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A triazole-conjugated benzoxazone induces reactive oxygen species and promotes autophagic apoptosis in human lung cancer cells

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Abstract

Numerous approaches suggested that compounds with conjugated triazole moieties or benzoxazone pharmacores are effective to antagonize proliferation of human tumors. The current study reported that a synthetic triazole-conjugated benzoxazone, 4-((5-benzyl-1H-1,2,3-triazol-3-yl)-methyl)-7-methoxy-2H-benzo[b][1,4]-oxazin-3(4H)-one (BTO), inhibited growth rates of human non-small cell lung cancer cells. The cytotoxicity can be enhanced with increasing drug concentrations. More evidence supported that the induced reactive oxygen species lead to ultimate apoptotic cell death by recruiting autophagy. The mechanistic pathway as elucidated involved tumor suppressor p53 activation and LC3-1 conversion followed by PARP and procaspase-3 cleavage. Autophagy inhibition reverted apoptotic death and restored cell viabilities. BTO suppressed the development of A549 cell xenograft tumors by activating autophagy and apoptosis simultaneously. As an efficient tumor growth inhibitor with relatively small molecular weight, BTO is a viable addition to the existing list of lung cancer treatment.

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Abbreviations

Bax:

B-cell lymphoma-2-associated X-protein

BTO:

4-((5-Benzyl-1H-1,2,3-triazol-3-yl)-methyl)-7-methoxy-2H-benzo[b][1,4]-oxazin-3(4H)-one

DAPI:

4′,6-Diamidino-2-phenylindole

DCFH-DA:

2′,7′-Dichlorofluorescein diacetate

DMEM:

Dulbecco’s modified eagle medium

DMSO:

Dimethyl sulfoxide

EDTA:

Ethylenediaminetetraacetic acid

FBS:

Fetal bovine serum

FITC:

Fluorescein isothiocyanate

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

GFP:

Green fluorescence protein

HE:

Hematoxylin and eosin

HRP:

Horseradish peroxidase

LC3:

Microtubule-associated protein light chain 3

3-MA:

3-Methyladenine

MTT:

3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbromide

NAC:

N-Acetylcysteine

NSCLC:

Non-small-cell lung carcinomas

PARP:

Poly(adenosine diphosphate ribose) polymerase

PBS:

Phosphate-buffered saline

PCNA:

Proliferating cell nuclear antigen

PI:

Propidium iodide

PI3K:

Phosphoinositide 3-kinase

ROS:

Reactive oxygen species

SD:

Standard deviation

TRITC:

Tetramethylrhodamine isocyanate

TUNEL:

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling

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Acknowledgements

The construct GFP-LC3 was kindly provided by professor Huang Wei-Bang, Department of Life Science, National Taiwan University, Taipei, Taiwan. The work is supported by grants from National Taiwan Normal University (106T3040B2, 106T3040C2 and 106T3040D2). Technical assistance of College of Life Science and Instrumentation Center, National Taiwan Normal University with the confocal laser microscopy is appreciated.

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Authors and Affiliations

  1. Department of Life Science, National Taiwan Normal University, 88 Ting-Chow Road, Section 4, Taipei, 116, Taiwan

    Chang-Heng Hsieh, Jing-Ping Wang, Chun-Yen Liu & Kang Fang

  2. Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan

    Chien-Chih Chiu

  3. Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan

    Ching-Fa Yao

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  1. Chang-Heng Hsieh
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  2. Jing-Ping Wang
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Correspondence to Kang Fang.

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All authors declare that they have no conflict of interests.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

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Scheme of BTO synthesis (JPG 49 KB)

Experimental protocols of BTO synthesis (DOCX 94 KB)

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Hsieh, CH., Wang, JP., Chiu, CC. et al. A triazole-conjugated benzoxazone induces reactive oxygen species and promotes autophagic apoptosis in human lung cancer cells. Apoptosis 23, 1–15 (2018). https://doi.org/10.1007/s10495-017-1432-9

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