Enhanced chemoimmunotherapy of breast cancer in mice by apolipoprotein A1-modified doxorubicin liposomes combined with interleukin-21.

Backgroud: Breast cancer is a prevalent malignancy among women, with triple-negative breast cancer (TNBC) comprising approximately 15-20% of all cases, possessing high invasiveness, drug resistance and poor prognosis. Chemotherapy, the main treatment for TNBC, is limited by toxicity and drug resistance. Apolipoprotein A1 modified doxorubicin liposome (ApoA1-lip/Dox) was constructed in our previous study, with promising anti-tumour effect and improved safety been proved. However, during long-term administration, the problem of cumulative toxicity and insufficient tumour inhibition is still inevitable. Interleukin-21 is a small molecule protein secreted by T cells with various immune regulatory functions. IL-21 has significantly curative effects in numerous solid tumours, but it has the disadvantages of low response rate and short half-life. The combination of chemotherapy and immunotherapy has received increasing attention.Purpose: In this study, ApoA1 drug loading system and long-acting IL-21 are innovatively combined for tumour treatment.Methods: We combined ApoA1-lip/Dox and IL-21 for treatment and evaluated their impact on tumor-infiltrating lymphocytes and CD8+ T and NK cell cytotoxicity.Results: Combined administration significantly improved the tumour-infiltrating lymphocytes and enhanced the cytotoxicity of CD8+ T and NK cells. The combination of ApoA1-lip/Dox and IL-21 exhibits significantly enhanced anti-tumour efficacy with lower toxicity of ApoA1-lip/Dox, providing a new strategy for TNBC treatment with enhanced anti-tumour response and reduced toxicity.

Inhibition of PCSK9 enhances the antitumor effect of PD-1 inhibitor in colorectal cancer by promoting the infiltration of CD8+ T cells and the exclusion of Treg cells.

Immunotherapy especially immune checkpoint inhibitors (ICIs) has brought favorable clinical results for numerous cancer patients. However, the efficacy of ICIs in colorectal cancer (CRC) is still unsatisfactory due to the poor median progression-free survival and overall survival. Here, based on the CRC models, we tried to elucidate novel relapse mechanisms during anti-PD-1 therapy. We found that PD-1 blockade elicited a mild antitumor effect in these tumor models with both increased CD8+ T cells and Treg cells. Gene mapping analysis indicated that proprotein convertase subtilisin/kexin type 9 (PCSK9), low-density lipoprotein receptor, transforming growth factor-ß (TGF-ß), and CD36 were unexpectedly upregulated during PD-1 blockade. To investigate the critical role of these proteins especially PCSK9 in tumor growth, anti-PCSK9 antibody in combination with anti-PD-1 antibody was employed to block PCSK9 and PD-1 simultaneously in CRC. Data showed that neutralizing PCSK9 during anti-PD-1 therapy elicited a synergetic antitumor effect with increased CD8+ T-cell infiltration and inflammatory cytokine releases. Moreover, the proportion of Treg cells was significantly reduced by co-inhibiting PCSK9 and PD-1. Overall, inhibiting PCSK9 can further enhance the antitumor effect of anti-PD-1 therapy in CRC, indicating that targeting PCSK9 could be a promising approach to potentiate ICI efficacy.

An engineered IL-21 with half-life extension enhances anti-tumor immunity as a monotherapy or in combination with PD-1 or TIGIT blockade.

Interleukin-21 (IL-21) has exhibited anti-tumor activity in preclinical and clinical studies; however, its modest efficacy and short half-time has limited its therapeutic utility as a monotherapy. Therefore, we engineered a fusion protein (IL-21-αHSA) in which a nanobody targeting human serum albumin (HSA) was fused to the C-terminus of rhIL-21. The αHSA nanobody displayed broad species cross-reactivity and bound to a HSA epitope that does not overlap with the FcRn binding site, thus providing a strategic design for half-life extension. The IL-21-αHSA fusion protein showed increased stability compared to rhIL-21, while retaining its bioactivity in a liquid solution for at least 6 months. Moreover, IL-21-αHSA showed a dramatically extended half-life and prolonged exposure in cynomolgus monkeys, with the t1/2 and AUC nearly 10 and 50 times greater than that of rhIL-21, respectively. Furthermore, IL-21-αHSA displayed enhanced anti-tumor efficacy in two syngeneic mouse models. Notably, IL-21-αHSA increased the anti-tumor effect of programmed cell death protein 1 (PD-1) and T cell immunoglobulin and ITIM domain (TIGIT) blockades when used in combination, with a protection against tumor rechallenge, suggesting the formation of long-term anti-tumor memory response. KEGG analysis identified significantly enriched pathways associated with anti-tumor immune response, with increased expression of genes associated with CD8+ T and NK cell cytotoxicity. Overall, these data support further clinical evaluation of IL-21-αHSA as a monotherapy or in combination with immune checkpoint blockades.

Reversal of Multidrug Resistance by Apolipoprotein A1-Modified Doxorubicin Liposome for Breast Cancer Treatment.

Multidrug resistance (MDR) remains a major problem in cancer therapy and is characterized by the overexpression of p-glycoprotein (P-gp) efflux pump, upregulation of anti-apoptotic proteins or downregulation of pro-apoptotic proteins. In this study, an Apolipoprotein A1 (ApoA1)-modified cationic liposome containing a synthetic cationic lipid and cholesterol was developed for the delivery of a small-molecule chemotherapeutic drug, doxorubicin (Dox) to treat MDR tumor. The liposome-modified by ApoA1 was found to promote drug uptake and elicit better therapeutic effects than free Dox and liposome in MCF-7/ADR cells. Further, loading Dox into the present ApoA1-liposome systems enabled a burst release at the tumor location, resulting in enhanced anti-tumor effects and reduced off-target effects. More importantly, ApoA1-lip/Dox caused fewer adverse effects on cardiac function and other organs in 4T1 subcutaneous xenograft models. These features indicate that the designed liposomes represent a promising strategy for the reversal of MDR in cancer treatment.

Fungichromin production by Streptomyces sp. WP-1, an endophyte from Pinus dabeshanensis, and its antifungal activity against Fusarium oxysporum.

In this study, we isolated an endophytic Streptomyces sp. strain, WP-1, from surface-sterilized barks of Pinus dabeshanensis, an endangered Chinese plant. WP-1 showed strong antifungal activity against diverse pathogenic fungi, such as Fusarium oxysporum, Rhizoctonia solani, Phytophthora infestan, and Candida albicans. Based on phylogenetic analyses, preliminary identification suggested that the WP-1 strain belonged to the genus Streptomyces. Column chromatogram and HPLC were employed to isolate the primary antifungal component from the culture medium of WP-1, and it was identified as the methylpentaene macrolide antibiotic, fungichromin (FC). In this study, for the first time, using in vitro bioassay studies, we revealed that FC strongly inhibited mycelial growth and conidia germination in Fusarium oxysporum. The median inhibitory concentration of FC was found to be 3.80 mg/L. The fermentation conditions of the WP-1 strain were further investigated to improve FC production. We found that supplementation of the synthetic medium with oils (soybean oil, oleic acid, and so on), particularly during the initial stage of fermentation, significantly increased the FC yield. Ammonium-trapping agent (magnesium phosphate) was used as an additive to increase FC yield to 5741.7 mg/L. It was 2.9-fold more as compared to the highest FC yield reported so far where Streptomyces padanus PMS-702 was used for FC production. KEY POINTS: • Isolation and identification of a fungichromin-producing endophytic actinomycete WP-1 strain. • Fungichromin production was significantly improved via oils and ammonium-trapping agents addition. • Discovery of the antifungal activity of fungichromin against Fusarium oxysporum.

Fenofibrate improves vascular endothelial function in diabetic mice.

Microvascular and macrovascular complications are major causes of disability and death in diabetic patients. High levels of blood glucose sabotage the integrity of blood vessels and induce endothelial dysfunction. Fenofibrate is an agonist of peroxisome proliferator-activated receptor α and can reduce the incidence of cardiovascular events in diabetic patients. This study tested the hypothesis that fenofibrate could ameliorate endothelium-dependent vasodilation in diabetic mice and relieve high glucose-induced endothelial dysfunction via activating endothelial nitric oxide synthase (eNOS) and adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. A streptozotocin (STZ)-induced diabetic model was established by intraperitoneal injection of STZ (dissolved in sodium citrate buffer) at a dose of 60 mg/kg for 5 consecutive days. Mice were administered fenofibrate (100 mg/kg/d, i.g.) for 14 days. The endothelial function of extracted mouse aortae was examined by evaluating acetylcholine induced endothelium-dependent relaxation combined with phenylephrine-induced vasoconstriction and sodium nitroprusside-induced endothelium-independent relaxation. Superoxide onion (O2-) was determined using dihydroethidium staining of aortae. Functions of mouse aortic endothelial cells (MAECs) were assessed, and expression levels of eNOS and AMPK were determined by Western blotting. Fenofibrate ameliorated the impaired endothelium-dependent relaxation in diabetic mice and decreased the level of intracellular O2- in diabetic mouse aortae. In-vitro, fenofibrate treatment improved the impaired function of MAECs, increased nitric oxide production, and decreased the O2- level, as well as activated eNOS and AMPK phosphorylation in cultured MAECs by high glucose. Fenofibrate could ameliorate endothelium-dependent vasodilation in diabetic mice and relieve high glucose-induced endothelial dysfunction, which was possibly related to the activation of eNOS and AMPK phosphorylation.

Improving dermal delivery of hydrophilic macromolecules by biocompatible ionic liquid based on choline and malic acid.

The aim of this work was to develop biocompatible ionic liquids (ILs) for enhancement of dermal delivery of hydrophilic macromolecules. The ILs were prepared from malic acid and choline, which are commonly used as food additives and generally regarded as safe. The choline malate IL (CM-IL) was formed via ionic interactions, which has been validated by Fourier transform infrared spectroscopy and nuclear magnetic resonance. Analysis by differential scanning calorimetry confirmed a melting point of -65.2 °C for CM-IL. In skin penetration study, CM-IL was proved to be able to deliver a model hydrophilic macromolecule dextran into deep skin. The amount of dextran delivered to epidermis and dermis by CM-IL is approximately two folds that of by dextran solution. Enhanced in vivo skin penetration by CM-IL was proved as well. The fluorescence of FITC-dextran could be observed permeating pervasively throughout the skin from 4 h to 24 h, while in the control groups it was mainly concentrated in stratum cornea (SC) and hair follicles. In addition, CM-IL did not shown any irritation to mice skin within 7 days of successive treatment and any toxicity to human epidermal cells (HEK-A) within 24 h. In conclusion, CM-IL could be potentially used as enhancers or vehicles for dermal delivery of hydrophilic macromolecules.

C2-ceramide enhances sorafenib-induced caspase-dependent apoptosis via PI3K/AKT/mTOR and Erk signaling pathways in HCC cells.

Sorafenib as an effective multikinase inhibitor has been approved for the clinical treatment against advanced hepatocellular carcinoma (HCC). HCC treatment requires usually combined therapy because of its complex pathogenesis. Ceramide has been confirmed to induce remarkable apoptosis in human tumor cells and has attracted increasing attention in investigations on combination therapy. In this paper, the anti-HCC effect of sorafenib combined with C2-ceramide was investigated on cell vitality, apoptosis, and migration, and the underlying mechanism was examined using flow cytometry and western blot. Bel7402 cells coincubated with sorafenib and C2-ceramide exhibited lower cell vitality and more irregular cellular morphology and cell cycle arrest. Sorafenib plus C2-ceramide stimulated significantly the production of reactive oxygen species (ROS) and mitochondrial depolarization, which promoted caspases-dependent cell apoptosis as illustrated by related protein expression including caspase 3, caspase 9, Bax, Bcl-2, and cytochrome c. Combination treatment of sorafenib and C2-ceramide inhibited obviously cell growth and proliferation via PI3K/AKT/mTOR and Erk signaling pathways. Furthermore, the combination treatment was proved to inhibit cell migration and epithelial-mesenchymal transition (EMT). These findings indicated that the combination of C2-ceramide and sorafenib provided synergistic inhibitory effects on HCC cells.

The Activity of Fungichromin against the Formation of Candida albicans Biofilm.

The effect of fungichromin (FC) on the formation of Candida albicans biofilm was assessed using 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction method, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Results showed that FC revealed an inhibitory effect on the formation of C. albicans biofilm in a dose-dependent manner with a minimum inhibitory concentration (MIC) of 10 µg/mL. Over 80% of biofilm formation was inhibited by FC at the concentration of 40 µg/mL when compared with the control. Similarly, real-time PCR showed that the expression of the genes such as ALS1, ALS3, HWP1, EFG1, HYR1, CPH1 and BCR1 appeared to be remarkably affected by FC at the concentration of 20 µg/mL during the biofilm formation. In addition, FC could also induce the apoptosis of C. albicans cells in a dose-dependent manner. Therefore, FC displayed potent activity against the formation of C. albicans biofilm in vitro and played an important role in reducing the incidence of device-associated infections.

Diosgenin induces ROS-dependent autophagy and cytotoxicity via mTOR signaling pathway in chronic myeloid leukemia cells.

BACKGROUND: Diosgenin, a steroidal saponin isolated from legumes and yams, has been confirmed to possess potent anticancer effect on multifarious tumors including chronic myeloid leukemia (CML). PURPOSE: We aimed to further determine the anti-cancer activity of diosgenin and its mechanisms in CML cells. METHODS: The cell vitality was detected by MTT assay. Autophagic flux and reactive oxygen species (ROS) production were analyzed by laser scanning confocal microscope. Apoptosis was observed by flow cytometry. All proteins expression was examined by western blotting. RESULTS: Autophagy induction was demonstrated by examination of autophagic flux including autophagosomes accumulation, autophagosome-lysosome fusion and degradation of autophagosomes. Moreover, blocking autophagy with inhibitor chloroquine (CQ) and 3-methyladenine (3-MA), enhanced diosgenin-induced apoptosis, indicating the protective effect of autophagy in diosgenin-treated CML cells. Further study suggested that diosgenin-induced autophagy and cytotoxicity were accompanied by reactive oxygen species (ROS) generation and mammalian target of rapamycin (mTOR) signaling pathway inhibition. N-acetyl-L-cysteine (NAC) administration, a scavenger agent of ROS, could down-regulate diosgenin-induced autophagy via reversion of mTOR pathway inhibition. CONCLUSION: These results indicate that diosgenin obviously generates ROS and this oxidative pressure not only produces cytotoxic effect on CML cells but also induces autophagy. What's more, autophagy functions as a cytoprotective mechanism to overcome cytotoxicity of diosgenin in tumor cells and inhibition of autophagy can enhance the anti-CML activity of diosgenin.