Antitumor effect of kurarinone and underlying mechanism in small cell lung carcinoma cells

Abstract

Background: Kurarinone, a prenylated flavonone isolated from the roots of Sophora flavescens, is known to be cytotoxic against many human cancer cells but not human small cell lung carcinoma (SCLC) yet. Also, the exact molecular mechanism of kurarinone for induction cytotoxicity remains unknown.

Material and methods: We investigated the effects of kurarinone on cell proliferation, apoptosis, and migration in H1688 SCLC cells. Cell viability was determined by the MTT assay. Apoptotic indices such as cell cycle, mitochondrial membrane potential, cytochrome c release, caspase activity, and death receptors were evaluated by flow cytometry. Transwell migration and invasion assays were also included.

Results: Our results indicated that kurarinone significantly decreased H1688 cell viability and induced the accumulation of sub-G1 fractions by activating caspase-3, -9, and PARP cleavage accompanied by the elevated release of cytochrome c and mitochondrial dysfunction in H1688 cells. Additionally, kurarinone promoted Fas and TRAIL receptor-1 and -2 expression via the caspase-8/Bid pathway, suggesting that kurarinone triggered apoptosis via the mitochondria-mediated and receptor-mediated apoptotic pathways. We also observed that kurarinone repressed migration and invasion capabilities of SCLC cells by suppressing the expression of epithelial-mesenchymal transition-related proteins and matrix metalloproteinases.

Conclusion: Our findings provided evidence that kurarinone can induce apoptosis in SCLC cells via multiple mechanisms and delayed the cell migration and invasion of SCLC cells.

Keywords: apoptosis; caspase; invasiveness; kurarinone; migration; small cell lung carcinoma.

Figure 1

(A) Cell viability of kurarinone-treated H1688, H146, and BEAS-2B cells. Cells were treated with 0.1% DMSO (0 μM of kurarinone), or kurarinone at 3.125, 6.25, 12.5, 25, or 50 μM for 24 h. Cell viability was examined by MTT assay. *P<0.05, ***P<0.001 versus 0.1% DMSO group. (B) Colony formation assay of H1688 cells following treatment with kurarinone for one week. Data are presented as the mean ± SD in 3 replicates for each treatment. Different letters (a,b,c) indicate statistically significant differences between groups (one way ANOVA followed by Tukey test, p<0.05).

Figure 2

Apoptosis induced by kurarinone on H1688 cells. Cells were treated with or without kurarinone at indicated concentrations for 24 h. (A) Expression levels of cleaved poly (ADP-ribose) polymerase (PARP) were investigated by Western blotting using GAPDH as a loading control. (B) Cell apoptosis was determined via flow cytometry using the Annexin V-FITC apoptosis detection kit and PI. The percentage of necrotic (Annexin V⁻/PI⁺), early apoptotic (Annexin V⁺/PI⁻), and late apoptotic (Annexin V⁺/PI⁺) (mean ± SD) from triplicate samples of each treatment were plotted in the bar graphs. (C) The cell cycle was investigated using flow cytometry analysis via PI staining. The percentages of sub-G1 cells (mean ± SD) were plotted based on triplicated samples. Different letters (a,b,c,d) indicate statistically significant differences between groups (one way ANOVA followed by Tukey test, p<0.05).

Figure 3

Changes on the mitochondrial membrane permeability (Δψm) and intrinsic mitochondrial apoptotic pathway in H1688 cells after kurarinone treatments. (A) Cells stained with the JC-1 probe for analyzing Δψm by flow cytometry. (B) Cytochrome c release was quantified with FITC-conjugated antibody via flow cytometry analysis. The activities of (C) caspase-3 and (D) caspase-9 were measured via flow cytometry. (E) Expression levels of cleaved caspase-3, Bcl-2, Bcl-XL, and Bax were determined by Western blot analysis along with loading control GAPDH Data were presented as mean ± SD from triplicate samples for each treatment and plotted as the bar graphs. Different letters (a,b,c) indicate statistically significant differences between groups (one way ANOVA followed by Tukey test, p<0.05).

Figure 4

Activation of caspase-8/tBid pathway by kurarinone in H1688 cells. (A) caspase-8 activity measured by flow cytometry in the presence or absence of kurarinone. (B) Protein expression levels of cleaved caspase-8 (43/41 kDa and 18 kDa) and truncated Bid 50 μM of caspase-8 inhibitor Z-IETD-FMK was applied 1 h before 25 μM of kurarinone treatment for next 24 h and followed by measuring (C) cell viability of H1688 cells, (D) cell apoptosis, (E) protein expression levels of cleaved Bid, (F) mitochondrial membrane potential, and activities of (G) caspase-8, and (H) caspase-9. Bar graphs present the mean ± SD of 3 replicates for each treatment. Different letters (a,b,c) indicate statistically significant differences between groups (one way ANOVA followed by Tukey test, p<0.05).

Figure 5

Death receptors were up-regulated in H1688 cells after kurarinone treatment. Flow cytometric analysis of (A) TRAIL, DR4, and DR5, (B) FAS and FASL expression after 24-h exposure to kurarinone. Bar graphs present the mean ± SD of 3 replicates for each treatment. Different letters (a,b,c) indicate statistically significant differences between groups (one way ANOVA followed by Tukey test, p<0.05).

Figure 6

Cell migration and invasion of H1688 cells after 24-h treatment with kurarinone (3.125, 6.25, or 12.5 μM). Cell migratory capacity was detected by (A) wound scratch assays as described in Materials and Methods, presenting representative images at 100× magnification and (B) transwell assays. (C) Cell invasiveness of H1688 cells was also evaluated by transwell assays. The wound area (mean ± SEM) is shown from 3 independent experiments and bar graphs represents the mean ± SD of 3 replicates for each treatment. Different letters (a,b,c) indicate statistically significant differences between groups (one way ANOVA followed by Tukey test, p<0.05).

Figure 7

EMT-related proteins and MMPs in H1688 cells were modulated by kurarinone. Cells were treated with 0.1% DMSO or kurarinone (3.125, 6.25, or 12.5 μM) for 24 h followed by examining expression levels of (A) vimentin, N-cadherin, E-cadherin, and (B) MMP2, MMP-3, and MMP-9 via Western blotting along with GAPDH as a loading control.

Figure 8

Schematic overview of cancer inhibitory pathways initiated by kurarinone. The cleaved form of a protein is labeled with a lower case c ahead of the protein name and a t indicates a truncated form. Red arrows indicate up-regulation or increase whereas green arrows signify down-regulation or decrease.