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.

Cisplatin is the most commonly used chemotherapeutic agent in cancer treatment. However, it is only moderately effective in most cancer types and is highly toxic (1). Cisplatin-based first-line combination therapy is associated with a response rate of approximately 50% for metastatic bladder cancer and induces complete remission in less than 40% at the neoadjuvant setting for this disease (2). Therefore, there is a great unmet need to develop novel therapies to potentiate efficacy and mitigate the toxicity of cisplatin (3).

One potential strategy to improve cisplatin therapy involves modulation of the arachidonic acid (ARA) pathway, which plays numerous roles in inflammation and tumorigenesis. Eicosanoids are lipid mediators derived from ARA by cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450s (CYP). Among them, a COX-2–mediated metabolite, prostaglandin E2 (PGE2), is proinflammatory and proangiogenic (4). COX inhibitors, both nonsteroidal anti-inflammatory drugs (NSAID) and COX-2–selective inhibitors (coxibs), have been widely used to treat inflammation and pain. Separately, epoxyeicosatrienoic acids (EET), derived from the metabolism of ARA by CYP epoxygenases, have potent anti-inflammatory, analgesic, antihypertensive, cardio-protective, and organ-protective properties (5–8). However, EETs are rapidly metabolized to inactive diols by soluble epoxide hydrolase (sEH; ref. 9). sEH inhibitors (sEHI) such as trans-4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxyl)-benzoic acid (t-AUCB) maintain the level of EETs in vivo and are now in development for treatment of various diseases. In preclinical studies as well as in clinical trials, sEHIs have displayed excellent safety profiles (10, 11).

EETs transcriptionally inhibit the expression of COX-2 and thus decrease the production of PGE2 (12). Interestingly, COX-2 overexpression in tumor or stromal cells leads to tumor angiogenesis (13) and coxibs block the production of angiogenic factors, leading to inhibition of proliferation, migration, and vascular tube formation. However, targeting this single component of the ARA pathway with coxibs has failed in human clinical trials for several cancers (14–16). Furthermore, sEHIs synergize the analgesic and anti-inflammatory effects of coxibs (17, 18), prevent gastrointestinal erosion (19), and alter prostacyclin (PGI2) and thromboxane A2 (TBX2) ratios associated with blood clotting (17). Therefore, it is desirable to inhibit both COX-2 and sEH in order to maximize antitumor activity and reduce toxic effects of selective COX-2 inhibition. This dual COX-2/sEH inhibition strategy may also have the potential to protect normal tissues from cisplatin toxicity.

We recently demonstrated that a combination treatment of celecoxib and the sEHI inhibitor t-AUCB has synergistic effects for blocking angiogenesis and tumorigenesis in two mouse models of cancer (20). A compound that concurrently inhibits both COX-2 and sEH called (4-(5-phenyl-3-{3-[3-(4-trifluoromethyl-phenyl)-ureido]-propyl}-pyrazol-1-yl)-benzenesulfonamide; PTUPB; Supplementary Fig. S1; ref. 21) is more effective at inhibiting primary tumor growth and metastasis compared with inhibitors selective to either pathway, either as single agents or in combination. PTUPB acts, in part, by suppressing tumor angiogenesis via selective inhibition of endothelial cell proliferation, without any obvious cytotoxic effects in mice (20).

Here, we report assessment of the interaction of cisplatin or gemcitabine plus cisplatin (GC) with PTUPB. We hypothesize that the combination of PTUPB and cisplatin-based therapy potentiates antitumor activity without increasing cisplatin toxicity. We extended our work to include recently developed immunodeficient nod scid gamma (NSG) mice bearing patient-derived xenografts (PDX) of bladder cancer (22) and conducted additional mechanistic studies. We observed that in vivo PTUPB potentiated cisplatin efficacy without increasing toxicity. PTUPB also improved in vivo response to GC therapy. Platinum–DNA adducts were not modulated by PTUPB exposure, indicating an orthogonal mechanism of action compared with DNA alkylation. However, PTUPB enhances apoptosis and downregulates proliferation signaling, especially when combined with cisplatin.

Materials and supplies

Bladder cancer patient-derived xenograft (PDX) models were provided by The Jackson Laboratory (JAX). PDXs were developed through subcutaneous implantation from clinical tumor tissues into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG; JAX strain #5557) female mice, followed by serial in vivo passaging as previously described (22). All experiments utilized PDX models within the first five passages. Cisplatin was purchased from Fresenius Kabi USA, LLC. Gemcitabine was purchased from LC Laboratories. [14C]carboplatin was purchased from GE Healthcare and was prepared as described (23, 24). Celecoxib was a gift from Pfizer. PTUPB and t-AUCB were synthesized as previously described (21, 24). The bladder cancer cell lines 5637, J82, T24, and TCCSUP were purchased from ATCC in 2007. Multiple frozen aliquots were established upon the acquisition and all experimental cells were passaged for fewer than 20 passages after reviving from liquid N2. The cell lines were not tested and authenticated by the authors. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco) and incubated at 37°C in 5% CO2 incubator.

PDX bladder cancer

NSG PDX studies were performed at the University of California Davis with IACUC approval. Experiments were carried out with 6- to 9-week-old female NSG mice bearing bladder cancer PDX models (UC Davis ID# BL0293 or JAX Model # TM00016; UC Davis ID#BL0269 or JAX Model #TM00015). When tumors achieved volumes of 100 to 200 mm3, mice were randomized to different treatment groups as follows: vehicle control (PEG 300, 10 mL/kg, oral), PTUPB, cisplatin, t-AUCB, celecoxib, combinations of PTUPB and cisplatin, t-AUCB and cisplatin, celecoxib and cisplatin, gemcitabine and cisplatin (GC), PTUPB and GC. PTUPB (30 mg/kg in PEG 300), celecoxib (30 mg/kg in PEG 300), and t-AUCB (3 mg/kg in PEG 300) were administered once per day by oral gavage for up to 30 days. Cisplatin (1 mg/mL) was diluted in 0.9% saline and administered at a dose of 2 mg/kg (i.v., tail vein, once per day) on days 1, 2, 3, 15, 16, and 17. Gemcitabine was dissolved in 0.9% saline and administered at a dose of 150 mg/kg (i.p., weekly) for 4 weeks. Animal weight and tumor size were measured twice per week. The tumor volume was calculated with the following formula: length (mm) × width (mm) × width (mm) × 0.5. The percentage of tumor growth inhibition (TGI) was calculated as follows: 100% × (1 − [(Vtreated(final day) − Vtreated(initial day))/(Vcontrol(final day) − Vcontrol(initial day))]), where V is the tumor volume.

Blood samples from mice were collected and analyzed for complete blood count (CBC), blood urea nitrogen (BUN), aspartate aminotransferase (AST), creatinine, and total bilirubin at the Veterinary Medicine Comparative Pathology Laboratory of University of California Davis. The tumor, heart, liver, spleen, lung, and kidney were harvested and the tissue samples were fixed in formalin or frozen at −80°C. Tumor sections were stained with hematoxylin and eosin (H&E) or were used for immunohistochemistry analysis. A board-certified pathologist provided detailed interpretation of tumor histomorphology and scoring of immunohistochemical staining. Some of the tumor sections were lysed and chromatographed using SDS-PAGE followed by transfer onto a PVDF membrane. The membranes were blocked in 5% nonfat dry milk for 1 hour at room temperature, and probed with p-AKT(S473), p-ERK(Thr202/Tyr204), total-AKT, total-ERK, and rabbit monoclonal anti-GAPDH antibodies (Cell Signaling Technology). The membranes were then probed with horseradish peroxidase (HRP) tagged secondary antibodies and epitopes were detected using the ECL Plus Western Blotting Detection Reagent (GE Healthcare). Cell proliferation, apoptosis, and angiogenesis assessed with Ki-67, cleaved caspase-3, and CD31 antibodies (Cell Signaling Technology) using an immunohistochemistry kit per the manufacturer's instructions (BioGenex).

Accelerator mass spectrometry to determine platinum–DNA adduct formation

The ATCC 5637 bladder cancer cell line and NSG-PDX mice were used to assess the impact of PTUPB on [14C]carboplatin –DNA adduct levels as a surrogate of cisplatin–DNA adducts.

Carboplatin–DNA adduct formation in vitro.

For cell culture studies, 60-mm dishes of 5637 cell cultures were either pretreated with 10 μmol/L PTUPB for 5 hours followed by 100 μmol/L [14C]carboplatin (36,000 dpm/mL), or simultaneously dosed with PTUPB and [14C]carboplatin. Four hours after carboplatin was added, the cells were washed with PBS. The 4-hour incubation time was chosen due to the in vivo carboplatin half-life (1.3–6 hours) in patients. Cells were harvested at the 4-hour time point in one group of dishes and another group was washed and further incubated for 20 hours with fresh drug-free medium before cell harvest in order to determine DNA repair. Cell pellets were stored at −80°C until DNA extraction.

Carboplatin–DNA adduct formation in vivo.

NSG PDX mice bearing BL0293 tumors were dosed at the volume of 10 μL/g of [14C]carboplatin (37.5 mg/kg, 14C at 50,000 dpm/g) via i.v. bolus injection. PTUPB (30 mg/kg in PEG 400) was administered via oral gavage 1 or 16 hours before carboplatin dosing. Mice were sacrificed and tumor tissues harvested 24 hours after carboplatin dosing. DNA was extracted using a Promega Wizard genomic DNA purification kit according to manufacturer's instructions. Ten micrograms of DNA per sample was submitted to Lawrence Livermore National Laboratory (LLNL) for accelerator mass spectrometry (AMS) analysis using previously reported protocol (25).

Median effect analysis to determine in vitro drug-drug interaction

The method published by Chou and Talalay was used to determine the extent and nature (synergism, additivity, and antagonism) of PTUPB and cisplatin interaction in cell culture (26, 27). PTUPB was dissolved in dimethyl sulfoxide (DMSO) to a final stock concentration of 10 mmol/L. Cisplatin was dissolved in PBS to a final stock concentration of 10 mmol/L. Cells were seeded at 2,000 to 3,000 cells and 100 μL of medium per well into 96-well plates (Becton Dickinson) and incubated overnight. Different concentrations of these two drugs were diluted in culture media and added to each well. The plates were then incubated for an additional 72 hours. The control group was dosed with 0.2% DMSO. Cell viability assays (MTS) were performed according to the manufacturer's protocol (Promega). The absolute 50% inhibitory concentrations (IC50) were calculated as previously described (28). Dose–response curves were generated with GraphPad Prism 5 software (GraphPad Software Inc.). The combination indices (CI) were determined based on the method of Chou and colleagues (26). CI values were calculated with CompuSyn software (http://www.combosyn.com/).

Oxylipin profile analysis

Lipid extraction and analysis was performed as previously reported (20). Briefly, for tumor lipid mediator extraction, ∼100 mg of tumor tissues was mixed with an antioxidant solution (0.2 mg/mL butylated hydroxytoluene and 0.2 mg/mL triphenylphosphine in methanol), the surrogate solution, and 400 μL of extract solution (0.1% acetic acid with 0.2 mg/mL butylated hydroxytoluene in methanol), and then homogenized. The resulting homogenates were kept overnight at −80°C. Next day, the homogenates were centrifuged and supernatants were collected. The pellets were washed with 0.1% butylated hydroxytoluene and 0.1% acetic acid in methanol and the supernatants were collected and combined. LC-MS/MS analysis of the extract was carried out on an Agilent 1200SL liquid chromatographic system coupled to a 4000 QTRAP MS/MS instrument (AB Sciex) as described (29).

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM) or mean ± standard deviation (SD). Group comparisons were carried out using one-way analysis of variance or Student t test. Survival analysis was performed using the Kaplan–Meier method. A P value of less than 0.05 was considered statistically significant.

Coadministration of PTUPB potentiated the antitumor activity of cisplatin

We previously showed PTUPB had antitumor activity in mouse Lewis lung cancer (LLC) and NDL (Her2+, Ki67+, ER/PR negative) breast carcinoma models (20). Here, we determined whether PTUPB possessed antitumor activity in human bladder cancer cell lines and PDXs, and synergized with cisplatin treatment. We used bladder cancer PDX models BL0293 and BL0269. These tumor types, like most bladder cancers in the clinic, are only moderately sensitive (BL0293) or resistant (BL0269) to cisplatin (22). Treatment with single-agent PTUPB or cisplatin exhibited moderate antitumor activity in mice bearing BL0293 tumors (Fig. 1). The time required to reach a 7.5-fold increase in tumor volume was used as a reasonably achievable endpoint to evaluate tumor growth among treatment groups. The vehicle-only control had a median time to a 7.5-fold increase in tumor volume of 20.0 days, whereas the median times to this endpoints were 24.4 days (P = 0.085) and 35.8 days (P = 0.0003) for the PTUPB and cisplatin monotherapy groups, respectively. The median time to a 7.5-fold increase in tumor volume in the cisplatin plus PTUPB combination group was significantly longer (60.9 days) than that of either PTUPB (P = 0.007) or cisplatin (P = 0.02) single-agent treatment groups (Fig. 1A). Analysis of overall survival showed that single-agent PTUPB did not significantly increase survival time compared with control (39.4 days vs. 31.3 days, P = 0.201), whereas single-agent cisplatin treatment extended survival to 47.0 days (P = 0.004). The survival time could be further significantly increased by cotreatment of mice with PTUPB and cisplatin to 60.9 days, which was longer than that of either the PTUPB (P = 0.007) or cisplatin (P = 0.02) monotherapy groups (Fig. 1B). In PDX model BL0269, which is resistant to cisplatin and gemcitabine monotherapy (Fig. 1C), tumor growth was significantly inhibited in the PTUPB plus cisplatin combination group (P = 0.006). Furthermore, addition of PTUPB to GC resulted in the best inhibition of tumor growth (Fig. 1D). We also performed experiments to examine the efficacy of combination treatments of celecoxib with cisplatin and t-AUCB with cisplatin in the bladder PDX model BL0269. These experiments were performed in order to assess whether concurrent inhibition of COX-2 and sEH by PTUPB was advantageous compared with the use of inhibitors specific to either pathway. We did not observe any potentiation of cisplatin by celecoxib with respect to inhibiting tumor growth, but we observed moderate additive effect of t-AUCB with cisplatin (Supplementary Fig. S2). PTUPB has the best and most statistically significant potentiation of cisplatin efficacy amongst these treatment groups.

Figure 1.

PTUPB potentiates cisplatin antitumor 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., days 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 fold of baseline (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 with 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., days 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 1.

PTUPB potentiates cisplatin antitumor 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., days 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 fold of baseline (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 with 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., days 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.

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Even though PTUPB potentiated the antitumor efficacy of cisplatin, we did not observe any significant increase in toxicity. In the BL0293 model, compared with vehicle control, PTUPB monotherapy slightly decreased body weight (P = 0.086 at day 23; P = 0.118 at day 30) while cisplatin treatment led to significant weight loss (P < 0.001 at day 23; P = 0.008 at day 30). The addition of PTUPB to cisplatin therapy did not further increase weight loss (Supplementary Fig. S3A). We did not observe any significant weight loss in the BL0269 groups, regardless of the treatment (Supplementary Fig. S3B and S3C). We also determined complete blood cell count (CBC) and chemistry panels at days 6 and 20 of treatment (Supplementary Fig. S4). No significant difference in blood panel data was observed among all treatment groups compared with the controls. Histology examination of major organs at day 20 revealed cisplatin and combination treatment induced swollen distal tubule cells in kidneys, and cytoplasmic vacuolization (microvesicular steatosis) in hepatocytes. Although these changes were consistent with cisplatin toxicity, they were modest and could be due to normal variations in tissue morphology. However, no such morphology changes were observed in the control and PTUPB monotherapy groups, suggesting that the changes were caused by cisplatin. No other histological changes were observed in other organs (Supplementary Fig. S5).

Combination treatment of cisplatin and PTUPB induced apoptosis but inhibited proliferation and angiogenesis in bladder cancer PDX

Ki-67 is a nuclear nonhistone protein that is preferentially expressed in dividing cells and is frequently used to assess the proliferation state of tissues. The determination of cleaved caspase 3 is commonly used as an indicator of apoptosis. CD31 is another maker being widely used to evaluate angiogenesis. The combination of cisplatin with PTUPB treatment led to a significant decrease of Ki-67 and CD31 expression and substantial increase of cleaved caspase-3 in stained BL0293 tumor tissues when compared with single treatment with PTUPB or cisplatin (Fig. 2 and Supplementary Fig. S6). These data demonstrate that the antitumor activity of the combination treatment with PTUPB and cisplatin was, at least in part, due to decreased cell proliferation and angiogenesis with increased apoptosis.

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 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 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 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.

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Combination treatment of cisplatin and PTUPB significantly reduced the activity of signaling pathways essential for cell growth

The MAPK/ERK and PI3K/AKT/mTOR signaling pathways are shared by many receptor tyrosine kinases and often essential for tumor growth and survival (Fig. 3A). To determine how the different treatments affected these two signaling pathways, tumor tissues were collected at day 3 after treatment started, and at day 17 when tumors started to regrow in the PTUPB and cisplatin groups or were stabilized as in the combination group. While treatment with either PTUPB alone or cisplatin alone did not significantly diminish levels of either phosphorylated activated ERK (p-ERK) or AKT (p-AKT), the combination treatment of PTUPB and cisplatin substantially decreased levels of both p-ERK and p-AKT at day 3. On day 17, increased levels of p-ERK and p-AKT were observed in the PTUPB and cisplatin combination group (Fig. 3B). These data confirmed that combined therapy suppressed bladder cancer growth, at least in part, through these two pathways, while pathway reactivation was associated with tumor adaptation and regrowth.

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 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.

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PTUPB did not alter platinum–DNA adduct formation

As alkylating agents, platinum-based drugs (including cisplatin and carboplatin) kill cancer cells through formation of covalent drug–DNA adducts. We determined whether PTUPB potentiated the antitumor activity of platinum agents via increasing DNA adducts by using [14C]carboplatin-DNA adducts as a surrogate marker that is amenable to AMS analysis. AMS is ultrasensitive for quantification of 14C in the biological sample and was used to measure carboplatin–DNA adduct formation under physiologically relevant drug concentrations (30). Because cisplatin does not have any carbon atoms in the molecule, it cannot be labeled with 14C. Because both cisplatin and carboplatin form the same therapeutically relevant drug–DNA diadducts and share a similar resistance spectrum (31), we used [14C]carboplatin for this part of the study.

First, we determined the effect of PTUPB on carboplatin–DNA adduct formation in cell culture with the bladder cancer cell line 5637 (32). Cultures of 5637 cells were treated with either carboplatin (100 μmol/L) alone or a combination of carboplatin (100 μmol/L) and PTUPB (10 μmol/L). The 100 μmol/L concentration of carboplatin was used based on its maximum blood concentration in patients after chemotherapy and the treatment duration of 4 hours was chosen to simulate carboplatin plasma half-life of 1.5–6.0 hours in patients. PTUPB exposure did not significantly alter platinum-DNA adduct formation after 4 h (528 ± 41 adducts per 108 nt with the carboplatin alone versus 593 ± 282 adducts per 108 nt with the combination treatment, P = 0.713) (Fig. 4A). Similarly, pretreatment of cells with 10 μmol/L PTUPB for 5 hours followed by the addition of carboplatin did not alter the carboplatin induced DNA adduct formation (706 ± 26 adducts per 108 nt with the carboplatin alone versus 606 ± 66 adducts per 108 nt with the PTUPB pretreatment (P = 0.071; Fig. 4B). Clearly, PTUPB did not impact drug–target binding and metabolism of carboplatin in cell culture.

Figure 4.

PTUPB does not alter carboplatin–DNA adduct levels. A, Cultures of the ATCC bladder cancer cell line 5637 were incubated with 100 μmol/L [14C]carboplatin in the presence (gray bar) or absence (white bar) of 10 μmol/L PTUPB for either 4 hours or 4 hours then washed and further incubated 20 hours with fresh drug-free culture medium. B, 5637 cells were pretreated (gray bar) with 10 μmol/L PTUPB for 5 hours before cells were exposed to 100 μmol/L [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 i.v. bolus, and tissue was harvested after 24 hours. PTUPB (30 mg/kg in PEG400) was administered via oral gavage 16 hours (gray bar) or 1 hour (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 4.

PTUPB does not alter carboplatin–DNA adduct levels. A, Cultures of the ATCC bladder cancer cell line 5637 were incubated with 100 μmol/L [14C]carboplatin in the presence (gray bar) or absence (white bar) of 10 μmol/L PTUPB for either 4 hours or 4 hours then washed and further incubated 20 hours with fresh drug-free culture medium. B, 5637 cells were pretreated (gray bar) with 10 μmol/L PTUPB for 5 hours before cells were exposed to 100 μmol/L [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 i.v. bolus, and tissue was harvested after 24 hours. PTUPB (30 mg/kg in PEG400) was administered via oral gavage 16 hours (gray bar) or 1 hour (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.

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We next determined whether PTUPB affected the repair of carboplatin–DNA adducts because increased DNA repair is one of the major mechanisms of cellular resistance to platinum-based cancer therapy. To perform this experiment, 5637 cell cultures were treated with carboplatin alone or with PTUPB plus carboplatin combination for 4 hours followed by removal of both drugs, washing and additional culture with drug-free medium for 20 hours. At 24 hours, the platinum–DNA adduct levels were not significantly different in the two treatment groups, suggesting no difference of DNA repair between two treatments (104.1 ± 17.7 vs. 90.8 ± 9.1 adducts/108 nt with the carboplatin alone or PTUPB plus carboplatin treatment; P = 0.312). The DNA repair rates were 21.2 ± 1.3 and 25.1 ± 8.1 adducts/108 nt per hour for the carboplatin alone and PTUPB plus carboplatin treatment groups (P = 0.648), suggesting no difference of DNA repair between these two treatments.

We also determined whether PTUPB influenced carboplatin–DNA adduct levels in vivo (Fig. 4C). PTUPB was administered either 16 hours or 1 hour before carboplatin injection, and tumors were collected 24 hours after carboplatin treatment. Carboplatin–DNA adduct levels from isolated tumor DNA showed no significant difference between tumors that were treated with carboplatin alone, 16 hours of PTUPB (P = 0.856) or 1 hour PTUPB (P = 0.362) pretreatment (1070 ± 317 adducts/108 nt, 1,019 ± 434 adducts/108 nt, and 1,334 ± 384 adducts/108 nt, respectively). The in vivo data are fully consistent with the cell line data, and support PTUPB having a fully orthogonal mechanism of action compared with carboplatin and likely cisplatin.

PTUPB and the platinum drug cisplatin showed modest synergistic drug–drug interaction

Because we showed PTUPB potentiated the antitumor effect of cisplatin in vivo in bladder PDX models, we wanted to further study the mechanism of the combination effect of these two drugs in vitro. To address this question, the combination index (CI) method (27) was used to determine the drug–drug interaction of PTUPB and cisplatin. First, we determined the effect of single-drug treatment on 5637 bladder cancer cells (Fig. 5A). Cultures of 5637 cells were treated with increasing concentrations of PTUPB or cisplatin (0, 0.01, 0.1, 1, 2, 5, 10, 20, 50, 100 μmol/L). The IC50 of cisplatin and PTUPB on 5637 cells are 4.5 μmol/L and 90.4 μmol/L, respectively. Next, we determined the combination drug effect of PTUPB and cisplatin (Fig. 5B). 5637 cells were treated with different concentrations of cisplatin (0, 0.01 0.1, 0.5, 1, 2, 5, 10, 100 μmol/L) in combination with different concentrations of PTUPB (1, 2, 5, 10 μmol/L). The CI values of cisplatin and PTUPB were calculated indicating that PTUPB at concentrations of 1, 2, 5, and 10 μmol/L showed modest synergistic effects in combination with cisplatin at 5 μmol/L. In addition to the 5637 cell line, we also assessed the cytotoxicity of PTUPB and cisplatin in other human bladder cancer cell lines T24, J82, TCCSUP. A modest cisplatin potentiation was observed only in the 5637 cell line but not J82, T24 and TCCSUP cell lines (Supplementary Fig. S7). Low or no direct effects on these cell lines is not surprising since we now know that the mechanism of action for PTUPB is predominantly antiangiogenesis (ref. 20; Fig. 2).

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 72-hour 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 μmol/L). 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 μmol/L) in combination with different concentrations of PTUPB (1, 2, 5, and 10 μmol/L). Sample size for the cell line experiments was N = 3. *,CI, combination index. 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 72-hour 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 μmol/L). 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 μmol/L) in combination with different concentrations of PTUPB (1, 2, 5, and 10 μmol/L). Sample size for the cell line experiments was N = 3. *,CI, combination index. The results are expressed as mean ± SD.

Close modal

Molecular correlative studies of COX-2/sEH inhibitor PTUPB

To test whether PTUPB targets COX-2/sEH and show that inhibition of COX-2 and sEH pathways is involved in the mode of action of PTUPB in vivo, we analyzed oxylipin profiles using LC-tandem MS-based lipidomics (33). PTUPB treatment reduced the levels of COX-dependent prostaglandins PGE2, PGD2, TXB2, 6-keto-PGF in BL0269 tumors by ∼50% (P < 0.05), indicating that PTUPB inhibited the COX-2 pathway in vivo (Fig. 6A). For the sEH-dependent metabolites, PTUPB treatment caused an approximately 2-fold increase of 12,13-EpOME, and about a 2-fold decrease on the corresponding diol metabolite 12,13-DiHOME. PTUPB also caused an approximately 2-fold increase of 10,11-EpDPE, 15,16-EpODE in BL0269 tumors, whereas it had no effect on the corresponding diol metabolites 10,11-DiHDPE and 15,16-DiHODE (Fig. 6B). These results indicate that PTUPB inhibited both the COX-2 and sEH pathways in tumor tissue. The lipid mediators from other pathways were not significantly changed (Supplementary Table S1). Together, these data support that PTUPB inhibits both COX-2 and sEH, although it may have effects on other cellular targets.

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 on the 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 the sEH pathway. The results are expressed as mean ± SD. *, P < 0.05.

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 on the 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 the sEH pathway. The results are expressed as mean ± SD. *, P < 0.05.

Close modal

As a dual inhibitor of COX-2 and sEH, PTUPB potentiated the antitumor activity of cisplatin without increasing the toxicity in mice bearing bladder cancer PDXs. We also performed experiments to examine the efficacy of combination treatments of celecoxib plus cisplatin or t-AUCB plus cisplatin in a bladder PDX model. We did not observe any potentiation of cisplatin by celecoxib with respect to inhibiting tumor growth. It was reported by Kurtova and colleagues that blocking tumor expression of PGE2 with celecoxib modulates tumor repopulation after several cycles and abrogates bladder cancer chemoresistance (34). However, these results are not contradictory with our studies. Kurtova and colleagues used a single PDX from a GC-resistant patient (which was paradoxically quite responsive to GC in the mouse); and a different dosing regimen showing that celecoxib did not have a pronounced effect on GC response in their PDX model until the fourth cycle of GC treatment. Our protocol only had two cycles of GC and was not designed to assess long-term tumor repopulation by cancer stem cells.

We not only showed that PTUPB enhanced cisplatin and GC efficacy, but also began to define the underlying mechanisms of potentiation. The increased efficacy was not due to increased PTUPB–DNA adduct formation. We gathered evidence that the potentiation is possibly due to in vivo factors, such as angiogenesis, and reduced activation of proliferation signaling including the AKT and ERK signaling pathways. Treatment with cisplatin and PTUPB in vivo decreased the levels of both p-ERK and p-AKT in tumor tissues, suggesting that these two major signaling pathways were downregulated. We previously reported the evidence of antiangiogenic properties of PTUPB (20).

PTUPB has the potential for improving platinum-based chemotherapy in the clinic. Even though targeted therapy and immunotherapy have emerged as promising therapeutic modalities, cytotoxic chemotherapy will still be the mainstay in the foreseeable future. For example, targeted and immunotherapies currently benefit only a minority of patients with non–small cell lung and bladder cancers. The response rate of immunotherapy in both cancers is approximately 20% or less (35, 36).

In conclusion, the COX2/sEH dual inhibitor PTUPB potentiates cisplatin and GC, possibly synergistically, in bladder cancer PDXs in vivo without increasing toxicity. PTUPB and cisplatin treatment increases apoptosis and decreases the activity of the AKT and ERK pathways, but does not increase the formation of platinum–DNA adducts, the most critical step of platinum-induced cell death.

P.T. Henderson has ownership interest (including patents) in Oomni Inc. No potential conflicts of interest were disclosed by the other authors.

Conception and design: H. Zhang, A.-H. Ma, S.H. Hwang, K.W. Turteltaub, R.W. de Vere White, C.-X. Pan

Development of methodology: H. Zhang, A.-H. Ma, S.H. Hwang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): F. Wang, H. Zhang, A.-H. Ma, W. Yu, M. Zimmermann, J. Yang, T.-y. Lin, M. Malfatti, S. Airhart, C.-X. Pan

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): F. Wang, H. Zhang, A.-H. Ma, W. Yu, M. Zimmermann, J. Yang, T.-y. Lin, M. Malfatti, R.W. de Vere White, C.-X. Pan

Writing, review, and/or revision of the manuscript: F. Wang, H. Zhang, A.-H. Ma, M. Zimmermann, J. Yang, S.H. Hwang, T.-y. Lin, M. Malfatti, K.W. Turteltaub, P.T. Henderson, S. Airhart, B.D. Hammock, R.W. de Vere White, C.-X. Pan

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.-H. Ma, W. Yu, D. Zhu, B.D. Hammock, R.W. de Vere White, C.-X. Pan

Study supervision: T.-y. Lin, K.W. Turteltaub, P.T. Henderson, J. Yuan, C.-X. Pan

Other (provided compound used in tests, carried out analytical chemistry): B.D. Hammock

We would like to thank Dr. Mingyi Chen for providing pathological diagnosis of PDX mice tissue samples; Tsung-Chieh Shih for technical assistance in IHC staining; Christopher Morisseau, Dipak Panigrhy, and Ted Ognibene for technical support in conducting the experiments; and George Cimino for help during manuscript preparation. This work was supported in part by Merit Review (Award # I01 BX001784, C.-X. Pan) from the United States (U.S.) Department of Veterans Affairs Biomedical Laboratory Research and Development Program (The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government); NCI Cancer Center Support Grant (PI: de Veer White; Grant #: 2 P30 CA 0933730); The Laney Foundation (PI: de Veer White); American Cancer Society Institutional Research Grant (PI: Lin); NIEHS grant R01 ES002710, NIEHS Superfund Research Program grant P42 ES04699, SBIR Phase II contract HHSN261201200048C (P.T. Henderson); and NIDDK grant R01 DK103616, National Institute of Neurological Disorders and Stroke (NINDS) U54 NS079202.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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