BackgroundProstate‐specific membrane antigen (PSMA)‐based imaging and therapy are increasingly used for prostate cancer management. However, limitations are a low PSMA expression in certain patients. Androgen receptor axis inhibition can induce PSMA expression in vitro. We hypothesized that different approved compounds upregulate PSMA expression and tested their effect in vitro.MethodsAndrogen receptor (AR) expressing prostate cancer (LNCaP) and epithelial prostate cells (PNT1A) were treated for 7 days with enzalutamide, dutasteride, rapamycin, metformin, lovastatin, and acetylsalicylic acid (ASA). PSMA and AR protein expression was assessed using flow cytometry, immunocytochemistry and immunoblotting. Furthermore, uptake and internalization of 177Lu‐PSMA‐617 was performed.ResultsEnzalutamide and dutasteride led to a significant (both P < 0.05) upregulation of PSMA surface levels in LNCaP cells. In addition, treatment with rapamycin showed a non‐significant trend toward PSMA upregulation. No changes were detected after treatment with vehicle, metformin, lovastatin, and ASA. Total PSMA protein expression was significantly enhanced after treatment with enzalutamide and rapamycin (both P < 0.05), whereas dutasteride led to a non‐significant upregulation. Uptake of 177Lu‐PSMA‐617 was significantly increased after treatment of LNCaP with enzalutamide, dutasteride, and rapamycin (P < 0.05). In addition, internalization was significantly increased by enzalutamide and rapamycin (P < 0.05), and non‐significantly increased by dutasteride.ConclusionIn conclusion, our data provide new insights into the effect of different approved pharmacological compounds that can markedly upregulate PSMA expression and radioligand uptake in vitro. Pharmacologically induced PSMA expression may prove useful to improve prostate cancer detection and to enhance anticancer effects in PSMA‐based therapy.