COX-2/sEH Dual Inhibitor PTUPB Potentiates the Antitumor Efficacy of Cisplatin

Abstract

Cisplatin-based therapy is highly toxic, but moderately effective in most cancers. Concurrent inhibition of cyclooxygenase-2 (COX-2) and soluble epoxide hydrolase (sEH) results in antitumor activity and has organ-protective effects. The goal of this study was to determine the antitumor activity of PTUPB, an orally bioavailable COX-2/sEH dual inhibitor, in combination with cisplatin and gemcitabine (GC) therapy. NSG mice bearing bladder cancer patient-derived xenografts were treated with vehicle, PTUPB, cisplatin, GC, or combinations thereof. Mouse experiments were performed with two different PDX models. PTUPB potentiated cisplatin and GC therapy, resulting in significantly reduced tumor growth and prolonged survival. PTUPB plus cisplatin was no more toxic than cisplatin single-agent treatment as assessed by body weight, histochemical staining of major organs, blood counts, and chemistry. The combination of PTUPB and cisplatin increased apoptosis and decreased phosphorylation in the MAPK/ERK and PI3K/AKT/mTOR pathways compared with controls. PTUPB treatment did not alter platinum-DNA adduct levels, which is the most critical step in platinum-induced cell death. The in vitro study using the combination index method showed modest synergy between PTUPB and platinum agents only in 5637 cell line among several cell lines examined. However, PTUPB is very active in vivo by inhibiting angiogenesis. In conclusion, PTUPB potentiated the antitumor activity of cisplatin-based treatment without increasing toxicity in vivo and has potential for further development as a combination chemotherapy partner. Mol Cancer Ther; 17(2); 474-83. ©2017 AACR.

Figure 1. PTUPB potentiates cisplatin anti-tumor activity

A, Tumor growth in NSG-PDX bladder cancer mouse model BL0293. When tumor volume of the tumor reached ∼100-200 cm³, mice were administered by i.v. with PEG 300 control, single agent cisplatin (2 mg/kg, i.v., Day 1, 2, 3, 15, 16, and 17, black arrows), single agent PTUPB (30 mg/kg, orally, once daily for up to 30 days), or cisplatin (2 mg/kg) plus PUTUB (30 mg/kg) in combination. The tumor dimensions were measured every 3∼4 days. The tumor volume was calculated using the formula: 0.5 × length × width² (mm³). Mice were euthanized when the tumor length reached 20 mm in any direction. The median time of the tumor growth to 7.5× BL (blacked dotted line) was 20 days for the control and 24.4 days in the PTUPB group (p=0.085) and 35.8 days in the cisplatin group (p=0.0003). The median time of the cisplatin and PTUPB combination group was significantly increased to 47.8 days compared to PTUPB (p<0.0001) or cisplatin (p=0.002) monotherapy groups. B, Overall survival with statistical analysis. Overall survival of the combination treatment group was 60.9 days, significantly longer than that of either PTUPB (39.4 days, p=0.007) or cisplatin (47 days, p=0.02) monotherapy groups. C, Tumor growth in the NSG-PDX bladder cancer mouse model BL0269. Mice were euthanized on day 29 and the tumors were collected. The representative images of the excised tumors are shown. D, Tumor growth in the NSG-PDX bladder cancer mouse model BL0269. When the size of the tumor xenografts reached around 0.1∼0.2 cm³, the NSG mice were treated with PEG 300 control, PTUPB (30 mg/kg, orally, once daily for up to 30 days), cisplatin (2 mg/kg, i.v., Day 1, 2, 3, 15, 16, and 17, black arrows), gemcitabine (150 mg/kg, i.p. weekly for 4 weeks), and cisplatin (2 mg/kg) plus gemcitabine (150 mg/kg) plus PTUPB (30 mg/kg) combination. The tumor sizes were measured every 3∼4 days. The tumor volume was calculated using the formula: 0.5 × length × width² (mm³). N=8-10 mice per group. The results are expressed as mean±SD.

Figure 2. Cisplatin plus PTUPB decreases proliferation and angiogenesis but increases apoptosis as determined by immunohistochemical (IHC) analysis

Formalin-fixed paraffin-embedded PDX BL0293 tumor sections were stained for Hematoxylin and Eosin (H&E), Ki-67, cleaved caspase-3 and CD31. More Ki-67 positive cells were observed in the control group, but significantly decreased in the combination group. Compared with the control group, increasing numbers of cells stained positive for cleaved caspase-3 were observed in the PTUPB, cisplatin, and PTUPB plus cisplatin combination groups. CD31 staining was decreased in PTUPB and combination groups. Quantitative data of Ki67, cleaved caspase-3 and CD31 staining in each group were generated from randomly selected 20 fields and are shown along with the images. *: p<0.05.

Figure 3. PTUPB combined with cisplatin modulates p-ERK and p-AKT in tumor tissue

A, Illustration of relevant signaling pathways indicating possible roles for sEH and COX-2. B, Western blot analysis of protein expression of indicated phospho-proteins, total proteins and loading control GAPDH. Protein was extracted at indicated times from PDX BL0293 tumors treated with cisplatin, PTUPB or cisplatin plus PTUPB combination therapy.

Figure 4. PTUPB does not alter carboplatin-DNA adduct levels

A, Cultures of the ATCC bladder cancer cell line 5637 were incubated with 100 μM [¹⁴C]carboplatin in the presence (gray bar) or absence (white bar) of 10 μM PTUPB for 4h or 4h then washed and further incubated 20hr with fresh drug-free culture medium. B, 5637 cells were pretreated (grey bar) with 10 μM PTUPB for 5h before cells were exposed to 100 μM [¹⁴C]carboplatin for indicated amount of time. C, NSG mice carrying BL0293 tumors were treated with 37.5 mg/kg (therapeutic dose) carboplatin (50,000 dpm/g) via IV bolus and tissue was harvested after 24hr. PTUPB (30 mg/kg in PEG400) was administered via oral gavage 16hr (grey bar) or 1hr (black bar) before carboplatin dosing. Sample size for the cell line experiments was N=3, sample size for PDX in experiments was N = 6 (carbo alone) or 3 (in both PTUPB groups). The results are expressed as mean±SD.

Figure 5. PTUPB increases cisplatin cytotoxicity in the 5637 bladder cancer cell line

Dose-response curves of 5637 cells treated with cisplatin and PTUPB at different concentrations as determined in a 72hr cell viability assay. A, Single drug treatment. Cultures of 5637 cells were treated with different concentrations of PTUPB or cisplatin (0, 0.01, 0.1, 1, 2, 5, 10, 20, 50, and 100 μM). B, Combination drugs treatment. 5637 cells were treated with different concentrations of cisplatin (0, 0.01 0.1, 0.5, 1, 2, 5, 10, and 100 μM) in combination with different concentrations of PTUPB (1, 2, 5, and 10 μM). Sample size for the cell line experiments was N=3. *CI: Combination Index. The results are expressed as mean±SD.

Figure 6. Molecular correlative studies of PTUPB showing inhibition of both COX-2 and sEH pathways in PDX BL0269 tumor tissues

A, PTUPB reduces the levels of prostaglandins PGE₂, PGD₂, TXB₂, 6-keto-PGF_1α on COX-2 pathway. B, PTUPB increased levels of sEH substrates 10,11-EpDPE, 12,13-EpOME, 15,16-EpODE and decreased levels of sEH product 12,13-DiHOME on sEH pathway. The results are expressed as mean± SD. *P<0.05.