δ-tocotrienol induces human bladder cancer cell growth arrest, apoptosis and chemosensitization through inhibition of STAT3 pathway

Abstract

Vitamin E intake has been implicated in reduction of bladder cancer risk. However, the mechanisms remain elusive. Here we reported that δ-tocotrienol (δ-T3), one of vitamin E isomers, possessed the most potent cytotoxic capacity against human bladder cancer cells, compared with other Vitamin E isomers. δ-T3 inhibited cancer cell proliferation and colonogenicity through induction of G1 phase arrest and apoptosis. Western blotting assay revealed that δ-T3 increased the expression levels of cell cycle inhibitors (p21, p27), pro-apoptotic protein (Bax) and suppressed expression levels of cell cycle protein (Cyclin D1), anti-apoptotic proteins (Bcl-2, Bcl-xL and Mcl-1), resulting in the Caspase-3 activation and cleavage of PARP. Moreover, the δ-T3 treatment inhibited ETK phosphorylation level and induced SHP-1 expression, which was correlated with downregulation of STAT3 activation. In line with this, δ-T3 reduced the STAT3 protein level in nuclear fraction, as well as its transcription activity. Knockdown of SHP-1 partially reversed δ-T3-induced cell growth arrest. Importantly, low dose of δ-T3 sensitized Gemcitabine-induced cytotoxic effects on human bladder cancer cells. Overall, our findings demonstrated, for the first time, the cytotoxic effects of δ-T3 on bladder cancer cells and suggest that δ-T3 might be a promising chemosensitization reagent for Gemcitabine in bladder cancer treatment.

Fig 1. Effects of vitamin E isomers on bladder cancer cells.

(A) Human bladder cancer cells T24, 5637, J82 and UMUC-3, as well as non-malignant human urothelial SV-HUC-1 cells were treated with each vitamin E isomers (α-TP, α-T3, γ-T3 and δ-T3) ranging from 0 to 200 μM for 72 h, and cell viability were detected by MTT assay. (B) Colony formation assay of T24 5637, J82 and UMUC-3 cells with 100 μM α-TP, α-T3, γ-T3 and δ-T3 for 14 days. Vertical bars indicate the mean cell count ±SD in each treatment group. *, P < 0.05; ***, P < 0.001, compared to vehicle treatment group.

Fig 2. δ-T3 induced cell cycle arrest in T24 (A, B) and 5637 cells (C, D) bladder cancer cells.

Cells were treated by δ-T3 ranging from 0–150 μM for 48 h. Cell cycle distribution and Sub-G1 ratios were assessed by flow cytometry. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 3. δ-T3 induces apoptosis in bladder cancer cells.

Annexin V/PI analysis showed that δT3 induced apoptosis in T24 (A) and 5637 (B) bladder cancer cells, as compared to vehicle treated control cells.

Fig 4. δ-T3 treatment changes protein expression levels involved in cell cycle arrest and apoptosis.

Western blotting analysis of the cell cycle (A), apoptosis (B) and other apoptosis-related (C) protein levels in T24 and 5637 cells, upon the δ-T3 treatment for 24 h. β-Actin was used as the loading control.

Fig 5. δ-T3 suppresses STAT3 signaling pathways in human bladder cancer cells.

Western blotting analysis of the STAT3/ETK signaling pathway-related (A) and SHP-1 (B) protein levels in T24 and 5637 cells under the δ-T3 treatment for 24 h. Western bands were quantified by Image J software and the digits shown below the upper panel were the relative STAT3 expression levels normalized by loading controls. (C) Reduction of nuclear STAT3 protein level upon the treatment of 150 μM δ-T3 in T24 cells. LaminB1 was used as a nuclear loading control. Tubulin was used as a cytoplasmic loading control. (D) Genomic structure of bclxl gene was shown, with the labels of three primer sets for ChIP assay. Primer set 1 and 2 contain STAT3 binding element; whereas primer set 3 (NC) serves as negative control. STAT3 occupancy in the bclxl promoter in bladder cancer cell line T24 treated with 150 μM δ-T3 or vehicle for 18 h were assayed by ChIP assay. Input DNA and immunoprecipitated DNA were analyzed by qPCR analyses using primer sets depicted above and normalized by IgG control. The error bar indicates the means ± SD. ***, P < 0.001. (E) δ-T3 treatment reduced the STAT3 downstream target genes (bcl2, bclxl and mcl-1) expression at mRNA level. (F) Luciferase activity analysis of STAT3-Luc upon the treatment of 150 μM δ-T3 in T24 cells for 24 h. TK-Renilla luciferase plasmid was used as internal control. (G) Knockdown efficiency of SHP-1 in T24 cells by Western blotting assay. β–Actin was used as internal control. siNC, negative control siRNA. siSHP-1, siRNA targeting to SHP-1. (H) T24 cells were treated with siRNA to SHP-1 or siNC, followed by treatment with δ-T3 or vehicle. Cell viability was tested by MTT assay. **, P < 0.01; ***, P < 0.001.

Fig 6. Low concentration of δ-T3 enhanced the anti-cancer effects of Gemcitabine (GEM) on bladder cancer cell growth.

(A) T24 and 5637 cells were incubated for 48 h in the presence of 25 μM δ-T3 and/or 0.08 μM GEM. Then, the percentage of cell viability was determined by MTT assay. (B) T24 and 5637 cells were cultured for 48 h in the absence or presence of 25 μM δ-T3 and/or 0.08 μM GEM, respectively. Apoptotic rates were analyzed by Annexin V/PI staining assay. (C) Combined treatment with GEM (0.08 μM) and δ-T3 (25 μM) for 14 days completely eliminated the colony formation capacity of T24 cells. (D) Western blotting analysis of the apoptosis-related and STAT3 signaling-related protein levels in T24 cells, treated with δ-T3 and/or GEM for 48 h. (E) Western blotting analysis of the STAT3 signaling-related protein levels in T24 cells, treated with δ-T3 and/or GEM for 24h. **, P < 0.01; ***, P < 0.001.