cell culture human nsclc cell lines  (ATCC)

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Silibinin is a suppressor of the metastasis-promoting transcription factor ID3.

doi: 10.1016/j.phymed.2024.155493

Figure Lengend Snippet: Fig. 3. Silibinin prevents constitutive, acquired, and inducible ID3 expression in NSCLC cells. A. Top panel. Basal expression levels of the ID3 protein were detected by immunoblotting in lysates from NSCLC cell lines using a specific anti-ID3 antibody. Shown is a representative immunoblot from multiple (n ≥3) independent experiments. Bottom panels. The intensity of the ID3 protein bands was measured using the ImageJ software and the fold-change relative to untreated cells was calculated using GAPDH as a loading control. ID3 transcript abundance was calculated using the ΔΔCt method and presented as relative expression. B. Left panels. Expression levels of ID3 were detected by immunoblotting in NSCLC cell models grown in the absence/presence of graded concentrations of silibinin for 24 or 48 h. The intensity of the ID3 protein bands was measured using the ImageJ software. The fold change of each protein relative to untreated samples was calculated using GAPDH as a loading control. The figure shows representative immunoblots from multiple (n ≥3) independent experiments. Right panels. ID3 transcript abundance of was calculated using the ΔΔCt method and presented as either relative or fold-change expression (compared to untreated H2228 parental cells) expression. *p < 0.05, ** p < 0.005, statistically significant differences. n.s. not significant. C. Top panel. Expression levels of P-SMAD1/5 and ID3 proteins were detected by immunoblotting in lysates from H2228 NSCLC cells exposed to the ALK-TKIs crizotinib, brigatinib, and lorlatinib (1 μM/l each) for 24 h in the absence or presence of either silibinin (100 μM/l) or K02288 (1 μM/l). Shown are representative immunoblots from multiple (n ≥3) independent experiments. Bottom panels. The intensity of the P- SMAD1/5 and ID3 protein bands was measured using the ImageJ software and the fold-change relative to untreated cells was calculated using GAPDH as a loading control. ID3 transcript abundance was calculated using the ΔΔCt method and presented as relative expression; *p < 0.05, ** p < 0.005, statistically significant differences. n.s. not significant.

Article Snippet: Cell lines and culture conditions Human NSCLC cell lines A549 (ATCC CCL-185), H460 (ATCC HTB177), H1993 (ATCC CRL-5909), and H1975 (ATCC CRL-5908), and HEK293T (ATCC CRL-3216) were obtained from the ATCC (Manassas, VA, USA).

Techniques: Expressing, Western Blot, Software, Control

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Silibinin is a suppressor of the metastasis-promoting transcription factor ID3.

doi: 10.1016/j.phymed.2024.155493

Figure Lengend Snippet: Fig. 7. Silibinin is a novel suppressor of the metastasis-promoting transcription factor ID3. Interfering with the regulatory actions of the metastasis-promoting transcription factor ID3 in primary lung cancer (e.g., metastasis-initiating and angiogenic/immunosuppressive capacity) and lung cancer brain metastasis (e.g., BBB cell crossing, brain vascular edema) may lead to additive or even synergistic anti-metastatic effects. Our research combined bioinformatic analyses, immu noblotting, qRT-PCR, luciferase reporter assays, computational modeling, and kinase assays to elucidate how the flavonolignan silibinin might modulate ID3 expression in endothelial and NSCLC cells. Results from NSCLC patient datasets indicate a strong correlation between ID3 expression and BMP9/ACVRL1/ALK1 and BMP6 levels, with silibinin effectively inhibiting the ALK1-phospho-SMAD1/5-ID3 axis in brain endothelial cells. Notably, silibinin disrupts ID3 expression by targeting BMP-responsive elements within the ID3 gene enhancers and demonstrates direct inhibition of BMPR kinase activity in vitro, with particular potency against ACVRL1/ALK1 and BMPR2. In vivo, oral silibinin was found to significantly reduce ID3 overexpression in NSCLC xenograft models. Collectively, these findings altogether suggest that silibinin may serve as a novel therapeutic to reduce the metastatic spread of NSCLC by suppressing ID3 in endothelial and tumor cells.

Article Snippet: Cell lines and culture conditions Human NSCLC cell lines A549 (ATCC CCL-185), H460 (ATCC HTB177), H1993 (ATCC CRL-5909), and H1975 (ATCC CRL-5908), and HEK293T (ATCC CRL-3216) were obtained from the ATCC (Manassas, VA, USA).

Techniques: Quantitative RT-PCR, Luciferase, Expressing, Inhibition, Activity Assay, In Vitro, In Vivo, Over Expression