Salinomycin inhibits prostate cancer growth and migration via induction of oxidative stress

Abstract

Background: We have shown that a sodium ionophore monensin inhibits prostate cancer cell growth. A structurally related compound to monensin, salinomycin, was recently identified as a putative cancer stem cell inhibitor.

Methods: The growth inhibitory potential of salinomycin was studied in a panel of prostate cells. To get insights into the mechanism of action, a variety of assays such as gene expression and steroid profiling were performed in salinomycin-exposed prostate cancer cells.

Results: Salinomycin inhibited the growth of prostate cancer cells, but did not affect non-malignant prostate epithelial cells. Salinomycin impacted on prostate cancer stem cell functions as evidenced by reduced aldehyde dehydrogenase activity and the fraction of CD44(+) cells. Moreover, salinomycin reduced the expression of MYC, AR and ERG, induced oxidative stress as well as inhibited nuclear factor-κB activity and cell migration. Furthermore, profiling steroid metabolites revealed increased levels of oxidative stress-inducing steroids 7-ketocholesterol and aldosterone and decreased levels of antioxidative steroids progesterone and pregnenolone in salinomycin-exposed prostate cancer cells.

Conclusion: Our results indicate that salinomycin inhibits prostate cancer cell growth and migration by reducing the expression of key prostate cancer oncogenes, inducing oxidative stress, decreasing the antioxidative capacity and cancer stem cell fraction.

Figure 1

Salinomycin inhibits aldehyde dehydrogenase (ALDH) activity in prostate cancer cells. ALDH activity was measured with Aldefluor assay in response to 1-mmol l⁻¹ exposures of salinomycin or control for 48 h in VCaP and LNCaP cells. ALDH inhibitor diethylaminobenzaldehyde (DEAB) was used as negative control. Asterisks indicate statistical significance. ^*P<0.05; ^**P<0.01; ^***P<0.001.

Figure 2

Salinomycin reduces the fraction of CD44⁺ cells in prostate cancer cells. VCaP, LNCaP, PC-3 and DU-145 cells were stained with CD44 antibody and the fluorescence intensities were identified for cells exposed to 6 h of salinomycin or DMSO control.

Figure 3

Salinomycin reduces the expression levels of key prostate cancer oncogenes, androgen receptor (AR), ERG and MYC in prostate cancer cells. (A) AR, (B) PSA, (C) ERG and (D) MYC mRNA expression in response to 3-, 6-, and 24-h salinomycin exposure in VCaP and LNCaP cells. Asterisks indicate statistical significance. ^*P<0.05; ^**P<0.01; ^***P<0.001.

Figure 4

(A) Salinomycin induces the gene expression signature characteristic of oxidative stress induction in prostate cancer cells. The expressions of MT1F, MT1G, ATF3, KLF6, DDIT3, DDIT4 and TXNIP mRNAs in VCaP and LNCaP cells in response to salinomycin exposure for 3, 6 and 24 h. (B) Salinomycin induces the level of intracellular reactive oxygen species in prostate cancer cells. Reactive oxygen species generation in response to salinomycin exposure in VCaP and LNCaP cells detected with carboxy-H2DCFDA. Hydrogen peroxide (400 mmol l⁻¹) exposure for 4 h was used as a positive control. Asterisks indicate statistical significance. ^*P<0.05; ^**P<0.01; ^***P<0.001.

Figure 5

Salinomycin reduces NF-κB activity in prostate cancer cells. The activity of NF-κB and negative control was measured using cancer reporter array in (A) VCaP and in (B) LNCaP cells in response to 100 nM salinomycin exposure for 24 h and the results were compared with control exposures. Asterisks indicate statistical significance. ^*P<0.05; ^**P<0.01.

Figure 6

Salinomycin reduces PC-3 prostate cancer cell migration and the effect is antagonised with antioxidant vitamin C. (A) Relative wound density in response to salinomycin alone (100 nM, P<0.001 for control vs salinomycin) and in combination with vitamin C (10 μM) as well as appropriate controls monitored for 24 h. (B) The morphological pictures from wound density after 24-h exposures of salinomycin alone and in combination vitamin C. The cells in the beginning of the experiment are marked in black.

Figure 7

Salinomycin induces the levels of 7-ketocholesterol and aldosterone and reduces the levels of progesterone and pregnenolone. The cells were exposed to salinomycin (1 μM) for 6 h and the steroid profile was measured with gas chromatography-mass spectrometry.