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. 2017 Apr 27;7(6):798-810.
doi: 10.1002/2211-5463.12223. eCollection 2017 Jun.

Evaluation of cell death pathways initiated by antitumor drugs melatonin and valproic acid in bladder cancer cells

Siwei Liu  1 , Bilin Liang  1 , Huiting Jia  1 , Yuhan Jiao  1 , Zhongqiu Pang  1 , Yongye Huang  1
Affiliations

Evaluation of cell death pathways initiated by antitumor drugs melatonin and valproic acid in bladder cancer cells

Siwei Liu et al. FEBS Open Bio. .

Abstract

Effective drug combinations have the potential to strengthen therapeutic efficacy and combat drug resistance. Both melatonin and valproic acid (VPA) exhibit antitumor activities in various cancer cells. The aim of this study was to evaluate the cell death pathways initiated by anticancer combinatorial effects of melatonin and VPA in bladder cancer cells. The results demonstrated that the combination of melatonin and VPA leads to significant synergistic growth inhibition of UC3 bladder cancer cells. Gene expression studies revealed that cotreatment with melatonin and VPA triggered the up-regulation of certain genes related to apoptosis (TNFRSF10A and TNFRSF10B), autophagy (BECN, ATG3 and ATG5) and necrosis (MLKL, PARP-1 and RIPK1). The combinatorial treatment increased the expression of endoplasmic reticulum (ER)-stress-related genes ATF6, IRE1, EDEM1 and ERdj4. Cotreatment with melatonin and VPA enhanced the expression of E-cadherin, and decreased the expression of N-cadherin, Fibronectin, Snail and Slug. Furthermore, the Wnt pathway and Raf/MEK/ERK pathway were activated by combinatorial treatment. However, the effects on the expression of certain genes were not further enhanced in cells following combinatorial treatment in comparison to individual treatment of melatonin or VPA. In summary, these findings provided evidence that cotreatment with melatonin and VPA exerted increased cytotoxicity by regulating cell death pathways in UC3 bladder cancer cells, but the clinical significance of combinatorial treatment still needs to be further exploited.

Keywords: ER stress; apoptosis; autophagy; epithelial–mesenchymal transition; melatonin; valproic acid.

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Figures

Figure 1
Figure 1
Cell viability of UC3 bladder cancer cells. (A) Cell proliferation and viability with melatonin (10−6 M) and/or VPA treatment (5 mM) were determined by an MTT assay at 0, 24, 48, 72 and 96 h. (B) Cytotoxicity among control (ctrl), melatonin (mel), VPA and combinatorial (comb) treated UC3 cells by crystal violet staining at 24 h. (C) LDH release assay among control (ctrl), melatonin (mel), VPA and combinatorial (comb) treated UC3 cells at 24 h. Error bars represent standard deviation. a,b,cValues with different superscripts are significantly different.
Figure 2
Figure 2
Apoptosis and cell‐cycle analysis of UC3 bladder cancer cells treated with melatonin and/or VPA for 24 h. (A) Cells were double‐stained with annexin V and propidium iodide (PI) and analyzed by flow cytometry. The gate settings distinguished living (bottom left), necrotic (top left), early apoptotic (bottom right) and late apoptotic (top right) cells. (B) Cells were stained with PI and the DNA content was analyzed by flow cytometry. G1, S and G2 indicate cell‐cycle phases.
Figure 3
Figure 3
(A–J) Comparison of cell death‐related gene expression among UC3 cells with control (ctrl), melatonin (mel), VPA and combinatorial (comb) treatment by qPCR. (K) Gene expression was determined by western blotting. The data are presented as mean ± SD, n = 3. *P < 0.05 vs. control, **P ≤ 0.01 vs. control, and ***P ≤ 0.001 vs. control.
Figure 4
Figure 4
Expression of endoplasmic reticulum stress‐associated genes induced by melatonin and/or VPA treatment in UC3 bladder cancer cells. The data are presented as mean ± SD, n = 3. *P < 0.05 vs. control, **P ≤ 0.01 vs. control, and ***P ≤ 0.001 vs. control. Control (ctrl), melatonin (mel), and combinatorial treatment with both melatonin and VPA (comb).
Figure 5
Figure 5
Exploration of epithelial–mesenchymal transition (EMT) at the gene level by quantitative real‐time PCR. The data are presented as mean ± SD, n = 3. *P < 0.05, as compared with the control group; **P ≤ 0.01, as compared with the control group. Control (ctrl), melatonin (mel), combinatorial treatment with both melatonin and VPA (comb).
Figure 6
Figure 6
Expression of genes involved in Wnt signaling pathway. The data are presented as mean ± SD, n = 3. *P < 0.05 vs. control, **P ≤ 0.01 vs. control, and ***P ≤ 0.001 vs. control. Control (ctrl), melatonin (mel), combinatorial treatment with both melatonin and VPA (comb).
Figure 7
Figure 7
Expression of genes involved in Raf/MEK/ERK signaling pathway. (A–E) Gene expression was determined by quantitative real‐time PCR. (F) Gene expression was determined by western blotting. The data are presented as mean ± SD, n = 3. **P ≤ 0.01 vs. control, and ***P ≤ 0.001 vs. control. Control (ctrl), melatonin (mel) and combinatorial treatment with both melatonin and VPA (comb).
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