Long-chain monounsaturated fatty acids improve endothelial function with altering microbial flora

Fish oil-derived long-chain monounsaturated fatty acids (LCMUFAs) with a carbon chain length longer than 18 units ameliorate cardiovascular risk in mice. In this study, we investigated whether LCMUFAs could improve endothelial functions in mice and humans. In a double-blind, randomized, placebo-controlled, parallel-group, multi-center study, healthy subjects were randomly assigned to either an LCMUFA oil (saury oil) or a control oil (olive and tuna oils) group. Sixty subjects were enrolled and administrated each oil for 4 weeks. For the animal study, ApoE−/− mice were fed a Western diet supplemented with 3% of either gadoleic acid (C20:1) or cetoleic acid (C22:1) for 12 weeks. Participants from the LCMUFA group showed improvements in endothelial function and a lower trimethylamine-N-oxide level, which is a predictor of coronary artery disease. C20:1 and C22:1 oils significantly improved atherosclerotic lesions and plasma levels of several inflammatory cytokines, including IL-6 and TNF-α. These beneficial effects were consistent with an improvement in the gut microbiota environment, as evident from the decreased ratio of Firmicutes and/ or Bacteroidetes, increase in the abundance of Akkermansia, and upregulation of short-chain fatty acid (SCFA)-induced glucagon-like peptide-1 (GLP-1) expression and serum GLP-1 level. These data suggest that LCMUFAs alter the microbiota environment that stimulate the production of SCFAs, resulting in the induction of GLP-1 secretion. Fish oil-derived long-chain monounsaturated fatty acids might thus help to protect against cardiovascular disease

Macrolide Antibiotics Exhibit Cytotoxic Effect under Amino Acid-Depleted Culture Condition by Blocking Autophagy Flux in Head and Neck Squamous Cell Carcinoma Cell Lines

<div><p>Autophagy, a self-digestive system for cytoplasmic components, is required to maintain the amino acid pool for cellular homeostasis. We previously reported that the macrolide antibiotics azithromycin (AZM) and clarithromycin (CAM) have an inhibitory effect on autophagy flux, and they potently enhance the cytocidal effect of various anticancer reagents <i>in vitro</i>. This suggests that macrolide antibiotics can be used as an adjuvant for cancer chemotherapy. Since cancer cells require a larger metabolic demand than normal cells because of their exuberant growth, upregulated autophagy in tumor cells has now become the target for cancer therapy. In the present study, we examined whether macrolides exhibit cytotoxic effect under an amino acid-starving condition in head and neck squamous cancer cell lines such as CAL 27 and Detroit 562 as models of solid tumors with an upregulated autophagy in the central region owing to hypovascularity. AZM and CAM induced cell death under the amino acid-depleted (AAD) culture condition in these cell lines along with CHOP upregulation, although they showed no cytotoxicity under the complete culture medium. CHOP knockdown by siRNA in the CAL 27 cells significantly suppressed macrolide-induced cell death under the AAD culture condition. CHOP^-/- murine embryonic fibroblast (MEF) cell lines also attenuated AZM-induced cell death compared with CHOP^+/+ MEF cell lines. Using a tet-off <i>atg5</i> MEF cell line, knockout of <i>atg5</i>, an essential gene for autophagy, also induced cell death and CHOP in the AAD culture medium but not in the complete culture medium. This suggest that macrolide-induced cell death via CHOP induction is dependent on autophagy inhibition. The cytotoxicity of macrolide with CHOP induction was completely cancelled by the addition of amino acids in the culture medium, indicating that the cytotoxicity is due to the insufficient amino acid pool. These data suggest the possibility of using macrolides for “tumor-starving therapy”.</p></div

CHOP induction by macrolides under AAD culture condition in accordance with intracellular amino acid starvation in CAL 27 cells.

<p>(A) After treatment of CAL 27 cells with/without macrolides in the AAD culture medium containing 10% FBS for various lengths of time, the cells were lysed and cellular proteins were separated by 11.25% SDS-PAGE and immunoblotting with anti-phosphor-p70S6K (Thr389) and anti-p70S6K Abs. The positive control was the cell lysate derived from Detroit 562 cells having constitutively activated PI3K mutation. Immunoblotting with anti-GAPDH mAb was used as an internal control. (B) Addition of amino acids into the culture medium canceled CHOP induction: After 4 hrs of depletion of amino acids in the culture medium in the presence of AZM/CAM (50 μM), amino acids were added at final concentrations of 1% MEM non-essential amino acids and 2% MEM essential amino acids, and subsequently cultured for the indicated time periods. Thereafter, cellular proteins were separated by 11.25% SDS-PAGE and immunoblotting with anti-CHOP mAb. The positive control was the CAL 27 cell lysates cultured in the complete culture medium with thapsigargin (300 nM) for 6 hrs. Immunoblotting with anti-GAPDH mAb was used as an internal control. (C) CAL 27 cells were cultured with thapsigargin (300 nM) for 6 hrs with/without the addition of amino acids into the cell culture medium as described above, and immunoblotted with anti-CHOP mAb. Immunoblotting with β-actin mAb was used as an internal control.</p

Effects of autophagy inhibition on macrolide-induced cytotoxicity in tet-off <i>atg5</i> MEF cell line.

<p>(A) m5-7 cells were pre-treated with/without doxycycline (Dox, 10 ng/mL) for 7 days for complete autophagy inhibition. Subsequently, the cells were further treated with/without macrolides for 24 hrs under the complete culture or AAD culture conditions. The cellular proteins were separated by 15% SDS-PAGE, and immunoblotted with LC3B Ab. Immunoblotting with anti-GAPDH mAb was used as an internal control. The positive control was the cell lysate derived from PANC-1 cells treated with AZM (50 μM) for 24 hrs and used in a previous report [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164529#pone.0164529.ref013" target="_blank">13</a>]. (B) After Dox treatment, m5-7 cells were cultured in the complete culture medium or AAD culture medium containing 10% FBS for 24 hrs. Viable cell numbers were assessed and expressed as percentage to the viable cells cultured in the complete culture medium at each indicated culture period. Data are presented as means ± SEM. *p < 0.05 (Dox (-) vs Dox (+)). (C) Cell growth inhibition of m5-7 cells cultured under the AAD culture condition with or without macrolides for 24 hrs. Data are presented as means ± SEM. *p < 0.05 (vs control). ‘n.s.’ indicates ‘not significant’.</p

Proposed scheme for cell death induction by macrolide antibiotics under amino acid-depleted culture condition.

<p>Under the amino acid-depleted culture condition, autophagy is induced as an adaptive response. AZM/CAM block autophagy flux leading to the shortage of the intracellular amino acid pool and failure of cellular amino acid homeostasis. This results in the induction of an ER-stress-related pro-apoptotic transcription factor, CHOP.</p

Apoptosis induction after treatment with macrolides under AAD culture condition.

<p>(A) CAL 27 cells in the AAD culture medium containing 10% FBS with or without macrolides were treated with various concentrations of either Z-VAD-fmk (upper panel) or necrostatin-1 (lower panel). Data are presented as means ± SEM. *p < 0.05 (vs without Z-VAD-fmk/necrostatin-1). (B) Immunoblotting with PARP Ab from the cell lysates of CAL 27 cells cultured in the complete culture medium or AAD culture medium with or without macrolides for 12 hrs. Immunoblotting with anti-GAPDH mAb was used as an internal control. Cell lysate derived from HL-60 cells treated with vitamin K2 for 48 hrs was used as a positive control for apoptosis induction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164529#pone.0164529.ref017" target="_blank">17</a>]. (C) Flow cytometry with annexin V/PI double staining after 24-hr treatment of CAL 27 cells with AZM/CAM (50 μM) in the complete culture medium or AAD culture medium containing 10% FBS. Numbers indicate the percentage of the cells in each area.</p

Antigen delivery targeted to tumor-associated macrophages overcomes tumor immune resistance

Immune checkpoint inhibitors and adoptive transfer of gene-engineered T cells have emerged as novel therapeutic modalities for hard-to-treat solid tumors; however, many patients are refractory to these immunotherapies, and the mechanisms underlying tumor immune resistance have not been fully elucidated. By comparing the tumor microenvironment of checkpoint inhibition-sensitive and -resistant murine solid tumors, we observed that the resistant tumors had low immunogenicity. We identified antigen presentation by CD11b+F4/80+ tumor-associated macrophages (TAMs) as a key factor correlated with immune resistance. In the resistant tumors, TAMs remained inactive and did not exert antigen-presenting activity. Targeted delivery of a long peptide antigen to TAMs by using a nano-sized hydrogel (nanogel) in the presence of a TLR agonist activated TAMs, induced their antigen-presenting activity, and thereby transformed the resistant tumors into tumors sensitive to adaptive immune responses such as adoptive transfer of tumor-specific T cell receptor-engineered T cells. These results indicate that the status and function of TAMs have a significant impact on tumor immune sensitivity and that manipulation of TAM functions would be an effective approach for improving the efficacy of immunotherapies