Tubuloside B, a major constituent of Cistanche deserticola, inhibits migration of hepatocellular carcinoma by inhibiting Hippo-YAP pathway.

BACKGROUND: Studies have shown that phenylethanoid glycosides (PhGs) have multiple pharmacological effects such as anti-inflammatory, hepatoprotective or neuroprotective functions, whereas their anti-tumor effects are rarely studied. Tubuloside B (Tub B) is a PhG isolated from Cistanche deserticola, a traditional Chinese medicine. To date, there is a lack of comprehensive research regarding the biological activity of Tub B. PURPOSE: The subject of the current study was to investigate the anti-hepatocellular carcinoma (HCC) cell activity and the underlying mechanism of Tub B. METHODS: We evaluated the in vitro anti-migratory effect of Tub B by scratch and transwell assays. RNA-seq was employed to identify the differential genes by Tub B. Besides, the functional mechanism of Tub B was investigated by distinct molecular biology techniques including immunofluorescent staining, quantitative PCR, as well as western blot analysis. Subsequently, we utilized Hep3B cells for in vivo metastasis assays through spleen injection and evaluated the anti-migratory effect of Tub B in hepatocellular carcinoma (HCC). RESULTS: Tub B exhibited in vitro and in vivo inhibition of HCC cell migration. Tub B decreased the expression of transcriptional target genes downstream of the Hippo pathway, including CTGF, CYR61, and N-cadherin as determined by RNA-seq. Furthermore, mechanistic studies confirmed that Tub B increased phosphorylation of YAP at S127, which contributes to YAP cytoplasmic localization. Additionally, overexpression of YAP abrogated Tub B-induced inhibition of HCC migration and the mRNA levels of CTGF, CYR61, and N-cadherin. CONCLUSIONS: Taken together, these results illustrated that Tub B demonstrated great potential in inhibiting migration of HCC, and a portion of its impact can be attributed to the modulation of the Hippo-YAP pathway.

Isoginkgetin, a potential CDK6 inhibitor, suppresses SLC2A1/GLUT1 enhancer activity to induce AMPK-ULK1-mediated cytotoxic autophagy in hepatocellular carcinoma.

Isoginkgetin (ISO), a natural biflavonoid, exhibited cytotoxic activity against several types of cancer cells. However, its effects on hepatocellular carcinoma (HCC) cells and mechanism remain unclear. Here, we revealed that ISO effectively inhibited HCC cell proliferation and migration in vitro. LC3-II expression and autophagosomes were increased under ISO treatment. In addition, ISO-induced cell death was attenuated by treatment with chloroquine or knockdown of autophagy-related genes (ATG5 or ULK1). ISO significantly suppressed SLC2A1/GLUT1 (solute carrier family 2 member 1) expression and glucose uptake, leading to activation of the AMPK-ULK1 axis in HepG2 cells. Overexpression of SLC2A1/GLUT1 abrogated ISO-induced autophagy. Combining molecular docking with thermal shift analysis, we confirmed that ISO directly bound to the N terminus of CDK6 (cyclin-dependent kinase 6) and promoted its degradation. Overexpression of CDK6 abrogated ISO-induced inhibition of SLC2A1/GLUT1 transcription and induction of autophagy. Furthermore, ISO treatment significantly decreased the H3K27ac, H4K8ac and H3K4me1 levels on the SLC2A1/GLUT1 enhancer in HepG2 cells. Finally, ISO suppressed the hepatocarcinogenesis in the HepG2 xenograft mice and the diethylnitrosamine+carbon tetrachloride (DEN+CCl4)-induced primary HCC mice and we confirmed SLC2A1/GLUT1 and CDK6 as promising oncogenes in HCC by analysis of TCGA data and human HCC tissues. Our results provide a new molecular mechanism by which ISO treatment or CDK6 deletion promotes autophagy; that is, ISO targeting the N terminus of CDK6 for degradation inhibits the expression of SLC2A1/GLUT1 by decreasing the enhancer activity of SLC2A1/GLUT1, resulting in decreased glucose levels and inducing the AMPK-ULK1 pathway.

Tubuloside B, isolated from Cistanche tubulosa, a promising agent against M1 macrophage activation via synergistically targeting Mob1 and ERK1/2.

Targeting macrophage M1 polarization is a promising strategy with fewer detrimental effects in COVID-19 curation. Phenylethanoid glycosides (PhGs) of Cistanche tubulosa are a botanical drug to possess various anti-inflammation-related functions, such as immunomodulating, hepatoprotective or neuroprotective functions, whereas their anti-inflammatory activity is rarely understood. A search into their anti-inflammatory characteristics led to the isolation of 49 PhGs along with 15 new PhGs. Their inhibitory effects against M1 polarization induced by LPS plus IFN-γ were explored in RAW264.7 macrophages. Of these PhGs, tubuloside B (Tub B) exerted substantial NO scavenging effect both in chemical- and cell-based assays, and it inhibited massive production of cytokines and chemokines. Tub B decreased ERK1/2 phosphorylation via direct binding and inhibited the MAPK signaling pathway. Tub B also directly binded to Mob1 protein, thereby increased the stability and level of Mob1 protein by inhibiting ubiquitinated degradation. Mob1 was pivotal for the anti-inflammatory activity of Tub B, and it acted independently of the canonical Hippo-YAP pathway. Moreover, ERK1/2 and Mob1 also had a synergic effect on modulating the inflammatory response. Finally, these effects of Tub B were verified in mice with LPS-induced systemic inflammatory response syndrome. Taken together, these results indicated that Tub B acted as a promising agent against M1 macrophage activation by synergistically targeting ERK1/2 and Mob1, and that it may potentially be a drug candidate to prevent/treat inflammatory diseases, especially in COVID-19.