Page 61 - I. Chemical biology and drug discovery

DD induced apoptosis in human non-small cell lung cancer cells

with different EGFR mutation status

Hyun-Ji Park and Shin-Hyung Park *
1
1

1 Department of Pathology, College of Korean Medicine, Dong-eui University, Busan, 614-052, Korea.

DD is a marine brown alga collected from Jeju island. The aim of the current

study is to examine the anticancer effects of DD in human non-small cell lung

cancer (NSCLC) cells. We used three kinds of NSCLC cell lines including H1299,

an epidermal growth factor receptor (EGFR)-wildtype, PC9, an EGFR-mutant

(delE746-A750) and EGFR tyrosine kinase inhibitor (TKI)-sensitive, and H1975,

an EGFR-mutant (L858R/T790M) and EGFR TKI-resistant cell lines. The ethanol

extract of DD (EDD) decreased the cell viability in these cell lines at 25-100

µg/ml. In addition, EDD stimulated chromatin condensation and increased the

cleavages of PARP and caspase-3. The enhanced proportion of sub-G1 phase

cells and the annexin V-positive cells following EDD treatment collectively

demonstrated that EDD triggered apoptosis in NSCLC cells regardless of EGFR - A type of programmed cell death.

mutation status. In addition, phosphorylation of signal transducer and activator - Used during early development to
eliminate unwanted cells.
of transcription 3 (STAT3), Src, AKT and ERK, key regulators of cell growth, - A marine brown alga found mainly in Japan, China, Korea, and some - Used to rid the body of cells

was rapidly decreased by EDD. Taken together, we demonstrate that EDD Atlantic islands. that have been damaged

exhibits anticancer effects in NSCLC cells. We propose that DD could be a - DD has been widely used as food and medicine. beyond repair.

novel candidate for managing EGFR-TKIs resistant as well as EGFR-TKI naive - Plays a role in preventing

NSCLC patients (NRF-2019R1F1A1059588). - DD extracts have been indicated to exhibit anti-inflammatory, cancer. If apoptosis is

Key words: Apoptosis, DD, non-small cell lung cancer, EGFR mutation antioxidant, antibacterial, neuroprotective , and antitumor properties. prevented, it can lead to

- Its anticancer effects in NSCLC cells have not been explored yet. uncontrolled cell division

and the development of a tumor.

https://www.genome.gov/genetics-glossary/apoptosis

Figure 1. (A) Various human

NSCLC cell lines were

treated with EDD for different

incubation times. Cell viability

was evaluated by MTT assay.

Data are expressed as the

mean ± S.D. of three

independent experiments.

NSCLC, non-small cell lung

cancer; EDD, ethanol extract

of DD.

Figure 3. H1299, PC9 and H1975 human NSCLC cells were treated with the

indicated concentrations of EDD for 48 h. The expression levels of cleaved PARP

and cleaved caspase-3 were evaluated by western blot analysis. Actin was used

as a loading control. Representative blot images were shown. NSCLC, non-small

cell lung cancer; EDD, ethanol extract of DD; Cl-PARP, cleaved PARP; Cl-Cas3,

cleaved caspase-3. Figure 2. H1299, PC9 and H1975 cells were treated with EDD for 48 h.
Nuclei were then stained with DAPI solution. Stained nuclei were

observed under a fluorescence microscope (x200 magnification). NSCLC,

non-small cell lung cancer; EDD, ethanol extract of DD.

Figure 4. Human NSCLC cells were treated with the indicated concentrations of EDD for 48 h. (A and B) Cells were stained with propidium iodide solution. The

relative DNA content in each phase of the cell cycle was determined using a flow cytometer. (A) Representative flow cytometry plots of 3 independent Figure 5. H1299, PC9 and H1975 cells were treated with

experiments are shown. (B) The relative DNA content in sub-G1 phase was evaluated by CellQuest Pro software (version 5.1). (C and D) Cells were EDD (100 μg/ml) for indicated time periods. The

double-stained with Annexin V-FITC and PI and analyzed using a flow cytometer. (C) Representative flow cytometry plots of 3 independent experiments are expressions of the indicated proteins were evaluated by

shown. (D) Annexin V-positive cells were identified as apoptotic cells. The data are expressed as the means ± SD of 3 independent experiments. * P < 0.05, *** western blot analysis. Actin was used as a loading control.

P < 0.001 vs. untreated controls. NSCLC, non-small cell lung cancer; EDD, ethanol extract of DD. EDD, ethanol extract of DD.

1. EDD decreased the cell viability of H1299, PC9 and H1975 human NSCLC cells with different EGFR mutation status.

2. EDD induced apoptotic cell death in human NSCLC cells regardless of the EGFR mutation status.

3. EDD suppressed the phosphorylation of STAT3, Src, ERK and AKT, key regulators of cell growth, in human NSCLC cells regardless of the EGFR mutation status.

4. We suggest that EDD could be a novel candidate for managing EGFR-TKIs resistant as well as EGFR-TKI naive NSCLC patients