Tempol effect on oxidative and mitochondrial markers in preclinical models for prostate cancer

Abstract

Background: Tempol is a redox-cycling nitroxide considered a potent antioxidant. The present study investigated the tempol effects on oxidative stress and mitochondrial markers on prostate cancer (PCa).

Methods: PC-3 and LnCaP cells were exposed to tempol. Cell viability test, western blot and Amplex Red analyses were performed. In vivo, five experimental groups evaluated tempol effects in the early (CT12 and TPL12 groups) and late stages (CT20, TPL20-I, and TLP20-II) of PCa development. The TPL groups were treated with 50 or 100 mg/kg tempol doses. Control groups received water as the vehicle. The ventral lobe of the prostate and the blood were collected and submitted to western blotting or enzymatic activity analyses.

Results: In vitro, tempol decreased cell viability and differentially altered the H₂O₂ content for PC-3 and LNCaP. Tempol increased SOD2 levels in both cell lines and did not alter Catalase protein levels. In vivo, tempol increased SOD2 levels in the early stage and did not change Catalase levels in the different PCa stages. Systemically, tempol decreased SOD2 levels in the late-stage and improved redox status in the early and late stages, which was confirmed by reduced LDH in tempol groups. Alterations on energetic metabolism and oxidative phosphorylation were observed in TRAMP model.

Conclusion: Tempol can be considered a beneficial therapy for PCa treatment considering its antioxidant and low toxicity properties, however the PCa progression must be evaluated to get successful therapy.

Keywords: TRAMP model; mitochondria; oxidative stress; prostate cancer; tempol.

Fig. 1

Trypan blue cell viability test for PC-3 and LNCaP cells. The treatment occurred at concentrations of 0 (or control), 0.5, 1.0, 2.0 mM of Tempol in the culture medium, during 24, 48 and 72 h of exposure. Statistical significance was given by comparison with the control column (^**P < 0.01, ^***P < 0.001).

Fig. 2

H₂O₂ analyses content by Amplex red ® for PC-3 and LNCaP cells. The treatment occurred at 1.0 mM for PC-3 and 2.0 mM for LNCaP, during 48 and 72 h of exposure. Statistical significance was considered between the treated and non-treated group of the same time point (^*P < 0,05, ^**P < 0,01).

Fig. 3

PC-3 and LNCaP protein levels after Tempol treatment, representative bands of protein blots and the respective fold-change related to control. For 4-HNE, all the bands were represented. statistical significance was considered between the treated and non-treated group of the same time point (^*P < 0,05, ^**P < 0,01, ^***P < 0,0001).

Fig. 4

TRAMP model protein levels in early and late-stage of cancer progression, representative bands of protein blots and the respective fold-change related to control. For 4-HNE, all the bands were represented. For CT12 and TPL12, statistical significance was considered between the treated and non-treated group (^*P < 0,05, ^**P < 0,01, ^***P < 0,0001). For CT20, TPL20-I and TPL20-II, letter “a” denotes statistical significance when compared to CT20 group and letter “b” denotes statistical significance when compared to TPL20-I group (^*P < 0,05, ^**P < 0,01, ^*** < 0,0001).

Fig. 5

TRAMP model enzymatic activity in serum. For CT12 and TPL12, statistical significance was considered between the treated and non-treated group (^*P < 0,05, ^***P < 0,0001). For CT20, TPL20-I and TPL20-II, letter “a” denotes statistical significance when compared to CT20 group and letter “b” denotes statistical significance when compared to TPL20-I group (^*P < 0,05, ^**P < 0,01, ^*** < 0,0001).

Fig. 6

TRAMP model protein levels in early stage of cancer progression, representative bands of protein blots and the respective fold-change related to control. For CT12 and TPL12, statistical significance was considered between the treated and non-treated group (^*P < 0,05).

Fig. 7

TRAMP model protein levels in late stage of cancer progression, representative bands of protein blots and the respective fold-change related to control. For CT20, TPL20-I and TPL20-II, letter “a” denotes statistical significance when compared to CT20 group and letter “b” denotes statistical significance when compared to TPL20-I group (^*P < 0,05, ^**P < 0,01, ^*** < 0,0001).