Hernandonine-mediated autophagic cell death in hepatocellular carcinoma: Interplay of p53 and YAP signaling pathways.

Hepatocellular carcinoma (HCC), the primary form of liver cancer, is the third leading cause of cancer-related death globally. Hernandonine is a natural alkaloid derived from Hernandia nymphaeifolia that has been shown to exert various biological functions. In a previous study, hernandonine was shown to suppress the proliferation of several solid tumor cell lines without affecting normal human cell lines. However, little is known about the effect of hernandonine on HCC. Therefore, this study aimed to investigate the effect and mechanism of hernandonine on HCC in relation to autophagy. We found that hernandonine inhibited HCC cell growth in vitro and in vivo. In addition, hernandonine elicited autophagic cell death and DNA damage in HCC cells. RNA-seq analysis revealed that hernandonine upregulated p53 and Hippo signaling pathway-related genes in HCC cells. Small RNA interference of p53 resulted in hernandonine-induced autophagic cell death attenuation. However, inhibition of YAP sensitized HCC cells to hernandonine by increasing the autophagy induction. This is the first study to illustrate the complex involvement of p53 and YAP in the hernandonine-induced autophagic cell death in human HCC cells. Our findings provide novel evidence for the potential of hernandonine as a therapeutic agent for HCC treatment.

Cryptocaryone induces apoptosis in human hepatocellular carcinoma cells by inhibiting aerobic glycolysis through Akt and c-Src signaling pathways.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, with the second highest mortality rate in all cancer. Energy reprogramming is one of the hallmarks of cancer, and emerging evidence showed that targeting glycolysis is a promising strategy for HCC treatment. Cryptocaryone has been shown to display promising anti-cancer activity against numerous types of cancer. Previous study also indicated that cryptocaryone induces cytotoxicity by inhibiting glucose transport in cancer cells, but the detailed mechanism still needs to be elucidated. Therefore, this study aimed to investigate the relationship between the anti-cancer effect and glycolytic metabolism of cryptocaryone in human HCC cells. In this study, we found that cryptocaryone potently induced growth inhibition by apoptotic cell death in HCC cells. Cryptocaryone also suppressed the ATP synthesis, lactate production and glycolytic capacity of HCC cells. Mechanistic investigations showed that phosphorylation of Akt and c-Src, as well as the expression of HK1 were impeded by cryptocaryone. Moreover, cryptocaryone markedly increased the expression level of transcription factor FoxO1. Importantly, clinical database analysis confirmed the negative correlation between HK1 and FoxO1. High expression levels of HK-1 were positively correlated with poorer survival in patients with HCCs. These results suggest that cryptocaryone may promote cell apoptosis by inhibiting FoxO1-mediated aerobic glycolysis through Akt and c-Src signaling cascades in human HCC cells. This is the first study to indicate that cryptocaryone exerts anti-cancer property against human HCC cells. Cryptocaryone is a potential natural product worthy of further development into a promising candidate for HCC treatment.

RGD4C peptide mediates anti-p21Ras scFv entry into tumor cells and produces an inhibitory effect on the human colon cancer cell line SW480.

BACKGROUND: We prepared an anti-p21Ras scFv which could specifically bind with mutant and wild-type p21Ras. However, it cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate the scFv penetration into tumor cells and produce antitumor effects. METHODS: RGD4C-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed to express fusion proteins in E. coli, then the fusion proteins were purified with HisPur Ni-NTA. RGD4C-EGFP was used as reporter to test the factors affecting RGD4C penetration into tumor cell. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. The ability of RGD4C-p21Ras-scFv to penetrate SW480 cells and colocalization with Ras protein was detected by immunocytochemistry and immunofluorescence. The antitumor activity of the RGD4C-p21Ras-scFv was assessed with the MTT, TUNEL, colony formation and cell migration assays. Chloroquine (CQ) was used an endosomal escape enhancing agent to enhance endosomal escape of RGD4C-scFv. RESULTS: RGD4C-p21Ras-scFv fusion protein were successfully expressed and purified. We found that the RGD4C fusion protein could penetrate into tumor cells, but the tumor cell entry of was time and concentration dependent. Endocytosis inhibitors and a low temperature inhibited RGD4C fusion protein endocytosis into cells. The change of the cell membrane potential did not affect penetrability. RGD4C-p21Ras-scFv could penetrate SW480 cells, effectively inhibit the growth, proliferation and migration of SW480 cells and promote this cells apoptosis. In addition, chloroquine (CQ) could increase endosomal escape and improve antitumor activity of RGD4C-scFv in SW480 cells. CONCLUSION: The RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of ras-driven cancers.

Determination of organophosphorus flame retardants in fish by freezing-lipid precipitation, solid-phase extraction and gas chromatography-mass spectrometry.

An analytical method has been developed for measuring 12 organophosphorus flame retardants (PFRs) in fish tissue samples. After the Soxhlet extraction of PFRs with dichloromethane. The experimental parameters of the clean-up were systematically optimized. Methanol was found to be a more effective solvent than acetonitrile used in freezing-lipid precipitation. Methanol (5%) in ultrapure water, was finally selected to perform solid-phase extraction (SPE, Oasis HLB cartridge), with mean lipid removal efficiency of 94% after freezing-lipid precipitation. Further purification followed by 200mg of Z-Sep and C18 dispersant to eliminate the remaining interferences. Quantification was performed using gas chromatography-mass spectrometry in selective ion monitoring mode. The recovery, precision, and the method detection limits (MDLs) were verified by spiking experiments. All chemicals except triethyl phosphate (TEP) showed satisfactory recoveries in the range of 73-107% and 56-108% in the spiked blanks samples and spiked fish tissue samples, respectively. MDLs for PFRs in the biological samples ranged from 0.004 to 0.059ng/g. The proposed method successfully applied to the determination of PFRs in real fish samples with recoveries of four internal standards varying from 75 to 97%. The results demonstrated that the proposed method is highly effective for analyzing PFRs in fish samples.