Oxaliplatin induces immunogenic cells death and enhances therapeutic efficacy of checkpoint inhibitor in a model of murine lung carcinoma

Abstract

Background: Platinum compounds are commonly used for lung cancer treatment. However, the severe side effects and relatively poor prognosis limit their therapeutic effect. Therefore, developing novel platinum derivative and treatment strategy are critical for current lung cancer therapy.

Methods: Flow cytometry, HMGB1 and ATP release, and immunoblotting were performed to evaluate the Oxaliplatin-induced immunogenic cell death (ICD) in two lung carcinoma cells. Vaccination approach and subcutaneous tumor models were created to analyze the tumor regression effect of Oxaliplatin. PD-L1 mRNA and protein levels were detected in LLC (Lewis lung carcinoma). Enhanced therapeutic efficacy of LLC was assessed by co-administration Oxaliplatin and aPD-L1 in murine lung tumor model.

Results: Oxaliplatin induced robust ICD in LLC cells, activated dendritic cells (DCs, CD80⁺CD86⁺) and enhanced cytotoxic T cells (CD8⁺) in LLC tumor tissues, which resulted in tumor regression. Co-administration of Oxaliplatin and checkpoint inhibitor, aPD-L1, could enhance the therapeutic efficacy of LLC in murine lung carcinoma.

Conclusion: This study reveals Oxaliplatin can induce robust ICD in tumor tissues and suppress tumor growth by activating DCs and enhancing T-cell infiltration. Notably, the Oxaliplatin-induced ICD provides an immunogenic microenvironment, which enhances the checkpoint inhibitor therapeutic efficacy of LLC.     

Development of a novel chemiluminescence method for the determination of cefazolin sodium in injectable powder and human urine based on a luminol‐Cu(III) complex reaction in alkaline medium

A novel chemiluminescence (CL) method was developed for the determination of cefazolin sodium based on the CL reaction between the [Cu(HIO₆)₂]^5‐Cu(III) complex and luminol in alkaline solution. Results showed that CL emission of Cu(III) complex–luminol in alkaline medium was significantly different from that in acidic medium. A possible mechanism of the enhanced effect of cefazolin on CL emission of the [Cu(HIO₆)₂]^5‐‐ luminol system was proposed. The effect of the reaction conditions on CL emissions was examined. Under optimized conditions, a good linear relationship was obtained between CL intensity and concentrations of cefazolin sodium in the range of 2.0 x 10^‐8 to 2.0 x 10^‐6 g/mL with a correlation coefficient of R² = 0.9978. The limit of detection was 4.58 x 10^‐9 g/mL. The proposed method was applied for the determination of cefazolin sodium in real samples with recoveries of 82.0‐109% with an RSD of 0.7‐2.1%. The proposed method was successfully used for the determination of cefazolin sodium in injectable powder preparations and human urine with satisfactory results. Copyright © 2012 John Wiley & Sons, Ltd.     

Lipopolysaccharide neutralization by a novel peptide derived from phosvitin

Lipopolysaccharide (LPS), also known as endotoxin, is the primary trigger of sepsis, which is associated with high mortality in patients. No therapeutic agents are currently efficacious enough to protect patients from sepsis characterized by LPS-mediated tissue damage and organ failure. Previously, a phosvitin-derived peptide, Pt5, which consists of the C-terminal 55 residues of zebrafish phosvitin, has been shown to function as an antibacterial agent. In this study, we have generated six mutants by site-directed mutagenesis based on the sequence of Pt5, and found that one of the six mutants, Pt5e, showed the strongest bactericidal activities against Escherichia coli and Staphylococcus aureus. We then demonstrated that Pt5e was able to bind to LPS and lipoteichoic acid (LTA). More importantly, we showed that Pt5e significantly inhibited LPS-induced tumor-necrosis factor (TNF)-α and interleukin (IL)-1β release from murine RAW264.7 cells and considerably reduced serum TNF-α and IL-1β levels in mice. Additionally, Pt5e protected the liver from damage by LPS, and remarkably promoted the survival rate of the endotoxemia mice. Furthermore, Pt5e displayed no cytotoxicity to murine RAW264.7 macrophages and no hemolytic activity toward human red blood cells. These data together indicate that Pt5e is an endotoxin-neutralizing agent with a therapeutic potential in clinical treatment of LPS-induced sepsis.     

Rapid Identification of Antifungal Compounds against Exserohilum rostratum Using High Throughput Drug Repurposing Screens

A recent large outbreak of fungal infections by Exserohilum rostratum from contaminated compounding solutions has highlighted the need to rapidly screen available pharmaceuticals that could be useful in therapy. The present study utilized two newly-developed high throughput assays to screen approved drugs and pharmaceutically active compounds for identification of potential antifungal agents. Several known drugs were found that have potent effects against E. rostratum including the triazole antifungal posaconazole. Posaconazole is likely to be effective against infections involving septic joints and may provide an alternative for refractory central nervous system infections. The anti-E. rostratum activities of several other drugs including bithionol (an anti-parasitic drug), tacrolimus (an immunosuppressive agent) and floxuridine (an antimetabolite) were also identified from the drug repurposing screens. In addition, activities of other potential antifungal agents against E. rostratum were excluded, which may avoid unnecessary therapeutic trials and reveals the limited therapeutic alternatives for this outbreak. In summary, this study has demonstrated that drug repurposing screens can be quickly conducted within a useful time-frame. This would allow clinical implementation of identified alternative therapeutics and should be considered as part of the initial public health response to new outbreaks or rapidly-emerging microbial pathogens.     

Reduced Levels of NR1 and NR2A with Depression-Like Behavior in Different Brain Regions in Prenatally Stressed Juvenile Offspring

Adolescence is a time of continued brain maturation, particularly in limbic and cortical regions, which undoubtedly plays a role in the physiological and emotional changes. Juvenile rats repeatedly exposed to prenatal stress (PS) exhibit behavioral features often observed in neuropsychiatric disorders including depression. However, to date the underlying neurological mechanisms are still unclear. In the current study, juvenile offspring rats whose mothers were exposed to PS were evaluated for depression-related behaviors in open field and sucrose preference test. NMDA receptor subunits NR1 and NR2A in the hippocampus, frontal cortex and striatum were assayed by western blotting. The results indicated that PS resulted in several behavioral anomalies in the OFT and sucrose preference test. Moreover, reduced levels of NMDA receptor subunits NR1 and NR2A in the hippocampus, and NR1 in prefrontal cortex and striatum of prenatally stressed juvenile offspring were found. Treatment with MK-801 to pregnant dams could prevent all those changes in the juvenile offspring. Collectivity, these data support the argument that PS to pregnant dams could induce depression-like behavior, which may be involved with abnormal expression of NR1 and NR2A in specific brain regions, and MK-801 may have antidepressant-like effects on the juvenile offspring.     

Intratumoral Hepatic Stellate Cells as a Poor Prognostic Marker and a New Treatment Target?for?Hepatocellular Carcinoma

Hepatic stellate cells (HSCs), a specialized stromal cytotype in the liver, have been demonstrated to actively contribute to hepatocellular carcinoma (HCC) development. However, the previous studies were performed using HSC cell lines, and the prognostic value of intratumoral HSCs (tHSCs) was unclear. Here we isolated tHSCs from fresh human HCC tissues, and analyzed the abilities of tHSCs to promote HCC progression by using in vitro assays for cell viability, migration and invasion as well as epithelial-mesenchymal transition (EMT) phenotype. 252 HCC patients who underwent hepatectomy were enrolled for analysis of tHSCs and E-cadherin expression in tumor tissues, and 55 HCC patients for analysis of tHSCs in tumor tissues and circulating tumor cells (CTCs) in blood. Prognostic factors were then identified. The results showed that coculture of tHSCs with HCC cells had a stronger effect on HCC cell viability, migration and invasion, accompanied with the acquisition of epithelial-mesenchymal transition (EMT) phenotype. In vivo cotransplantation of HCC cells with tHSCs into nude mice more efficiently promoted tumor formation and growth. Icaritin, a known apoptosis inducer of HSCs, was demonstrated to effectively inhibit tHSC proliferation in vitro and tHSC-induced HCC-promoting effects in vivo. Clinical evidence indicated that tHSCs were rich in 45% of the HCC specimens, tHSC-rich subtypes were negatively correlated either with E-cadherin expression in tumor tissues (r = -0.256, p < 0.001) or with preoperative CTCs in blood (r = -0.287, p = 0.033), and were significantly correlated with tumor size (p = 0.027), TNM staging (p = 0.018), and vascular invasion (p = 0.008). Overall and recurrence-free survival rates of tHSC-rich patients were significantly worse than those for tHSC-poor patients. Multivariate analysis revealed tHSC-rich as an independent factor for overall and recurrence-free survival. In conclusion, tHSCs provide a promising prognostic biomarker and a new treatment target for HCC.     

Ursolic Acid Inhibits Adipogenesis in 3T3-L1 Adipocytes through LKB1/AMPK Pathway

Background

Ursolic acid (UA) is a triterpenoid compound with multiple biological functions. This compound has recently been reported to possess an anti-obesity effect; however, the mechanisms are less understood.

Objective

As adipogenesis plays a critical role in obesity, the present study was conducted to investigate the effect of UA on adipogenesis and mechanisms of action in 3T3-L1 preadipocytes.

Methods and Results

The 3T3-L1 preadipocytes were induced to differentiate in the presence or absence of UA for 6 days. The cells were determined for proliferation, differentiation, fat accumulation as well as the protein expressions of molecular targets that regulate or are involved in fatty acid synthesis and oxidation. The results demonstrated that ursolic acid at concentrations ranging from 2.5 µM to 10 µM dose-dependently attenuated adipogenesis, accompanied by reduced protein expression of CCAAT element binding protein β (C/EBP_β), peroxisome proliferator-activated receptor γ (PPAR_γ), CCAAT element binding protein α (C/EBP_α) and sterol regulatory element binding protein 1c (SREBP-1c), respectively. Ursolic acid increased the phosphorylation of acetyl-CoA carboxylase (ACC) and protein expression of carnitine palmitoyltransferase 1 (CPT1), but decreased protein expression of fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Ursolic acid increased the phosphorylation of AMP-activated protein kinase (AMPK) and protein expression of (silent mating type information regulation 2, homolog) 1 (Sirt1). Further studies demonstrated that the anti-adipogenic effect of UA was reversed by the AMPK siRNA, but not by the Sirt1 inhibitor nicotinamide. Liver kinase B1 (LKB1), the upstream kinase of AMPK, was upregulated by UA. When LKB1 was silenced with siRNA or the inhibitor radicicol, the effect of UA on AMPK activation was diminished.

Conclusions

Ursolic acid inhibited 3T3-L1 preadipocyte differentiation and adipogenesis through the LKB1/AMPK pathway. There is potential to develop UA into a therapeutic agent for the prevention or treatment of obesity.     

Salt-Restriction-Spoon Improved the Salt Intake among Residents in China

Objective

To evaluate the effect of an improved salt-restriction spoon on the attitude of salt-restriction, the using rate of salt-restriction-spoon, the actual salt intake, and 24-hour urinary sodium excretion (24HUNa).

Design

A community intervention study.

Setting

Two villages in Beijing.

Participants

403 local adult residents being responsible for home cooking.

Intervention

Participants were randomly assigned to the intervention group or the control group. Those in the intervention group were provided with an improved salt-restriction-spoon and health education, and were informed of their actual salt intake and 24HUNa. Not any intervention was given to those in the control group.

Main Outcome Measures

The scores on the variables of Health Belief Model, the using rate of salt-restriction-spoon, the actual salt intake, and 24HUNa.

Analysis

Covariance analyses, Chi-square tests, Student’s t tests, and repeated measures analyses of variance.

Results

After 6 months of intervention, the intervention group felt significantly less objective barriers, and got access to significantly more cues to action as compared to the control group. The using rate and the correctly using rate of salt-restriction-spoon were significantly higher in the intervention group. The daily salt intake decreased by 1.42 g in the intervention group and by 0.28 g in the control group, and repeated measures analysis of variance showed significant change over time (F = 7.044, P<0.001) and significant difference between groups by time (F = 2.589, P = 0.041). The 24HUNa decreased by 34.84 mmol in the intervention group and by 33.65 mmol in the control group, and repeated measures analysis of variance showed significant change over time (F = 14.648, P<0.001) without significant difference between groups by time (F = 0.222, P = 0.870).

Conclusions

The intervention effect was acceptable, therefore, the improved salt-restriction-spoon and corresponding health education could be considered as an alternative for salt reduction strategy in China and other countries where salt intake comes mainly from home cooking.     

Conditional Deletion of the Pten Gene in the Mouse Prostate Induces Prostatic Intraepithelial Neoplasms at Early Ages but a Slow Progression to Prostate Tumors

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancer. Using Osr1 (odd skipped related 1)-Cre mice, we generated a novel conditional Pten knockout mouse strain, Pten^LoxP:Osr1-Cre. Conditional biallelic and monoallelic Pten knockout mice were viable. Deletion of Pten expression was detected in the prostate of Pten^LoxP/LoxP:Osr1-Cre mice as early as 2 weeks of age. Intriguingly, Pten^LoxP/LoxP:Osr1-Cre mice develop high-grade prostatic intraepithelial neoplasms (PINs) with high penetrance as early as one-month of age, and locally invasive prostatic tumors after 12-months of age. Pten^LoxP/+:Osr1-Cre mice show only mild oncogenic changes after 8-weeks of age. Castration of Pten^LoxP/LoxP:Osr1-Cre mice shows no significant regression of prostate tumors, although a shift of androgen receptor (AR) staining from the nuclei to cytoplasm is observed in Pten null tumor cells of castrated mice. Enhanced Akt activity is observed in Pten null tumor cells of castrated Pten^LoxP/LoxP:Osr1-Cre. This study provides a novel mouse model that can be used to investigate a primary role of Pten in initiating oncogenic transformation in the prostate and to examine other genetic and epigenetic changes that are required for tumor progression in the mouse prostate.