Mitochondria, which is essential for adequate innate immune response, energy metabolism and mitochondria reactive oxygen species (ROS) production, might be in the cross fire of Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and host cell defense. However, little is known about interactions between mitochondria and SARS-CoV-2. We performed fluorescent microscopy and found an enrichment of SARS-CoV-2 replication products double stranded RNA (dsRNA) within mitochondria. The entry process of dsRNA might be mediated by Tom20 as observed by reduced mitochondrial localization of SARS-CoV-2 dsRNA in Tom20 knockdown cells. Importantly, decreased mitochondrial localization of dsRNA, as well as mitochondrial membrane stabilizers mdivi-1 and cyclosporin A, inhibited viral load in cells. Next, we detected mitochondrial dysfunction caused by SARS-CoV-2 infection, including mitochondrial membrane depolarization, mitochondrial permeability transition pore opening and increased ROS release. In response to mitochondrial damage, we observed an increase in expression and mitochondrial accumulation of Pink1 and Parkin proteins, as well as Pink-1-mediated recruitment of P62 to mitochondria, suggesting initiated mitophagy for mitochondrial quality control and virus clearance. Nevertheless, we observed that mitophagy was inhibited and stayed in early stage with an unchanged Hsp60 expression post SARS-CoV-2 infection. This might be one of the anti-autophagy strategies of SARS-CoV-2 and we used co-immunoprecipitation to found that SARS-CoV-2 infection inhibited P62 and LC3 binding which plays a critical role in selective envelopment of substrates into autophagosomes. Our results suggest that mitochondria are closely involved in SARS-CoV-2 replication and mitochondrial homeostasis is disrupted by SARS-CoV-2 in the virus-cell confrontation.

Ferritinophagy is associated with tumor occurrence, development, and therapy effects. Ferritinophagy and ferroptosis are regulated by iron metabolism and are closely connected. LC3 protein is a key protein in autophagy. Following the binding of NCOA4 to FTH1, it links to LC3Ⅱ in lysosomes, a symbol of ferritinophagy. A ferritinophagy’s inducer is likely to open new avenues for anticancer medication research and development. In this study, we discovered that caryophyllene oxide has a substantial inhibitory effect on HCCLM3 and HUH7 cells, by regulating the level of cellular oxidative stress, and the levels of autophagy and iron metabolism in HCCLM3 and HUH7 cells, leading to a ferritinophagy-related phenomenon. Furthermore, the results of T-AOC, DPPH free radical scavenging rate, and hydroxyl radical inhibition indicated that caryophyllene oxide can inhibit cell anti-oxidation. The examination of the ferritinophagy-related process revealed that caryophyllene oxide promotes the production and accumulation of intracellular reactive oxygen species and lipid peroxidation. NCOA4, FTH1, and LC3Ⅱ were found to be targeted regulators of caryophyllene oxide. Caryophyllene oxide regulated NCOA4, LC3 Ⅱ, and FTH1 to promote ferritinophagy. In vivo, we discovered that caryophyllene oxide can lower tumor volume, significantly improve NCOA4 and LC3 protein levels in tumor tissue, and raise Fe²⁺ and malondialdehyde levels in serum. The compound can also reduce NRF2, GPX4, HO-1, and FTH1 expression levels. The reduction in the expression levels of NRF2, GPX4, HO-1, and FTH1 by caryophyllene oxide also inhibited GSH and hydroxyl radical’s inhibitory capacities in serum, and promoted iron deposition in tumor tissue resulting in the inhibition of tumor growth. In summary, our study revealed that caryophyllene oxide mostly kills liver cancer cells through ferritinophagy-mediated ferroptosis mechanisms. In conclusion, caryophyllene oxide may be used as a ferritinophagy activator in the field of antitumor drug research and development.

Neobavaisoflavone (NBIF), a natural active ingredient isolated from Psoralea, possesses anti-inflammatory, anti-cancer, and antioxidant properties; however, the anti-tumor mechanism of NBIF has not been thoroughly investigated, and the inhibitory effect and inhibitory pathway of NBIF on liver cancer are still unknown. Our study aimed to explore the effects of NBIF on hepatocellular carcinoma and its potential mechanisms. First, we detected the inhibition of NBIF on HCC cells by the CCK8 assay and then observed the morphological changes of the cells under the microscope. Besides, we analyzed the changes in the pyroptosis level of NBIF when inhibiting the cells through flow cytometry, immunofluorescence, and a western blot assay. Finally, we used a mouse tumor-bearing model to explore the effects of NBIF in vivo on HCCLM3 cells. NBIF-treated HCC cells exhibited specific features of pyroptosis. Analysis of pyroptosis-related protein levels revealed that NBIF primarily induced pyroptosis in HCC cells via the caspase-3-GSDME signaling pathway. Then, we demonstrated that NBIF impacted the protein expression of Tom20 by producing ROS in HCC cells, hence promoting the recruitment of Bax to mitochondria, activating caspase-3, cutting GSDME, and triggering pyroptosis. By activating ROS, NBIF was able to trigger pyroptosis in HCC cells, providing an experimental basis for the future study of new treatments for liver cancer.

Pyroptosis is related to the occurrence, development, and therapeutic response of tumors, mediated by the proteins of the Gasdermin family. These proteins have become potential biomarkers for cancer treatment, and their agonists are likely to become a new direction in research and development of antitumor drugs. In this study, we found that myricetin has an inhibitory effect on lung cancer cells of the activation of pyroptosis. Analysis of the expression of Gasdermin family proteins revealed that this phenomenon was caused by the cleavage of GSDME. Subsequently, specific inhibitors, we found that caspase-3 was its upstream activation factor. In addition, mitochondrial and endoplasmic reticulum (ER) analysis showed that myricetin can cause endoplasmic reticulum stress and increase reactive oxygen species (ROS) levels. Subsequent inhibition of caspase-12 revealed that the expression levels of cleaved-caspase-3 and cleaved-GSDME were significantly reduced, resulting in the inhibition of pyroptosis. Using in vivo experiments, we also found that the treatment with myricetin can reduce tumor volume and significantly increase the level of pyroptosis-related proteins in tumor tissues. Overall, our findings show that myricetin induces cell death of lung cancer cells primarily through an ER stress pathway-induced pyroptosis. Therefore, myricetin has the potential to be used as a pyroptosis agonist in research and development of antitumor drugs.

Background: Apoptin, as a tumor-specific pro-apoptotic protein, plays an important anti-tumoral role, but its mechanism of autophagy activation and the interaction between autophagy and apoptosis have not been accurately elucidated. Here, we studied the mechanism of apoptin-induced apoptosis and autophagy and the interaction between two processes. Methods: Using crystal violet staining and the CCK-8 assay, we analyzed the effect of apoptin in the inhibition of liver cancer cells in vitro and analyzed the effect of inhibiting liver cancer in vivo by establishing a nude mouse tumor model. Flow cytometry and fluorescence staining were used to analyze the main types of apoptin-induced apoptosis and autophagy. Subsequently, the relationship between the two events was also analyzed. Flow cytometry was used to analyze the effect of ROS on apoptin-mediated apoptosis and autophagy mediated by apoptin. The effect of ROS on two phenomena was analyzed. Finally, the role of key genes involved in autophagy was analyzed using gene silencing. Results: The results showed that apoptin can significantly increase the apoptosis and autophagy of liver cancer cells, and that apoptin can cause mitophagy through the increase in the expression of NIX protein. Apoptin can also significantly increase the level of cellular ROS, involved in apoptin-mediated autophagy and apoptosis of liver cancer cells. The change of ROS may be a key factor causing apoptosis and autophagy. Conclusion: The above results indicate that the increase in ROS levels after apoptin treatment of liver cancer cells leads to the loss of mitochondrial transmembrane potential, resulting in endogenous apoptosis and mitophagy through the recruitment of NIX. Therefore, ROS may be a key factor connecting endogenous apoptosis and autophagy induced by apoptin in liver cancer cells. Graphical

Autophagy is a cellular self-defense mechanism by which cells can kill invading pathogenic microorganisms and increase the presentation of components of pathogens as antigens. Contrarily, pathogens can utilize autophagy to enhance their own replication. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) NSP6 can interact with ATPase proton pump component to inhibit lysosomal acidification, which was implicated in the autophagy process. However, research on how SARS-CoV-2 NSP6 affected autophagy, and its impact on virus replication is still lacking. Coronavirus NSP6 has been reported to promote coronavirus replication by limiting autophagosome expansion. However, this finding has not been confirmed in coronavirus disease 2019 (COVID-19). We investigated the effect of NSP6 protein on autophagosomes in different mutant strains of SARS-CoV-2 and revealed that the size of autophagosomes was reduced by NSP6 of the wild-type and Delta variant of SARS-CoV-2. In addition, we found that SARS-CoV-2 NSP6 localized to the lysosome and had an inhibitory effect on the binding of autophagosomes to the lysosome, which blocked the autophagy flux; this may be related to endoplasmic reticulum (ER)-related pathways. We also found that sigma-1 receptor (SIGMAR1) knock out (KO) reversed NSP6-induced autophagosome abnormality and resisted SARS-CoV-2 infection, which responds to the fact that SIGMAR1 is likely to be used as a potential target for the treatment of SARS-CoV-2 infection. In summary, we have provided a preliminary explanation of the effects on autophagy of the SARS-CoV-2 NSP6 protein from the pre-autophagic and late stages, and also found that SIGMAR1 is likely to be used as a potential target for SARS-CoV-2 therapy to develop relevant drugs. IMPORTANCE: We have provided a preliminary explanation of the effects on autophagy of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) non-structure protein 6 from the pre-autophagic and late stages, and also found that sigma-1 receptor is likely to be used as a potential target for SARS-CoV-2 therapy to develop relevant drugs.