Journal: Frontiers in Pharmacology

Article Title: Nodosin Exerts an Anti-Colorectal Cancer Effect by Inhibiting Proliferation and Triggering Complex Cell Death in Vitro and in Vivo

doi: 10.3389/fphar.2022.943272

Figure Lengend Snippet: Nodosin suppressed CRC cells in dose- and time-dependent way in vitro . (A) The chemical structural formula of nodosin ( http://www.chemspider.com ). (B) CRC SW480 cells were treated with 0–16 μM nodosin for 48 h. The relative viability of cells was detected by CCK-8 kit. Each group was repeated three times. The significant difference between the nodosin treated and control group (without nodosin) was evaluated by the two-tailed t -test, * p < 0.05, **0.01 < p < 0.05, *** p < 0.01. (C) HT-29 cells were treated with 0–16 μM nodosin for 48 h. The cell viability was tested by CCK-8 kit. (D) LoVo cells were treated with 0–16 μM nodosin for 48 h. The cell viability was tested by CCK-8 kit. (E) NCM460 cells were treated with 0–16 μM nodosin for 48 h. The cell activity was tested by CCK-8 kit. (F) SW480 cells were treated with nodosin for 48 h, and the morphological changes of SW480 cells were recorded by fluorescence inverted microscope. (G) The repressive effect of nodosin on the migration of SW480 cells was tested by cell scratch method. SW480 cells were treated with different concentrations of nodosin for 24 h to measure the scratch width and calculate the cell scratch closure rate. The relative migration rate significant difference between the nodosin treated group and the control group (without nodosin) was evaluated by the two-tailed t -test, * p < 0.05, ** p < 0.01, *** p < 0.001. (H) Nodosin reduced the relative activity of SW480 cells in a dose- and time-dependent manner. The significant difference was evaluated with one-way ANOVA analysis followed by LSD Post Hocnultiple comparison, and p -value < 0.05 was considered significant. (I) Nodosin reduced the relative activity of HT-29 cells in a dose-dependent and time-dependent way. The significance difference was evaluated with one-way ANOVA analysis followed by LSD Post Hocnultiple comparison, and p -value < 0.05 was considered significant.

Article Snippet: The changes of ROS in nodosin-induced SW480 cells were detected with the cell-permeable ROS probe H 2 DCFDA (HY-D0940, MCE, United States).

Techniques: In Vitro, CCK-8 Assay, Control, Two Tailed Test, Activity Assay, Fluorescence, Inverted Microscopy, Migration, Comparison

Journal: Frontiers in Pharmacology

Article Title: Nodosin Exerts an Anti-Colorectal Cancer Effect by Inhibiting Proliferation and Triggering Complex Cell Death in Vitro and in Vivo

doi: 10.3389/fphar.2022.943272

Figure Lengend Snippet: Nodosin supressed cell proliferation and triggered cell death. (A) Nodosin-treated SW480 cells were stained with Dio and Hoechst 33342 (Dio stained membranes in green and Hoechst 33342 stained nucleus in blue). (B) Different concentrations of nodosin treated SW480 cells for 12 days. Crystal violet stained cell communities were observed with an inverted microscope. (C) Nodosin-treated SW480 cells were stained with Hoechest 33342, Calcein AM and PI. The stained results were recorded by fluorescence inverted microscope (Hoechest 33342 stained cells in blue, Calcein AM stained cells in green and PI stained cells in red).

Article Snippet: The changes of ROS in nodosin-induced SW480 cells were detected with the cell-permeable ROS probe H 2 DCFDA (HY-D0940, MCE, United States).

Techniques: Staining, Inverted Microscopy, Fluorescence

Journal: Frontiers in Pharmacology

Article Title: Nodosin Exerts an Anti-Colorectal Cancer Effect by Inhibiting Proliferation and Triggering Complex Cell Death in Vitro and in Vivo

doi: 10.3389/fphar.2022.943272

Figure Lengend Snippet: Nodosin repressed the proliferation of CRC cells by inhibiting DNA synthesis. (A) Nodosin-treated SW480 cells were stained with EdU and Hoechst 33342, and then cell proliferation was observed with fluorescence inverted microscope (EdU stained cells in red, Hoechst 33342 stained cells in blue). (B) Nodosin-treated SW480 cells were stained with PI. The cell cycle of staining cells was detected by flow cytometry. The blue area was G1 phase cells, the red area was S phase cells, and the green area was G2 phase cells. The significant difference was evaluated with one-way ANOVA analysis followed by LSD Post Hocmultiple comparison, and p -value < 0.05 was considered significant. (C) SW480 cells were treated with different concentrations of nodosin for 24 h. The Ki67 was detected by IF. F-actin was red fluoreacence, Ki67 was green fluorescence, and DAPI was blue fluorescence. The significance difference was evaluated with one-way ANOVA analysis followed by LSD Post Hocmultiple comparison, and p -value < 0.05 was considered significant.

Article Snippet: The changes of ROS in nodosin-induced SW480 cells were detected with the cell-permeable ROS probe H 2 DCFDA (HY-D0940, MCE, United States).

Techniques: DNA Synthesis, Staining, Fluorescence, Inverted Microscopy, Flow Cytometry, Comparison

Journal: Frontiers in Pharmacology

Article Title: Nodosin Exerts an Anti-Colorectal Cancer Effect by Inhibiting Proliferation and Triggering Complex Cell Death in Vitro and in Vivo

doi: 10.3389/fphar.2022.943272

Figure Lengend Snippet: Nodosin triggered cell death in CRC in a manner of oxidative stress, apoptosis and autophagy. (A) Intracellular ROS changes in SW480 caused by nodosin were determined by H 2 DCFDA, red indicated the blank control (no ROS probe), light green indicated the solvent control (DMSO treatment, including the ROS probe), purple indicated the negative control (8 μM nodosin treatment, but with no ROS probe available), orange indicated the 5 μM nodosin treated and containing the ROS probe, light blue indicated the 8 μM nodosin treated and containing the ROS probe, dark blue indicated the 12 μM nodosin treated and containing the ROS probe, brown indicated the 16 μM nodosin treated and containing the ROS probe, dark green indicated a positive control (treated with 1 mM H 2 O 2 ). (B) SW480 cells were pretreated with ROS inhibitor NAC for 1 h and treated with nodosin for 48 h. The relative viability of cells was detected by CCK-8 kit. (C) Flow cytometry was employed to determine the apoptosis rate. X axis represented FITC intensity of Annexin-V on the extracellular membrane, Y axis represented PI staining intensity of cells, early apoptotic cell populations were in Q1-LR, late apoptotic cell populations were in Q1-UR. (D) The rate of SW480 cells apoptosis. The significant difference was evaluated with one-way ANOVA analysis followed by LSD Post Hocmultiple comparison, and p -value < 0.05 was considered significant. (E) SW480 cells (transfected with lamp1 OFP, ER, and mito) were treated with different concentrations of nodosin. The intracellular changes in lysosomes, endoplasmic reticulum, and mitochondria were recorded by laser confocal microscopy. Orange represented lysosomes, blue represented mitochondria, and green representsed endoplasmic reticulum. (F) After SW480 cells were treated with nodosin for an appropriate period, different concentrations of chloroquine were added to rescue the nodosin-inactivated cells with the CCK-8 kit.

Article Snippet: The changes of ROS in nodosin-induced SW480 cells were detected with the cell-permeable ROS probe H 2 DCFDA (HY-D0940, MCE, United States).

Techniques: Control, Solvent, Negative Control, Positive Control, CCK-8 Assay, Flow Cytometry, Membrane, Staining, Comparison, Transfection, Confocal Microscopy