The data presented here conclusively demonstrate that androgen-induced activation of SSAT, the first enzyme of one major polyamine oxidation pathway, is a key source of oxidative stress in the prostatic lumen and is one of the major factors in prostate tumor progression. The data establish that CPC-200, a small molecule specific inhibitor of polyamine oxidase, can not only block androgen-induced oxidative stress in cultured LNCaP human prostate cancer cells, but can also significantly delay prostate carcinogenesis in TRAMPxFVB animals. These results open up a new avenue for research in prostate cancer therapy.
The fold increase of SSAT mRNA level estimated using qRT-PCR in siSSAT clone is nearly 80% less than that observed for wild-type LNCaP cells (). This decrease in SSAT mRNA level is sufficient to completely block androgen induced ROS (). Casero et al
) have reported the detection, isolation and characterization of another inducible polyamine oxidase (PAOh1) in human breast, colon, lung and prostate tumors. PAOh1 oxidizes unacetylated polyamines and also produces H2
. PAOh1 induced ROS production, however, does not go through APAO pathway and has been reported to be the primary source of ROS in breast cancer cells and not specific for prostate cancer metabolism. Almost complete block of androgen induced ROS production in SSAT mRNA silenced cells () suggests that PAOh1 probably plays a minor role in the androgen-induced ROS production specifically in the prostate cancer cells.
The ability of CPC-200 treatment to reduce cellular ROS production () also demonstrates that an overexpression of SSAT that initiates enhanced polyamine catabolism is one of the major causes of androgen-induced oxidative stress in androgen dependent human prostate cancer cells. Since the enzyme PAOh1 is also inhibited by CPC-200 (21
), some contribution of PAOh1 activity in the cellular oxidative stress cannot be completely ruled out. It is to be noted, however, that androgen treatment decreases cellular spermine levels and increases cellular acetyl-polyamine level (). The polyamine levels in R1881 () and the reduction of ROS levels in cells treated with 25 μM CPC-200 () suggest that most of the ROS production in R1881-treated cells is due to SSAT induction followed by oxidation of acetylated polyamines by constitutively expressed APAO, rather than a direct oxidation of spermine by PAOh1.
It is also to be noted that CPC-200 pretreated LNCaP cells growing in the presence of 1 nM R1881 have even less ROS than do untreated cells (). This suggests that the basal level of ROS produced in LNCaP cells growing in the absence of androgen may also be due to low grade oxidation of cellular polyamines that is now blocked by CPC-200 treatment.
As spermine acts as a scavenger for ROS (30
), androgen- induced decrease in polyamine levels may also cause the observed increase in ROS levels. Twenty-five μM CPC-200 pretreatment, however, does not reverse androgen-induced reduction of cellular spermine levels () even though it completely blocks the ROS production (). Therefore, R1881 induced depletion of the cellular spermine level can be ruled out as a major contributor to the increase in the ROS levels.
Our Western analysis for androgen receptor protein expression showed no effect of CPC-200 treatment on androgen receptor levels (data not shown). Thus, we rule out the possibility of CPC-200-induced changes in androgen receptor expression as a cause for the changes in growth and/or ROS.
We have established pre-clinical efficacy of CPC-200 using the TRAMPxFVB mouse model of prostate carcinogenesis. CPC-200 at a well-tolerated dose of 25 mg/kg given intra peritoneal once every two weeks for a total of six treatments significantly inhibited the growth of tumors in this model as evidenced by improved survival (). In our studies, the majority of mice (>90%) were sacrificed due to reaching a pre-defined tumor size per our animal protocol, thus survival is a surrogate for tumor burden, and improvement in survival thus equates with an inhibition of tumor growth by CPC-200 in our TRAMPxFVB model. Therefore, the significant 5.5 week improvement (p=0.03) in median survival for CPC-200 using this model () demonstrates the ability of CPC-200 to slow prostate tumor growth. The efficacy of CPC-200 against prostate tumor progression in this animal model supports the hypothesis that polyamine oxidation and resultant increase in ROS play an important role in prostate carcinogenesis and strongly implicates the potential of CPC-200 as a new therapeutic agent for prostate cancer.
Several mechanisms such as expression (or nuclear translocation) of specific transcription factors such as hypoxia-induced transcription factor (HIF-1α), NF-κB, AP-1, etc. (9
) have been suggested as a probable mode of regulation of specific genes that may control cellular redox status. Enhanced mitochondrial activity (13
), suppression of glutathione S-transferase-π expression with a reduction in the level of total glutathione specifically in prostate cancer cells (8
) have also been suggested as probable pathways for an increase in ROS production in prostate cancer. A direct effect of androgen in regulating any of these pathways has yet to be demonstrated. To the best of our knowledge, this is the first report of androgen-induced regulation of a rate-limiting enzyme of a specific biochemical pathway (polyamine catabolism) that is directly related to cellular oxidative stress induction. Because of the high levels of polyamines present in human prostate and prostate cancer cells, this pathway seems all the more important in regulating oxidative stress in the prostate gland and specifically in prostate cancer cells. The spermine level in LNCaP cells reported here is by far the highest among most of the cell lines reported thus far (31
and related references therein). A higher basal metabolism of spermine may be one reason for the relatively higher ROS level in LNCaP cells as compared to other cell lines (12
A close inspection of the SSAT gene sequence reveals that there are four glucocorticoids response elements (GRE), but no androgen response element (ARE), upstream of the SSAT transcription start site. It has been reported that androgen receptor binds and activates GRE containing promoters (32
). Its efficiency of activating promoters containing GRE, however, is much lower than that of activating promoters containing ARE. This may be one reason why the SSAT activation and the consequent ROS production was observed only when the cells were treated with high concentration (≥ 0.5 nM) of R1881, but not with low concentration (≤ 0.05 nM) of R1881 (12
Lastly, a decrease in cellular polyamine levels has been related to a delay in tumor growth and progression both in cell culture as well as in animals and humans (33
). In this report, we observed a significant delay in tumor growth by CPC-200 () even though there is an increase
and not a decrease in cellular spermidine and putrescine levels and no observable change in cellular spermine level. Therefore, change in cellular polyamine levels is probably not a cause for the delay in tumor progression in the TRAMPxFVB animals developing spontaneous prostate tumor. The detail study of the polyamine and acetyl polyamine levels in the TRAMP animal and tumor tissue have now been undertaken to confirm this point.
To the best of our knowledge, this is the first report of SSAT induction by a hormone (hormone analog) R1881. Identification of androgen-induced polyamine catabolism leading to enhanced oxidative stress in the prostate cells and significant inhibition of prostate cancer progression by blocking this pathway should open up a new avenue for prostate cancer chemoprevention.