Page 63 - I. Chemical biology and drug discovery

Anti-cancer effects of the root of TK in human non-small cell

lung cancer cells with resistance to epidermal growth factor

receptor-tyrosine kinase inhibitors

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

1 Department of Pathology, College of Korean Medicine, Dong-eui University, Busan, 47227, Korea.

Although epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) has - In up to 60%–80% of

become the standard treatment for non-small cell lung cancer (NSCLC), acquired patients treated with

resistance to these drugs remains a major obstacle for managing NSCLC. This study an EGFR TKI, there is

explored the anti-cancer effects of the root of TK in NSCLC cells with EGFR TKIs a meaningful tumor

resistance. We first established erlotinib-resistant PC9 (PC9/ER) and gefitinib-resistant regression.

PC9 (PC9/GR) cell lines using a dose-escalating maintenance of drugs in PC9 cells. - After 9–12 months, all

We also used H1975 cell line (L858R/T790M) as the EGFR-TKI resistant cell line. Our patients develop

results showed that ethanol extract of TK (ETK) suppressed the cell proliferation in acquired resistance.

EGFR-TKI resistant cell lines. In addition, ETK enhanced the sub-G1 DNA content,

annexin V-positive cell population as well as chromatin condensation. The expression - Among the different

of cleaved PARP and cleaved caspase-3 was also increased by ETK. These findings mechanisms of

demonstrate that ETK induced apoptosis in EGFR TKI-resistant NSCLC cell lines. - Widely used as a medicinal plant in Oriental medicine acquired resistance, a

Furthermore, the activities of general activators of cancer cell proliferation were for the treatment of lung diseases accompanied by secondary mutation,
T790M, in the exon 20
significantly decreased by ETK in H1975 cells. In conclusion, the current study dry cough, fever and thirst. of the EGFR gene is

suggests the potential use of TK for managing the advanced NSCLC with EGFR-TKIs - It is also applied to injury, abscess, and hemorrhoids. the most frequent

resistance (NRF-2019R1F1A1059588). - Have anticancer, antibacterial, antiviral, antidiabetic Appl Clin Genet. 2017 Jul 26;10:49-56. event, occurring in

and immunomodulating activities.
Key words: Apoptosis, TK, EGFR-TKI resistance, NSCLC ~50%–60% of cases.

Figure 1. EGFR TKI resistant cell lines were treated with ETK for 72 h. The cell viability was measured by MTT assay. Figure 2. EGFR TKI resistant cell lines were treated with ETK for the indicated time periods. At each time point, the live cells were counted by

The data are expressed as the mean ± S.D. of three independent experiments. ** P < 0.01, *** P < 0.001 vs. untreated trypan blue exclusion assay. The data are expressed as the mean ± S.D. of three independent experiments. ** P < 0.01, *** P < 0.001 vs.

controls. untreated controls

Figure 4. EGFR TKI resistant cell lines were treated with the indicated concentration of ETK for 72 h. The

cells were double-stained with Annexin V-FITC and PI and analyzed using a flow cytometer. (A) The Figure 7. H1975 cells were treated with ETK (100

representative plots are presented. (B) Annexin V-positive apoptotic cells were evaluated by BD μg/ml) for the indicated time periods. The expression

CellQuest Pro software (version 5.1). The data are expressed as the mean ± S.D. of three independent of the indicated proteins was measured by western

experiments. *** P < 0.001 vs. untreated controls. blot analysis. Actin was used as a loading control.

Figure 3. EGFR TKI resistant cell lines were treated with the indicated concentration of ETK for 72 h.

The cells were stained with PI solution, and the cell cycle was measured using a flow cytometer. (A)

The representative plots are presented. (B) The sub-G1 DNA content was evaluated by BD CellQuest

Pro software (version 5.1). The data are expressed as the mean ± S.D. of three independent

experiments. *** P < 0.001 vs. untreated controls. Figure 6. EGFR TKI resistant cell lines were treated with the indicated

concentrations of ETK for 72 h. The expression of cleaved PARP was

detected by western blot analysis. Actin was used as an internal control.

Figure 5. EGFR TKI resistant cell

lines were treated with the indicated

concentrations of ETK for 72 h. To

observe the changes of nuclear

morphology, cells were stained with

DAPI solution. The stained nuclei

were observed under fluorescent

microscope (×200). The apoptotic 1. ETK exhibited a concentration-dependent decrease in cell viability and cell proliferation in EGFR TKI

nuclei were indicated as white arrow.
resistant cell lines.

2. ETK triggered apoptosis in EGFR TKI resistant NSCLC cell lines.

3. ETK suppressed the phosphorylation of general activators of cancer cell proliferation.

4. We suggest a potential use of ETK as a second-line therapy for the advanced NSCLC patients with the

acquired resistance to EGFR TKIs.