Isobutyrylshikonin has a potentially stronger cytotoxic effect in oral cancer cells than its analogue shikonin in vitro

Dae-Gun Park; Da Jeong Kim; Bok Hee Woo; Hye Jung Kim; Young Whan Choi; Hae Ryoun Park

doi:10.1016/j.archoralbio.2020.104774

Isobutyrylshikonin has a potentially stronger cytotoxic effect in oral cancer cells than its analogue shikonin in vitro

Arch Oral Biol. 2020 Aug:116:104774. doi: 10.1016/j.archoralbio.2020.104774. Epub 2020 May 20.

Authors

Dae-Gun Park¹, Da Jeong Kim¹, Bok Hee Woo¹, Hye Jung Kim², Young Whan Choi³, Hae Ryoun Park⁴

Affiliations

¹ Department of Oral Pathology & BK21 PLUS Project, School of Dentistry, Pusan National University, 49 Busandaehak-Ro, Yangsan 626-870, South Korea.
² Periodontal Disease Signaling Network Research Center, Dental & Life Science Institute, Pusan National University, 49 Busandaehak-Ro, Yangsan 626-870, South Korea.
³ Department of Horticultural Bioscience, Pusan National University, Samnangjin-Ro 1268-50, Samnangjin-eup, Miryang 627-706, South Korea.
⁴ Department of Oral Pathology & BK21 PLUS Project, School of Dentistry, Pusan National University, 49 Busandaehak-Ro, Yangsan 626-870, South Korea; Periodontal Disease Signaling Network Research Center, Dental & Life Science Institute, Pusan National University, 49 Busandaehak-Ro, Yangsan 626-870, South Korea. Electronic address: parkhr@pusan.ac.kr.

PMID: 32470830
DOI: 10.1016/j.archoralbio.2020.104774

Abstract

Objectives: The aim of the present study was to identify the anticancer effects and the mechanisms of action of shikonin and its analogue isobutyrylshikonin in oral squamous carcinoma cells.

Designs: The cytotoxic effects of isobutyrylshikonin and shikonin in Ca9-22 and SCC-25 cells were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry analysis of Annexin V/Propidium Iodide (PI) staining, western blot analysis and immunohistochemistry.

Results: Treatment with both isobutyrylshikonin and shikonin induced dose- and time-dependent apoptotic cell death in Ca9-22 cells, although the IC₅₀ of isobutyrylshikonin was less than that of shikonin. The induction of apoptosis by both isobutyrylshikonin and shikonin was accompanied by activation of caspase-8, -9, -3, and PARP, loss of mitochondrial trans-membrane potential, and release of cytochrome c from the mitochondria. ROS mediated the apoptosis induced by isobutyrylshikonin and shikonin, indicating that ROS may play a critical role in the distinctive cytotoxic effects of isobutyrylshikonin and shikonin in Ca9-22. Isobutyrylshikonin showed a similar cytotoxic effect in SCC-25 cells at concentrations showing the effects in Ca9 cells, but not in human normal keratinocyte cells. Although there is no biological difference between isobutyrylshikonin and shikonin, isobutyrylshikonin exerts the same cytotoxic effect at a concentration 6 times lower than shikonin.

Conclusions: The present study suggest that isobutyrylshikonin may be a more potent chemotherapeutic agent against oral cancer cells than shikonin. In addition, our data exhibit that both isobutyrylshikonin and shikonin induce caspase-dependent apoptosis via the mitochondrial pathway through accumulation of ROS in oral squamous carcinoma cells.

Keywords: Apoptosis; Isobutyrylshikonin; Oral cancer; Oral squamous cell carcinoma; Reactive oxygen species; Shikonin.

MeSH terms

Apoptosis
Cell Line, Tumor
Humans
Membrane Potential, Mitochondrial
Mouth Neoplasms* / drug therapy
Naphthoquinones* / pharmacology
Naphthoquinones* / toxicity
Reactive Oxygen Species
Tumor Cells, Cultured

Substances

Naphthoquinones
Reactive Oxygen Species
isobutyrylshikonin
shikonin