Obstet Gynecol Sci.  2016 Jul;59(4):261-268. 10.5468/ogs.2016.59.4.261.

The effect of salinomycin on ovarian cancer stem-like cells

• Chung H
• Kim YH
• Kwon M
• Shin SJ
• Kwon SH
• Cha SD
• Cho CH

• KMID: 2329046
• DOI: http://doi.org/10.5468/ogs.2016.59.4.261

Abstract

OBJECTIVE
The identification of cancer stem-like cells is a recent development in ovarian cancer. Compared to other cancer cells, cancer stem-like cells present more chemo-resistance and more aggressive characteristics. They play an important role in the recurrence and drug resistance of cancer. Therefore, the target therapy of cancer stem-like cell may become a promising and effective approach for ovarian cancer treatment. It may also help to provide novel diagnostic and therapeutic strategies.
METHODS
The OVCAR3 cell line was cultured under serum-free conditions to produce floating spheres. The CD44⁺CD117⁺ cell line was isolated from the human ovarian cancer cell line OVCAR3 by using immune magnetic-activated cell sorting system. The expression of stemness genes such as OCT3/4, NANOG and SOX2 mRNA were determined by reverse transcription polymerase chain reaction. OVCAR3 parental and OVCAR3 CD44⁺CD117⁺ cells were grown in different doses of paclitaxel and salinomycin to evaluate the effect of salinomycin. And growth inhibition of OVCAR3 CD44+CD117+ cells by paclitaxel combined with salinomycin was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay.
RESULTS
Tumor spheroids generated from the OVCAR3 cell line are shown to have highly enriched CD44 and CD117 expression. Treatment with a combination of paclitaxel and salinomycin demonstrated growth inhibition of OVCAR3 CD44+CD117+ cells.
CONCLUSION
The present study is a detailed investigation on the expression of CD44 and CD117 in cancer stem cells and evaluates their specific tumorigenic characteristics in ovarian cancer. This study also demonstrates significant growth inhibition of cancer stem-like cells by paclitaxel combined with salinomycin. Identification of these cancer stem-like cell markers and growth inhibition effect of salinomycin may be the next step to the development of novel target therapy in ovarian cancer.

Keyword

Ovarian neoplasms; Salinomycin; Stem-like cell

MeSH Terms

Cell Line
Drug Resistance
Humans
Neoplastic Stem Cells
Ovarian Neoplasms*
Paclitaxel
Parents
Polymerase Chain Reaction
Recurrence
Reverse Transcription
RNA, Messenger
Paclitaxel
RNA, Messenger

Figure

• 

  Fig. 1 (A) Increased expression of CD44 in OVCAR3 sphere forming cells. The expression of ovarian cancer stem cell marker CD44 was increased in OVCAR3 sphere forming cells as observed under fluorescence microscopy. Nuclei were stained with Hoechst (×100). (B) Analysis of surface marker expressions by flow cytometry. CD44 and CD117 were increased approximately two folds in OVCAR3 sphere cells. FITC, fluorescein isothiocyanate; APC, allophysocyanin.

• 

  Fig. 2 The expressions of stemness genes in OVCAR3 CD44+CD117+ cells by Western blot (A) and by semiquantitative reverse transcription polymerase chain reaction (B). The amount of cDNA input was adjusted to equalize the expression level of GAPDH. Octamer-binding trascription factor 3/4 (OCT3/4), nanog homeobox (NANOG) and sex determining region Y-box 2 (SOX2) are known to be as stemness genes. The expressions of stemness genes were increased in OVCAR3 CD44+CD117+ cells than those in OVCAR3. Each band was quantified by densitometric analysis and presented in a bar graph. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 3 Effect of salinomycin in growth inhibition of ovarian cancer stem-like cells. The cells were exposed to various concentrations of (A) paclitaxel (1, 10, 100 and 200 nM) and (B) salinomycin (0.1, 0.5, 1 and 5 µM) in OVCAR3 and OVCAR3 CD44+CD117+ cells for 48 hours to evaluate effect in growth inhibition of ovarian cancer stem-like cells. (C) OVCAR3 CD44+CD117+ cells were treated with paclitaxel (10 nM) and/or salinomycin (0.1 µM) for 48 hours. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Values are mean±standard deviation of three measurements. CTL, control; PTX, paclitaxel; Sal, salinomycin. *P <0.05.

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