The recombinant defensin/HSA fusion protein that inhibits NF-κb associated with intensive macropinocytosis shows potent efficacy against pancreatic cancer.

KRAS mutation and NF-κB both play crucial role in pancreatic cancer; in addition, defensin, the peptide mediator in innate immunity, can inhibit NF-κB. Assuming a strategy that targets both NF-κB and concomitantly the mutated KRAS indirectly via intensive macropinocytosis, we designed and generated a recombinant protein DF2-HSA which consists of two molecules of human beta-defensin 2 (HBD2) and a moiety of human serum albumin (HSA). As shown, the recombinant protein DF2-HSA markedly down-regulated NF-κB in both KRAS mutant MIA PaCa-2 cells and wild type BxPC-3 cells. Determined by confocal microscopy, the uptake of DF2-HSA in MIA PaCa-2 cells was more intense than that in BxPC-3 cells. The uptake was blocked by the specific inhibitor EIPA, indicating that DF2-HSA internalized via macropinocytosis. DF2-HSA displayed more potent cytotoxicity to cancer cells than HBD2. DF2-HSA induced apoptosis in cancer cells. Notably, DF2-HSA inhibited tumor cell spheroid formation, an effect comparable to that of salinomycin. DF2-HSA inhibited tumor cell migration and invasion. As detected with scanning electron microscopy, DF2-HSA strongly depleted filopodia on cell surface; and salinomycin induced similar changes. By in vivo imaging, DF2-HSA displayed intense tumor-site accumulation and lasting retention for over 14 days; however, HBD2 showed much less tumor-site accumulation and a shorter retention time for only 24 h. DF2-HSA suppressed the growth of pancreatic cancer MIA PaCa-2 xenograft in athymic mice; and its combination with gemcitabine achieved higher antitumor efficacy. In summary, the recombinant defensin/HSA fusion protein that inhibits NF-κb associated with intensive macropinocytosis is highly effective against pancreatic cancer.

Pingyangmycin enhances the antitumor efficacy of anti-PD-1 therapy associated with tumor-infiltrating CD8+ T cell augmentation.

PURPOSE: To investigate the antitumor efficacy of pingyangmycin (PYM) in combination with anti-PD-1 antibody and determine the capability of PYM to induce immunogenic cell death (ICD) in cancer cells. METHODS: The murine 4T1 breast cancer and B16 melanoma models were used for evaluation of therapeutic efficacy of the combination of PYM with anti-PD-1 antibody. The ELISA kits were used to quantify the ICD related ATP and HMGB1 levels. The Transwell assay was conducted to determine the chemotaxis ability of THP-1 cell in vitro. The flow cytometry was used to measure reactive oxygen species level and analyze the ratio of immune cell subsets. RESULTS: PYM induced ICD in murine 4T1 breast cancer and B16 melanoma cells and increased the release of nucleic acid fragments that may further promote the monocytic chemotaxis. In the 4T1 murine breast cancer model, PYM alone, anti-PD-1 antibody alone, and their combination suppressed tumor growth by 66.3%, 16.1% and 77.6%, respectively. PYM markedly enhanced the therapeutic efficacy of anti-PD-1 antibody against 4T1 breast cancer. The calculated CDI (coefficient of drug interaction) indicated synergistic effect. Evaluated by graphic analysis, the nucleated cells intensity in the femur bone marrow remained unchanged. Histopathological observations revealed no noticeable toxico-pathological changes in the lung and various organs, indicating that the PYM and anti-PD-1 antibody combination exerted enhanced efficacy at well-tolerated dosage level. By the combination treatment, a panel of immunological changes emerged. The ratio of CD3+ cells, NK cells and NKT cells increased and Tregs decreased in peripheral blood. The DCs increased in the spleen. Prominent changes occurred in tumor infiltrating lymphocytes. The ratio of CD8+ cells increased, while that of CD4+ cells decreased; however, the ratio of CD3+ cells remained unchanged, implying that certain immunological responses emerged in the tumor microenvironment. PYM alone could also increase CD8+ cells and reduce CD4+ cells in tumor infiltrating lymphocytes. CONCLUSIONS: The studies indicate that PYM, as an ICD inducer with mild myelosuppression effect, may enhance the therapeutic efficacy of anti-PD-1 antibody in association with tumor infiltrating CD8+ T cell augmentation.

Silk fibroin peptide suppresses proliferation and induces apoptosis and cell cycle arrest in human lung cancer cells.

Silkworm cocoon was recorded to cure carbuncle in the Compendium of Materia Medica. Previous studies have demonstrated that the supplemental silk protein sericin exhibits anticancer activity. In the present study, we investigated the effects of silk fibroin peptide (SFP) extracted from silkworm cocoons against human lung cancer cells in vitro and in vivo and its possible anticancer mechanisms. SFP that we prepared had high content of glycine (~ 30%) and showed a molecular weight of ~ 10 kDa. Intragastric administration of SFP (30 g/kg/d) for 14 days did not affect the weights, vital signs, routine blood indices, and blood biochemical parameters in mice. MTT assay showed that SFP dose-dependently inhibited the growth of human lung cancer A549 and H460 cells in vitro with IC50 values of 9.921 and 9.083 mg/mL, respectively. SFP also dose-dependently suppressed the clonogenic activity of the two cell lines. In lung cancer H460 xenograft mice, intraperitoneal injection of SFP (200 or 500 mg/kg/d) for 40 days significantly suppressed the tumor growth, but did not induce significant changes in the body weight. We further examined the effects of SFP on cell cycle and apoptosis in H460 cells using flow cytometry, which revealed that SFP-induced cell cycle arrest at the S phase, and then promoted cell apoptosis. We demonstrated that SFP (20-50 mg/mL) dose-dependently downregulates Bcl-2 protein expression and upregulates Bax protein in H460 cells during cell apoptosis. The results suggest that SFP should be studied further as a novel therapeutic agent for the treatment of lung cancer.