Role of CYP3A5 in modulating androgen receptor signaling and its relevance to African American men with prostate cancer

Abstract

1Background: Androgen receptor signaling is crucial for prostate cancer growth and is regulated by intratumoral 2 CYP3A5. As African American (AA) men often carry the wild type CYP3A5 and express high level of CYP3A5 3 protein, we tested the effect of blocking the wild type CYP3A5 in prostate cancer cells from AA men on androgen 4 receptor signaling. CYP3A5 processes several commonly prescribed drugs and many of these are CYP3A5 5inducers (e.g. phenytoin and rifampicin) or inhibitors (e.g. ritonavir and amiodarone). In this study, we test the 6 effect of these commonly prescribed CYP3A5 inducers/inhibitors on AR signaling in prostate cancer cells. 7Methods: Cell fractionation and immunofluorescence studies were performed to study AR nuclear localization 8 and activation process using CYP3A5 siRNA and CYP3A5 inducers and inhibitors. A qPCR based array was 9 employed to examine expression of AR downstream regulated genes after blocking CYP3A5 expression using 10 a pool of CYP3A5 siRNA. Cell growth was monitored using MTS based assays. Since AAs tend to carry wild 11 type CYP3A5 and non-Hispanic White Americans (NHWA) carry mutated CYP3A5 two cell lines one of AA origin 12 (MDAPCA2b) carrying wt CYP3A5 and the other of NHWA origin (LNCaP) carrying mutant CYP3A5 were used 13 for above experiments. 14 Results: Similar to that observed in LNCaP (mutant CYP3A5) earlier, CYP3A5 siRNA treated MDACPA2b (AA, 15 wild type CYP3A5) cells showed decreased AR nuclear translocation and PSA production. q-PCR based profiler 16 assay identified several AR regulated genes which were downregulated with CYP3A5 siRNA pool treatment 17 performed with cDNA from CYP3A5 siRNA pool and NT treated MDAPCA2b cells. These downregulated genes 18 include SCL45A3, FKBP5, NCAPD3, MYC, MME, ELL2, PIK3R3, HPRT1 and SPDEF with p-value of ≤0.005. 19These genes are known to regulate AR nuclear translocation, cell cycle progression and cell growth. SCL45A3, 20 FKBP5, MYC, and ELL2 also showed decreased protein levels after CYP3A5 siRNA treatment. 21Commonly prescribed drugs which are either CYP3A5 inhibitors (amiodarone, ritonavir) or inducers (phenytoin, 22 rifampicin) were tested for their ability to alter AR signaling in both LNCaP and MDAPCa2b cells. The results 23show that the CYP3A5 inducers promoted AR nuclear translocation and downstream signaling whereas CYP3A5 24 inhibitors abrogated them. The increased nuclear AR observed with phenytoin and rifampicin (CYP3A inducers) 25 treatment is abrogated in CYP3A5 siRNA treated MDAPCa2b cells, confirming that the activation of AR activity 26 is specific to changes in CYP3A5 activity. Both the inducers tested demonstrated increased cell growth of 1 prostate cancer cells, whereas the inhibitors showed reduced cell growth. The difference in growth is more 2 pronounced in MDAPCa2b cells which carries a wild type CYP3A5 as compared to LNCaP with the exception of 3 ritonavir which also downregulates total AR levels. 4 Conclusions:Concomitantly prescribed CYP3A5 modulating drugs may alter downstream AR signaling...

Bergamottin a CYP3A inhibitor found in grapefruit juice inhibits prostate cancer cell growth by downregulating androgen receptor signaling causing cell cycle block and apoptosis

“…The lower IC 50 values for LNCaP may be attributed to lower levels of CYP3A5 protein due to *3/*3 mutation present as compared to MDAPCa2b which carries one copy of wild type of CYP3A5 (*1/*3) (20). The other major difference between the previously reported results in the DU145 AR negative prostate cancer cell line 1 (18) suggests bergamottin's action is CYP3A5 -AR signaling dependent (14).…”

“…Previously we have investigated how CYP3A5 inhibitors and inducers can modify AR signaling and effect the efficacy of commonly prescribed concomitant medications (20). CYP3A4/5 are hepatic enzymes and are involved in processing several commonly prescribed drugs, we do have to be cautious of drug-drug interactions while using bergamottin as an anticancer agent as it is a strong CYP3A inhibitor.…”

“…So far it has not been tested for its ability to block AR activation which is very relevant in prostate cancer. In our recently published work we have shown how CYP3A5 inhibitors and inducers can alter AR activation more so in AAs expressing wild type CYP3A5(20). respectively.…”

Bergamottin a CYP3A inhibitor found in grapefruit juice inhibits prostate cancer cell growth by downregulating androgen receptor signaling and promoting G0/G1 cell cycle block and apoptosis

“…We have genotyped these two cell lines used in our current study. The LNCaP carries only mutant CYP3A5 (*3/*3) where as MDAPCa2b carries one wild type and one mutant CYP3A5 (*1/*3), producing higher levels of CYP3A5 full length protein [ 21 ]. The *3 splice variant results in a truncated protein due to the presence of a stop codon.…”

“…So far it has not been tested for its ability to block AR activation which is very relevant in prostate cancer. In our recently published work we have shown how CYP3A5 inhibitors and inducers can alter AR activation more so in AAs expressing wild type CYP3A5 [ 21 ]. Understanding the biological mechanisms of how bergamottin regulates AR signaling in prostate cancer patients can help establish its mechanism based role in blocking prostate cancer growth especially in AAs expressing high CYP3A5 to reduce health disparity.…”