Background: Standard anti-proliferative chemotherapy is relatively ineffective against slowly proliferating androgen-independent prostate cancer cells within metastatic sites. In contrast, the lipophilic cytotoxin thapsigargin, which causes apoptosis by disrupting intracellular free Ca²⁺ levels, is effective against both proliferative and quiescent (i.e., G₀-arrested) cells. However, thapsigargin’s mechanism of action indicates that it is unlikely to be selective for cancer cells or prostate cells. Methods: We coupled a chemically modified form of thapsigargin, L12ADT, to a peptide carrier that is a substrate for the prostate-specific antigen (PSA) protease to produce a soluble, cell-impermeant latent prodrug that is specifically activated extracellularly within metastatic prostate cancer sites by PSA. We analyzed the kinetics of PSA hydrolysis of the prodrug, the in vitro cytoxicity of the prodrug against PSA-producing LNCaP human prostate cancer and PSA non-producing HCT-116 human colon cancer cells, and the in vivo pharmacokinetics of the prodrug in mice. We also analyzed antitumor efficacy of the prodrug in nude mice xenograft models of prostate cancer (using LNCaP cells) and renal carcinoma (using human SN12C cells). Results: The L12ADT peptide prodrug was hydrolyzed efficiently by PSA, was selectively toxic to PSA-producing prostate cancer cells in vitro, and was stable in human plasma. A single dose of 7 mg/kg resulted in a peak serum prodrug concentration of 15.4 ± 1.1 μM and a half-life of approximately 2.8 hours. Over 24 hours, less than 0.5% of free L12ADT was observed in plasma. Levels of prodrug and liberated L12ADT in prostate cancer xenograft tumors were approximately eightfold and sixfold, respectively, higher than the in vitro LD₅₀s. Prostate cancer xenograft tumors in mice treated with prodrug by intravenous administration were growth-inhibited without substantial host toxicity. Continuous subcutaneous prodrug administration in mice produced complete growth inhibition of established PSA-producing prostate cancer xenograft tumors but had no effect on PSA non-producing renal carcinoma xenograft tumors. Conclusion: Further development of PSA-activated thapsigargin prodrugs as therapy for metastatic prostate cancer is warranted.

Calcium (Ca²⁺) is a fundamental regulator of cell signaling and function. Thapsigargin (Tg) blocks the sarco/endoplasmic reticulum (ER) Ca²⁺-ATPase (SERCA), disrupts Ca²⁺ homeostasis, and causes cell death. However, the exact mechanisms whereby SERCA inhibition induces cell death are incompletely understood. Here, we report that low (0.1 μm) concentrations of Tg and Tg analogs with various long-chain substitutions at the O-8 position extensively inhibit SERCA1a-mediated Ca²⁺ transport. We also found that, in both prostate and breast cancer cells, exposure to Tg or Tg analogs for 1 day caused extensive drainage of the ER Ca²⁺ stores. This Ca²⁺ depletion was followed by markedly reduced cell proliferation rates and morphological changes that developed over 2–4 days and culminated in cell death. Interestingly, these changes were not accompanied by bulk increases in cytosolic Ca²⁺ levels. Moreover, knockdown of two key store-operated Ca²⁺ entry (SOCE) components, Orai1 and STIM1, did not reduce Tg cytotoxicity, indicating that SOCE and Ca²⁺ entry are not critical for Tg-induced cell death. However, we observed a correlation between the abilities of Tg and Tg analogs to deplete ER Ca²⁺ stores and their detrimental effects on cell viability. Furthermore, caspase activation and cell death were associated with a sustained unfolded protein response. We conclude that ER Ca²⁺ drainage and sustained unfolded protein response activation are key for initiation of apoptosis at low concentrations of Tg and Tg analogs, whereas high cytosolic Ca²⁺ levels and SOCE are not required.