The Effects Of ABL-N On Proliferation And Apoptosis Of Lewis Lung Cancer Cell And Investigation Of The Mechanisms

Lung cancer is the most common cancer diagnosed and the leading cause of death from cancer in males in 2008 globally. It is the fourth most common cancer diagnosed and the second leading cause of cancer death among females. Lung cancer accounts for 13% (1.6 million) of the diagnosis and 18% (1.4 million) of the deaths in 2008. Lung cancer serious threatens the human health. Although with the development of the surgery and chemical therapy techniques, the survival rates in patients with lung cancer have not been improved. Chinese Traditional and Herbal Drugs have been paid considerable attention for the prevention and treatment of certain types of cancer in clinical studies.Several phytochemicals have been used in clinical cancer chemotherapy, such as paclitaxel, camptothecin and vinca alkaloids. Acetylbritannilactone (ABL) is a sesquiterpene lactone, is abundant in Inula britannica L, and has anticancer activity. ABL-N used in the present study is a derivative of ABL, 5-(5-(ethylperoxy)pentan-2-yl)-6-methyl-3-methylene-2-oxo-2, 3, 3a, 4, 7, 7a -hexahydrobenzofuran-4-yl2-(6-methoxynaphthalen-2-yl) propanoate.Objective: To study the inhibitory effects of ABL-N on proliferation and apoptosis of Lewis lung cancer cells and the mechanisms.Methods:1 Lewis lung cancer cells were cultured and treated with different concentrations of ABL-N for 12, 24 and 48 hours respectively. The growth-inhibitory rate and IC₅₀ were measured by MTT assay.2 The morphological features of the apoptosis of Lewis lung cancer cells induced by ABL-N.(1) Inverted microscope was used to observe growth of cells in control group and the group treated with ABL-N 10μg/mL for 48 hours. (2) Hoechst 33258 staining: Fluorescence microscope was used to observe the change of cell nuclei of the control group and the group treated with ABL-N 10μg/mL for 48 hours.3 Gel electrophoresis of DNA fragment: Lewis lung cancer cells were cultured and treated with different concentrations of ABL-N for 24, 48 and 72 hours, and then the cells were collected and the DNA was abstracted for gel electrophorosis.4 Analysis of apoptosis rate and cell cycle of Lewis lung cancer cells treated with different concentrations of ABL-N through flow cytometry: Cells treated with different concentrations of ABL-N for 48 hours were collected, and the apoptosis rate and cell cycle distribution were examined through flow cytometry after PI staining.5 Western blot was used to detect the expression of Bax, Bcl-2 and p53 proteins after Lewis lung cancer cells were treated by different concentrations of ABL-N for 48 hours.Results:1 Lewis lung cancer cells were cultured and treated with ABL-N 2.5, 5, 10 and 20μg/mL. The inhibitory rate was 7.6%, 8.9%, 16.5% and 73.4%, and the IC₅₀ was 14.0μg/mL after cells were treated for 12 hours. The inhibitory rate was 17.7%, 25.6%, 60.3% and 89.2%, and the IC₅₀ was 7.3μg/mL after cells were treated for 24 hours. The inhibitory rate was 15.3%, 29.9%, 89.9% and 94.2%, and the IC₅₀ was 5.7μg/mL after cells were treated for 48 hours.2 Inverted microscope was used to observe the morphological features of cells treated with ABL-N 10μg/mL for 48 hours. The cell volume decreased, and the cell gaps were enlarged.Fluorescence microscope was used to observe the change of cell nuclei of the cells treated with ABL-N 10μg/mL for 48 hours. Chromatin aggregation, condensation or fracture, and apoptotic body formation were seen in the cells treated with ABL-N.3 Gel electrophoresis analysis of DNA fragment: ABL-N induced DNA fragmentation in a time- and dose-dependent manner. DNA ladder appeared in groups treated with different concentrations(5,10,20μg/mL) of ABL-N for 48 h and 72 h. The DNA ladder is a typical characteristic of apoptosis biochemical.4 Analysis of apoptosis rate and cell cycle of Lewis lung cancer cells treated with ABL-N 10 and 20μg/mL for 48 hours through flow cytometry: The apoptosis rate was 21.2% and 50.9% in ABL-N-treated groups. The cell cycle arrested at G₁ phase and led to an accumulation of sub-G₁ population.5 Western blot results showed that ABL-N down-regulated the expression of Bcl-2 protein and up-regulated the expression of Bax and p53 proteins.Conclusion:1 ABL-N could inhibit the growth of Lewis lung cancer cells and promote apoptosis in a time- and dose-dependent manner. The cell cycle arrested at G₁ phase.2 The effect of ABL-N on apoptosis could relate to down- regulating the expression of Bcl-2 protein and up-regulating the expression of Bax and p53 proteins.