Rationally designed catalytic nanoplatform for enhanced chemoimmunotherapy via deploying endogenous plus exogenous copper and remodeling tumor microenvironment.

Chemodynamic therapy represents a novel tumor therapeutic modality via triggering catalytic reactions in tumors to yield highly toxic reactive oxygen species (ROS). Nevertheless, low efficiency catalytic ability, potential systemic toxicity and inefficient tumor targeting, have hindered the efficacy of chemodynamic therapy. Herein, a rationally designed catalytic nanoplatform, composed of folate acid conjugated liposomes loaded with copper peroxide (CP) and chloroquine (CQ; a clinical drug) (denoted as CC@LPF), could power maximal tumor cytotoxicity, mechanistically via maneuvering endogenous and exogenous copper for a highly efficient catalytic reaction. Despite a massive autophagosome accumulation elicited by CP-powered autophagic initiation and CQ-induced autolysosomal blockage, the robust ROS, but not aberrant autophagy, underlies the synergistic tumor inhibition. Otherwise, this combined mode also elicits an early onset, above all, long-term high-level existence of immunogenic cell death markers, associated with ROS and aberrant autophagy -triggered endoplasmic reticulum stress. Besides, CC@LPF, with tumor targeting capability and selective tumor cytotoxicity, could elicit intratumor dendritic cells (mainly attributed to CQ) and tumor infiltrating CD8+ T cells, upon combining with PD-L1 therapeutic antibody, further induce significant anti-tumor effect. Collectively, the rationally designed nanoplatform, CC@LPF, could enhance tumor chemoimmunotherapy via deploying endogenous plus exogenous copper and remodeling tumor microenvironment.

Glucagon Enhances Chemotherapy Efficacy By Inhibition of Tumor Vessels in Colorectal Cancer.

Chemotherapy is widely used to treat colorectal cancer (CRC). Despite its substantial benefits, the development of drug resistance and adverse effects remain challenging. This study aimed to elucidate a novel role of glucagon in anti-cancer therapy. In a series of in vitro experiments, glucagon inhibited cell migration and tube formation in both endothelial and tumor cells. In vivo studies demonstrated decreased tumor blood vessels and fewer pseudo-vessels in mice treated with glucagon. The combination of glucagon and chemotherapy exhibited enhanced tumor inhibition. Mechanistic studies demonstrated that glucagon increased the permeability of blood vessels, leading to a pronounced disruption of vessel morphology. Signaling pathway analysis identified a VEGF/VEGFR-dependent mechanism whereby glucagon attenuated angiogenesis through its receptor. Clinical data analysis revealed a positive correlation between elevated glucagon expression and chemotherapy response. This is the first study to reveal a role for glucagon in inhibiting angiogenesis and vascular mimicry. Additionally, the delivery of glucagon-encapsulated PEGylated liposomes to tumor-bearing mice amplified the inhibition of angiogenesis and vascular mimicry, consequently reinforcing chemotherapy efficacy. Collectively, the findings demonstrate the role of glucagon in inhibiting tumor vessel network and suggest the potential utility of glucagon as a promising predictive marker for patients with CRC receiving chemotherapy.

A Multifunctional Vanadium-Iron-Oxide Nanoparticle Eradicates Hepatocellular Carcinoma via Targeting Tumor and Endothelial Cells.

Nanoparticles are widely used in biological research and cancer therapy. In hepatocellular carcinoma, several nanoplatforms have been synthesized and studied to improve the drug efficacy; however, these nanoplatforms are still insufficient to eradicate tumors. Herein, we have synthesized a novel vanadium (V)-iron-oxide (ION) nanoparticle (VIO) that combines chemodynamic, photothermal, and diagnostic capacities to enhance the tumor suppression effect in one agent with multiple functions. In the in vitro models, hepatocellular carcinoma cells are significantly inhibited by VIO-based nanoagents. The mechanistic study validates that VIO increases reactive oxygen species (ROS), which led to apoptosis and ferroptosis resulting in cell death. To our surprise, VIO targets not only tumor cells but also endothelial cells. In addition to inducing cell death, VIO also blocks tube formation and cell migration in human umbilical vein endothelial cell (HUVEC) and C166 models, indicating an antiangiogenic potential. In mouse tumor models, VIO retards tumor growth and induces apoptosis in tumor tissues. Furthermore, a significant blood vessel regression is seen in VIO-treated groups accompanied with larger necrotic areas. More interestingly, the activation of photothermal therapy completely eradicates tumor tissues. Taken together, this VIO nanoplatform could be a powerful anticancer candidate for nanodrug development.

Identification of prothymosin alpha (PTMA) as a biomarker for esophageal squamous cell carcinoma (ESCC) by label-free quantitative proteomics and Quantitative Dot Blot (QDB).

BACKGROUND: Esophageal cancer (EC) is one of the malignant tumors with a poor prognosis. The early stage of EC is asymptomatic, so identification of cancer biomarkers is important for early detection and clinical practice. METHODS: In this study, we compared the protein expression profiles in esophageal squamous cell carcinoma (ESCC) tissues and adjacent normal esophageal tissues from five patients through high-resolution label-free mass spectrometry. Through bioinformatics analysis, we found the differentially expressed proteins of ESCC. To perform the rapid identification of biomarkers, we adopted a high-throughput protein identification technique of Quantitative Dot Blot (QDB). Meanwhile, the QDB results were verified by classical immunohistochemistry. RESULTS: In total 2297 proteins were identified, out of which 308 proteins were differentially expressed between ESCC tissues and normal tissues. By bioinformatics analysis, the four up-regulated proteins (PTMA, PAK2, PPP1CA, HMGB2) and the five down-regulated proteins (Caveolin, Integrin beta-1, Collagen alpha-2(VI), Leiomodin-1 and Vinculin) were selected and validated in ESCC by Western Blot. Furthermore, we performed the QDB and IHC analysis in 64 patients and 117 patients, respectively. The PTMA expression was up-regulated gradually along the progression of ESCC, and the PTMA expression ratio between tumor and adjacent normal tissue was significantly increased along with the progression. Therefore, we suggest that PTMA might be a potential candidate biomarker for ESCC. CONCLUSION: In this study, label-free quantitative proteomics combined with QDB revealed that PTMA expression was up-regulated in ESCC tissues, and PTMA might be a potential candidate for ESCC. Since Western Blot cannot achieve rapid and high-throughput screening of mass spectrometry results, the emergence of QDB meets this demand and provides an effective method for the identification of biomarkers.

PDLIM5 identified by label-free quantitative proteomics as a potential novel biomarker of papillary thyroid carcinoma.

In order to better understand the mechanisms underlying the development of papillary thyroid carcinoma (PTC), and to identify new potential biomarkers, high-resolution label-free mass spectrometry was performed on PTC tissues and adjacent normal thyroid tissues from six patients. In this process, 2788 proteins were identified, out of which 49 proteins presented significant differences between PTC tissues and adjacent normal thyroid tissues. Gene ontology revealed that the majority of these proteins are involved in the catalytic activity and binding. We selected three proteins with differential expressions: PDZ and LIM domain 5 (PDLIM5), PDLIM1 and ALDH1A1; Protein expressions were further verified by RT-PCR and western blot. Among these, expression of PDLIM5 and PDLIM1 was up-regulated, while that of ALDH1A1 was down-regulated in PTC tissues. Next, we confirmed their expression through quantitative dot blot (QDB) technique. We found that knockdown of PDLIM5 expression in the B-CPAP cell line could inhibit the migration, invasion and proliferation of PTC cells. In addition, PDLIM5 knockdown reduced Ras and Phospho-ERK1/2 expression. Thus, we suggested that PDLIM5 promotes PTC via activation of the Ras-ERK pathway. Our research provides new molecular insight into the function of PDLIM5, which may assist in studying the mechanism of PTC. In addition, PDLIM5 could be further explored as a potential candidate for PTC treatment.

Survivin regulated by autophagy mediates hyperglycemia-induced vascular endothelial cell dysfunction.

Diabetic vascular complications are often defined by vascular endothelial lesions. However, as a plastic cell type, whether endothelial cells could transit from quiescence to hyper-active status and hamper vascular stability upon hyperglycemia stimulation and whether this process is involved in diabetic vascular complications remain obscure. Survivin has been identified as an anti-apoptotic protein in tumor or epithelial cells by either promoting proliferation or inhibiting apoptosis. Therefore, this study aims at investigating the effects of hyperglycemia on endothelial cell status and the potential involvement of survivin. We found that high glucose (25 mM) did not cause endothelial injuries, instead, it evidently promotes endothelial proliferation and tube formation capacity indicating endothelial cell dysfunction upon hyperglycemia characterized by its preference to hyper-active status. Concomitantly, an upregulation of survivin was detected accompanied by the key component elevations of autophagy pathway including LC3, Beclin1, and p62. YM155, a specific inhibitor of survivin, could abrogate hyperglycemia-induced endothelial hyper-activation. Application of the autophagy inhibitor (3MA) and agonist (rapamycin) supported that survivin could be as a downstream effect or of autophagy. Thus, our results suggested that survivin/autophagy axis a potential therapeutic target in treatment of diabetic vascular complications.

Differential expression and function of survivin during the progress of pterygium.

PURPOSE: To investigate the expression pattern and function of survivin in the development of pterygium. METHODS: Primary pterygia at quiescent or advanced clinical stage and normal human conjunctival tissues were used in this study. Pterygium epithelial cells (PECs) were cultured in keratinocyte serum-free defined medium and harvested at different growth stages. Tissue sections and cultured cells were detected with survivin, phosphorylated survivin (Thr43), p63, p57, and p21 on protein, and/or mRNA level. Cell Counting Kit (CCK)-8 assay was performed to measure proliferation status of primary cultured PECs. Small interfering (si) RNA specific for survivin was transfected on PECs at subconfluence stage. RESULTS: Survivin was highly expressed in all pterygium tissues, but not in normal human conjunctiva, at mRNA and protein levels. It was mainly present in the epithelial cytoplasm of pterygium at quiescent stage, while present in the nucleus of pterygium at advanced stage. Phosphorylated survivin was upregulated in pterygium at advanced stage. Pterygium epithelial cells cultured under subconfluence stage showed higher expression of survivin and p63, but lower expression of p57 and p21, compared with PECs reached confluence. Both total and phosphorylated survivin was mainly expressed in the nuclei of PECs under subconfluence, and there was cytoplasmic translocation of survivin when PECs reached confluence. The knockdown of survivin by siRNA inhibited proliferation of PECs, accompanied by downregulation of p63, and upregulation of p57 and p21. CONCLUSIONS: Higher subcellular expression and phosphorylation of survivin may play roles in the development of pterygium. Survivin could be targeted for the treatment of pterygium.

Amniotic membrane extract ameliorates benzalkonium chloride-induced dry eye in a murine model.

Human amniotic membrane (AM) is avascular but contains various beneficial bioactive factors, its extract (AE) is also effective in treating many ocular surface disorders. In this study, we for the first time evaluated the therapeutic effects of AE on dry eye induced by benzalkonium chloride in a BALB/c mouse model. Topical application of AE (1.5 and 3 µg/eye/day) resulted in significantly longer tear break-up time on Day 3 and 6, lower fluorescein staining scores on Day 3, and lower inflammatory index on Day 6. AE reduced corneal epithelial K10 expression, inflammatory infiltration, and levels of TNF-α, IL-1ß and IL-6 in BAC treated mice than that in the control mice. Moreover, decreased TUNEL positive cells in cornea and increased goblet cells in conjunctiva were also observed in AE treated corneas. Finally, AE induced more Ki-67 positive cells in corneal epithelium of dry eye mouse. Taken together, our data provide further support for BAC induced dry eye model as a valuable for dry eye study and suggest a great potential for AE as a therapeutic agent in the clinical treatment of dry eye.

Regulation of Cigarette Smoke (CS)-Induced Autophagy by Nrf2.

Cigarette smoke (CS) has been reported to induce autophagy in airway epithelial cells. The subsequent autophagic cell death has been proposed to play an important pathogenic role in chronic obstructive pulmonary disease (COPD); however, the underlying molecular mechanism is not entirely clear. Using CS extract (CSE) as a surrogate for CS, we found that it markedly increased the expressions of both LC3B-I and LC3B-II as well as autophagosomes in airway epithelial cells. This is in contrast to the common autophagy inducer (i.e., starvation) that increases LC3B-II but reduces LC3B-I. Further studies indicate that CSE regulated LC3B at transcriptional and post-translational levels. In addition, CSE, but not starvation, activated Nrf2-mediated adaptive response. Increase of cellular Nrf2 by either Nrf2 overexpression or the knockdown of Keap1 (an Nrf2 inhibitor) significantly repressed CSE-induced LC3B-I and II as well as autophagosomes. Supplement of NAC (a GSH precursor) or GSH recapitulated the effect of Nrf2, suggesting the increase of cellular GSH level is responsible for Nrf2 effect on LC3B and autophagosome. Interestingly, neither Nrf2 activation nor GSH supplement could restore the repressed activities of mTOR or its downstream effctor-S6K. Thus, the Nrf2-dependent autophagy-suppression was not due to the re-activation of mTOR-the master repressor of autophagy. To search for the downstream effector of Nrf2 on LC3B and autophagosome, we tested Nrf2-dependent genes (i.e., NQO1 and P62) that are also increased by CSE treatment. We found that P62, but not NQO1, could mimic the effect of Nrf2 activation by repressing LC3B expression. Thus, Nrf2->P62 appears to play an important role in the regulation of CSE-induced LC3B and autophagosome.