Euphorbia factor L2 induces apoptosis in A549 cells through the mitochondrial pathway

Abstract

Euphorbia factor L2, a lathyrane diterpenoid isolated from caper euphorbia seed (the seeds of Euphorbia lathyris L.), has been traditionally applied to treat cancer. This article focuses on the cytotoxic activity of Euphorbia factor L2 against lung carcinoma A549 cells and the mechanism by which apoptosis is induced. We analyzed the cytotoxicity and related mechanism of Euphorbia factor L2 with an MTT assay, an annexin V-FITC/PI test, a colorimetric assay, and immunoblotting. Euphorbia factor L2 showed potent cytotoxicity to A549 cells. Euphorbia factor L2 led to an increase in reactive oxygen species (ROS) generation, a loss of mitochondrial electrochemical potential, release of cytochrome c, activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase, suggesting that Euphorbia factor L2 induced apoptosis through a mitochondrial pathway. The cytotoxic activity of Euphorbia factor L2 in A549 cells and the related mechanisms of apoptotic induction provide support for the further investigation of caper euphorbia seeds.

Keywords: Anticancer agent; Apoptosis; Caper euphorbia seed; Euphorbia Factor L2; Euphorbia lathyris L.; Mitochondrial pathway.

Graphical abstract

Figure 1

Structure of EFL2 and its cytotoxicity to A549 cells. (A) Chemical structure of Euphorbia factor L2 (EFL2); (B) EFL2 inhibited viability of A549 cells with log scale of concentration.

Figure 2

EFL2-induced cell apoptosis in A549 cells. (A) A549 cells were treated with 0, 40 and 80 μmol/L of EFL2 for 48 h, apoptosis rates were then detected by annexin V-FITC/PI double staining and flow cytometer. D4 quadrant represented cells stained mainly by annexin-V (early apoptotic cells) and D2 quadrant represented cells stained by both PI and annexin-V (late apoptotic). D1 quadrant represented cells stained mainly by PI and viable cells negative for both annexin-V and PI appeared in the D3 quadrant. (B) The apoptosis rate was showed in the bar graph.

Figure 3

EFL2 increased ROS levels in A549 cells. (A) ROS generation was increased in A549 cells; (B) ROS levels in A549 cells were calculated as percentage of control.

Figure 4

EFL2 decreased ΔΨ_m in A549 cells. (A) The decrease of ΔΨ_m level was observed in A549 cells; (B) ΔΨ_m level in A549 cells was calculated as percentage of control.

Figure 5

EFL2 activates the expression of cytochrome c in A549 cells. (A) Release of cytochrome c was discovered in A549 cells; (B) activation of caspase-9 and caspase-3 were detected in A549 cells.

Figure 6

EFL2 activates the expression of caspase-9 and caspase-3, and the cleavage of PARP in A549 cells in a time-dependent manner. (A) Activation of caspase-9 and caspase-3, and the cleavage of PARP were detected in A549 cells; (B) densitometric analysis of Western blot results of (A). The molecular weight of activated (cleaved) caspase-9, caspase-3 and cleaved PARP were 37, 17 and 89 kD, respectively. GAPDH (36 kD) was used to confirm equal protein loading. Results were expressed as mean±SD of at least three determinations.