ANTITUMOR AND APOPTOTIC EFFECTS OF CUCURBITACIN A IN A-549 LUNG CARCINOMA CELLS IS MEDIATED VIA G2/M CELL CYCLE ARREST AND M-TOR/PI3K/AKT SIGNALLING PATHWAY

Abstract

Background: The main aim of this study was to demonstrate the antitumor potential of cucurbitacin A on A-549 NSCLC (non-small cell lung cancer cells). The effects of Cucurbitacin A on apoptotic induction, cell physic, cell cycle failure and m-TOR/PI3K/Akt signalling pathway were also investigated in the present study.

Materials and methods: MTT assay and clonogenic assay were carried out to study effects of this compound on cell cytotoxicity and colony forming tendency in A-549 cells. Moreover, phase and fluorescence microscopic techniques were used to examine the effects on cell morphology and induction of apoptosis. The effects on cell cycle phase distribution were investigated by flow cytometry and effects on m-TOR/PI3K/Akt signalling proteins were assessed by western blot analysis.

Results: Results showed that cucurbitacin A induced dose-dependent cytotoxic effects along with suppressing the colony forming tendency in these cells. Cucurbitacin A also induced morphological changes in these cells featuring chromatin condensation, cell shrinkage and apoptotic body formation. G2/M phase cell cycle collapse was also induced by Cucurbitacin A along with inhibition of expression levels of m-TOR/PI3K/Akt proteins.

Conclusions: In conclusion, cucurbitacin A inhibits cancer growth in A-549 NSCLC cells by inducing apoptosis, targeting m-TOR/PI3K/Akt signalling pathway and G2/M cell cycle.

Keywords: Non-small cell lung cancer; antitumor activity; apoptosis; cell cycle; cucurbitacin A.

Figure 1

Cucurbitacin A: Chemical structure.

Figure 2

Evaluation of cell cytotoxicity induced by cucurbitacin B in A-549 human lung cancer cells by MTT assay. The cells were processed with 0, 10, 20, 40, 100, 150 and 200 μM dose of the drug and then incubated for 24 and 48 time intervals. The data of all the three independent experiments are shown as the mean ± SD. *, P < 0.05, **, P < 0.01, vs 0 μM (control).

Figure 3

Cucurbitacin A decreases the potential of colony formation of A-549 human lung cancer cells. These cells were treated with 0 (A), 40 (B), 100 (C) and 200 (D) μM dose of cucurbitacin A for 48 h and then analyzed using a light microscope.

Figure 4

Effects of cucurbitacin A on the cellular morphology of A-549 human lung cancer cells (NSCLC). The cells were treated with 0 (A), 40 (B), 100 (C) and 200 (D)μM (unreacted control cells) dose of cucurbitacin A for 48 h and then stained with hoechst 33258. Fluorescence microscopy was then used to visualize the changes in cellular and nuclear morphology and apoptotic body formation. Arrows indicate apoptotic cells.

Figure 5

Impact of cucurbitacin A on the cell physic or morphology of A-549 NSCLC cells. Physical (Morphological) changes were examined under phase-contrast microscope after the cells were treated with 0 (A), 40 (B), 100 (C) and 200 (D) μM of cucurbitacin A. Arrows indicate changes in cell morphology.

Figure 6

Cucurbitacin A induces G2/M cell cycle arrest in A-549 human non-small cell lung cancer cells. The cells were reacted with 0, 40, 100 and 200 μM dose of cucurbitacin A and then analyzed by flow cytometry. There was a significant increase in the number of G2/M phase cells as the dose of cucurbitacin A increased.

Figure 7

Effect of cucurbitacin A on the m-TOR, PI3K/ Akt pathways in human A-549 NSCLC cells (non-small cell lung). Western blot analysis was used evaluate the expressions of these proteins. GAPDH- served as a control.