A novel DDIT3 activator dehydroevodiamine effectively inhibits tumor growth and tumor cell stemness in pancreatic cancer.

BACKGROUND: The existence of pancreatic cancer stem cells (PCSCs) results in limited survival benefits from current treatment options. There is a scarcity of effective agents for treating pancreatic cancer patients. Dehydroevodiamine (DeHE), a quinazoline alkaloid isolated from the traditional Chinese herb Evodiae fructus, exhibited potent inhibition of pancreatic ductal adenocarcinoma (PDAC) cell proliferation and tumor growth both in vitro and in vivo. METHODS: The cytotoxic effect of DeHE on PDAC cells was assessed using CCK-8 and colony formation assays. The antitumor efficacy of DeHE were appraised in human PANC-1 xenograft mouse model. Sphere formation assay and flow cytometry were employed to quantify the tumor stemness. RNA-Seq analysis, drug affinity responsive target stability assay (DARTS), and RNA interference transfection were conducted to elucidate potential signaling pathways. Western blotting and immunohistochemistry were utilized to assess protein expression levels. RESULTS: DeHE effectively inhibited PDAC cell proliferation and tumor growth in vitro and in vivo, and exhibited a better safety profile compared to the clinical drug gemcitabine (GEM). DeHE inhibited PCSCs, as evidenced by its suppression of self-renewal capabilities of PCSCs, reduced the proportion of ALDH+ cells and downregulated stemness-associated proteins (Nanog, Sox-2, and Oct-4) both in vitro and in vivo. Furthermore, there is potential involvement of DDIT3 and its downstream DDIT3/TRIB3/AKT/mTOR pathway in the suppression of stemness characteristics within DeHE-treated PDAC cells. Additionally, results from the DARTS assay indicated that DeHE interacts with DDIT3, safeguarding it against degradation mediated by pronase. Notably, the inhibitory capabilities of DeHE on PDAC cell proliferation and tumor stemness were partially restored by siDDIT3 or the AKT activator SC-79. CONCLUSION: In summary, our study has identified DeHE, a novel antitumor natural product, as an activator of DDIT3 with the ability to suppress the AKT/mTOR pathway. This pathway is intricately linked to tumor cell proliferation and stemness characteristics in PDAC. These findings suggest that DeHE holds potential as a promising candidate for the development of innovative anticancer therapeutics.

Erianin inhibits the growth and metastasis through autophagy-dependent ferroptosis in KRASG13D colorectal cancer.

Colorectal cancer (CRC) is the third most common cause of cancer mortality worldwide. Approximately 40% of CRC patients are KRAS sequence variation, including KRAS G13D mutation (KRASG13D) CRC patients, accounting for approximately 8% of all KRAS mutations in CRC patients and showing little benefit from anti-EGFR therapy. Therefore, there is an urgent need for new and effective anticancer agents in patients with KRASG13D CRC. Here, we identified a natural product, erianin, that directly interacted with purified recombinant human KRASG13D with a Kd of 1.1163 µM, which also significantly improve the thermal stability of KRASG13D. The cell viability assay showed that KRASG13D cells were more sensitive to erianin than KRASWT or KRASG12V cells. In vitro, results showed that erianin suppressed the migration, invasion and epithelial-mesenchymal transition (EMT) of KRASG13D CRC cells. Furthermore, erianin induced ferroptosis, as evidenced by the accumulation of Fe2+ and reactive oxygen species (ROS), lipid peroxidation, and changes in the mitochondrial morphology of KRASG13D CRC cells. Interestingly, we also found that erianin-induced ferroptosis was accompanied by autophagy. Moreover, the occurrence of erianin-induced ferroptosis is reversed by autophagy inhibitors (NH4Cl and Bafilomycin A1) and ATG5 knockdown, suggesting that erianin-induced ferroptosis is autophagy-dependent. In addition, we evaluated the inhibition of tumor growth and metastasis by erianin in vivo using a subcutaneous tumor model and a spleen-liver metastasis model, respectively. Collectively, these data provide novel insights into the anticancer activity of erianin, which is valuable for the further discussion and investigation of the use of erianin in clinical anticancer chemotherapy for KRASG13D CRC.

Periplocin inhibits hepatocellular carcinoma progression and reduces the recruitment of MDSCs through AKT/NF-κB pathway.

AIMS: We aimed to evaluate the effect of periplocin on inhibiting hepatocellular carcinoma (HCC) and further determine its mechanisms. MAIN METHODS: Cytotoxic activity of periplocin against HCC cells was tested by CCK-8 and colony formation assays. The antitumor effects of periplocin were evaluated in human HCC SK-HEP-1 xenograft and murine HCC Hepa 1-6 allograft mouse models. Flow cytometry was used to measure cell cycle distribution, apopotosis, and the number of myeloid-derived suppressor cells (MDSCs). Hoechst 33258 dye was applied to observe the nuclear morphology. Network pharmacology was performed to predict possible signaling pathways. Drug affinity responsive target stability assay (DARTS) was used to evaluate AKT binding of periplocin. Western blotting, immunohistochemistry, and immunofluorescence were used to examine the protein expression levels. KEY FINDING: Periplocin inhibited cell viability with IC50 values from 50 nM to 300 nM in human HCC cells. Periplocin disrupted cell cycle distribution and promoted cell apoptosis. Moreover, AKT was predicted as the target of periplocin by network pharmacology, which was confirmed by that AKT/NF-κB signaling was inhibited in periplocin-treated HCC cells. Periplocin also inhibited the expression of CXCL1 and CXCL3, leading to decreased accumulation of MDSCs in HCC tumors. SIGNIFICANCE: These findings reveal the function of periplocin in inhibiting HCC progression by G2/M arrest, apoptosis and suppression of MDSCs accumulation through blockade of the AKT/NF-κB pathway. Our study further suggests that periplocin has the potential to be developed as an effective therapeutic agent for HCC.

Ectopic expression of FGF5s induces wool growth in Chinese merino sheep.

Fibroblast growth factor 5 (FGF5) has been recognized as an inhibitor to cease animal hair growth, while in contrary, FGF5 short alternative transcript (FGF5s) can induce hair growth by antagonizing FGF5 function. To investigate the role of FGF5s in wool growth in Chinese Merino sheep, we generated transgenic sheep of ectopic expression of FGF5s by injection of recombinant lentivirus into zygote. Totally 20 transgenic sheep were obtained and 12 were alive after birth. Characterization of the transgene revealed that the transgenic sheep showed variety of integrant, ranged from 2 to 11 copies of transgene. The ectopic expression of FGF5s was observed in all transgenic sheep. Further study on the effect of ectopic expression of FGF5s revealed that the wool length of transgenic sheep were significantly longer than that of non-transgenic control, with 9.17cm of transgenic lambs versus 7.58cm of control animals. Notably, besides the increase of wool length, the yearling greasy fleece weight was also concordantly greater than that of wild-type (p<0.01), with 3.22kg of transgenic sheep versus 2.17kg of control lambs (p<0.01) in average. Our results suggested that overexpression of FGF5s could stimulate wool growth and resulted in increase of wool length and greasy wool weight.

CRISPR/Cas9-mediated loss of FGF5 function increases wool staple length in sheep.

Fibroblast growth factor 5 (FGF5) regulates hair length in humans and a variety of other animals. To investigate whether FGF5 has similar effects in sheep, we used clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) to generate loss-of-function mutations with the FGF5 gene in Chinese Merino sheep. A total of 16 lambs were identified with genetic mutations within the targeting locus: 13 lambs had biallelic modifications and three lambs had monoallelic modifications. Characterization of the modifications revealed that 13 were frameshift mutations that led to premature termination, whereas the other three were in-frame deletions. Thus, CRISPR/Cas9 efficiently generated loss-of-function mutations in the sheep FGF5 gene. We then investigated the effect of loss of FGF5 function on wool traits in 12 lambs and found that wool staple length and stretched length of genetically modified (GM) yearling sheep were significantly longer compared with that of wild-type (WT) control animals. The greasy fleece weight of GM yearling sheep was also significantly greater compared with that of WT sheep. Moreover, the mean fiber diameter in GM sheep showed no significant difference compared with WT sheep, suggesting that the increase in greasy fleece weight was likely attributed to the increase in wool length. The results of this study suggest that CRISPR/Cas9-mediated loss of FGF5 activity could promote wool growth and, consequently, increase wool length and yield.