Anlotinib Reverses Multidrug Resistance (MDR) in Osteosarcoma by Inhibiting P-Glycoprotein (PGP1) Function In Vitro and In Vivo

Overexpression of the multidrug resistance (MDR)-related protein P-glycoprotein (PGP1), which actively extrudes chemotherapeutic agents from cells and significantly decreases the efficacy of chemotherapy, is viewed as a major obstacle in osteosarcoma chemotherapy. Anlotinib, a novel tyrosine kinase inhibitor (TKI), has good anti-tumor effects in a variety of solid tumors. However, there are few studies on the mechanism of anlotinib reversing chemotherapy resistance in osteosarcoma. In this study, cellular assays were performed in vitro and in vivo to evaluate the MDR reversal effects of anlotinib on multidrug-resistant osteosarcoma cell lines. Drug efflux and intracellular drug accumulation were measured by flow cytometry. The vanadate-sensitive ATPase activity of PGP1 was measured in the presence of a range of anlotinib concentrations. The protein expression level of ABCB1 was detected by Western blotting and immunofluorescence analysis. Our results showed that anlotinib significantly increased the sensitivity of KHOSR2 and U2OSR2 cells (which overexpress PGP1) to chemotherapeutic agents in vitro and in a KHOSR2 xenograft nude mouse model in vivo. Mechanistically, anlotinib increases the intracellular accumulation of PGP1 substrates by inhibiting the efflux function of PGP1 in multidrug-resistant cell lines. Furthermore, anlotinib stimulated the ATPase activity of PGP1 but affected neither the protein expression level nor the localization of PGP1. In animal studies, anlotinib in combination with doxorubicin (DOX) significantly decreased the tumor growth rate and the tumor size in the KHOSR2 xenograft nude mouse model. Overall, our findings suggest that anlotinib may be useful for circumventing MDR to other conventional antineoplastic drugs.

Cang-Ai Volatile Oil Ameliorates Depressive Behavior Induced by Chronic Stress Through IDO-Mediated Tryptophan Degradation Pathway

Background: Cang-ai volatile oil (CAVO) is a Chinese herbal volatile oil. Previous studies report that CAVO exhibits of anti-depressant and anti-inflammatory effects, and modulates activity of monoamine neurotransmitter. The current study sought to explore whether CAVO exhibits anti-depressant effects of CAVO through inhibition of inflammatory response and regulation of indoleamine 2 and 3-dioxygenase (IDO) mediated tryptophan degradation pathway.Methods: The study established chronic unpredictable mild stress (CUMS) depression-like model using rats. Body weight and food intake of animals were determined, and open field test (OFT), forced swim test (FST), and sucrose preference test (SPT) were performed to explored the behavioral changes of animals. Expression levels of interleukin-6 (IL-6), interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-4 (IL-4), interleukin-10 (IL-10), kynurenine (KYN), quinolinic acid (QUIN), tryptophan (Trp), kynurenic acid (KYNA), serotonin (5-HT), and 5-hydroxyindole acetic acid (5-HIAA) in the prefrontal cortex of CUMS rats were determined by ELISA. Co-localization of the microglia markers, Iba1 and IL-6 was determined by immunofluorescence. Western blotting was performed to determine the protein expression level of IDO1.Results: The findings of the current study showed that CAVO increased the body weight and food intake of rats and alleviated depression-like behaviors as shown in OFT, FST, and SPT analysis. ELISA assay showed that CAVO decreased IL-6, IL-1β, TNF-α, and IFN-γ levels and increased levels of IL-4 and IL-10 in the prefrontal cortex of CUMS rats. Analysis showed that CAVO significantly reduced KYN and QUIN levels and the ratio of KYN/Trp, whereas it increased the levels of Trp, KYNA, 5-HT, and 5-HIAA. Immunofluorescence analysis showed that CAVO reduced the number of positive cells with co-localization of microglia markers, Iba1 and IL-6. Western blot analysis showed that CAVO decreased the protein expression level of IDO1 in rats.Conclusion: The findings show that the anti-depressant effects of CAVO are mainly attributed to inhibition of the activation of microglia and downregulation of IDO expression, thus inhibiting the kynurenine pathway and reversing the effects exerted on the 5-HT system.

Renocardiac protective effects of SGLT2 inhibitor combined with angiotensin receptor blocker in salt sensitive Dahl rats

Background Kidney plays a central role in regulating salt-sensitivity of blood pressure (BP) to governs sodium excretion via several mechanisms including pressure natriuresis and the actions of renal sodium transporters. Purpose We clarified the effects of combination treatment of sodium-glucose cotransporter 2 (SGLT2) inhibitor and angiotensin receptor blocker (ARB) on BP and the pathogenesis of renocardiac injuries, and elucidated underlying molecular mechanisms involved in the regulation of renal sodium handling in the development of salt-sensitivity by comparing with each monotreatment in Dahl salt-sensitive (DSS) hypertensive rats. Methods DSS rats were treated orally for 8-weeks with normal salt diet (0.3% NaCl) (NS/Cont group), high salt diet (8% NaCl) (HS/Cont group), high salt diet with ipragliflozin (0.04%) (HS/Ipra group), high salt diet with losartan (0.05%) (HS/Los group), or high salt diet with combination of ipragliflozin and losartan (HS/Ipra+Los group). Results The combination group significantly reduced systolic BP compared with either high salt diet control group, losartan or ipragliflozin monotreatment groups (HS/Ipra+Los: 182.5±18.4mmHg vs HS/Cont: 227.7±26.1; HS/Ipra: 216.6±26.9; HS/Los: 208.6±21.6, at 8-weeks of treatment, P<0.05, respectively) (Figure 1A). The slope of pressure-natriuresis curve was significantly increased in the HS/Ipra+Los group compared to that in the HS/Cont group (interaction P=0.024), HS/Ipra group (P=0.009), and HS/Los group (P=0.084) using the linear regression model (Figure 1B), which indicated that only the combination treatment of ipragliflozin and losartan improved salt-sensitivity. The combined treatment significantly improved creatinine clearance (HS/Ipra+Los: 3.3±0.9mL/min vs HS/Cont: 1.1±0.5; HS/Ipra: 1.7±0.6; HS/Los: 1.9±0.8, P<0.05, respectively). The combination treatment also significantly ameliorated glomerulosclerosis, and improved cardiomyocyte hypertrophy and perivascular fibrosis (Figure 1C). Angiotensin II type 1 receptor (AT1R) protein expression level in the kidney was remarkably suppressed in the combination treatment group compared to the other high salt diet groups. The protein expression level of Na+/H+ exchanger isoform 3 (NHE3) and Na+-K+-Cl– cotransporter 2 (NKCC2), two of major sodium transports in the renal tubules, were significantly decreased with losartan monotreatment and combination treatment, but not with ipragliflozin monotreatment (Figure 2). Conclusions The dual inhibition of SGLT2 and AT1R effectively improved salt-sensitivity via reducing renal expression levels of the sodium transporters, which eventually lead to renocardiac protection. Thus, the combination treatment could be a novel and useful therapeutic strategy for treating salt-sensitive hypertension and renal injury in non-diabetic patients. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research

Latanoprost eye drops induce conjunctival lymphatic vessel development

AIM: To investigate the effect of latanoprost eye drops on the conjunctival lymphatics. METHODS: Twenty-four healthy New Zealand White rabbits weighing 1.5 to 2.0 kg were randomly divided into three groups: latanoprost group (n=8) administered with latanoprost eye drops once a day for 2mo, carteolol group (n=8) administered with carteolol eye drops once a day for 2mo, and control group (n=8) without any treatment. The conjunctival tissues in the three groups were extracted to investigate the expression levels of 5’-nucleotidase (5’-Nase) by Western blot, reverse transcription-polymerase chain reaction (RT-PCR), and immunofluorescence staining, respectively. RESULTS: The protein expression level of 5’-Nase was significantly higher in latanoprost group than carteolol group (F=231.175, P<0.001) and control group (P<0.001), while there was no significant difference between the carteolol group and the control group (P>0.05). The mRNA expression level of 5’-Nase in the latanoprost group was also significantly higher than carteolol group (F=71.169 P<0.005) and control group (P<0.005). The conjunctival lymphatics were positive immunofluorescence stained with the 5’-Nase antibodies in the latanoprost group and not stained in the control group. CONCLUSION: Latanoprost eye drops can induce conjunctival lymphangiogenesis which may be concerned in clinical implications.

New insights into the role of PTCH1 protein in serous ovarian carcinomas

Background The Hedgehog (Hh) signaling pathway is essential for normal embryonic development, while its hyperactivation in adult organism is associated with development of various cancers, including ovarian cancer. The role of the Hh signaling pathway in ovarian cancer, as well as in certain histological subtypes of ovarian cancer, is poorly understood. Therefore, we investigated the role of PTCH1 protein and changes in the promoter methylation status of the corresponding gene, in a cohort of low- (LGSC) and high-grade (HGSC) serous ovarian carcinomas and HGSC cell lines (OVCAR5, OVCAR8 and OVSAHO). Methods PTCH1 protein expression level was analyzed using immunohistochemistry in tissue samples, and by immunofluorescence and Western blot in cell lines. DNA methylation pattern of PTCH1 gene were analyzed by methylation-specific PCR (MSP). Mann-Whitney U test was used to compare differences in expression of PTCH1 protein among ovarian tumor samples compared with normal tissue samples, while Spearman’s correlation was used to test the association between DNA promoter methylation of the PTCH1 gene and expression of the corresponding protein. Results PTCH1 protein expression level was significantly higher in HGSCs and LGSCs compared with control tissues (healthy ovaries and fallopian tubes). Similarly, cancer cell lines exhibited significantly higher PTCH1 protein expression in comparison with normal fallopian tube non-ciliated epithelium cell line (FNE1). Nuclear localization of the PTCH1 protein in tumor tissue and cultured tumor cells suggests that this protein could play an active tumor promoter role in the nuclei of serous ovarian carcinoma cells. PTCH1 protein fragments of different molecular weights were detected in the cell lines, indicating possible proteolytic cleavage of this protein, resulting in the generation of soluble N-terminal fragments that are translocated to the nucleus. DNA methylation of the PTCH1 gene promoter was not in line with the expression level of this protein, suggesting that possibly other mechanisms, either epigenetic or posttranslational, regulate PTCH1 gene expression and protein level in serous ovarian carcinomas. Conclusions Our results indicate that PTCH1 protein could play an active tumor promoter role in the pathogenesis of serous ovarian carcinoma.

Phosphorylation at Serines 157 and 161 Is Necessary for Preserving Cardiac Expression Level and Functions of Sarcomeric Z-Disc Protein Telethonin

Aims: In cardiac myocytes, the sarcomeric Z-disc protein telethonin is constitutively bis-phosphorylated at C-terminal residues S157 and S161; however, the functional significance of this phosphorylation is not known. We sought to assess the significance of telethonin phosphorylation in vivo, using a novel knock-in (KI) mouse model generated to express non-phosphorylatable telethonin (TcapS157/161A).Methods and Results:TcapS157/161A and wild-type (WT) littermates were characterized by echocardiography at baseline and after sustained β-adrenergic stimulation via isoprenaline infusion. Heart tissues were collected for gravimetric, biochemical, and histological analyses. At baseline, TcapS157/161A mice did not show any variances in cardiac structure or function compared with WT littermates and mutant telethonin remained localized to the Z-disc. Ablation of telethonin phosphorylation sites resulted in a gene-dosage dependent decrease in the cardiac telethonin protein expression level in mice carrying the S157/161A alleles, without any alteration in telethonin mRNA levels. The proteasome inhibitor MG132 significantly increased the expression level of S157/161A telethonin protein in myocytes from TcapS157/161A mice, but not telethonin protein in myocytes from WT mice, indicating a role for the ubiquitin–proteasome system in the regulation of telethonin protein expression level. TcapS157/161A mice challenged with sustained β-adrenergic stimulation via isoprenaline infusion developed cardiac hypertrophy accompanied by mild systolic dysfunction. Furthermore, the telethonin protein expression level was significantly increased in WT mice following isoprenaline stimulation but this response was blunted in TcapS157/161A mice.Conclusion: Overall, these data reveal that telethonin protein turnover in vivo is regulated in a novel phosphorylation-dependent manner and suggest that C-terminal phosphorylation may protect telethonin against proteasomal degradation and preserve cardiac function during hemodynamic stress. Given that human telethonin C-terminal mutations have been associated with cardiac and skeletal myopathies, further research on their potential impact on phosphorylation-dependent regulation of telethonin protein expression could provide valuable mechanistic insight into those myopathies.

Umbilical Cord Mesenchymal Stem Cell Exosomes Alleviate the Progression of Kidney Failure by Modulating Inflammatory Responses and Oxidative Stress in an Ischemia-Reperfusion Mice Model

The efficacy of stem cells for the treatment of renal failure is widely recognized; however, an excessive volume of stem cells can block the capillaries; thus, the potential risks should not be ignored. Stem cell exosomes are secretory extracellular vesicles with a size of 30–150 nm, which have similar functions to stem cells but are much smaller in size. This study aims to investigate the role of human umbilical cord mesenchymal stem cells (UCMSCs)-derived exosomes in the treatment of renal failure caused by ischemia-reperfusion. Fifty 8-week-old female C57 mice underwent bilateral renal ischemia-reperfusion surgery for 30 minutes. After 4 weeks, the treated group received UCMSCs-derived exosomes treatment, and the control group was solely injected with the same amount of PBS. At the age of 16 weeks, the kidney function, kidney damage, inflammatory responses and oxidative stress were measured. Moreover, the effect of UCMSCs-derived exosomes on the phenotype of M1 macrophages was also tested. The results showed that UCMSCsderived exosomes significantly reduced the levels of blood urea nitrogen (BUN), serum creatinine (SCR), and urinary albumin and creatinine (ACR) and 8-isoprostane. UCMSCs-derived exosomes also improved the atrophy of the kidney and glomerulus, decreased kidney pro-inflammatory factors expression (mRNA of II-1β, II-6, Tnf-α, and Mcp-1) and oxidative stress (malondialdehyde), and increased glutathione level. However, F4/80 immunohistochemistry did not show significant differences between the two groups. In systemic inflammation measurement, UCMSCs-derived exosomes decreased proinflammatory factors TNF-α, IL-6, and IL-1β levels, and increased anti-inflammatory factor IL-10 level. In vitro experiments showed that UCMSCs-derived exosomes decreased the protein expression level of TNF-α and increased the protein expression level of IL-10 in M1 macrophages. UCMSCs-derived exosomes reduce kidney inflammation and oxidative stress by improving systemic inflammation, and thus reduce kidney damage and improve kidney function. This study shows the potential application value of exosomes in the treatment of renal failure.

Expression Analysis of α5 Integrin Subunit Reveals Its Upregulation as a Negative Prognostic Biomarker for Glioblastoma

Integrin α5β1 was suggested to be involved in glioblastoma (GBM) aggressiveness and treatment resistance through preclinical studies and genomic analysis in patients. However, further protein expression data are still required to confirm this hypothesis. In the present study, we investigated by immunofluorescence the expression of integrin α5 and its prognostic impact in a glioblastoma series of patients scheduled to undergo the Stupp protocol as first-line treatment for GBM. The integrin α5 protein expression level was estimated in each tumor by the mean fluorescence intensity (MFI) and allowed us to identify two subpopulations showing either a high or low expression level. The distribution of patients in both subpopulations was not significantly different according to age, gender, recursive partitioning analysis (RPA) prognostic score, molecular markers or surgical and medical treatment. A high integrin α5 protein expression level was associated with a high risk of recurrence (HR = 1.696, 95% CI 1.031–2.792, p = 0.0377) and reduced overall survival (OS), even more significant in patients who completed the Stupp protocol (median OS: 15.6 vs. 22.8 months; HR = 2.324; 95% CI 1.168–4.621, p = 0.0162). In multivariate analysis, a high integrin α5 protein expression level was confirmed as an independent prognostic factor in the subpopulation of patients who completed the temozolomide-based first-line treatment for predicting OS over age, extent of surgery, RPA score and O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation (p = 0.029). In summary, for the first time, our study validates that a high integrin α5 protein expression level is associated with poor prognosis in GBM and confirms its potential as a therapeutic target implicated in the Stupp protocol resistance.

MTF1 Is Essential for the Expression of MT1B, MT1F, MT1G, and MT1H Induced by PHMG, but Not CMIT, in the Human Pulmonary Alveolar Epithelial Cells

The inhalation of humidifier disinfectants (HDs) is linked to HD-associated lung injury (HDLI). Polyhexamethylene guanidine (PHMG) is significantly involved in HDLI, but the correlation between chloromethylisothiazolinone (CMIT) and HDLI remains ambiguous. Additionally, the differences in the molecular responses to PHMG and CMIT are poorly understood. In this study, RNA sequencing (RNA-seq) data showed that the expression levels of metallothionein-1 (MT1) isoforms, including MT1B, MT1E, MT1F, MT1G, MT1H, MT1M, and MT1X, were increased in human pulmonary alveolar epithelial cells (HPAEpiCs) that were treated with PHMG but not in those treated with CMIT. Moreover, upregulation of MT1B, MT1F, MT1G, and MT1H was observed only in PHMG-treated HPAEpiCs. The protein expression level of metal regulatory transcription factor 1 (MTF1), which binds to the promoters of MT1 isoforms, was increased in PHMG-treated HPAEpiCs but not in CMIT-treated HPAEpiCs. However, the expression of early growth response 1 (EGR1) and nuclear receptor superfamily 3, group C, member 1 (NR3C1), other transcriptional regulators involved in MT1 isomers, were increased regardless of treatment with PHMG or CMIT. These results suggest that MTF1 is an essential transcription factor for the induction of MT1B, MT1F, MT1G, and MT1H by PHMG but not by CMIT.