Liposomal β-Sitosterol Suppresses Metastasis of CT26/luc Colon Carcinoma via Inhibition of MMP-9 and Evoke of Immune System

β-sitosterol (SITO) has been reported with anticancer effects; however, with poor bioavailability. The current study aimed to investigate whether liposomal encapsulated β-sitosterol (LS) has a better inhibition effect on tumor metastasis than β-sitosterol in a CT26/luc lung metastasis mouse model and the possible underlying mechanism. LS was liposomal-encapsulated SITO and was delivered to mice by oral gavage. The cell viability was determined by the MTT assay, and invasiveness of the tumor cells and related protein expression were evaluated with the invasion assay and Western blotting. For therapeutic efficacy evaluation, male BALB/c mice were treated with PBS, SITO, and LS once a day for 7 days prior to intravenous injections of CT26/luc cells; treatments were continued twice a week post-cell inoculation throughout the entire experiment. Tumor growth inhibition was monitored by bioluminescent imaging (BLI). IL-12, IL-18, and IFN-γ in the intestinal epithelium were determined by ELISA. The results show that LS treatment had a better invasion inhibition with lower cytotoxicity than SITO when the same dose was utilized. Notably, mice treated with LS significantly exhibited fewer metastases to the lungs and other tissues/organs compared with the Control and SITO groups. Additionally, the IL-12, IL-18, and IFN-γ levels were significantly increased in the LS-treated mice compared with the Control and SITO groups. The underlying mechanism may be through the inhibition of MMP-9 and elicitation of the antitumoral Th1 immune response, such as increasing CD4+ and CD8+ T cells, IL-12, IL-18, and IFN-γ

Guidelines for the use and interpretation of assays for monitoring autophagy

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field