immunotherapy.

Bispecific T cell engagers (TCEs) show promising clinical efficacy in blood tumors, but their application to solid tumors remains challenging. Here, we show that Fc-fused IL-7 (rhIL-7-hyFc) changes the intratumoral CD8 T cell landscape, enhancing the efficacy of TCE immunotherapy. rhIL-7-hyFc induces a dramatic increase in CD8 tumor-infiltrating lymphocytes (TILs) in various solid tumors, but the majority of these cells are PD-1-negative tumor non-responsive bystander T cells. However, they are non-exhausted and central memory-phenotype CD8 T cells with high T cell receptor (TCR)-recall capacity that can be triggered by tumor antigen-specific TCEs to acquire tumoricidal activity. Single-cell transcriptome analysis reveals that rhIL-7-hyFc-induced bystander CD8 TILs transform into cycling transitional T cells by TCE redirection with decreased memory markers and increased cytotoxic molecules. Notably, TCE treatment has no major effect on tumor-reactive CD8 TILs. Our results suggest that rhIL-7-hyFc treatment promotes the antitumor efficacy of TCE immunotherapy by increasing TCE-sensitive bystander CD8 TILs in solid tumors.

Metformin induces muscle atrophy by transcriptional regulation of myostatin via HDAC6 and FoxO3a.

BACKGROUND: Skeletal muscle atrophy is a severe condition that involves loss of muscle mass and quality. Drug intake can also cause muscle atrophy. Biguanide metformin is the first-line and most widely prescribed anti-diabetic drug for patients with type 2 diabetes. The molecular mechanism of metformin in muscle is unclear. METHODS: Myostatin expression was investigated at the protein and transcript levels after metformin administration. To investigate the pathways associated with myostatin signalling, we used real-time polymerase chain reaction, immunoblotting, luciferase assay, chromatin immunoprecipitation assay, co-immunoprecipitation, immunofluorescence, primary culture, and confocal microscopy. Serum analysis, physical performance, and immunohistochemistry were performed using our in vivo model. RESULTS: Metformin induced the expression of myostatin, a key molecule that regulates muscle volume and triggers the phosphorylation of AMPK. AMPK alpha2 knockdown in the background of metformin treatment reduced the myostatin expression of C2C12 myotubes (-49.86 ± 12.03%, P < 0.01) and resulted in increased myotube diameter compared with metformin (+46.62 ± 0.88%, P < 0.001). Metformin induced the interaction between AMPK and FoxO3a, a key transcription factor of myostatin. Metformin also altered the histone deacetylase activity in muscle cells (>3.12-fold ± 0.13, P < 0.001). The interaction between HDAC6 and FoxO3a induced after metformin treatment. Confocal microscopy revealed that metformin increased the nuclear localization of FoxO3a (>3.3-fold, P < 0.001). Chromatin immunoprecipitation revealed that metformin induced the binding of FoxO3a to the myostatin promoter. The transcript-level expression of myostatin was higher in the gastrocnemius (GC) muscles of metformin-treated wild-type (WT) (+68.9 ± 10.01%, P < 0.001) and db/db mice (+55.84 ± 6.62%, P < 0.001) than that in the GC of controls (n = 4 per group). Average fibre cross-sectional area data also showed that the metformin-treated C57BL/6J (WT) (-31.74 ± 0.75%, P < 0.001) and C57BLKS/J-db/db (-18.11 ± 0.94%, P < 0.001) mice had decreased fibre size of GC compared to the controls. The serum myoglobin level was significantly decreased in metformin-treated WT mice (-66.6 ± 9.03%, P < 0.01). CONCLUSIONS: Our results demonstrate that metformin treatment impairs muscle function through the regulation of myostatin in skeletal muscle cells via AMPK-FoxO3a-HDAC6 axis. The muscle-wasting effect of metformin is more evident in WT than in db/db mice, indicating that more complicated mechanisms may be involved in metformin-mediated muscular dysfunction.

B7-H3×4-1BB bispecific antibody augments antitumor immunity by enhancing terminally differentiated CD8+ tumor-infiltrating lymphocytes.

Cancer immunotherapy with 4-1BB agonists has limited further clinical development because of dose-limiting toxicity. Here, we developed a bispecific antibody (bsAb; B7-H3×4-1BB), targeting human B7-H3 (hB7-H3) and mouse or human 4-1BB, to restrict the 4-1BB stimulation in tumors. B7-H3×m4-1BB elicited a 4-1BB-dependent antitumor response in hB7-H3-overexpressing tumor models without systemic toxicity. BsAb primarily targets CD8 T cells in the tumor and increases their proliferation and cytokine production. Among the CD8 T cell population in the tumor, 4-1BB is solely expressed on PD-1+Tim-3+ "terminally differentiated" subset, and bsAb potentiates these cells for eliminating the tumor. Furthermore, the combination of bsAb and PD-1 blockade synergistically inhibits tumor growth accompanied by further increasing terminally differentiated CD8 T cells. B7-H3×h4-1BB also shows antitumor activity in h4-1BB-expressing mice. Our data suggest that B7-H3×4-1BB is an effective and safe therapeutic agent against B7-H3-positive cancers as monotherapy and combination therapy with PD-1 blockade.

Niche-specific MHC II and PD-L1 regulate CD4+CD8αα+ intraepithelial lymphocyte differentiation.

Conventional CD4+ T cells are differentiated into CD4+CD8αα+ intraepithelial lymphocytes (IELs) in the intestine; however, the roles of intestinal epithelial cells (IECs) are poorly understood. Here, we showed that IECs expressed MHC class II (MHC II) and programmed death-ligand 1 (PD-L1) induced by the microbiota and IFN-γ in the distal part of the small intestine, where CD4+ T cells were transformed into CD4+CD8αα+ IELs. Therefore, IEC-specific deletion of MHC II and PD-L1 hindered the development of CD4+CD8αα+ IELs. Intracellularly, PD-1 signals supported the acquisition of CD8αα by down-regulating the CD4-lineage transcription factor, T helper-inducing POZ/Krüppel-like factor (ThPOK), via the Src homology 2 domain-containing tyrosine phosphatase (SHP) pathway. Our results demonstrate that noncanonical antigen presentation with cosignals from IECs constitutes niche adaptation signals to develop tissue-resident CD4+CD8αα+ IELs.

Cancer immunotherapy with T-cell targeting cytokines: IL-2 and IL-7.

Clinical trials have demonstrated that an increased number of effector cells, especially tumor-specific T cells, is positively linked with patients' prognosis. Although the discovery of checkpoint inhibitors (CPIs) has led to encouraging progress in cancer immunotherapy, the lack of either T cells or targets for CPIs is a limitation for patients with poor prognosis. Since interleukin (IL)-2 and IL-7 are cytokines that target many aspects of T-cell responses, they have been used to treat cancers. In this review, we focus on the basic biology of how these cytokines regulate T-cell response and on the clinical trials using the cytokines against cancer. Further, we introduce several recent studies that aim to improve cytokines' biological activities and find the strategy for combination with other therapeutics. [BMB Reports 2021; 54(1): 21-30].

Hybrid Fc-fused interleukin-7 induces an inflamed tumor microenvironment and improves the efficacy of cancer immunotherapy.

OBJECTIVES: Emerging oncotherapeutic strategies require the induction of an immunostimulatory tumor microenvironment (TME) containing numerous tumor-reactive CD8+ T cells. Interleukin-7 (IL-7), a T-cell homeostatic cytokine, induces an antitumor response; however, the detailed mechanisms underlying the contributions of the IL-7 to TME remain unclear. Here, we aimed to investigate the mechanism underlying the induction of antitumor response by hybrid Fc-fused long-acting recombinant human IL-7 (rhIL-7-hyFc) through regulation of both adaptive and innate immune cells in the TME. METHODS: We evaluated rhIL-7-hyFc-mediated antitumor responses in murine syngeneic tumor models. We analysed the cellular and molecular features of tumor-infiltrating lymphocytes (TILs) and changes in the TME after rhIL-7-hyFc treatment. Furthermore, we evaluated the antitumor efficacy of rhIL-7-hyFc combined with chemotherapy and checkpoint inhibitors (CPIs). RESULTS: Systemic delivery of rhIL-7-hyFc induced significant therapeutic benefits by expanding CD8+ T cells with enhanced tumor tropism. In tumors, rhIL-7-hyFc increased both tumor-reactive and bystander CD8+ TILs, all of which displayed enhanced effector functions but less exhausted phenotypes. Moreover, rhIL-7-hyFc suppressed the generation of immunosuppressive myeloid cells in the bone marrow of tumor-bearing mice, resulting in the immunostimulatory TME. Combination therapy with chemotherapy and CPIs, rhIL-7-hyFc elicited a strong antitumor response and even under a T lymphopenic condition by restoring CD8+ T cells. When combined with chemotherapy and CPIs, rhIL-7-hyFc administration enhanced antitumor response under intact andlymphopenic conditions by restoring CD8+ T cells. CONCLUSION: Taken together, these data demonstrate that rhIL-7-hyFc induces antitumor responses by generating T-cell-inflamed TME and provide a preclinical proof of concept of immunotherapy with rhIL-7-hyFc to enhance therapeutic responses in the clinic.

Development and validation study of Game Overuse Screening Questionnaire.

The aim of this study was to develop a screening questionnaire to distinguish high-risk individuals associated with game overuse from casual internet users. Reliability, validity, and diagnostic ability were evaluated for the newly developed Game Overuse Screening Questionnaire (GOS-Q). Preliminary items were assessed by 50 addiction experts online and 30 questions were selected. A total of 158 subjects recruited from six community centers for internet addiction participated in this study. Finally, 150 people were used in the analysis after excluding eight non-respondents. GOS-Q, Young's internet addiction scale, and Korean scale for internet addiction were used to assess concurrent validity. Internal consistency and item-total correlations were favorable (α= 0.96, r= 0.47-0.82). Test-retest reliability was moderate in size (r= 0.74). GOS-Q showed superior concurrent validity, and the highest correlation with Y-Scale (r= 0.77). The construct validity was marginally supported by a six-factor model using exploratory factor analysis. The area under the Receiver Operating Characteristic curve was 0.945. The high-risk addiction group was effectively characterized by a cut-off point of 38.5, with a sensitivity of 0.87 and a specificity of 0.88. Overall, the current study supports the use of GOS-Q as a reliable screening tool in a variety of settings.

Metformin overcomes resistance to cisplatin in triple-negative breast cancer (TNBC) cells by targeting RAD51.

BACKGROUND: Chemotherapy is a standard therapeutic regimen to treat triple-negative breast cancer (TNBC); however, chemotherapy alone does not result in significant improvement and often leads to drug resistance in patients. In contrast, combination therapy has proven to be an effective strategy for TNBC treatment. Whether metformin enhances the anticancer effects of cisplatin and prevents cisplatin resistance in TNBC cells has not been reported. METHODS: Cell viability, wounding healing, and invasion assays were performed on Hs 578T and MDA-MB-231 human TNBC cell lines to demonstrate the anticancer effects of combined cisplatin and metformin treatment compared to treatment with cisplatin alone. Western blotting and immunofluorescence were used to determine the expression of RAD51 and gamma-H2AX. In an in vivo 4T1 murine breast cancer model, a synergistic anticancer effect of metformin and cisplatin was observed. RESULTS: Cisplatin combined with metformin decreased cell viability and metastatic effect more than cisplatin alone. Metformin suppressed cisplatin-mediated RAD51 upregulation by decreasing RAD51 protein stability and increasing its ubiquitination. In contrast, cisplatin increased RAD51 expression in an ERK-dependent manner. In addition, metformin also increased cisplatin-induced phosphorylation of γ-H2AX. Overexpression of RAD51 blocked the metformin-induced inhibition of cell migration and invasion, while RAD51 knockdown enhanced cisplatin activity. Moreover, the combination of metformin and cisplatin exhibited a synergistic anticancer effect in an orthotopic murine model of 4T1 breast cancer in vivo. CONCLUSIONS: Metformin enhances anticancer effect of cisplatin by downregulating RAD51 expression, which represents a novel therapeutic target in TNBC management.

Comparing the screening property of the shortened versions of the Hypomania Checklist-32 (HCL-32): Cross-validation in Korean patients with bipolar disorder and major depressive disorder.

BACKGROUND: To date, shortened versions of the Hypomania Checklist-32 (HCL-32) were proposed to overcome the limitation of a lengthy format; however, a cross-validation study is currently needed to identify which shorter version may function optimally in a clinical sample. METHODS: In a Korean patient sample with bipolar disorder (BD) and major depressive disorder (MDD) (BD-I n = 84, BD = II n = 145, MDD n = 285), we examined the reliability and screening property of three shorter versions of the HCL (HCL-20, -16, -8) in comparison with the full HCL-32. Diagnosis was confirmed by the structured clinical interview (SCID-I). RESULTS: All three shortened HCLs demonstrated a fair screening ability (Area Under the Curve = .72~.74) to discriminate BD patients from MDD patients, which was comparable to that of the HCL-32. When sensitivity and specificity were considered, the HCL-20 showed relatively superior performance among the shortened versions. LIMITATIONS: The shorter versions were not administered in a 'stand-alone' manner. CONCLUSIONS: This is the first cross-validation study in a large clinical sample with an increased statistical power to compare the screening property of the shortened HCLs. Our results suggest that briefer versions of the HCL could be reliably and economically utilized in clinical and research settings to enhance detection of BD.

Validation Study for the Korean Version of Fear of Cancer Recurrence Inventory.

Fear of cancer recurrence (FCR) is one of the most prevalent unmet psychosocial needs. This study aimed to confirm the cultural equivalence, reliability, and validity of the Korean version of Fear of Cancer Recurrence Inventory (K-FCRI). We conducted a forward-backward translation of the English version FCRI to Korean version through meticulous process including transcultural equivalence test. The psychometric property of the K-FCRI was then validated in 444 survivors from cancers at various sites. The Korean translation was accepted well by participants. There was a good cultural equivalence between the Korean version and the English version of FCRI. Confirmatory factor analysis supported the original seven-factor structure with slightly insufficient level of goodness-of-fit indices (comparative fit index = 0.900, non-normed fit index = 0.893, root mean square error of approximation = 0.060). The K-FCRI had high internal consistency (α = 0.85 for total scale and α = 0.77-0.87 for subscales) and test-retest reliability (r = 0.90 for total scale and r = 0.54-0.84 for subscales). The K-FCRI had significant correlations with the Korean version of Fear of Progression Questionnaire, European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Version 3.0, Hospital Anxiety and Depression Scale, and Fatigue Severity Score, supporting the good construct validity and psychometric properties of K-FCRI. The K-FCRI was confirmed as a valid and reliable psychometric test for measuring FCR of Korean survivors from cancers at various sites.

Altered Gut Microbiota Composition in Rag1-deficient Mice Contributes to Modulating Homeostasis of Hematopoietic Stem and Progenitor Cells.

Hematopoietic stem and progenitor cells (HSPCs) can produce all kind of blood lineage cells, and gut microbiota that consists of various species of microbe affects development and maturation of the host immune system including gut lymphoid cells and tissues. However, the effect of altered gut microbiota composition on homeostasis of HSPCs remains unclear. Here we show that compositional change of gut microbiota affects homeostasis of HSPCs using Rag1 (-/-) mice which represent lymphopenic condition. The number and proportions of HSPCs in Rag1 (-/-) mice are lower compared to those of wild types. However, the number and proportions of HSPCs in Rag1 (-/-) mice are restored as the level of wild types through alteration of gut microbiota diversity via transferring feces from wild types. Gut microbiota composition of Rag1 (-/-) mice treated with feces from wild types shows larger proportions of family Prevotellaceae and Helicobacterceae whereas lower proportions of family Lachnospiraceae compared to unmanipulated Rag1 (-/-) mice. In conclusion, gut microbiota composition of lymphopenic Rag1 (-/-) mice is different to that of wild type, which may lead to altered homeostasis of HSPCs.

Paclitaxel suppresses the viability of breast tumor MCF7 cells through the regulation of EF1α and FOXO3a by AMPK signaling.

Paclitaxel (Taxol), a potent drug of natural origin isolated from the bark of the Pacific yew, is widely used for treating ovarian, lung and breast cancers. Currently, there is little information regarding the specific mechanism underlying the anticancer activity of paclitaxel. In the present study, we found that 5-amino-1-ß-D-ribofuranosyl-imidazole-4-carboxamide (AICAR), a well-known activator of adenosine monophosphate (AMP)-activated protein kinase (AMPK), downregulated the protein and mRNA levels of elongation factor 1 α (EF1α) in breast cancer MCF7 cells. Paclitaxel increased the phosphorylation of AMPK and also downregulated the expression of EF1α in MCF7 cells. In addition, paclitaxel increased the expression, as well as the phosphorylation of forkhead box O3a (FOXO3a). Phosphorylation of FOXO3a was suppressed in the presence of compound C, a specific AMPK inhibitor, suggesting the involvement of AMPK in paclitaxel-induced FOXO3a phosphorylation. The induction and phosphorylation of FOXO3a by paclitaxel were not observed in EF1α and AMPK knockdown cells. Co-treatment with AICAR resulted in increased susceptibility of cancer cells to paclitaxel-induced suppression of their viability and further enhanced paclitaxel-induced FOXO3a phosphorylation. These results suggest that the antitumor effects of paclitaxel in breast cancer are mediated by activation of the AMPK/EF1α/FOXO3a signaling pathway.

Demethylation of RUNX3 by vincristine in colorectal adenocarcinoma cells.

BACKGROUND: Methylation-mediated inactivation of tumor-suppressor genes is a critical event during the pathogenesis of many malignancies. Vincristine is a conventional anticancer drug used to treat various types of cancers. However, few studies describe the epigenetic-based effects of vincristine. In this study, changes in the methylation of runt-related transcription factor-3 (RUNX3) were investigated in CCD18Co normal colon cells and DLD-1 colorectal adenocarcinoma cells. MATERIALS AND METHODS: CCD18Co and DLD-1 cells were treated with vincristine, and the methylation status was assessed using quantitative methylation-specific polymerase chain reaction (QMSP). Eleven normal colon tissues and 105 colorectal cancer tissues were investigated by methylation and mRNA expression of RUNX3 using QMSP and real-time reverse transcription polymerase chain reaction (real time-PCR). RESULTS: RUNX3 was demethylated after vincristine treatment in DLD-1 cells. The expression of RUNX3 mRNA was down-regulated in DLD-1 cells because of DNA hypermethylation, but was restored after vincristine treatment. In addition, hypermethylation of RUNX3 was detected in 70 out of 105 colorectal carcinomas (66.7%). RUNX3 hypermethylation was greater in colon cancer tissues than in rectal cancer tissues. The expression of RUNX3 mRNA was reduced in 68 out of 105 colorectal cancer tissues (64.8%). CONCLUSION: These results demonstrate that vincristine demethylates RUNX3 in colorectal adenocarcinoma cells, and restores its expression.

Tiam-1, a GEF for Rac1, plays a critical role in metformin-mediated glucose uptake in C2C12 cells.

Metformin is known to stimulate glucose uptake, but the mechanism for this action is not fully understood. In this study, AMPK activators (AICAR and metformin) increased the expression of T-lymphoma invasion and metastasis-inducing protein-1 (Tiam-1), a Rac1 specific guanine nucleotide exchange factor (GEF), mRNA and protein in skeletal muscle C2C12 cells. Metformin increases the serine-phosphorylation of Tiam-1 by AMPK and induces interaction between Tiam-1 and 14-3-3. Pharmacologic inhibition of AMPK blocks this interaction, indicating that 14-3-3 may be required for induction of Tiam-1 by AMPK. Metformin also increases the phosphorylation of p21-activated kinase 1 (PAK1), a direct downstream target of Rac1, dependent on AMPK. Tiam-1 is down-regulated at high glucose concentrations in cultured cells and in the db/db mouse model of hyperglycemia. Furthermore, Tiam-1 knock-down blocked metformin-induced increase in glucose uptake. These findings suggest that metformin promotes cellular glucose uptake in part through Tiam-1 induction.

Health-related quality of life and cognitive functioning at on- and off-treatment periods in children aged between 6-13 years old with brain tumors: a prospective longitudinal study.

PURPOSE: Our study aimed to examine the relationship between intelligence and health-related quality of life (HRQOL) in children (6-13 years old) diagnosed as having a brain tumor. MATERIALS AND METHODS: We administered a Korean version of the Wechsler Intelligence Scale for Children-III, the Pediatric Quality of Life Inventory, version 4.0 (PedsQL), the Korean version of the Parenting Stress Index-Short Form, and the Korean Version of the Parenting Sense of Competence (K-PSOC) scale before or after initial radiotherapy (T1) and after treatment termination (T2). In total, 13 patients completed both the T1 and T2 interviews. RESULTS: Scores significantly declined between T1 and T2 on the full-scale intelligence quotients (FIQ), verbal intelligence quotients (VIQ), performance intelligence quotients (PIQ), similarity and coding tests, as well as the K-PSOC, which measures parental anxiety. FIQ scores at T1 were correlated with the self-reported PedsQL total scores (r=0.739) and the parent proxy-report PedsQL scores for school functioning (r=0.706) at T2. Also, the FIQ scores at T2 were correlated with the self-reported PedsQL total scores (r=0.748) and scores for physical health (r=0.728) at T2. CONCLUSION: The cognitive ability and intelligence level of the patients significantly declined between on and off treatment periods, and higher intelligence functioning at both on and off treatment was correlated with long-term higher HRQOL. Further investigations that monitor intelligence, HRQOL and parenting stress over a longer period, using a greater number of participants, are needed.

Celastrol suppresses breast cancer MCF-7 cell viability via the AMP-activated protein kinase (AMPK)-induced p53-polo like kinase 2 (PLK-2) pathway.

Celastrol, an anti-oxidant flavonoid that is widely distributed in the plant kingdom, has been suggested to have chemopreventive effects on cancer cells: however, the mechanism of this process is not completely understood. In this study, we found that celastrol suppressed the viability of breast cancer MCF-7 cells in an AMP-activated protein kinase (AMPK)-dependent fashion. Celastrol also induced an increase in reactive oxygen species (ROS) levels, leading to AMPK phosphorylation. Protein kinase C (PKC) zeta was also shown to play a role in celastrol-induced ROS generation. In addition, celastrol increased phosphorylation of the pro-apoptotic effector, p53. Inhibition of AMPK blocked celastrol-mediated p53 phosphorylation. Moreover, celastrol increased the expression of tumor suppressor polo like kinase-2 (PLK-2) in a p53-dependent manner. Neither celastrol-induced PLK-2 induction nor celastrol-mediated apoptosis inducing factor poly(ADP-ribose) polymerase-2 (PARP-2) induction was observed in p53 knock-out cells. Furthermore, add-back of PLK-2 resulted in an increase in both celastrol-mediated PARP-2 induction and celastrol-induced apoptotic index sub G1 population. Together, these results suggest that celastrol may have anti-tumor effects on MCF-7 cells via AMPK-induced p53 and PLK-2 pathways.

Metformin regulates glucose transporter 4 (GLUT4) translocation through AMP-activated protein kinase (AMPK)-mediated Cbl/CAP signaling in 3T3-L1 preadipocyte cells.

Metformin is a leading oral anti-diabetes mellitus medication and is known to stimulate GLUT4 translocation. However, the mechanism by which metformin acts is still largely unknown. Here, we showed that short time treatment with metformin rapidly increased phosphorylation of Cbl in an AMP-activated protein kinase (AMPK)-dependent fashion in 3T3-L1 preadipocytes. Metformin also increased phosphorylation of Src in an AMPK-dependent manner. Src inhibition blocked metformin-mediated Cbl phosphorylation, suggesting that metformin stimulates AMPK-Src-Cbl axis pathway. In addition, long term treatment with metformin stimulated the expression of Cbl-associated protein (CAP) mRNA and protein. Long term treatment with metformin stimulated phosphorylation of c-Jun N-terminal kinase (JNK) and its downstream molecule c-Jun, which is a critical molecule for CAP transcription. Knockdown of AMPK and JNK blocked metformin-induced expression of CAP, implying that metformin stimulates AMPK-JNK-CAP axis pathway. Moreover, AMPK knockdown attenuated metformin-induced Cbl/CAP multicomplex formation, which is critical for GLUT4 translocation. A colorimetric absorbance assay demonstrated that metformin-induced translocation of GLUT4 was suppressed in CAP or Cbl knockdown cells. Furthermore, the promoter activity of CAP was increased by metformin in an AMPK/JNK-dependent fashion. In summary, these results demonstrate that metformin modulates GLUT4 translocation by regulating Cbl and CAP signals via AMPK.

Recovery of pigmentation following selective photothermolysis in adult zebrafish skin: clinical implications for laser toning treatment of melasma.

In recent years, laser toning has gained popularity for the treatment of melasma, and tyrosinase inhibitors are conventionally used to prevent its recurrence after this treatment. The effectiveness of this treatment modality, however, is still questionable, and additional in vivo studies are needed to validate the method. In this study, we used adult zebrafish skin as an adult melanocyte regenerative system and examined the simulated human skin response to laser toning. Melanosomes regenerated after selective photothermolysis, and these organelles showed a bi-directional translocation pattern in accordance with the changes of intact melanosome patterns. Furthermore, a tyrosinase inhibitor, 1-phenyl-2-thiourea (PTU), completely blocked melanosome regeneration after laser irradiation, but this inhibitor failed to prevent melanosome regeneration after the medication was discontinued. Finally, arbutin and kojic acid, the commercially available tyrosinase inhibitors, slowed down but did not completely block melanosome regeneration. Based on these findings, we describe the limitations of laser toning treatment of melasma and the combined use of tyrosinase inhibitors. We suggest that melanosomes in adult zebrafish skin can be utilized for studying melanosome regeneration response to laser irradiation and for developing a system to assess the comparative efficacy of melanogenic regulatory compounds.

Streptococcus mutans GS-5 antigen I/II stimulates cell survival in serum deprived-cultures through PI3K/Akt pathways.

The antigen I/II (AgI/II) protein is a major surface protein that mediates the attachment of Streptococcus mutans (S. mutans) to the saliva-coated pellicle. Numerous studies have investigated not only the mechanisms by which AgI/II signaling is transduced within cells, but have also attempted to use AgI/II-specific antibodies to treat dental caries and host immune responses. However, little information is available about the effects of AgI/II on basic cellular events in bone cells. In this study, we examined the effects of the His-tagged recombinant N-terminal half of the AgI/II protein (rAgI/II-N) generated from S. mutans GS-5 on the viability, proliferation, and cell cycle progression of primary calvarial osteoblasts. We also investigated the mechanisms involved in the rAgI/II-N-mediated survival of serum-starved osteoblasts. We found that rAgI/II treatment attenuated the serum deprivation-induced decrease in cell viability and proliferation of osteoblasts. rAgI/II-N also prevented the loss of mitochondrial membrane potential (MMP), alterations in levels of two key mitochondrial Bcl-2 family proteins, and the accumulation of numerous cells into the sub-G(1) phase that were observed in serum-starved osteoblasts. Pharmacological inhibitors of phosphoinositide 3-kinase (PI3K), but not of extracellular signal-regulated kinase or Ras, blocked the rAgI/II-N-mediated protection against serum deprivation-induced cell death. Additional experiments revealed that the integrin α5ß1-mediated PI3K pathway is required for rAgI/II-N-mediated Akt phosphorylation in osteoblasts. Collectively, these results suggest that rAgI/II-N induces survival signals in serum-starved osteoblasts through integrin-induced PI3K/Akt signaling pathways.

Radish (Raphanus sativus L. leaf) ethanol extract inhibits protein and mRNA expression of ErbB(2) and ErbB(3) in MDA-MB-231 human breast cancer cells.

In this study, we investigated the effects of the ethanol extract of aerial parts of Raphanus sativus L. (ERL) on breast cancer cell proliferation and gene expression associated with cell proliferation and apoptosis in MDA-MB-231 human breast cancer cells. The MDA-MB-231 cells were cultured in the presence or absence of various concentrations (100, 200, or 300 µg/mL) of ERL. ERL significantly decreased cell proliferation after 48 h of incubation (P < 0.05). The protein and mRNA expression of ErbB(2) were decreased significantly in a dose-dependent manner (P < 0.05). The protein expression of ErbB(3) was decreased significantly at an ERL concentration of 300 µg/mL (P < 0.05), and mRNA expression of ErbB(3) was decreased significantly in a dose-dependent manner (P < 0.05). The protein expression of Akt was decreased significantly at the ERL concentration of 200 µg/mL (P < 0.05), and the protein expression of pAkt was decreased significantly in a dose-dependent manner (P < 0.05). The mRNA expression of Akt was decreased significantly at the ERL concentration of 200 µg/mL ERL (P < 0.05). The protein and mRNA expression of Bax were increased significantly at ERL concentrations of 200 µg/mL or higher (P < 0.05). The protein expression of Bcl(2) was increased significantly at ERL concentrations of 100 µg/mL or higher (P < 0.05), and mRNA expression of Bcl(2) was increased significantly at an ERL concentration of 300 µg/mL (P < 0.05). In conclusion, we suggest that Raphanus sativus, L. inhibits cell proliferation via the ErbB-Akt pathway in MDA-MB-231 cells.