The effects of Benjakul extract and its isolated compounds on cell cycle arrest and apoptosis in human non-small cell lung cancer cell line NCI-H226

Arunporn Itharat; Ruchilak Rattarom; Pintusorn Hansakul; Intouch Sakpakdeejaroen; Buncha Ooraikul; Neal M Davies

doi:10.4103/1735-5362.310520

The effects of Benjakul extract and its isolated compounds on cell cycle arrest and apoptosis in human non-small cell lung cancer cell line NCI-H226

Res Pharm Sci. 2021 Mar 5;16(2):129-140. doi: 10.4103/1735-5362.310520. eCollection 2021 Apr.

Authors

Arunporn Itharat^{1

2}, Ruchilak Rattarom³, Pintusorn Hansakul^{2

4}, Intouch Sakpakdeejaroen^{1

2}, Buncha Ooraikul⁵, Neal M Davies⁶

Affiliations

¹ Department of Applied Thai Traditional Medicine, Faculty of Medicine, Thammasat University, Klong Luang, Pathumthani 12120, Thailand.
² Center of Excellence in Applied Thai Traditional Medicine Research (CEATMR), Thammasat University, Klong Luang, Pathumthani 12120, Thailand.
³ Faculty of Pharmacy, Mahasarakham University, Kantarawichai, Maha Sarakham 44150, Thailand.
⁴ Department of Preclinical Science, Faculty of Medicine, Thammasat University, Klong Luang, Pathumthani 12120, Thailand.
⁵ Department of Agricultural Food and Nutritional Science, Faculty of Agricultural Life and Environmental Sciences, University of Alberta, Edmonton, Alberta T6G 2P5, Canada.
⁶ Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada.

Abstract

Background and purpose: Benjakul, a traditional Thai formulation for cancer treatment, is composed of five plants. This study aimed to assess the cytotoxicity of Benjakul, its five plants, and its isolated compounds against non-small cell lung cancer (NSCLC) by the sulforhodamine B (SRB) assay.

Experimental approach: Analyses of cell cycle and membrane asymmetry changes were performed with different fluorescent dyes and analyzed by flow cytometry in NCI-H226 cells. Activation of caspase-3 was measured using a caspase-3 colorimetric assay kit. The pan-caspase inhibitor Z-VAD-FMK was used in analyses of cell cycle and caspase-3 activity.

Findings/results: Benjakul exhibited cytotoxicity against NSCLC with IC50 between 5.56-5.64 μg/mL. Among its five ingredients, Benjakul displayed the highest selectivity with selectivity index values ranging from 2.93 to 6.88, with the exception of Plumbago indica, indicating its protective effects. Plumbagin and 6- shogaol displayed the highest cytotoxicity and underwent molecular studies in NCI-H226 cells. Flow cytometry analysis revealed that Benjakul and 6-shogaol dose-dependently induced G2/M phase arrest, and plumbagin dose-dependently induced S-G2/M phase arrest with the highest percentage in early incubation time (12-24 h). At the highest doses, Benjakul extract, 6-shogaol, and plumbagin time-dependently increased the population of sub-G1 apoptotic cells with the highest percentage in longer incubation time (60-72 h). Similarly, membrane asymmetry changes showed time-dependent increases in the percentage of early and late apoptotic cells. Moreover, the apoptosis-inducing effect of Benjakul, 6-shogaol, and plumbagin at the highest dose, via the caspase cascade was confirmed by time-dependent induction of caspase-3 activity, followed by its complete reduction and abolished sub-G1 peaks upon addition of Z-VAD-FMK.

Conclusion and implication: Our findings demonstrated for the first time the effects of Benjakul and its compounds on S-G2/M or G2/M phase arrest and caspase-dependent apoptosis in lung cancer cells.

Keywords: 6-Shogaol; Apoptosis; Benjakul; Cytotoxic activity; Plumbagin.