Juglone-ascorbate treatment enhances reactive oxygen species mediated mitochondrial apoptosis in pancreatic cancer.

BACKGROUND: Treatment of Pancreatic Cancer (PC) is challenging due to its aggressiveness and acquired resistance to conventional chemotherapy and radiotherapy. Therefore, the discovery of new therapeutic agents and strategies is essential. Juglone, a naphthoquinone, is a secondary metabolite produced naturally in walnut-type trees having allelopathic features in its native environment. Juglone was shown to prevent cell proliferation and induce ROS-mediated mitochondrial apoptosis. Ascorbate with both antioxidant and oxidant features, shows selective cytotoxicity in cancer cells. METHODS AND RESULTS: In this study, we evaluated the anticancer effects of Juglone in combination with ascorbate in PANC-1 and BxPC-3 PC cells. The MTT assay was used to determine the IC50 dose of Juglone with 1 mM NaAscorbate (Jug-NaAsc). Subsequently, the cells were treated with 5, 10, 15 and 20 µM Jug-NaAsc for 24 h. Apoptotic effects were evaluated by analyzing the following genes using qPCR; proapoptotic Bax, antiapoptotic Bcl-2 related to the mitochondrial apoptotic pathway and apoptosis inhibitor Birc5 (Survivin). Immunofluorescence analysis was performed using Annexin V-FITC in PC cells. As an antioxidant enzyme, Trx2 protein levels were determined by a commercial ELISA test kit. Jug-NaAsc treatment decreased the expressions of antiapoptotic genes Bcl-2 and Birc5 while the apoptotic gene Bax expression increased at all doses. Additionally, a dose-dependently increase of apoptosis according to immunofluorescence analysis and the decreases of Trx2 enzyme levels at all treatments in both cell lines supported gene expression results. CONCLUSION: Our results suggest that Juglone is a potential anticancer agent especially when combined with ascorbate.

Piceatannol induces caspase-dependent apoptosis by modulating intracellular reactive oxygen species/mitochondrial membrane potential and enhances autophagy in neuroblastoma cells.

The aim of this study was to evaluate the anticancer effects of piceatannol, a natural stilbenoid, on human neuroblastoma cells. In order to accomplish this goal, we performed various cellular assays, including the XTT cell proliferation assay for cell viability, colony formation assay for colony formation capacity, FITC Annexin V and cell death detection kit for apoptosis, matrigel invasion assay for invasion capacity, intracellular reactive oxygen species (ROS) red dye for intracellular ROS levels, TMRM staining method for mitochondrial membrane potential (MMP), and the CYTO-ID autophagy detection kit for autophagy. Furthermore, we analyzed the expression levels of genes associated with apoptosis and autophagy using RT-qPCR. Based on our findings, piceatannol exhibited cytotoxic effects on neuroblastoma cells. Besides, treatment with piceatannol at both 50 and 100 µM concentrations for 72 h decreased colony formation, induced apoptosis and autophagy, inhibited cell invasion, decreased MMP, and increased ROS levels in SH-SY5Y cells. In addition, we observed significant upregulation in the expression levels of CASP8, BECLIN, ATG5, ATG7, and MAPILC3A genes between the two doses. These results suggest that piceatannol enhances autophagic activity and induces caspase-dependent apoptosis, indicating its potential as a therapeutic agent against neuroblastoma cells.

Upregulation of Immune checkpoint PD-L1 in Colon cancer cell lines and activation of T cells by Leuconostoc mesenteroides.

Globally colorectal cancer ranks as the third most widespread disease and the third leading cause of cancer-associated mortality. Immunotherapy treatments like PD-L1 blockade have been used to inhibit the PD-L1 legend, which boosts the activity of cytotoxic T lymphocytes. Recently, studies suggest that some probiotics could potentially enhance the effectiveness of immunotherapy treatments for cancer patients. We found that in Caco-2 and HT-29 cells, the live Leuconostoc mesenteroides treatment resulted an increase in the PD-L1 expression and this treatment stimulated interferon-gamma (IFN-γ) production in Jurkat T-cells. Due to the well-established ability of IFN-γ to enhance PD-L1 expression, the combination of IFN-γ and L. mesenteroides was used in colon cancer cell lines and a resulting remarkable increase of over tenfold in PD-L1 expression was obtained. Interestingly, when L. mesenteroides and IFN-γ are present, the blockage of PD-L1 using PD-L1 antibodies not only improved the viability of Jurkat T-cells but also significantly boosted the levels of IFN-γ and IL-2, the T-cells activation marker cytokines. In addition to upregulating PD-L1, L. mesenteroides also activated Toll-like receptors (TLRs) and NOD-like receptors (NODs) pathways, specifically through TLR2 and NOD2, while also exerting a suppressive effect on autophagy in colon cancer cell lines. In conclusion, our findings demonstrate a significant upregulation of PD-L1 expression in colon cancer cells upon co-culturing with L. mesenteroides. Moreover, the presence of PD-L1 antibodies during co-culturing activates Jurkat T cells. The observed enhancement in PD-L1 expression may be attributed to the inhibition of the Autophagy pathway or activation of the hippo pathway. KEY POINTS: Co-culturing L. mesenteroides increases PD-L1 gene and protein transaction in colon cancer. L. mesenteroides existing enhances T cells viability and activity. GPCR41/42 is a possible link between L. mesenteroides, YAP-1 and PD-L1.