Translocator protein blockade reduces prostate tumor growth

Abstract

Purpose: The transmembrane molecule, translocator protein (TSPO), has been implicated in the progression of epithelial tumors. TSPO gene expression is high in tissues involved in steroid biosynthesis, neurodegenerative disease, and in cancer, and overexpression has been shown to contribute to pathologic conditions including cancer progression in several different models. The goal of our study was to examine the expression and biological relevance of TSPO in prostate cancer and show that the commonly prescribed benzodiazepine lorazepam, a ligand for TSPO, exhibits anticancer properties.

Experimental design: Immunohistochemical analysis using tissue microarrays was used to determine the expression profile of TSPO in human prostate cancer tissues. To show the effect of TSPO ligands (lorazepam and PK11195) in prostate cancer, we used cell proliferation assays, apoptosis ELISA, prostate cancer xenograft study, and immunohistochemistry.

Results: TSPO expression is increased in prostatic intraepithelial neoplasia, primary prostate cancer, and metastases compared with normal prostate tissue and benign prostatic hyperplasia. Furthermore, TSPO expression correlates with disease progression, as TSPO levels increased with increasing Gleason sum and stage with prostate cancer metastases demonstrating the highest level of expression among all tissues examined. Functionally, we have shown that lorazepam has antiproliferative and proapoptotic properties in vitro and in vivo. Additionally, we have shown that TSPO overexpression in nontumorigenic cells conferred susceptibility to lorazepam-induced growth inhibition.

Conclusion: These data suggest that blocking TSPO function in tumor cells induces cell death and denotes a survival role for TSPO in prostate cancer and provides the first evidence for the use of benzodiazepines in prostate cancer therapeutics.

Figure 1. Increased TSPO expression in prostate cancer

(A) relative expression of TSPO by IHC in normal donor prostate, normal prostate tissue adjacent to tumor (NAT), benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), primary prostate cancer (PCa), and prostate cancer metastases (met) by scoring of TMA cores. TSPO expression is significantly (p<0.05) increased in PIN, PCa, and PCa metastases, compared to normal prostate tissue and BPH. (B-G) representative results of IHC staining of TSPO in normal donor prostate (B) NAT (C), BPH (D), PIN (E), PCa (F), and PCa metastasis (G). (H) total cell lysates (10ug) were used for Western blot analysis to determine expression levels of TSPO in human prostate cancer cell lines PPC-1, DU145, LAPC4, LA98, and LNCaP, LN05, compared to human embryonic kidney cells (HEK293), human T lymphocytes (Jurkat), human cervical cancer cells (HeLa), and human liver hepatocytes (Hep). * indicates p<0.05

Figure 2. TSPO antagonism decreases cell proliferation and increases apoptosis in prostate cancer cells in vitro

(A) MTT assay following 48 hour treatment of human prostate cancer cells, PPC-1 with PK11195 or Lorazepam (0.1uM-100uM) or vehicle (EtOH or DMSO, respectively). (B) direct cell counting of PPC-1 and LN97 cells treated with varying concentrations of PK11195 or Lorazepam or vehicle for 48 hours. (C) Cell death ELISA following 18 hour treatment of PPC-1 and DU145 cells with varying concentrations of PK11195 or Lorazepam or vehicle. * indicates p<0.05

Figure 3. Increased Susceptibility to Lorazepam in 293 Cells Overexpressing TSPO

(A) Western blot analysis of 293 cells overexpressing TSPO (293-TSPO A/B) compared to the parental cell line (293) and empty vector control cells (293-C). (B) Percent of 293 and 293-TSPO cells treated with 50uM lorazepam (black bars) compared to vehicle treated cells (grey bars). * indicates p<0.05

Figure 4. TSPO antagonism decreases cell proliferation and increases apoptosis in prostate cancer cells in vivo

(A) Average tumor volume over time of athymic nude mice bearing PPC-1 xenograft tumors treated daily with Lorazepam (40mg/kg) or DMSO. Tumor volume was measured twice weekly as described in the Materials and Methods section. Data are shown as means + the standard error. (B) immunohistochemical staining of lorazepam or vehicle treated PPC-1 xenograft tumors for cell proliferation (ki67), microvascular density (CD31), and apoptosis (TUNEL). Bars graphs represent average values of positive signal counted in four random fields (40X magnification) * indicates p<0.05