Fig 1.
Chemical structure of rubiarbonol B (Ru-B) and 3-O-acetylrubiarbonol B (ARu-B).
Table 1.
IC50 values of ARu-B and Ru-B in inhibiting the growth of NSCLC and HEKa cells (48 h, µM).
Fig 2.
Growth inhibition of NSCLC cells by Ru-B and ARu-B.
(A) Viability of NSCLC cells (HCC827 and HCC827GR) treated for 24 (black) and 48 h (white) with Ru-B (2, 4, 6, and 8 µM), ARu-B (2, 4, and 6 µM), gefitinib (GEF, 1 µM), and savolitinib (SAV, 2 nM) as monitored by the MTT assay. Data are shown as the mean ± SD (n = 3). (B) Cell viability of HEKa cells treated as in (A). (C) and (D) The soft agar assay was used to determine anchorage-independent colony growth in NSCLC cells (14 days of incubation). (C) Micrograph of the cells on day 14 and (D) colony number. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to vehicle only. Scale bar, 400 µm.
Fig 3.
Inhibitory effect of Ru-B and ARu-B treatment on protein kinases.
(A) In vitro ADP-Glo kinase activity assay for EGFR, MET, AKT1, and AKT2 to determine the inhibitory effects of Ru-B, ARu-B, gefitinib (GEF), savolitinib (SAV), or capivasertib (AZD5363). Data are shown as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control group. (B) Molecular modeling of ARu-B binding with EGFR and MET protein kinases. Bird’s eye view: Protein kinases are shown as surface and cartoon with ARu-B in spheres. Zoomed in: ARu-B (pink) and surrounding amino acids (purple).
Fig 4.
Inhibitory effects of Ru-B and ARu-B treatment on EGFR, MET, and AKT signaling.
(A) HCC827 and HCC827GR non-small cell lung cancer (NSCLC) cells were treated with Ru-B (6 µM) or ARu-B (4 and 6 µM) for 48 h before western blotting to detect p-EGFR (Y1068), EGFR, p-MET (Y1234/1235), MET, p-AKT (S473), and AKT. β-actin was used as the loading control. (B)-(D) Histograms of protein expression analysis. Data are shown as the mean ± SD (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control group.
Fig 5.
HCC827 and HCC827GR non-small cell lung cancer (NSCLC) cells were treated with ARu-B (0, 2, 4, and 6 µM) for 48 h before flow cytometric analysis of propidium iodide (PI) staining. (A) Flow cytometry plots. (B) Proportion of sub-G1 cells. (C, D) Cell cycle distribution. (E) Western blots of cyclin D1, CDK4, CDK6, and p27 proteins. β-actin was used as the loading control. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control group.
Fig 6.
Induction of excessive ROS generation by ARu-B treatment.
HCC827 and HCC827GR non-small cell lung cancer (NSCLC) cells were treated with ARu-B (0, 2, 4, and 6 µM) for 48 h before flow cytometric analysis using a Muse™ Oxidative Stress Kit. (A) Flow cytometry plots (left) showing ROS levels detected using Muse™ Oxidative Stress Kit, which identifies ROS-positive cells based on DCF fluorescence intensity. M1 represents ROS-negative, and M2 indicates ROS-positive cell populations. Quantification (right) represents the percentage of M2-gated ROS-positive cells. (B, C) NSCLC cells pretreated with N-acetyl-L-cysteine (NAC, 4 mM) or vehicle were incubated with ARu-B for 48 h. (B) Cell viability was measured using the MTT assay. (C) Western blot analysis of phosphoproteins and proteins: p-EGFR, p-MET, p-AKT and poly (ADP-ribose) polymerase (PARP). β-actin was used as the loading control. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the control group. ###p < 0.001 compared with ARu-B treatment.
Fig 7.
ARu-B-induced apoptosis mediated by mitochondrial membrane depolarization.
HCC827 and HCC827GR NSCLC cells were treated with ARu-B (0, 2, 4, and 6 µM) for 48 h before flow cytometric analysis with annexin V/7-AAD double staining and Mitopotential/7-AAD double staining assay. (A) Annexin V/7-AAD double staining assay. Early and late apoptotic cells were distinguished using Annexin V-FITC/7-AAD staining; early apoptotic cells are Annexin V⁺/7-AAD⁻, and late apoptotic cells are Annexin V⁺/7-AAD⁺. (B) Proportion of apoptotic cells. (C) Mitopotential/7-AAD double staining assay. Left side: cells with depolarized mitochondrial membrane. (D) Proportion of depolarized cells. *p < 0.05,**p < 0.01, and ***p < 0.001 compared with the control group.
Fig 8.
ARu-B-induced apoptosis mediated by caspase activation.
HCC827 and HCC827GR NSCLC cells were treated with ARu-B (0, 2, 4, and 6 µM) for 48 h before flow cytometric analysis using a Muse™ Multi-Caspase Kit. (A) Multi-Caspase assay. (B) Proportion of cells with caspase activation. (C) Western blot analysis of Bcl-2, Mcl-1, Bad, Bax, caspase 3, and PARP. β-actin was used as the control. (D) NSCLC cells pretreated with Z-VAD-FMK (12 µM) or vehicle for 3 h were incubated with ARu-B for 48 h. Cell viability was measured using the MTT assay. *p < 0.05 and ***p < 0.001 compared with the control group. ###p < 0.001 compared with the ARu-B treatment group.
Fig 9.
Proposed mechanism of apoptosis induced by ARu-B and Ru-B.
ARu-B and Ru-B, isolated from Rubia philippinensis, induce apoptosis in non-small cell lung cancer (NSCLC) cells by targeting EGFR, MET, and AKT signaling pathways.