Methaneseleninic acid and γ-Tocopherol combination inhibits prostate tumor growth in Vivo in a xenograft mouse model - PubMed (original) (raw)

Methaneseleninic acid and γ-Tocopherol combination inhibits prostate tumor growth in Vivo in a xenograft mouse model

Chandra K Singh et al. Oncotarget. 2014.

Abstract

Studies have shown that vitamin E and selenium possess antiproliferative effects against prostate cancer (PCa). However, results from the Selenium and Vitamin E Cancer Prevention Trial (SELECT) suggest that vitamin E (α-tocopheryl acetate; 400 mg) and/or selenium (L-selenomethionine; 200 μg) were ineffective against PCa in humans. It is arguable that the selected dose/formulation of vitamin E/selenium were not optimal in SELECT. Thus, additional studies are needed to define the appropriate formulations/dose regimens of these agents. Here, we investigated the effect of methaneseleninic acid (MSA; 41 µg/kg) and/or γ-tocopherol (γT; 20.8 mg/kg or 41.7 mg/kg) in Nu/J mice implanted with 22Rν1 tumors. MSA (41 µg/kg) and γT (20.8 mg/kg) combination was most consistent in imparting anti-proliferative response; resulting in a significant decrease in i) tumor volume/weight, ii) serum PSA, and iii) Ki-67 immunostaining. Further, we observed i) an upregulation of pro-apoptosis Bax and a down-regulation of the pro-survival Bcl2, and ii) an increase in pro-apoptosis Bad. Furthermore, the combination resulted in a modulation of apolipoprotein E, selenoprotein P and Nrf2 in a fashion that favors antiproliferative responses. Overall, our study suggested that a combination of MSA and γT, at lower dose regimen, could be useful in PCa management.

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Figures

Figure 1

Figure 1. Effects of oral MSA and/or γT on prostate tumor growth in athymic nude mice

Mice were subcutaneously implanted with 22Rν1 cells, and after 12 days MSA and/or γT were administered as described in ‘Materials and Methods’. Effects of MSA and/or γT were assessed for tumor size, tumor volume, tumor wet weight and serum PSA levels. Following sacrifice, tumors were resected. Representative images of each treatment group are shown (A). Tumor volume was measured at the start of the experiment and subsequently at 7th and 14th days after starting treatment (B). At the end of the experiment, tumor volume was determined (C); Average wet weights of resected tumors were taken (D); and serum PSA levels were measured (E). All are represented as mean value ± 2 standard errors of mean (*P<0.05, **P<0.01 and ***P<0.001).

Figure 2

Figure 2. Effects of MSA and/or γT on tumor cell proliferation

At the termination of xenograft experiment, tumors were excised and processed for Ki-67 immunostaining as detailed in ‘Materials and Methods’. Immunohistochemical analysis was performed in tumor tissue of control and treatment groups under bright field microscope. Representative images (400x magnification) of each treatment group are shown.

Figure 3

Figure 3. Effects of MSA and γT on Bax and Bcl2 proteins

At the termination of the experiment, the effects of the treatments on Bax and Bcl2 proteins were determined using immunoblot analysis. 40 μg protein was separated on SDS-PAGE and immunobloted for Bax (A) and Bcl2 (B) as detailed in ‘Materials and Methods’. The blots were reprobed for β-actin for loading control. Representative blots are shown and the data (relative density normalized to β-actin) are expressed as mean ± 2 standard error of three replicates (representing 6 mice). Ratio of Bax/Bcl2 was also calculated and plotted (C) (*P<0.05).

Figure 4

Figure 4. Effects of MSA and/or γT on Bax and Bcl2 mRNA

At the termination of xenograft experiment, effects of treatments on Bax and Bcl2 transcription were assessed by qRT-PCR analyses. RNA from tumor tissue samples was isolated and cDNA was made. qRT-PCR was run for Bax and Bcl2 as detailed in ‘Materials and Methods’. GAPDH was used as endogenous control. The qRT-PCR data are represented as relative mRNA levels for Bax (A) and Bcl2 (B). Ratio of Bax/Bcl2 mRNA was calculated and plotted (C). The data represented are mean ± 2 standard error of three replicates (representing 6 mice) (*P<0.05).

Figure 5

Figure 5. Effects of MSA and/or γT on Bad protein and mRNA

The effects of MSA and γT on pro-apoptosis Bad was analyzed at protein as well as mRNA levels. For immunoblot analysis, 40 μg protein was separated on SDS-PAGE and immunobloted as detailed in ‘Materials and Methods’. The blots were reprobed for β-actin for loading control. Relative protein expression of Bad was calculated and plotted (A). Relative mRNA levels of Bad were assessed using qRT-PCR assay as detailed in ‘Materials and Methods’ (B). The data represented are mean ± 2 standard error of three replicates (representing 6 mice) (*P<0.05).

Figure 6

Figure 6. Effects of MSA and/or γT on oxidative stress markers

To assess the effects of MSA and/or γT on modulation of oxidative stress, ApoE, SepP and Nrf2 mRNA level were analyzed. RNA isolation from tumor tissue followed by cDNA synthesis and then qRT-PCR was performed as detailed in ‘Materials and Methods’. The qRT-PCR data are represented as relative quantity (normalized to GAPDH) for ApoE (A), SepP (B) and Nrf2 (C) transcriptional levels. Representative data are expressed as mean ± 2 standard error of three replicates (representing 6 mice) (*P<0.05).

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