Evaluation of unmodified human cell-derived extracellular vesicle mitochondrial deoxyribonucleic acid-based biodistribution in rodents.

Recently, extracellular vesicles (EVs) have been developed as therapeutic targets for various diseases. Biodistribution is crucial for EVs intended for therapeutic purposes because it can determine the degree of on- and off-target effects. This study aimed to explore techniques to evaluate the biodistribution of unmodified EVs. We devised a novel quantitative polymerase chain reaction (qPCR)-based assay to detect unmodified EVs by targeting mitochondrial deoxyribonucleic acid (mtDNA), a constituent of EVs. We focused on specific mtDNA regions that exhibited homologous variations distinct from their rodent mtDNA counterparts to establish this analytical approach. Herein, we successfully designed primers and probes targeting human and rodent mtDNA sequences and developed a highly specific and sensitive qPCR method. Furthermore, the quantification range of EVs isolated from various cells differed based on the manufacturer and cell source. IRDye 800CW-labelled Expi293F EV mimetics were administered to the animals via the tail vein to compare the imaging test and mtDNA-qPCR results. The results obtained from imaging tests and mtDNA-qPCR to investigate EV biodistribution patterns revealed differences. The results revealed that our newly developed method effectively determined the biodistribution of unmodified EVs with high sensitivity and reproducibility.

CHI3L1 induces autophagy through the JNK pathway in lung cancer cells.

CHI3L1 is closely related to the molecular mechanisms of cancer cell migration, growth, and death. According to recent research, autophagy regulates tumor growth during various stages of cancer development. This study examined the association between CHI3L1 and autophagy in human lung cancer cells. In CHI3L1-overexpressing lung cancer cells, the expression of LC3, an autophagosome marker, and the accumulation of LC3 puncta increased. In contrast, CHI3L1 depletion in lung cancer cells decreased the formation of autophagosomes. Additionally, CHI3L1 overexpression promoted the formation of autophagosomes in various cancer cell lines: it also increased the co-localization of LC3 and the lysosome marker protein LAMP-1, indicating an increase in the production of autolysosomes. In mechanism study, CHI3L1 promotes autophagy via activation of JNK signaling. JNK may be crucial for CHI3L1-induced autophagy since pretreatment with the JNK inhibitor reduced the autophagic effect. Consistent with the in vitro model, the expression of autophagy-related proteins was downregulated in the tumor tissues of CHI3L1-knockout mice. Furthermore, the expression of autophagy-related proteins and CHI3L1 increased in lung cancer tissues compared with normal lung tissues. These findings show that CHI3L1-induced autophagy is triggered by JNK signals and that CHI3L1-induced autophagy could be a novel therapeutic approach to lung cancer.

PAUF Induces Migration of Human Pancreatic Cancer Cells Exclusively via the TLR4/MyD88/NF-κB Signaling Pathway.

PAUF, a tumor-promoting protein secreted by cancer cells, exerts paracrine effects on immune cells through TLR4 receptors expressed on immune cell surfaces. This study aimed to investigate if PAUF elicits autocrine effects on pancreatic cancer (PC) cells through TLR4, a receptor that is overexpressed on PC cells. In this study, TLR4 expression was detected in PC cells only, but not normal pancreatic cells. The migration of TLR4 high-expressing PC cells (i.e., BxPC-3) was reduced by a selective TLR4 inhibitor, in a dose-dependent manner. Using TLR4 overexpressed and knockout PC cell lines, we observed direct PAUF-TLR4 binding on the PC cell surfaces, and that PAUF-induced cancer migration may be mediated exclusively through the TLR4 receptor. Further experiments showed that PAUF signaling was passed down through the TLR4/MyD88 pathway without the involvement of the TLR4/TRIF pathway. TLR4 knockout also downregulated PC membrane PD-L1 expression, which was not influenced by PAUF. To the best of our knowledge, TLR4 is the first receptor identified on cancer cells that mediates PAUF's migration-promoting effect. The results of this study enhanced our understanding of the mechanism of PAUF-induced tumor-promoting effects and suggests that TLR4 expression on cancer cells may be an important biomarker for anti-PAUF treatment.

Bee Venom Triggers Autophagy-Induced Apoptosis in Human Lung Cancer Cells via the mTOR Signaling Pathway.

In oriental medicine, bee venom has long been used as a therapeutic agent against inflammatory diseases. Several studies have reported that isolated and purified bee venom components are effective in treating dementia, arthritis, inflammation, bacterial infections, and cancer. In previous studies, we reported that bee venom inhibits cell growth and induces apoptotic cell death in lung cancer cells. In the present study, we assessed whether bee venom affects autophagy and thereby induces apoptosis. Bee venom treatment increased the levels of autophagy-related proteins (Atg5, Beclin-1, and LC3-II) and the accumulation of LC3 puncta. We found that bee venom could induce autophagy by inhibiting the mTOR signaling pathway. In addition, we found that hydroxychloroquine (HCQ)- or si-ATG5-induced autophagy inhibition further demoted bee venom-induced apoptosis. Bee venom-induced autophagy promotes apoptosis in lung cancer cells and may become a new approach to cancer treatment.

A Natural CHI3L1-Targeting Compound, Ebractenoid F, Inhibits Lung Cancer Cell Growth and Migration and Induces Apoptosis by Blocking CHI3L1/AKT Signals.

Our previous big data analyses reported a strong association between CHI3L1 expression and lung tumor development. In this present study, we investigated whether a CHI3L1-inhibiting natural compound, ebractenoid F, inhibits lung cancer cell growth and migration and induces apoptosis. Ebractenoid F concentration-dependently (0, 17, 35, 70 µM) and significantly inhibited the proliferation and migration of A549 and H460 lung cancer cells and induced apoptosis. In the mechanism study, we found that ebractenoid F bound to CHI3L1 and suppressed CHI3L1-associated AKT signaling. Combined treatment with an AKT inhibitor, LY294002, and ebractenoid F synergistically decreased the expression of CHI3L1. Moreover, the combination treatment further inhibited the growth and migration of lung cancer cells and further induced apoptosis, as well as the expression levels of apoptosis-related proteins. Thus, our data demonstrate that ebractenoid F may serve as a potential anti-lung cancer compound targeting CHI3L1-associated AKT signaling.