A bifunctional fusion protein protected against diabetic nephropathy by suppressing NLRP3 activation.

Diabetic nephropathy (DN), the principal pathogeny of end-stage renal disease (ESRD), is related to metabolic disorders, chronic inflammation, and oxidative stress. It was reported that high expression of interleukin-17A (IL-17A) was intimately related to the progression of DN, and targeting IL-17A exhibited regulating effects on inflammation and autoimmunity but had only limited impact on the oxidative stress damage in DN. Recent studies showed that interleukin-22 (IL-22) could inhibit mitochondrial damage and inflammatory response. Thus, the cytokine IL-22 was first fused to anti-IL-17A antibody for endowing the antibody with the anti-hyperglycemia and anti-inflammation activity. Our study demonstrated that the fusion molecule, anti-IL17A/IL22 fusion protein, could not only lead to the increase of M1 macrophages and the decrease of M2 macrophages, further improving the immune microenvironment, but also prevent the loss of mitochondrial membrane potential by reducing the production of ROS in murine DN model. In addition, the fusion protein could block TRAF6/NF-κB and AKT/ROS/TXNIP signaling pathways, further synergistically restraining the production of NLRP3, thus suppressing the inflammatory response and playing beneficial effect on slowing down the progression of DN. In conclusion, our findings demonstrated that the bifunctional IL-17A antibody and IL-22 fusion protein were of great benefit to DN, which highlighted a potential therapeutic strategy. KEY POINTS: • Anti-IL17A/IL22 fusion protein could improve the immune microenvironment and reduce the production of ROS. • Anti-IL17A/IL22 fusion protein could block TRAF6/NF-κB and AKT/ROS/TXNIP signaling pathways and then restrain the activation of NLRP3.

Targeting CD47 enhanced the antitumor immunity of PD-L1 blockade in B-cell lymphoma.

Background: Only a subset of B-cell lymphoma (BCL) patients can benefit from immune checkpoint inhibitors targeting PD-1/PD-L1. Materials & methods: In the A20 model, SIRPα-Fc and anti-PD-L1 were employed to target CD47 and PD-L1 simultaneously. Flow cytometry, immunofluorescence and quantitative polymerase chain reaction were used to unravel the potential mechanisms. Results: Simultaneously targeting CD47 and PD-L1 activated CD8+ T cells with an increased release of effector molecules. Furthermore, infiltration of F4/80+iNOS+ M1 macrophages was enhanced by the dual therapy. Conclusion: Anti-CD47 therapy could sensitize BCL tumors to anti-PD-L1 therapy in a CD8+ T-cell- and M1-macrophage-dependent manner by promoting cytotoxic lymphocyte infiltration, which may provide a potential strategy for BCL treatment by simultaneously targeting CD47 and PD-L1.

Immune checkpoint inhibitors targeting PD-1/PD-L1 have become effective agents for cancer treatment. However, only a minority of patients benefit from this treatment in the clinic because of the limited response rate. Targeting CD47/SIRPα restores macrophage function and improves the response of antitumor immunity. Here, combination immunotherapy targeting CD47/SIRPα and PD-1/PD-L1 was investigated to increase the response rate and antitumor effect of PD-L1 monotherapy in B-cell lymphoma (BCL). This study broadens the application of the combination therapy and provided a promising strategy for B-cell lymphoma treatment by simultaneous targeting of PD-1/PD-L1 and CD47/SIRPα axis.

A novel nanobody-heavy chain antibody against Angiopoietin-like protein 3 reduces plasma lipids and relieves nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a metabolic disease mainly on account of hypercholesterolemia and may progress to cirrhosis and hepatocellular carcinoma. The discovery of effective therapy for NAFLD is an essential unmet need. Angiopoietin-like protein 3 (ANGPTL3), a critical lipid metabolism regulator, resulted in increased blood lipids and was elevated in NAFLD. Here, we developed a nanobody-heavy chain antibody (VHH-Fc) to inhibit ANGPTL3 for NAFLD treatment. RESULTS: In this study, we retrieved an anti-ANGPTL3 VHH and Fc fusion protein, C44-Fc, which exhibited high affinities to ANGPTL3 proteins and rescued ANGPLT3-mediated inhibition of lipoprotein lipase (LPL) activity. The C44-Fc bound a distinctive epitope within ANGPTL3 when compared with the approved evinacumab, and showed higher expression yield. Meanwhile, C44-Fc had significant reduction of the triglyceride (~ 44.2%), total cholesterol (~ 36.6%) and LDL-cholesterol (~ 54.4%) in hypercholesterolemic mice and ameliorated hepatic lipid accumulation and liver injury in NAFLD mice model. CONCLUSIONS: We discovered a VHH-Fc fusion protein with high affinity to ANGPTL3, strong stability and also alleviated the progression of NAFLD, which might offer a promising therapy for NAFLD.

Versatile ginsenoside Rg3 liposomes inhibit tumor metastasis by capturing circulating tumor cells and destroying metastatic niches.

Limited circulating tumor cells (CTCs) capturing efficiency and lack of regulation capability on CTC-supportive metastatic niches (MNs) are two main obstacles hampering the clinical translation of conventional liposomes for the treatment of metastatic breast cancers. Traditional delivery strategies, such as ligand modification and immune modulator co-encapsulation for nanocarriers, are inefficient and laborious. Here, a multifunctional Rg3 liposome loading with docetaxel (Rg3-Lp/DTX) was developed, in which Rg3 was proved to intersperse in the phospholipid bilayer and exposed its glycosyl on the liposome surface. Therefore, it exhibited much higher CTC-capturing efficiency via interaction with glucose transporter 1 (Glut1) overexpressed on CTCs. After reaching the lungs with CTCs, Rg3 inhibited the formation of MNs by reversing the immunosuppressive microenvironment. Together, Rg3-Lp/DTX exhibited excellent metastasis inhibition capacity by CTC ("seeds") neutralization and MN ("soil") inhibition. The strategy has great clinical translation prospects for antimetastasis treatment with enhanced therapeutic efficacy and simple preparation process.

Reprogramming the immunosuppressive microenvironment of IDH1 wild-type glioblastoma by blocking Wnt signaling between microglia and cancer cells.

The vast majority (>90%) of glioblastoma (GBM) patients belong to the isocitrate dehydrogenase 1 wild type (IDH1WT) group which exhibits a poor prognosis with a median survival of less than 15 months. This study demonstrated numerous immunosuppressive genes as well as ß-catenin gene, pivotal for Wnt/ß-catenin signaling, were upregulated in 206 IDH1WT glioma patients using the Chinese Glioma Genome Atlas (CGGA) database. The increase in microglia with an immunosuppressive phenotype and the overexpression of ß-catenin protein were further verified in IDH1WT GBM patients and IDH1WT GL261 glioma allografts. Subsequently, we found that IDH1WT GL261 cell-derived conditioned medium activated Wnt/ß-catenin signaling in primary microglia and triggered their transition to an immunosuppressive phenotype. Blocking Wnt/ß-catenin signaling not only attenuated microglial polarization to the immunosuppressive subtype but also reactivated immune responses in IDH1WT GBM allografts by simultaneously enhancing cytotoxic CD8+ T cell infiltration and downregulating regulatory T cells. Positron emission tomography imaging demonstrated enhanced proinflammatory activities in IDH1WT GBM allografts after the blockade of Wnt signaling. Finally, gavage administration of a Wnt signaling inhibitor significantly restrained tumor proliferation and improved the survival of model mice bearing IDH1WT GBM allografts. Depletion of CD8+ T cells remarkably abrogated the therapeutic efficacy induced by the Wnt signaling inhibitor. Overall, the present work indicates that the crosstalk between IDH1WT glioma cells and immunosuppressive microglia is important in maintaining the immunosuppressive glioma microenvironment. Blocking Wnt/ß-catenin signaling is a promising complement for IDH1WT GBM treatment by improving the hostile immunosuppressive microenvironment.

VEGF-B antibody and interleukin-22 fusion protein ameliorates diabetic nephropathy through inhibiting lipid accumulation and inflammatory responses.

Diabetic nephropathy (DN) is considered the primary causes of end-stage renal disease (ESRD) and is related to abnormal glycolipid metabolism, hemodynamic abnormalities, oxidative stress and chronic inflammation. Antagonism of vascular endothelial growth factor B (VEGF-B) could efficiently ameliorate DN by reducing renal lipotoxicity. However, this pharmacological strategy is far from satisfactory, as it ignores numerous pathogenic factors, including anomalous reactive oxygen species (ROS) generation and inflammatory responses. We found that the upregulation of VEGF-B and downregulation of interleukin-22 (IL-22) among DN patients were significantly associated with the progression of DN. Thus, we hypothesized that a combination of a VEGF-B antibody and IL-22 could protect against DN not only by regulating glycolipid metabolism but also by reducing the accumulation of inflammation and ROS. To meet these challenges, a novel anti-VEGFB/IL22 fusion protein was developed, and its therapeutic effects on DN were further studied. We found that the anti-VEGFB/IL22 fusion protein reduced renal lipid accumulation by inhibiting the expression of fatty acid transport proteins and ameliorated inflammatory responses via the inhibition of renal oxidative stress and mitochondrial dysfunction. Moreover, the fusion protein could also improve diabetic kidney disease by increasing insulin sensitivity. Collectively, our findings indicate that the bifunctional VEGF-B antibody and IL-22 fusion protein could improve the progression of DN, which highlighted a novel therapeutic approach to DN.

Synergistic Antitumor Effect of 5-Fluorouracil Combined with Constituents from Pleurospermum lindleyanum in Hepatocellular Carcinoma SMMC-7721 Cells.

BACKGROUND: A Chinese folk medicine plant Pleurospermum lindleyanum possesses pharmacological activities of heat-clearing, detoxifying and preventing from hepatopathy, coronary heart disease, hypertension, and high altitude sickness. We isolated and characterized its constituents to investigate its synergistic effects against human hepatoma SMMC-7721 cells. OBJECTIVE: The aim of this study was to explore the synergistic anti-cancer activities of isolates from P. lindleyanum with 5-FU on hepatoma SMMC-7721 cells in vitro and their primary mechanisms. METHODS: Sequential chromatographic techniques were conducted for the isolation studies. The isolate's structures were established by spectroscopic analysis as well as X-ray crystallographic diffraction. Growth inhibition was detected by MTT assay. The isobologram method was used to assess the effect of drug combinations. Flow cytometry and western blot were used to examine apoptosis and protein expression. RESULTS: A new coumarin (16), along with sixteen known compounds, were isolated from the whole plant of P. lindleyanum and their structures were elucidated by spectroscopic methods. Four coumarins (2, 3, 5, and 16), two flavonoids (8 and 9) and three phytosterols and triterpenes (12-14) were found to synergistically enhance the inhibitory effect of 5-FU against SMMC-7721 cells. Among them, compounds 3 and 16 exhibited the best synergistic effects with IC50 of 5-FU reduced by 16-fold and 22-fold possessing the minimum Combination Index (CI) 0.34 and 0.27. The mechanism of action of combinations might be through synergistic arresting for the cell cycle at G1 phases and the induction of apoptosis. Moreover, western blotting and molecular docking revealed that compounds 3 or 5 might promote 5-FU-induced apoptosis by regulating the expression of Caspase 9 and PARP. CONCLUSION: Constituents from P. lindleyanum may improve the treatment effectiveness of 5-FU against hepatocellular carcinoma cells.

Development of a kind of RG108-Fluorescein conjugates for detection of DNA methyltransferase 1 (DNMT1) in living cells.

DNA methyltransferase 1 (DNMT1) is one of the most essential proteins in propagating DNA methylation patterns during replication. Developing methods to assess the expression level of DNMT1 will enable study of gene methylation abnormalities. Thus, a series of fluorescein-conjugated RG108 derivatives were designed and synthesized in the current study. The affinity of the derivatives with DNMT1 was evaluated using surface plasmon resonance. Permeability of the derivatives through the cytomembrane and nuclear envelope was evaluated via confocal imaging. Probe 8a was found to compete with RG108 binding to DNMT1 in the nucleus of HeLa cells, suggesting that probe 8a and RG108 share the same binding site. A HeLa cell model with 4.05-fold overexpression of DNMT1 was constructed and used to evaluate probe 8a. Probe 8a was found to be significantly increased in the nucleus of DNMT1 overexpressing cells. These results indicate that fluorescent probes derived from RG108 have the potential to be used for evaluating the expression level of DNMT1 in living cells.

High Affinity of Chlorin e6 to Immunoglobulin G for Intraoperative Fluorescence Image-Guided Cancer Photodynamic and Checkpoint Blockade Therapy.

Cancer photodynamic therapy (PDT) represents an attractive local treatment in combination with immunotherapy. Successful cancer PDT relies on image guidance to ensure the treatment accuracy. However, existing nanotechnology for co-delivery of photosensitizers and image contrast agents slows the clearance of PDT agents from the body and causes a disparity between the release profiles of the imaging and PDT agents. We have found that the photosensitizer Chlorin e6 (Ce6) is inherently bound to immunoglobulin G (IgG) in a nanomolarity range of affinity. Ce6 and IgG self-assemble to form the nanocomplexes termed Chloringlobulin (Chlorin e6 + immunoglobulin G). Chloringlobulin enhances the Ce6 concentration in the tumor without changing its elimination half-life in blood. Utilizing the immune checkpoint inhibitor antiprogrammed death ligand 1 (PD-L1) (αPD-L1) to prepare αPD-L1 Chloringlobulin, we have demonstrated a combination of Ce6-based red-light fluorescence image-guided surgery, stereotactic PDT, and PD-L1 blockade therapy of mice bearing orthotopic glioma. In mice bearing an orthotopic colon cancer model, we have prepared another Chloringlobulin that allows intraoperative fluorescence image-guided PDT in combination with PD-L1 and cytotoxic T lymphocyte antigen 4 (CTLA-4) dual checkpoint blockade therapy. The Chloringlobulin technology shows great potential for clinical translation of combinatorial intraoperative fluorescence image-guided PDT and checkpoint blockade therapy.

APC/CCDH1 synchronizes ribose-5-phosphate levels and DNA synthesis to cell cycle progression.

Accumulation of nucleotide building blocks prior to and during S phase facilitates DNA duplication. Herein, we find that the anaphase-promoting complex/cyclosome (APC/C) synchronizes ribose-5-phosphate levels and DNA synthesis during the cell cycle. In late G1 and S phases, transketolase-like 1 (TKTL1) is overexpressed and forms stable TKTL1-transketolase heterodimers that accumulate ribose-5-phosphate. This accumulation occurs by asymmetric production of ribose-5-phosphate from the non-oxidative pentose phosphate pathway and prevention of ribose-5-phosphate removal by depleting transketolase homodimers. In the G2 and M phases after DNA synthesis, expression of the APC/C adaptor CDH1 allows APC/CCDH1 to degrade D-box-containing TKTL1, abrogating ribose-5-phosphate accumulation by TKTL1. TKTL1-overexpressing cancer cells exhibit elevated ribose-5-phosphate levels. The low CDH1 or high TKTL1-induced accumulation of ribose-5-phosphate facilitates nucleotide and DNA synthesis as well as cell cycle progression in a ribose-5-phosphate-saturable manner. Here we reveal that the cell cycle control machinery regulates DNA synthesis by mediating ribose-5-phosphate sufficiency.

Effect of five novel 5-substituted tetrandrine derivatives on P-glycoprotein-mediated inhibition and transport in Caco-2 cells.

Tetrandrine (Tet) is a potent inhibitor that reverses P-glycoprotein-mediated multidrug resistance (MDR). A number of novel 5-substituted tetrandrine derivatives were synthesized by the authors. The present study aimed at identifying potential P-gp inhibitor candidates, and intracellular uptake and efflux experiments and Caco-2 cell-based Transwell transport studies were performed. It was demonstrated that all five test compounds were able to inhibit efflux and increase intracellular uptake of the P-gp substrate, rhodamine-123 (Rho-123); the test compounds were P-gp inhibitors. The transepithelial transport experiment indicated that the secretory (basolateral-to-apical) of Rho-123 decreased, the absorption (apical-to-basolateral) increased and the transport efflux ratio (ER) reduced in the presence of the five compounds. Among the compounds, fluobenzene-Tet (TF) exhibited similar inhibitory effect as Tet. Although the other four test compounds exhibited weaker inhibitory effects than Tet and TF, the compounds exhibited stronger inhibitory effects compared with the reference compound verapamil. The study demonstrated that the five novel 5-substituted tetrandrine derivatives are able to act as inhibitors of P-gp to overcome P-gp-mediated drug resistance.

Endogenous albumin-mediated delivery of redox-responsive paclitaxel-loaded micelles for targeted cancer therapy.

Targeted delivery and accumulation of chemotherapeutics to tumor sites is in high demand and extremely challenging for anticancer therapy. Studies show that albumin is actively recruited into tumor tissue overexpressing albumin-binding proteins including secreted protein acidic and rich in cysteine (SPARC) and gp60. Herein, a novel redox-responsive paclitaxel-loaded micelle system with the modification of ABD035, which specifically and strongly binds to albumin, is designed to combine the strengths of albumin and micelles together for antitumor drug biomimetic delivery. The ABD035-modified micelle has a strong binding response to albumin and co-localization of BODIPY-labeled ABD035-modified micelle and SPARC is observed in tumor tissues. Furthermore, the ABD035-modified micelle significantly improves the therapeutic effect in the animal model bearing triple negative breast cancer by increasing drug accumulation in tumor tissue, enhancing cell uptake efficiency, rapidly releasing prototype drug under intracellular reductive conditions and increasing tumor cell apoptosis. These results together vote the biomimetic delivery of ABD035-modified micelle as a promising strategy for cancer therapy.

Mitochondria-targeting BODIPY-loaded micelles as novel class of photosensitizer for photodynamic therapy.

In this paper, a series of novel BODIPY-based photosensitizers have been designed and synthesized for photodynamic therapy. BODIPY3 was screened out as the most potential photosensitizer due to its excellent optical properties, high singlet oxygen efficiency and good photostability. However, as an organic photosensitizer, BODIPY3 still suffered from the drawbacks of insolubility and instability in aqueous system. In view of these problems, DSPE-PEG2000 was used to trap BODIPY3 into the hydrophobic core of micelles to obtain well-dispersing nano complexes BODIPY3-PEG3 in aqueous system. More importantly, BODIPY3-PEG3 not only has better solubility and stability in aqueous media but can generate significant singlet oxygen (1O2, one of the reactive oxygen species, the real cytotoxic agent in photodynamic therapy) in living cells and exhibit high light cytotoxicity to three cancer cell lines. The mechanism studies indicated the mitochondrial localization of BODIPY3-PEG3 was able to generate ROS in mitochondria, which further result in mitochondrial dysfunction and photoinduced apoptosis via caspase-8 and caspase-3 pathway.

Peptide-Functionalized Nanoinhibitor Restrains Brain Tumor Growth by Abrogating Mesenchymal-Epithelial Transition Factor (MET) Signaling.

Malignant gliomas are the most common primary brain tumors and are associated with aggressive growth, high morbidity, and mortality. Aberrant mesenchymal-epithelial transition factor (MET) activation occurs in approximately 30% of glioma patients and correlates with poor prognosis, elevated invasion, and increased drug resistance. Therefore, MET has emerged as an attractive target for glioma therapy. In this study, we developed a novel nanoinhibitor by conjugating MET-targeting cMBP peptides on the G4 dendrimer. Compared to the binding affinity of the free peptide ( KD = 3.96 × 10-7 M), the binding affinity of the nanoinhibitor to MET increased 3 orders of magnitude to 1.32 × 10-10 M. This nanoinhibitor efficiently reduced the proliferation and invasion of human glioblastoma U87MG cells in vitro by blocking MET signaling with remarkably attenuated levels of phosphorylated MET ( pMET) and its downstream signaling proteins, such as pAKT and pERK1/2. Although no obvious therapeutic effect was observed after treatment with free cBMP peptide, in vivo T2-weighted magnetic resonance imaging (MRI) showed a significant delay in tumor growth after intravenous injection of the nanoinhibitor. The medium survival in mouse models was extended by 59%, which is similar to the effects of PF-04217903, a small molecule MET inhibitor currently in clinical trials. Immunoblotting studies of tumor homogenate verified that the nanoinhibitor restrained glioma growth by blocking MET downstream signaling. pMET and its downstream proteins pAKT and pERK1/2, which are involved in the survival and invasion of cancer cells, decreased in the nanoinhibitor-treated group by 44.2%, 62.2%, and 32.3%, respectively, compared with those in the control group. In summary, we developed a peptide-functionalized MET nanoinhibitor that showed extremely high binding affinity to MET and effectively inhibited glioma growth by blocking MET downstream signaling. To the best of our knowledge, this is the first report of therapeutic inhibition of glioma growth by blocking MET signaling with a novel nanoinhibitor. Compared to antibodies and chemical inhibitors in clinical trials, the nanoinhibitor blocks MET signaling and provides a new approach for the treatment of glioma with the advantages of high efficiency, affordability, and, most importantly, potentially reduced drug resistance.

Proapoptotic Cyclic Peptide BC71 Targets Cell-Surface GRP78 and Functions as an Anticancer Therapeutic in Mice.

Glucose regulated protein 78 kDa (GRP78) is a recently emerged target for cancer therapy and a biomarker for cancer prognosis. Overexpression of GRP78 is observed in many types of cancers, with the cell-surface GRP78 being preferentially present in cancer cells and cancer blood vessel endothelial cells. Isthmin (ISM) is a secreted high-affinity proapoptotic protein ligand of cell-surface GRP78 that suppresses angiogenesis and tumor growth in mice. The C-terminal AMOP (adhesion-associated domain in MUC4 and other proteins) domain of ISM is critical in mediating its interaction with human umbilical vein endothelial cells (HUVECs). In this work, we report novel cyclic peptides harboring the RKD motif in the ISM AMOP domain that function as proapoptotic ligands of cell-surface GRP78. The most potent peptide, BC71, binds to GRP78 and converge to tumor in mice. Intravenous administration of BC71 suppressed xenograft tumor growth in mice as a single agent, with significant reduction in tumor angiogenesis and upsurge in apoptosis. Fluorescent-labeled BC71 accumulates in tumor in mice by targeting cell-surface GRP78. We show that BC71 triggers apoptosis via cell-surface GRP78 and activates caspase-8 and p53 signaling pathways in HUVECs. Using amide hydrogen-deuterium exchange mass spectrometry (HDXMS), we identified that BC71 preferentially binds to ATP-bound GRP78 via amino acid residues 244-257 of GRP78. Hence, BC71 serves as a valuable prototype for further development of peptidomimetic anticancer drugs targeting cell-surface GRP78 as well as PET imaging agents for cancer prognosis.

Autophagy suppression potentiates the anti-glioblastoma effect of asparaginase in vitro and in vivo.

Asparaginase has been reported to be effective in the treatment of various leukemia and several malignant solid cancers. However, the anti-tumor effect of asparaginase is always restricted due to complicated mechanisms. Herein, we investigated the mechanisms of how glioblastoma resisted asparaginase treatment and reported a novel approach to enhance the anti-glioblastoma effect of asparaginase. We found that asparaginase could induce growth inhibition and caspase-dependent apoptosis in U87MG/U251MG glioblastoma cells. Meanwhile, autophagy was activated as indicated by autophagosomes formation and upregulated expression of LC3-II. Importantly, abolishing autophagy using chloroquine (CQ) and LY294002 enhanced the cytotoxicity and apoptosis induced by asparaginase in U87MG/U251MG cells. Further study proved that Akt/mTOR and Erk signaling pathways participated in autophagy induction, while reactive oxygen species (ROS) served as an intracellular regulator for both cytotoxicity and autophagy in asparaginase-treated U87MG/U251MG cells. Moreover, combination treatment with autophagy inhibitor CQ significantly enhanced anti-glioblastoma efficacy of asparaginase in U87MG cell xenograft model. Taken together, our results demonstrated that inhibition of autophagy potentiated the anti-tumor effect of asparagine depletion on glioblastoma, indicating that targeting autophagy and asparagine could be a potential approach for glioblastoma treatment.

Acidity-Triggered Ligand-Presenting Nanoparticles To Overcome Sequential Drug Delivery Barriers to Tumors.

The success of cancer chemotherapy is impeded by poor drug delivery efficiency due to the existence of a series of pathophysiological barriers in the tumor. In this study, we reported a tumor acidity-triggered ligand-presenting (ATLP) nanoparticle for cancer therapy. The ATLP nanoparticles were composed of an acid-responsive diblock copolymer as a sheddable matrix and an iRGD-modified polymeric prodrug of doxorubicin (iPDOX) as an amphiphilic core. A PEG corona of the polymer matrix protected the iRGD ligand from serum degradation and nonspecific interactions with the normal tissues while circulating in the blood. The ATLP nanoparticles specifically accumulated at the tumor site through the enhanced permeability and retention (EPR) effect, followed by acid-triggered dissociation of the polymer matrix within the tumoral acidic microenvironment (pH ∼ 6.8) and subsequently exposing the iRGD ligand for facilitating tumor penetration and cellular uptake of the PDOX prodrug. Additionally, the acid-triggered dissociation of the polymer matrix induced a 4.5-fold increase of the fluorescent signal for monitoring nanoparticle activation in vivo. Upon near-infrared (NIR) laser irradiation, activation of Ce6-induced significant reactive oxygen species (ROS) generation, promoted drug diffusion inside the tumor mass and circumvented the acquired drug resistance by altering the gene expression profile of the tumor cells. The ATLP strategy might provide a novel insight for cancer nanomedicine.

A novel and promising therapeutic approach for NSCLC: recombinant human arginase alone or combined with autophagy inhibitor.

Recombinant human arginase (rhArg), an enzyme capable of depleting arginine, has been shown to be an effective therapeutic approach for various cancers. Non-small-cell lung cancer (NSCLC), a histological subtype of pulmonary carcinoma, has a high rate of morbidity and mortality in the world. Thus, the need for novel and more effective treatment is urgent. In this study, it is the first time to report that rhArg could induce significant cytotoxicity and caspase-dependent apoptosis in NSCLC cells. Subsequently, our research revealed that rhArg dramatically stimulated autophagic response in NSCLC cells, which was proved by the formation and accumulation of autophagosomes and the conversion of microtubule-associated protein light chain 3 (LC3) from LC3-I to LC3-II. Furthermore, blocking autophagy by chloroquine or LY294002 remarkably enhanced rhArg-induced cytotoxicity and caspase-dependent apoptosis, suggesting that autophagy acted a cytoprotective role in rhArg-treated NSCLC cells. Further experiments showed that two signaling pathways including the Akt/mTOR and extracellular signal-regulated kinase pathway, and mitochondrial-derived reactive oxygen species (ROS) production were involved in rhArg-induced autophagy and apoptosis. Meanwhile, N-acetyl-L-cysteine, a common antioxidant, was employed to scavenge ROS, and we detected that it could significantly block rhArg-induced autophagy and cytotoxicity, indicating that ROS played a vital role in arginine degradation therapy. Besides, xenograft experiment showed that combination with autophagy inhibitor potentiated the anti-tumor efficacy of rhArg in vivo. Therefore, these results provided a novel prospect and viewpoint that autophagy acted a cytoprotective role in rhArg-treated NSCLC cells, and treatment with rhArg alone or combined with autophagy inhibitor could be a novel and promising therapeutic approach for NSCLC in vivo and in vitro.

The development of a quantitative and qualitative method based on UHPLC-QTOF MS/MS for evaluation paclitaxel-tetrandrine interaction and its application to a pharmacokinetic study.

Paclitaxel is a broad-spectrum anti-cancer drug by targeting microtubulin. However, multidrug resistant (MDR) makes its clinical application more difficult and results in failure of chemotherapy. Tetrandrine as a potential multidrug resistant modulator could be combined with other anti-cancer drugs. In this study, ultra-performance liquid chromatography (UHPLC) combined with quadrupole time-of-flight mass spectrometry (QTOF) was applied to simultaneously qualitative and quantitative analysis of paclitaxel for the pharmacokinetic studies while combined with tetrandrine. This method was developed based on non-target screening mode IDA (Information Dependent Acquisition). As a result, the validated range was 0.25-64ng/ml (30µl plasma) for paclitaxel. Totally 33 metabolites of paclitaxel and tetrandine were identified in vivo and in vitro. The main metabolites of PTX were dose-dependent decreased with different amounts of tetrandine co-administration no matter in vivo and in vitro, the exposure of PTX increased in pharmacokinetic study. The verified method is sensitive accurate and effective for the simultaneous determination of paclitaxel and its metabolites in blood, urine and live microsome incubation samples and it was successfully applied to evaluate the pharmacokinetics and drug-drug interaction between paclitaxel and tetrandine. Furthermore, a biosensor technology, surface plasmon resonance (SPR) analysis was applied to preliminary evaluate the competitive protein binding of multiple components. The SPR analysis indicated that the affinity between 6-hydroxy-paclitaxel and micotubulin is similar to that between paclitaxel and micotubulin, and tetrandrine also does not form a competitive combination with paclitaxel. For human, 6-hydroxy-paclitaxel is the one of main metabolites of paclitaxel, so the results suggested that tetrandine has an influence on the metabolite of paclitaxel, but tetrandine and the main metabolites of PTX probably do not affect PTX's biological targeting, the effect of its pharmacological action needs to be further studied.

Targeting asparagine and autophagy for pulmonary adenocarcinoma therapy.

The mounting number of patients with pulmonary adenocarcinoma (ADCA) is subjected to poor prognosis and heavy mortality, which prompts us to explore new potential therapeutics for lung ADCA. Herein, we reported a novel approach for lung ADCA therapy by abolishing autophagy and asparagine. We demonstrated that deprivation of asparagine by asparaginase could induce significant cytotoxicity and apoptosis in A549 and H1975 cells. During this process, autophagy was triggered by the asparaginase treatment, characterized by the autophagic flux with three main stages including formation of autophagosomes, lysosomes fused with autophagosomes, and degradation of autophagosomes by lysosomes. Importantly, suppression of autophagy could notably enhance the cytotoxicity and accelerate the caspase 3-dependent apoptosis induced by asparaginase. Furthermore, suppression of reactive oxygen species (ROS) could attenuated both the cytotoxicity and autophagy induced by asparaginase, while inhibition of autophagy promoted the generation of ROS in A549 and H1975 cells, indicating the essential role of ROS in asparagine deprivation therapy in lung ADCA cells. Our results demonstrated that targeting cytoprotective autophagy and asparagine could potently kill the ADCA cells, which highlighted a novel approach for lung ADCA therapy in the clinics.