Orally administered MSeA and MSeC decreased indices of prostate tumor burden
In the TRAMP model, the activation of probasin-driven T-Ag expression leads to the accelerated growth of the DLP and ventral prostate where most prostate lesions arise and of the lower genital urinary (GU) tract organs including seminal vesicle, testes and bladder in addition to the prostate gland (25
). The weights of GU and DLP have therefore been used as quantitative indicators of tumor burden in early stage carcinogenesis (25
). In the first experiment, we initiated daily oral treatment (5 times per week) of MSeA, MSeC or water (vehicle) at 8 weeks of age and euthanized mice at 18 weeks and up to 26 weeks of age. For reference values, we obtained baseline GU weight of 8-week old wild-type and TRAMP mice and 18-week old wild-type mice. The GU weights for the 8 week-old wild-type and TRAMP mice were the same (0.52 g vs. 0.53 g, N = 10 mice per group; column 1 and 2) (). Over the next 10 weeks, the GU weight of the wild-type mice grew by 36% (from 0.52 g to 0.71 g, N = 10 mice, p < 0.01; column 3 vs. 1), whereas GU weight of the TRAMP mice increased by 136% (from 0.53 to 1.25 g, N =10, p < 0.001; column 4 vs. 2). The GU weight of TRAMP mice receiving MSeA or MSeC only increased by 60% and 49% (from 0.53 g to 0.85 g and 0.79 g, column 5 and 6 vs. 2), respectively. Overall, MSeA and MSeC treatment in the 10-week period resulted in a net inhibition of T-Ag driven expansion of GU weight by 76% and 87%, respectively (p<0.001 for each compound). There was no statistical difference between MSeA and MSeC treatments.
Fig. 1 Effect of daily orally-administered MSeA and MSeC starting from 8 weeks of age on prostate carcinogenesis surrogate indicators (i.e., weight of genitourinary tract [GU] and weight of the dorsolateral prostate [DLP]) of TRAMP mice at 18 weeks and 26 weeks (more ...)
The DL-prostate weight () of the TRAMP mice at 18 weeks (101 mg, N=9 mice, excluding 1 mouse with a tumor weighing 2.6 g (synaptophysin positive NE carcinoma); column 2) was 237% greater than that of the wild-type littermates (30 mg, N=10; column 1) (p < 0.001). The DLP weight of the TRAMP mice treated with MSeA or MSeC was significantly less than the water-treated mice, being 62 and 58 mg, respectively (p < 0.001 for each compound, columns 3 and 4 vs. 2). As a conservative estimate (excluding a 2.6 g tumor in the water-control group), the MSeA or MSeC treatment decreased the T-Ag driven DLP weight gain by 55% and 63%, respectively, after 10 weeks of administration.
At 26 weeks of age, the GU weight data were not normally distributed due to at least one very large tumor in each group and were therefore presented as ranking order, but were not analyzed by ANOVA (). The mathematical mean GU weight of the water-, MSeA- and MSeC-treated TRAMP mice was 3.03 g (N = 10), 1.73 g (N = 10) and 1.21g (N = 9), respectively. The DLP weight of the water-, MSeA- or MSeC-treated mice was 424 mg (N = 8, excluding 2 mice with large tumors), 119 mg (N = 9, excluding one mouse with a large tumor) and 76 mg (N = 8, excluding one mouse with a large tumor), respectively (). Together these data at 26 weeks of age indicated decreased growth of the DLP and GU organs by MSeA and MSeC treatment. Because of the demonstration of different lineages for NE-carcinomas (synaptophysin+, mostly arising from ventral prostate) and glandular epithelial lesions from the DLP (27
), we analyzed synaptophysin expression in the prostate tissue and tumor sections by immunohistochemistry staining (See supplementary Fig S1
for staining patterns) and immunobloting (). As shown in , synaptophysin(+) carcinomas represented the fast growing cancer in our model and their growth were seemingly inhibited by MSeA or MSeC.
The body weight of TRAMP mice at 18 weeks was not significantly affected by oral administration of 3 mg Se per kg body weight as MSeA, but was decreased by MSeC by 9.4% (). At 26 weeks of age, the final body weight was significantly lower (compare 34.3 g in water-control TRAMP mice with 30.0 g of MSeA-treated mice and 29.0 g of MSeC-treated mice, respectively) (). A portion of the body weight difference, however, was attributable to the heavier GU weight in the control mice at 26 weeks (). These data indicated that the dosage level of 3 mg Se/kg was marginally tolerated by the TRAMP mice. Because of the complication of GU weight difference in TRAMP mice, the long-term safety issue of MSeA in wild type male mice was assessed in a second experiment along with TRAMP-mouse survival evaluation (See long-term experiment later).
MSeA and MSeC delayed the lesion progression in DL-prostate and the occurrence of poorly differentiation NE-carcinomas
As shown in , at both 18 weeks and 26 weeks of age, the severity of DLP lesions (highest grade based on H&E for each mouse) showed that the MSeA or MSeC-treated groups had more mice with PIN lesions and fewer mice with more advanced poorly differentiated carcinomas (PD) than the control groups. These results suggest that both Se compounds when administered starting at 8 weeks of age inhibit lesion progression in the DLP from PIN and WD lesions to MD carcinomas () and also decreased the emergence of PD carcinomas (), which are mostly synaptophysin+ ().
Fig 2 Effect of daily orally-administered MSeA and MSeC starting from 8 weeks of age on prostate carcinogenesis lesion progression (A), epithelial proliferation index (B and E.a; Ki67 staining), apoptosis (C and E.b; TUNEL staining) and T-antigen transgene (more ...)
Effect of MSeA and MSeC on the proliferation and apoptosis indices in DL-prostates
To provide a mechanistic explanation of the delayed DLP lesion progression, we next analyzed indices of epithelial proliferation and apoptosis focusing on the 18-week sample cohorts. Ki67 is a nuclear protein highly expressed in proliferating cells. IHC analysis of DLP () showed a virtual absence of Ki67-positive staining in wild-type mice and greatly increased Ki-67 index in TRAMP mice in the water-control group. TRAMP mice receiving either MSeA or MSeC treatment showed decreased Ki67 expression compared to the water group (t-test, p < 0.05) (, graph a).
In vivo apoptotic response of MSeA- or MSeC-treated DL-prostate in the 18-week cohort was analyzed by TUNEL staining (). Wild-type mice showed minimal apoptosis in the DLP, and TRAMP mice had much increased apoptosis (~4.4%, , graph b). The TUNEL-positive cells in MSeA- and MSeC-treated TRAMP mice were greater than in the water-treated TRAMP mice, being 8.9 % (t-test, p<0.01) and 7.4 % (t-test, p<0.05), respectively (, graph b).
The expression of the oncogenic transgene T-Ag in the DLP was examined by immunohistochemistry staining (). As expected, the wild type DLP was devoid of this transgene product. The epithelial expression of T-Ag in the TRAMP mice was evident and was not appreciably affected by either MSeA or MSeC treatment. These data support the involvement of anti-proliferative as well as pro-apoptotic effects of MSeA and MSeC in the suppression of DLP lesion growths in the TRAMP mice, without affecting transgene product expression.
Effect of MSeA or MSeC on selected proteins in cell proliferation and apoptosis
To begin to define potential in vivo molecular mediators or targets associated with the cell proliferation and apoptosis signaling responses in the DLP affected by Se treatments, we focused on the 18-week tissues, because these early samples were less complicated by the emergence of large tumors than at later time points. As shown in , the abundance of CDK2 and CDK4, cyclin E, c-Jun and PCNA was greatly increased in the DLP of the TRAMP mice (lane 1) compared with the wild type mice (lane 4). MSeA and MSeC decreased the abundance of CDK2 and c-Jun and increased the abundance of CDK inhibitory protein p27Kip1, without appreciable effect on CDK4 and PCNA. MSeA treatment appeared to suppress cyclin E abundance (lane 2 vs. lane 1) whereas MSeC did not (lane 3 vs. lane 1).
Fig. 3 Analyses by Western blots of (A) cell cycle and (B) apoptosis regulatory proteins and assay of caspase-3/-7 enzyme activity (C) of the dorsolateral prostate of Water-treated, MSeA- or MSeC-treated TRAMP mice as well as from wild type mice at 18 weeks (more ...)
As far as cell death was concerned, cleavage of PARP was barely detectable in the wild type DLP (lane 4) and was higher in the water-treated control TRAMP DLP (lane 1) (). MSeA and MSeC-treated DLP contained elevated level of cleaved PARP in comparison with water-control group (lanes 2 and 3 vs. 1). Measurement of caspase-3/-7 enzymatic activity in the tissue lysate confirmed the increased activation of caspase-mediated execution in the MSeA- or MSeC-treated DLP (). The abundance and phosphorylation level of the survival kinase AKT were lower in both Se-groups (lanes 2 and 3) in comparison with the water control group (lane 1) (). The mitochondria integrity regulatory proteins Bax and Bcl-xL were not altered by the Se treatments. High expression of the caspase inhibitor protein XIAP was found to correlate with the low apoptosis in the wild type mice, in spite of low AKT and low Bcl-xL abundance in these mice (lane 4). These biochemical data support the possible involvement of CDK2, c-Jun and P27kip1 in regulating of DLP cell proliferation and of AKT inhibition in caspase-mediated apoptosis in vivo by MSeA or MSeC consumption.
MSeA and MSeC caused a significant decrease of serum IGF-1 levels in TRAMP mice
The IGF-1 axis plays a crucial role in prostate cancer progression (38
) and some studies suggest that serum IGF-1 level might be a better predictor of prostate cancer risk than serum prostate-specific antigen (PSA) (42
). We found that MSeA or MSeC treatment led to decreased serum IGF-1 in 18-week old TRAMP mice () (ANOVA, p<0.01), and 26-week old TRAMP mice () (ANOVA, p<0.05).
Fig. 4 Effect of daily orally-administered MSeA and MSeC starting from 8 weeks of age on serum IGF-1 levels at (A) 18 weeks and (B) 26 weeks of age and on (C) the IGF-1R phosphorylation status of the dorsolateral prostate of TRAMP mice at 18 weeks of age detected (more ...)
Western blot analyses of the DLP lysates did not reveal any change of IGF-1 receptor abundance resulting from Se treatment (). The phosphorylation of IGF-1R was increased in the water-treated control TRAMP mice (lane 1) when compared to wild type mice (lane 4). The treatment by either Se compound decreased the phosphorylation modestly (lanes 2 and 3 vs. 1). These data suggest that MSeA and MSeC might inhibit IGF-1 axis at both the ligand and receptor phosphorylation level, upstream of AKT phosphorylation.
MSeA improved the overall and the cancer-specific survival of TRAMP mice
Prompted by the data from the above experiment supporting a similar in vivo inhibitory efficacy of MSeA and MSeC on early stage prostate lesion progression in DLP and the emergence of synaptophysin (+) NE-carcinomas, we next did a long-term experiment to establish the survival benefit of oral MSeA administration. Suspecting that the overall efficacy would depend on the stages of the disease when MSeA intervention was started, we therefore compared the MSeA oral treatment at 10 weeks (early PIN stage) vs. 16 weeks of age (PIN to organ confined NE-carcinomas).
showed the Kaplan-Meier survival curves of TRAMP mice due to all causes of mortality (euthanasia or those found dead) among the three groups. The median survival time was 40.5, 40.5 and 45.0 weeks of age for the water group (N = 31), 16-week MSeA cohort (N = 30) and 10-week MSeA cohort (N = 31), respectively. Whereas nearly all water-treated TRAMP mice were dead by 50 weeks of age, approximately one third of mice treated with MSeA starting either at 10-weeks or 16-weeks of age survived to this age. Log rank tests show: water vs. 10-week MSeA cohort, p = 0.0225; water vs. 16- week MSeA cohort, p = 0.1793.
Fig. 5 Effect of daily orally-administered MSeA starting from 10 weeks or 16 weeks of age on the long-term survival of TRAMP mice. A. Kaplan-Meier survival curves of all causes of mortality. Median survival time was 40.5, 40.5 and 45.0 weeks for the water-treated (more ...)
There was some unexpected non-cancer-related mortality between 20-30 weeks in all 3 groups. Therefore, we excluded those mice that died of verifiable non-cancer causes before 30 weeks of age and re-plotted the survival curves (effective number of mice: water group, N = 28; 10-week MSeA cohort, N=25; 16-week MSeA cohort, N=23) (). The median survival time of the corrected cancer-specific mortality was 42, 43 and 47 weeks of age for the water-, 16-week MSeA and 10-week MSeA cohorts, respectively. Nevertheless, a greater number of mice in both MSeA-treated groups than the control mice survived cancer-specific mortality to 50 weeks of age. Two group log rank tests showed: water vs. 10-week MSeA cohort, p = 0.0078; water vs. 16-week MSeA cohort, p = 0.0385; and 16-week vs. 10-week MSeA cohort, p = 0.7269. These data therefore support an improvement of the long-term survival of TRAMP mice against all-causes of mortality and cancer-specific mortality by intervening with MSeA at either 10 weeks or 16-weeks of age. The lack of improvement of median survival time by MSeA treatment starting at 16-weeks of age might indicate a decreased ability for MSeA to inhibit pre-existing aggressive early carcinomas in some mice at that age.
Indeed, when we plotted the GU weight of mice vs. the age at death/euthanasia according to the synaptophysin-staining status, it became apparent that the early cancer-death (<30 weeks) in the control mice was due to the formation of the aggressive synaptophysin(+) NE-carcinomas (, water group) and the mice that were euthanized or died >40 weeks of age were due to enlarged seminal vesicles and their prostate lesions were mostly negative for synaptophysin-staining. When provided starting at 10-weeks of age, MSeA decreased and delayed not only death due to the synaptophysin(+) carcinomas, but also the death of mice bearing synaptophysin(-) staining lesions (, 10_wk MSeA group). However, when started at 16 weeks, MSeA did not suppress the early death due to synaptophysin (+) carcinomas (20-24 weeks), but delayed the deaths due to the remaining synaptophysin (+) carcinomas and synaptophysin (-) prostate lesions (, 16_wk MSeA group).
Consistent with improved long-term survival, the visible macro-metastasis to the lymph nodes, liver and lung were 43.8% (14/32) in the water group and 26.7% (8/30) and 22.6% (7/31) in the 16-week and 10-week MSeA cohorts, respectively (). When the MSeA groups were combined for comparison with the water group, Chi square = 4.51, 1 degree of freedom, 0.025< P <0.05.
Lack of growth inhibition and liver damage by long term MSeA administration in wild type littermates
The safety of long-term MSeA treatment was a concern and therefore was simultaneously evaluated in wild-type littermates. The mice given daily orally MSeA treatment from 10 weeks of age until 41 weeks of age showed similar rate of body weight gain in comparison to the water-treated mice (). MSeA treatment did not affect the weight of the GU when compared with the water control mice ().
Fig. 6 Lack of significant effect of orally administered MSeA from 10 weeks of age to 41 weeks of age on (A) body weight, (B) GU weight and (C) liver damage serum marker, ALT in wild type male littermates. Average body weights of water-treated (open symbols) (more ...)
As a biochemical indicator of liver damage, we measured the serum ALT level of the non-TRAMP littermates. There was no significant difference between the water- and the MSeA-treated groups (). These data support the well-tolerated nature of MSeA daily oral dosing regimen for long term chemoprevention use.