Altingia chinensis petroleum ether extract suppresses NSCLC via induction of apoptosis, attenuation of EMT, and downregulation of PI3K/Akt pathway.

Non-small-cell lung cancer (NSCLC) is the primary type of lung cancer with the leading cause of fatalities from cancer, and the effective treatment is minimal. Altingia chinensis is a medicinal plant utilized as a traditional folk remedy to alleviate rheumatism, punch injury and paralysis. APE is the petroleum ether extract from A. Chinensis, whose antitumor effects are rarely studied. To explore the antitumor effects of APE on NSCLC and its molecular mechanism. LLC and H1299 cells were used to explore the anticancer effect of APE on NSCLC in vitro. MTT assay and colony formation were employed to evaluate cell viability. Flow cytometry was used to evaluate apoptosis. Wound healing and transwell were employed to evaluate cell migration and invasive capacity. Meanwhile, an LLC tumor-bearing C57BL/6J mice model was established for assessing the anticancer effect of APE on NSCLC in vivo. H&E staining was used to assess histopathology. TUNEL assay was performed to assess apoptosis in tumor tissue. Network pharmacology, CESTA, and kinase assay were employed to analyze potential molecular mechanisms. Western blots were performed to detect proteins involved in apoptosis, EMT, and the PI3K/Akt pathway. This is the first investigation to identify APE's antitumor potential in both NSCLC cells and tumor-bearing mice models. Significantly, APE dose-dependently decreased cell viability and caused morphological changes both in LLC and H1299 cells. Furthermore, APE (31.25, 62.5, and 125 μg/ml) induced apoptosis in NSCLC cells, as demonstrated by increased Annexin V-FITC/PI-stained cells, the cleaved-caspase 3 levels, and the Bax/Bcl-2 ratio. Additionally, APE suppressed cell migration and invasion by the increase of E-cadherin expression and the downregulation of vimentin, implying that APE inhibited cell metastasis via attenuation of EMT. Importantly, intragastric administration of 100 mg/kg APE significantly inhibited tumor growth without apparent side effects. TUNEL assay confirmed the apoptosis in tumor tissue. Western blots validated the alteration of EMT and apoptotic markers in tumor tissue, which matched the in vitro findings. Moreover, APE directly bound to PI3Kα and inhibited its activity, leading to inhibition of the PI3K-Akt pathway. Overall, APE exhibits anti-tumor effects on NSCLC via induction of apoptosis, attenuation of EMT, and its mechanism involves the suppression of the PI3K/Akt pathway. Our study offers new insights for the identification of novel drug development for NSCLC.

Qin Y ，Zhang D ，Shangguan C ，Xu C ，Li S ，Li Y ，Liu Y ，Jiang S ... -  《-》

Rosmarinic acid attenuates glioblastoma cells and spheroids' growth and EMT/stem-like state by PTEN/PI3K/AKT downregulation and ERK-induced apoptosis.

Glioblastoma (GB) is a highly malignant type of brain cancer with a poor prognosis. Therapeutic strategies for GB are still limited. Rosmarinic acid (RA), a polyphenolic compound, is a promising experimental anticancer agent, but its specific protein targets for GB remain unclear. This study aimed to elucidate the anticancer effects of RA in 2D- and 3D-GB cells and the underlying mechanisms. 3D-tumor spheroids (mimics in vivo tumors) were obtained by the hanging-drop/agarose method. RA's anti-glioma activity on U-87MG (p53-wt/PTEN-mt) and LN229 (p53-mt/PTEN-wt) cells was evaluated through cell viability, colony-formation, migration/invasion/angiogenesis assays, fluorescence imaging, and spheroid growth analysis. The underlying mechanism of the anticancer effects of RA was investigated by Western blot and immunofluorescence analysis. The MEK inhibitor U0126 was used to block ERK phosphorylation. RA treatments exerted anti-proliferative and pro-apoptotic effects on human GB cells. RA dose-dependently reduced angiogenesis and intracellular ROS levels, suppressed glioma growth, and migration/invasion in 2D-culture and cancer stem cell (CSC)-like 3D-spheroid culture (SPC). Repeated therapy in SPC was more effective by leading to disrupted structure than a single treatment. Treatments in SPC also suppressed epithelial-mesenchymal transition (EMT) and CSC-like properties. Strikingly, RA downregulated the SIRT1/FOXO1/NF-κB axis independently of p53 or PTEN function in both gliomas. Immunofluorescence labeling revealed decreased SIRT1 and NF-κB-p65 and increased FOXO1 and GAPDH proteins in nuclear location (associated with apoptosis). Surprisingly, RA increased p-ERK1/2 levels, but priming with U0126 abolished RA-mediated p-ERK upregulation; thus, autophagy and apoptosis induction in GB cells were prevented, and the growth of GB spheroids accelerated. Specifically, RA also inhibited the PTEN/PI3K/AKT pathway in U-87MG cells. Due to genetic differences in cells, U-87MG cells were more sensitive to RA treatments than LN229 cells. Meanwhile, our positive control drug trial results with FDA-approved temozolomide (TMZ) used in GB treatment showed that our test compound rosmarinic acid exhibited higher therapeutic effects than TMZ at lower doses. Suppression of EMT, downregulation of SIRT1/FOXO1/NF-κB axis, inhibition of PTEN/PI3K/AKT signaling pathway, and ERK-induced apoptosis and autophagy were determined to be involved in stopping glioma progression. Our findings for the first time, revealed that RA may have potential therapeutic use by having multiple targets in human brain cancer with further clinical studies.

Şengelen A ，Önay-Uçar E 《-》

Hypocrellin A against intrahepatic Cholangiocarcinoma via multi-target inhibition of the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive and highly lethal cancer with an increasing incidence worldwide that lacks effective treatment regimens. Hypocrellin A (HA), a natural small compound isolated from S. bambusicola, has multiple biomedical activities, including antitumor activity. We intended to investigate the therapeutic effects of HA on ICC and its potential mechanisms. RBE and HuccT1 cell lines were utilized for in vitro experiments. CCK8 assay, colony formation analysis, RTCA, and immunofluorescence staining of ki67 were employed to evaluate the suppression effects of HA on proliferation. The inhibitory effects of HA on cell migration and invasion were evaluate through transwell and wound healing assays, and Hoechst 33,258 staining was performed to evaluate apoptosis. Additionally, we performed transcriptome sequencing and molecular docking for targeting identification, and immunoblotting and immunofluorescence of key molecules for validation. Two in vivo models, HuccT1 xenografts, and the primary ICC model (KRAS/P19/SB) established via hydrodynamic tail-vein injection were implemented. Multiplex immunohistochemistry (mIHC) was used to illustrate the multi-target inhibitory effects of HA. The IC50 values of HA against RBE and HuccT1 cells were 4.612 μM and 10.01 μM for 24 h, as determined through the CCK8 assay. Our results confirmed that HA significantly repressed the proliferation, migration, invasion, and promoted the apoptosis of ICC cells at low concentrations. Moreover, HA exerted its anti-cancer effects through multi-target inhibition of the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways. This inhibitory effect was rescued by Recilisib, an activator of the PI3K-AKT-mTOR pathway. Bioinformatics analysis of a multi-center RNA-Seq cohort (n = 90) demonstrated significant associations between these target pathways and the occurrence and poor prognosis of ICC. Animal studies suggested that HA strongly inhibited tumor growth in xenograft ICC models, and repressed the tumor number and size in the liver of primary ICC models by suppressing these three crucial pathways. HA, a novel natural small molecule, demonstrated promising therapeutic efficacy against ICC through its multi-target inhibitory effects on the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways. Moreover, it exhibited notable therapeutic benefits in a primary ICC model (KRAS/P19/SB), positioning it as a novel therapeutic agent for ICC.

Chen B ，Chen Q ，Lu M ，Zou E ，Lin G ，Yao J ，Wang L ，Gan Y ，Chen B ，Chen G ，Wu L ... -  《-》

[GINS1 Enhances Glycolysis, Proliferation and Metastasis in Lung Adenocarcinoma Cells by Activating the Notch/PI3K/AKT/mTORC1 Signaling Pathway].

Huo Y ，Xu X ，Ma X ，Feng Y ... -  《-》

Britannin inhibits hepatocellular carcinoma development and metastasis through the GSK-3β/β-catenin signaling pathway.

Hepatocellular carcinoma (HCC) stands out as a significant contributor to cancer-related death. Traditional Chinese Medicine (TCM) offers several advantages in the treatment of HCC. Britannin, a pivotal compound in Inulae Flos, has demonstrated pharmacological effects against various cancers, yet research on its specific anti-HCC effects remains limited. This study aims to explore the anti-HCC effects of britannin and its underlying mechanism. MTT assay, clone formation assay and flow cytometry were utilized to detect the cell activity, proliferation ability and apoptosis of britannin against HCC cell lines. Cell migration and invasion abilities of HCC cell lines treated with britannin were evaluated by wound-healing assay and transwell migration and invasion assay. H22 xenografted tumor mouse model was constructed and britannin treatment was performed to observe the effect of britannin on HCC tumors. The expression levels of liver cancer biomarkers AFP, AFP-L3, APT and TGF-β were detected by Elisa, and the histopathology was observed by HE staining. Network pharmacology and molecular docking were used to predict the possible signaling pathway of anti-HCC effect of britannin. The surface plasmon resonance (SPR) experiment was used to verify the interaction between britannin and proteins. The cell kinase activity function experiment was employed to detect the effect of britannin on enzyme activity. RT-qPCR and Western-Blot were used to verify the effect of britannin on the mRNA expressions of key genes and protein levels related to GSK-3β/β-catenin pathway in HCC cells and tumor tissues in mice. In vitro experiments showed that britannin could inhibit the activity, proliferation, migration and invasion abilities of HCC cells, while promoting their apoptosis. In vivo experiments revealed that britannin exerted inhibitory effects on the growth of transplanted liver cancer tumors, reducing the inflammatory infiltration and the expression levels of AFP, AFP-L3, APT and TGF-β of liver cancer markers in transplanted mice. Network pharmacology and molecular docking predicted that cell adhesion factors and GSK-3β/β-catenin pathway might be the related signaling pathway and had potential docking activity with key proteins. The SPR experiments elucidated the molecular interaction between britannin and GSK-3β. Enzyme activity assays indicated that britannin could modulate the functional activity of GSK-3β kinase. RT-qPCR suggested britannin could regulate the mRNA expressions of β-catenin, GSK-3β, E-cadherin and NCadherin. Western-Blot further verified that britannin could significantly up-regulate the expression of GSK-3β and down-regulate the expression of p-GSK-3β and β-catenin. At the same time, the expression of E-cadherin increased and NCadherin decreased, thereby reducing the occurrence of EMT and inhibiting the metastasis of HCC. In conclusion, britannin could inhibit the growth, development and metastasis of HCC, and its mechanism may be related to the regulation of GSK-3β/β-catenin signaling pathway to inhibit epithelial-mesenchymal transition of HCC.

Lu Q ，Zhu J ，Teng L ，Chen C ，Bi L ，Chen W ... -  《-》