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Molecular Cancer Therapeutics
Molecular Cancer Therapeutics
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Small Molecule Therapeutics

Addition of DHA Synergistically Enhances the Efficacy of Regorafenib for Kidney Cancer Therapy

Jeffrey Kim, Arzu Ulu, Debin Wan, Jun Yang, Bruce D Hammock and Robert H. Weiss
Jeffrey Kim
1Division of Nephrology, Department of Internal Medicine, University of California, Davis, California.
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Arzu Ulu
1Division of Nephrology, Department of Internal Medicine, University of California, Davis, California.
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Debin Wan
2Department of Entomology, University of California, Davis, California.
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Jun Yang
2Department of Entomology, University of California, Davis, California.
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Bruce D Hammock
2Department of Entomology, University of California, Davis, California.
3Cancer Center, University of California, Davis, California.
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Robert H. Weiss
1Division of Nephrology, Department of Internal Medicine, University of California, Davis, California.
3Cancer Center, University of California, Davis, California.
4Medical Service, Sacramento VA Medical Center, Sacramento, California.
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  • For correspondence: rhweiss@ucdavis.edu
DOI: 10.1158/1535-7163.MCT-15-0847 Published May 2016
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Abstract

Kidney cancer is the sixth most common cancer in the United States, and its incidence is increasing. The treatment of this malignancy took a major step forward with the recent introduction of targeted therapeutics, such as kinase inhibitors. Unfortunately, kinase inhibition is associated with the onset of resistance after 1 to 2 years of treatment. Regorafenib, like many multikinase inhibitors, was designed to block the activities of several key kinase pathways involved in oncogenesis (Ras/Raf/MEK/ERK) and tumor angiogenesis (VEGF-receptors), and we have recently shown that it also possesses soluble epoxide hydrolase (sEH) inhibitory activity, which may be contributing to its salutary effects in patients. Because sEH inhibition results in increases in the DHA-derived epoxydocosapentaenoic acids that we have previously described to possess anticancer properties, we asked whether the addition of DHA to a therapeutic regimen in the presence of regorafenib would enhance its beneficial effects in vivo. We now show that the combination of regorafenib and DHA results in a synergistic effect upon tumor invasiveness as well as p-VEGFR attenuation. In addition, this combination showed a reduction in tumor weights, greater than each agent alone, in a mouse xenograft model of human renal cell carcinoma (RCC), yielding the expected oxylipin profiles; these data were supported in several RCC cell lines that showed similar results in vitro. Because DHA is the predominant component of fish oil, our data suggest that this nontoxic dietary supplement could be administered with regorafenib during therapy for advanced RCC and could be the basis of a clinical trial. Mol Cancer Ther; 15(5); 890–8. ©2016 AACR.

Introduction

Renal cell carcinoma (RCC) arises from the renal tubular epithelium (1, 2), is the most common malignancy of the kidney, and is the sixth most common cancer in the United States. In contrast to many other cancers, the incidence of RCC is increasing likely due to smoking as well as the increased prevalence of the metabolic syndrome in the Western world (3–7). When localized to the kidney, surgical resection is usually curative; however, once the cancer metastasizes, the survival statistics, even with currently available novel therapies, are dismal. Among nonsurgical treatments of RCC, the immune modulators were historically associated with a very low success rate (8), likely related to immune suppression in the tumor microenvironment, possibly through local generation of tryptophan metabolites (9, 10). Enter the era of targeted therapeutics, which has resulted in the discovery of drugs possessing antiangiogenic activity via abrogation of vascular endothelial growth factor (VEGF) and other tyrosine kinase receptor signaling pathways involved in tumor growth and angiogenesis (11–14). However, while these approaches represented a major advance in the field, they are unfortunately associated with a high level of resistance after 1 to 2 years of treatment (15, 16), and furthermore, some are linked with a troublingly high rate of systemic hypertension (17). Therefore, novel approaches are urgently needed to improve the efficacy of these drugs. In the current study, we examined the use of the tyrosine kinase inhibitors in combination with compounds that we hypothesized would attenuate tumor resistance.

Regorafenib is a second-generation multikinase inhibitor that blocks the activity of kinases involved in the regulation of oncogenesis (Ras/Raf/MEK/ERK) and tumor angiogenesis (VEGF-R1, VEGF-R2, and VEGF-R3; ref.13). This drug is a marked improvement over the first-generation compounds (e.g., sorafenib) due to its higher specific activity leading to greater pharmacologic potency (13). The antitumor activity of regorafenib has been demonstrated in a variety of preclinical models and is associated with its kinase inhibitory effects, which results in suppression of cell proliferation, induction of apoptosis, and inhibition of tumor angiogenesis (13, 18, 19), the last being a key area of investigation for therapies of highly angiogenic RCC (20). We have recently shown that these multikinase inhibitors block soluble epoxide hydrolase (sEH; ref. 21), a key enzyme that metabolizes bioactive lipids of inflammation (22). Because inhibition of sEH stabilizes these lipids, thereby prolonging their beneficial effects on angiogenesis and inflammation, we asked whether it is possible to capitalize on this enzymatic activity to enhance the salutary effects of these specific kinase inhibitors in RCC.

sEH hydrolyzes epoxygenated fatty acids generated by the P450 metabolism of omega-3 and omega-6 polyunsaturated fatty acids (PUFA). Among these PUFAs, sEH metabolizes epoxyeicosatrienoic acids (EET), which are P450 products of arachidonic acid (ARA), and epoxydocosapentaenoic acid (EDPs), which are also P450 products but derive from docosahexaenoic acid (DHA), to their less bioactive diols (diols of EETs and EDPs, dihydroxyeicosatrienoic acids, DHETs) and dihydroxydocosapentaenoic acids, DiHDPEs, respectively; Fig. 1; ref. 23). While EETs possess anti-inflammatory (24) and antihypertensive (25) properties, they have been shown to be proangiogenic (26–28), a property that can clearly be detrimental in the treatment of highly angiogenic tumors, such as RCC. In addition, recent studies have suggested that EETs can promote the progression of cancer (29, 30), while other studies have contradicted these findings (31). In contrast, EDPs, which are also stabilized by sEH inhibitors (Fig. 1), have the opposite effect on angiogenesis (32); hence, we focus on the DHA metabolites of sEH in this study.

Figure 1.
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Figure 1.

EET and EDP synthesis occurs through cytochrome P450 family enzymes naturally via oxidation of unsaturated bonds of precursor fatty acids. The EET and EDP epoxygenated products are degraded by sEH into their respective diols. Inhibition of sEH by regorafenib or other means results in accumulation of epoxygenase metabolites.

We hypothesized that the sEH inhibitory activity of regorafenib will result in marked increases in the anti-angiogenic and anti-hypertensive EDPs, which will be enhanced in the presence of exogenously administered DHA, usually the most abundant component of dietary fish oil supplements. We now show that the combination of DHA and regorafenib causes a decrease in HuVEC cell invasion as a measure of tumor angiogenesis as well as synergistically decreasing cell viability across three human RCC lines. Furthermore, by using a xenograft model of RCC in athymic nude mice, we demonstrate a decrease in tumor mass in vivo associated with the expected target effects and plasma oxylipin changes. Thus, once validated in human studies, novel therapy based on the addition of the dietary supplement DHA to regorafenib has the potential to result in an enhanced therapeutic efficacy of this kinase inhibitor for treatment of advanced RCC.

Materials and Methods

Cell culture

Human umbilical vein endothelial cells (HuVEC; Lonza) were grown in endothelial basal medium (EBM-2) supplemented with growth factors. The RCC cell lines 786-O(VHL−/−), Caki-1(VHL+/+), and Renca (VHL+/+) were obtained from the American Type Culture Collection and the Renal proximal tubule epithelial cells (RPTEC or “normal human kidney, NHK”) were a primary (i.e., non-immortalized) line acquired from Lonza, which were cultured in renal epithelial cell growth medium (REGM; Lonza). All ATCC and Lonza cell lines undergo extensive authentication tests during the accessioning process as described on their Web site; in addition, all cells were frequently tested for mycoplasma in the author's laboratory. The 786–0 and Caki-1 and Renca cells were maintained in RPMI, and NHK cells were grown and cultured in DMEM, both supplemented with 10% FBS, 100 units/mL streptomycin, and 100 mg/mL penicillin. Cells were maintained at 5% CO2 and at 37°C. All cell lines were used with a passage number of two and confirmed to be free of mycoplasma, per monthly laboratory testing.

Animals and treatments

All animal studies were approved by the University of California Davis Animal Use and Care Committee and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Thirty-six 4-week-old male athymic nude Nu/Nu mice (Harlan Laboratories) were acclimated to housing conditions for 1 week and were kept under a 12-h light–dark cycle with free access to water and food for the duration of the experiment.

Subsequently, mice were injected a suspension containing 786-O cells at 0.5 × 106 mixed in 30% of non-growth factor reduced Matrigel (Corning Inc.) subcutaneously in the flank region as previously described (33). Tumor growth was monitored twice a week for each mouse using a digital caliper. Tumor volume (mm3) was calculated as length*(width2/2). When tumor volume reached approximately 100 mm3 (around 3–4 weeks of inoculation), treatments and diets began.

Mice were randomly divided into two experimental dietary groups: control diet (5% corn oil) or a 1% DHA-enriched diet (17.5 g DHA and 52.5 g corn oil/kg). DHA ethyl ester replaced corn oil to retain equal dietary fat between both isocaloric diets. The detailed composition of the diets is described in Supplementary Table S1. Half of the mice in each dietary group were given a daily administration of either 10 mg/kg regorafenib or vehicle (PEG400/125 mmol/L aqueous methanesulfonic acid; 80/20) via oral gavage. Treatments continued for 3 weeks. Body weights and tumor sizes were measured every 2 days. At the end of the experiment, plasma and tissues were harvested for immunohistochemistry and oxylipin analysis.

Endothelial cell invasion assay

HuVECs were grown in 24-well plates containing Transwell inserts of 8-μm pore polyvinylpyrrolidone-free polycarbonate filters coated with Matrigel on the upper compartment at a density of 1×105 cells in EBM-2 media containing 0.1% BSA (34). EBM-2 media consisting of 10% BSA were added in the bottom compartment of the well as a chemoattractant. Both upper and lower chambers contained one of the following treatments: 1 μmol/L ARA, 1 μmol/L DHA, 1 mol/L DHA plus 1 μmol/L regorafenib, 1 μmol/L regorafenib, 1 μmol/L linoleic acid (LA) or DMSO. Cells were incubated at 37°C for 20 hours to allow for migration. Afterward, Transwells containing cells were washed in PBS, fixed in 5% glutaraldehyde, and stained with 0.5% Toluidine Blue. Next, the upper wells were gently scraped to allow for imaging and quantification of cells that had migrated toward the lower compartment of the Transwell inserts.

MTT assay

Cell viability was assayed by plating cells in 96-well plates at a density of 3 × 103 cells. After 24 hours, NHK, 786-0, Caki-1, and Renca cells were treated with 1 μmol/L of the fatty acids LA, ARA, eicosapentaenoic acid (EPA), and DHA each with the presence or absence of 1 μmol/L regorafenib and DMSO control. After 24 hours of treatment, cells were quantified via hemocytometer and treated with media containing MTT solution (1 mg/mL thiazolyl blue tetrazolium bromide) for 3 hours. Afterward, the MTT solution was removed, and the blue crystalline precipitate internalized by the cells were dissolved with DMSO. Finally, plates were placed in a plate reader to measure visible absorbance at 570 nm.

Immunoblotting

HuVECs were grown at a density of 2 × 105 cells in six-well plates. After serum starvation for 6 hours in EBM-2 media containing 0.1% bovine serum albumin (BSA), cells were treated with 1 μmol/L omega-6 LA, 1 μmol/L LA + regorafenib, 1 μmol/L DHA, or 1 μmol/L DHA + 1 μmol/L regorafenib for 24 hours. Cells were then lysed, and total cell lysates were analyzed for proteins of interest using antibodies against phosphorylated VEGFR-2 and β-actin (Cell Signaling Technology).

Immunoblotting of tumor tissue was performed as previously described (35). Briefly, after the indicated treatments, the tissues were washed with PBS, lysed, and subjected to immunoblotting. For the xenograft tissue tumors, proteins were extracted with T-PER. The membranes were blocked in 5% nonfat dry milk for 1 hour at room temperature, incubated with antibodies (β-actin, pVEGFR-2, VEGFR-2, pERK1/2, and ERK1/2), and then probed with HRP-tagged anti-mouse or anti-rabbit IgG antibodies. The signal was detected using ECL solutions (Thermo Fisher Scientific). Densitometry was performed using ImageJ software.

Oxylipin analysis

The quantitative profiling of oxylipin was carried out as previously described (36). Briefly, plasma samples were extracted using solid-phase extraction cartridges. Samples were eluted through the cartridges, dried, and then reconstituted by adding 200 nmol/L 1-cyclohexyl-dodecanoic acid urea (CUDA) methanol solution. Oxylipins were then detected using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The optimized conditions of chromatographic separation have been reported previously (37) as have the instrument parameters, including MRM transitions (ref. 36; Applied Biosystems, 4000 QTRAP tandem mass spectrometer).

Statistical analysis and synergy calculations

All data were analyzed for significance in SAS version 9.3 (SAS Institute Inc.). Cell numbers from invasion assay, tumor weights, oxylipin quantification, and tumor volumes were analyzed for significance by one-way ANOVA at P < 0.05. Where significant differences were found, a Tukey multiple comparison test was performed at a probability of α = 0.05. The data are presented as means ± SEM. Different letters appearing above bars in bar graphs designate that significant differences were found, while bars sharing the same letter indicate that significance was not achieved. Bars having two letters (such as ‘bc’) indicate that significance was not achieved compared with group ‘b’ or group ‘c’.

Synergy was assessed by calculating the combination index (CI) values using CalcuSyn software, which provides a quantitative definition for additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1) in combination treatments.

Results and Discussion

Coadministration of regorafenib and DHA suppresses vascular endothelial cell invasion and is associated with attenuated angiogenesis markers

Because the addition of DHA in the presence of sEH inhibition provided by regorafenib would be expected to increase local EDP levels (Fig. 1) and thereby attenuate angiogenesis (32), we first evaluated this property in an in vitro model of angiogenesis (38, 39). Because we previously reported an increase in HuVEC proliferation and infiltration when treated with EETs, specifically 11,12-EET and 14,15-EET, which are generated from ARA (32), we utilized the omega-6 PUFA LA, the predominant PUFA found in corn oil, as an additional control. HuVEC were grown on matrigel in Transwell plates, in which cells that infiltrated the matrigel were enumerated in order to assay for invasive potential (see Materials and Methods). After treatment with 1 μmol/L ARA, 1 μmol/L DHA, 1 μmol/L DHA + 1 μmol/L regorafenib, 1 μmol/L regorafenib, 1 μmol/L LA or DMSO for 20 hours, invading HuVEC were imaged (Fig. 2A) and quantitated (Fig. 2B). The cells treated concurrently with DHA and regorafenib were found to be the least invasive of all conditions tested, with a reduction of ∼60% compared with DMSO control. This combination likely resulted in a higher amount of EDPs, which comes about with a high availability of DHA in concert with the inhibition of sEH afforded by regorafenib. To confirm target inhibition by regorafenib, we evaluated its kinase activity on the phosphorylated (kinase-active) form of VEGFR-2 (40) and showed that pVEGFR-2 expression was lower in cells treated with regorafenib after both LA and DHA treatments alone, but more pronounced with the combination (Fig. 2C), thereby confirming target engagement with regorafenib. Regorafenib alone demonstrated a nonsignificant decrease in VEGFR-2 expression (data not shown).

Figure 2.
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Figure 2.

Endothelial cell invasive potential was assayed using an in vitro Matrigel model with HuVECs. A, HuVECs were grown on Matrigel and treated with 1 μmol/L LA, 1 μmol/L ARA, 1 μmol/L DHA, 1 μmol/L DHA + 1 μmol/L regorafenib, 1 μmol/L regorafenib, or DMSO for 20 hours. The cells transiting the Matrigel were photographed (see Materials and Methods). B, quantification of cells was performed by counting the cells in wells (n = 3 for each treatment, repeated in triplicates). C, in a separate parallel experiment, HuVEC cells were grown to confluence and treated with the indicated compounds for 24 hours and immunoblotted for pVEGFR-2 and β-actin as a loading control. VEGFR-2 was also immunoblotted under the same conditions in a separate blot. These experiments were each repeated at least three times. Bars in graphs indicate mean ± SEM. Different letters above bars indicate statistical difference among groups (P < 0.05). ARA, arachidonic acid.

These results are consistent with previous data showing that EDPs inhibit VEGF-induced cell migration in HuVEC after being treated with 19,20-EDP (32). Further, these findings demonstrate a synergistic effect of DHA and regorafenib on endothelial cells to suppress angiogenesis, primarily via suppression of endothelial cell migration likely via high levels of EDP.

The combination of regorafenib and DHA synergistically decreases survival of kidney cancer cells in vitro

We next assessed cell viability in vivo utilizing two human kidney cancer lines (786-0 and Caki-1) and the mouse kidney cancer cell line Renca, as well as primary (non-immortalized) normal human kidney epithelial (NHK) cells as controls. All cells were treated with 1 μmol/L of the fatty acids LA, ARA, EPA, and DHA each, in the presence or absence of 1 μmol/L regorafenib and DMSO control. LA, which is the major polyunsaturated fatty acid comprising corn oil, served as the in vitro control treatment that would best mimic the conditions of corn oil administration in vivo such that the two experiments could be compared. EPA was used to discern if the mitigation of cell viability was due to an omega-3 effect or specifically to DHA. After 24 hours of treatment, both regorafenib + DHA and DHA alone decreased cell viability in all three of the cancer lines with no significant effect on NHK cells; however, a greater decrease in cell viability was found with the former treatment (Fig. 3). Furthermore, cells were quantified from an experiment performed in parallel to the MTT assay on the four cell types (inset, Fig. 3); these data demonstrate that the combination of regorafenib with DHA resulted in synergistic responses after 24 hours of incubation. The therapeutic efficacy was assessed by calculating CI values using CalcuSyn software (41). Analysis of combination therapeutic indexes revealed synergistic effects by demonstrating the CI values in the range of 0.61 to 0.85 (synergy defined as CI <1) with the combination of regorafenib and DHA alone among the three RCC lines. Antagonistic interactions (CI>1) were found with LA and ARA with CI calculations 1.14 and 1.23, respectively. These findings demonstrate that the combination of regorafenib and DHA produced a synergistic decrease in several RCC, but not in normal renal epithelial cell, viability.

Figure 3.
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Figure 3.

Cell viability was assayed via MTT in NHK, 786-0, Caki-1, and Renca cells. NHK, 786-0, Caki-1, and Renca cells were treated with 1 μmol/L of the fatty acids LA, ARA, EPA, and DHA each in the presence or absence of 1 μmol/L regorafenib and DMSO control. After 24 hours of treatment, an MTT assay was performed, and cells were counted via hemocytometer. The DHA and regorafenib CI was calculated using CalcuSyn software as discussed in Materials and Methods. These experiments were each repeated at least three times. Bars in the graph indicate mean ± SEM. *, statistical difference compared with DMSO treatment of the identical cell line (P < 0.05).

The combination of regorafenib and DHA decreases tumor growth in vivo

In light of previous data from one of our laboratories demonstrating that treatment with EDP concurrently with sEH inhibition attenuated both tumor growth and angiogenesis (32), we next asked whether the concurrent addition of regorafenib and DHA synergizes in an in vivo xenograft model of human RCC using the 786-0 (VHL−/−) human RCC cell line used in several previous studies (40, 42, 43). Male athymic Nu/Nu mice were started on the diets and pharmacologic treatments after the 786-0 xenografts achieved a volume of ∼100 mm3. The mice were given free access to either a diet with fat originating from corn oil, which is naturally high in the omega-6 PUFA LA, or a 1% enriched DHA diet. The DHA concentration in the diet was determined by metabolic body size using an average daily food intake of 5 g/day/mouse, which translates to ∼3.1 g/day of DHA in a 70-kg human. This amount is achievable through consuming fish oil supplementation and in fact has been recommended to decrease progression in IgA nephropathy, a common renal disease (44).

Mice were given either regorafenib (10 mg/kg/day) or vehicle control administered by oral gavage. Tumors and terminal plasma were collected after 18 days of intervention for immunoblot and oxylipin analysis, respectively. There was no significant difference between treatment groups in body weights after 18 days, indicating a lack of general toxicity (Fig. 4A); tumor weights (Fig. 4B) and volume (Supplementary Fig. S1) were found to be the smallest in the mice treated with regorafenib while ingesting the DHA diet (∼1.9-fold decrease) and there was a synergistic decrease of the combination as compared with DHA or regorafenib administered alone.

Figure 4.
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Figure 4.

The combination of regorafenib and DHA reduces tumor weight with the expected on-target effects. Male athymic Nu/Nu mice (n = 8 per group) were transplanted with 786-0 cells and subsequently administered either a corn oil or DHA-enriched diet (see Materials and Methods). Where indicated, mice were given 10 mg/kg of regorafenib or vehicle administered by oral gavage daily for 18 days. A, body weights were measured at the indicated times. B, tumor weights were determined. C, terminal tumors were immunoblotted with pVEGFR-2, pERK1/2, or ERK1/2, and β-actin in was used as a loading control. Lines and bars in graphs indicate mean ± SEM. Different letters above bars indicate statistical difference among groups (P < 0.05).

To evaluate the target effects of regorafenib in the xenografted animals, we evaluated the MAPK and VEGFR pathways, which are known receptor tyrosine kinase targets (13). Immunoblotting of the tumors for pVEGFR-2 demonstrated the most dramatic reduction in the tumors from the DHA+regorafenib-treated mice with minimal effects upon these proteins in the other animals (Fig. 4C), indicating that regorafenib attenuates the active forms of both MAPK and VEGFR species, consistent with the HuVEC data (see Fig. 2C). Because we have previously shown an sEH inhibitory effect of regorafenib similar to sorafenib (45), the influence of regorafenib and DHA in the in vivo model is likely specific to this combination.

The DHA diet resulted in an increase in all CYP450 metabolites of DHA in murine plasma

While the circulating plasma oxylipin profile can suggest the mechanism of the observations, these data do not always correlate with what is occurring at the local (i.e., tissue) level (46). The EDP species are rapidly metabolized to their diol constituents due to the actions of sEH; however, the inhibitory actions on this catabolic enzyme from an sEH-inhibitor, as we have shown for sorafenib (21, 47), were evident in the plasma (32). Terminal plasma oxylipin analysis showed the expected higher levels of 7(8)-EDP, 10(11)-EDP, 13(14)-EDP, 16(17)-EDP, and 19(20)-EDP in mice treated with the DHA diet compared with the corn oil diet groups (Fig. 5A). An increase in the corresponding diols was also observed as 10(11)-DiHDPE, 13(14)-DiHDPE, 16(17)-DiHDPE, and 19(20)-DiHDPE in the DHA-fed mice (Fig. 5B). The production of these diols was anticipated due to the enriched dietary DHA.

Figure 5.
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Figure 5.

Plasma oxylipin analysis shows increased levels of EDPs in mice fed with the DHA diet compared with the other mice. Oxylipins were measured by HPLC-ESI-MS/MS on terminal plasma. A, EDPs. B, DHA-derived diols. C, the epoxide-to-diol ratio as a measure of sEH inhibition. Bars in graphs indicate mean ± SEM. Different letters above bars indicate statistical difference among groups (P < 0.05).

To assess in vivo sEH inhibition, we examined the ratio of epoxide to their corresponding diol products in the plasma. The sum epoxide-to-diol ratio was found to be ∼2.2-fold in the corn oil diet + regorafenib treatment group compared with the corn oil diet alone, with greatest difference being ∼3.6-fold increase found in the 16(17)-EDP-to-16(17)-DiHDPE (Fig. 5C). Surprisingly, the epoxide-to-diol ratio in the plasma of the DHA-fed mice did not reflect sEH inhibition as the concentrations were found to be about the same for all of the measured species, and even lower in the 10(11)-EDP-to-10(11)-DiHDPE and 13(14)-EDP-to-13(14)-DiHDPE. This was also observed in an earlier experiment performed with sorafenib rather than regorafenib treatments (data not shown). Recently, it has been identified that the omega-3–derived EDPs are turned over more rapidly than the corresponding omega-6–derived EETs as sEH has a preference for these DHA-derived epoxygenated metabolites (47). Thus, it is conceivable that due to this preference in substrate and the abundance of EDP in the blood, sEH enzyme levels may be upregulated and higher in the DHA-diet fed mice, resulting in a greater amount of epoxide turnover to diols, leading to a decrease in the epoxide-to-diol ratio in the plasma, although this may not be representative of tissue. Future investigations may elucidate this observation by measuring circulating EET and EDP concentrations or measuring oxylipins in other tissues.

Conclusion

We have shown that combination treatment of DHA with regorafenib results in a synergistic efficacy over regorafenib or DHA alone in inhibiting growth in an in vivo xenograft model of VHL-mut RCC. We further show that there is a decrease in markers of angiogenesis and that this growth inhibition is accompanied by the expected target effects. We provide evidence that tumor growth attenuation likely occurs as a result of increasing levels of EDPs due to the sEH inhibitory property of regorafenib (Fig. 6). Until a clinical trial is accomplished, patient use of the common and readily available dietary supplement, fish oil, can therefore be recommended in individuals undergoing regorafenib treatment for advanced RCC.

Figure 6.
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Figure 6.

Proposed mechanism of combining regorafenib treatment with dietary DHA to inhibit RCC growth. sEH inhibition by regorafenib reduces degradation of DHA-derived epoxides and leads to increased EDP bioavailability resulting in attenuation of angiogenesis and likely an increase in vasodilation (which was not measured in this study). The kinase inhibitor activity of regorafenib impedes VEGF and MAPK/Raf signaling, which likely further decreases angiogenesis and concomitant RCC tumor growth.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: J. Kim, A. Ulu, B.D. Hammock, R.H. Weiss

Development of methodology: J. Kim, A. Ulu, B.D. Hammock

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Kim, A. Ulu, J. Yang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Kim, A. Ulu, D. Wan, J. Yang, R.H. Weiss

Writing, review, and/or revision of the manuscript: J. Kim, A. Ulu, D. Wan, J. Yang, B.D. Hammock, R.H. Weiss

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Kim, D. Wan, B.D. Hammock, R.H. Weiss

Study supervision: J. Kim, R.H. Weiss

Other (worked with Dr. Weiss to develop original hypothesis based on previously published collaborative work): B.D. Hammock

Grant Support

This work was supported by NIH grants 1R01CA135401-01A1, 1R03CA181837-01, and 1R01DK082690-01A1, the Medical Service of the US Department of Veterans' Affairs, and Dialysis Clinics, Inc. (DCI; all to R.H. Weiss). Partial support was provided by NIEHS R01 ES002710 and NIEHS Superfund Program P42 ES004699 (to B.D. Hammock.)

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.

Footnotes

  • Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

  • Received October 19, 2015.
  • Revision received February 11, 2016.
  • Accepted February 22, 2016.
  • ©2016 American Association for Cancer Research.

References

  1. 1.↵
    1. Escudier B,
    2. Kataja V,
    3. Group EGW
    . Renal cell carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2010;21:v137–9.
    OpenUrlFREE Full Text
  2. 2.↵
    1. Cohen HT,
    2. McGovern FJ
    . Renal-cell carcinoma. N Engl J Med 2005;353:2477–90.
    OpenUrlCrossRefPubMed
  3. 3.↵
    1. Luo J,
    2. Margolis KL,
    3. Adami HO,
    4. Lopez AM,
    5. Lessin L,
    6. Ye W,
    7. et al.
    Body size, weight cycling, and risk of renal cell carcinoma among postmenopausal women: the Women's Health Initiative (United States). Am J Epidemiol 2007;166:752–9.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    1. Haggstrom C,
    2. Rapp K,
    3. Stocks T,
    4. Manjer J,
    5. Bjorge T,
    6. Ulmer H,
    7. et al.
    Metabolic factors associated with risk of renal cell carcinoma. PloS One 2013;8:e57475.
    OpenUrlCrossRefPubMed
  5. 5.↵
    1. Chow WH,
    2. Dong LM,
    3. Devesa SS
    . Epidemiology and risk factors for kidney cancer. Nat Rev Urol 2010;7:245–57.
    OpenUrlCrossRefPubMed
  6. 6.↵
    1. Hunt JD,
    2. van der Hel OL,
    3. McMillan GP,
    4. Boffetta P,
    5. Brennan P
    . Renal cell carcinoma in relation to cigarette smoking: meta-analysis of 24 studies. Int J Cancer 2005;114:101–8.
    OpenUrlCrossRefPubMed
  7. 7.↵
    1. Weiss RH,
    2. Lin PY
    . Kidney cancer: identification of novel targets for therapy. Kidney Int 2006;69:224–32.
    OpenUrlCrossRefPubMed
  8. 8.↵
    1. Belldegrun AS,
    2. Klatte T,
    3. Shuch B,
    4. LaRochelle JC,
    5. Miller DC,
    6. Said JW,
    7. et al.
    Cancer-specific survival outcomes among patients treated during the cytokine era of kidney cancer (1989–2005): a benchmark for emerging targeted cancer therapies. Cancer 2008;113:2457–63.
    OpenUrlCrossRefPubMed
  9. 9.↵
    1. Wettersten HI,
    2. Hakimi AA,
    3. Morin D,
    4. Bianchi C,
    5. Johnstone ME,
    6. Donohoe DR,
    7. et al.
    Grade-dependent metabolic reprogramming in kidney cancer revealed by combined proteomics and metabolomics analysis. Cancer Res 2015;75:2541–52.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Ganti S,
    2. Taylor SL,
    3. Abu Aboud O,
    4. Yang J,
    5. Evans C,
    6. Osier MV,
    7. et al.
    Kidney tumor biomarkers revealed by simultaneous multiple matrix metabolomics analysis. Cancer Res 2012;72:3471–9.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. Molina AM,
    2. Motzer RJ
    . Clinical practice guidelines for the treatment of metastatic renal cell carcinoma: today and tomorrow. Oncologist 2011;16:45–50.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    1. Hutson TE,
    2. Davis ID,
    3. Machiels JP,
    4. De Souza PL,
    5. Rottey S,
    6. Hong BF,
    7. et al.
    Efficacy and safety of pazopanib in patients with metastatic renal cell carcinoma. J Clin Oncol 2010;28:475–80.
    OpenUrlAbstract/FREE Full Text
  13. 13.↵
    1. Wilhelm SM,
    2. Dumas J,
    3. Adnane L,
    4. Lynch M,
    5. Carter CA,
    6. Schutz G,
    7. et al.
    Regorafenib (BAY 73–4506): a new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity. Int J Cancer 2011;129:245–55.
    OpenUrlCrossRefPubMed
  14. 14.↵
    1. Chang YS,
    2. Adnane J,
    3. Trail PA,
    4. Levy J,
    5. Henderson A,
    6. Xue D,
    7. et al.
    Sorafenib (BAY 43–9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models. Cancer Chemother Pharmacol 2007;59:561–74.
    OpenUrlCrossRefPubMed
  15. 15.↵
    1. Motzer RJ,
    2. Michaelson MD,
    3. Rosenberg J,
    4. Bukowski RM,
    5. Curti BD,
    6. George DJ,
    7. et al.
    Sunitinib efficacy against advanced renal cell carcinoma. J Urol 2007;178:1883–7.
    OpenUrlCrossRefPubMed
  16. 16.↵
    1. Shepard DR,
    2. Garcia JA
    . Toxicity associated with the long-term use of targeted therapies in patients with advanced renal cell carcinoma. Expert Rev Anticancer Ther 2009;9:795–805.
    OpenUrlCrossRefPubMed
  17. 17.↵
    1. Wu S,
    2. Chen JJ,
    3. Kudelka A,
    4. Lu J,
    5. Zhu X
    . Incidence and risk of hypertension with sorafenib in patients with cancer: a systematic review and meta-analysis. Lancet Oncol 2008;9:117–23.
    OpenUrlCrossRefPubMed
  18. 18.↵
    1. Mross K,
    2. Frost A,
    3. Steinbild S,
    4. Hedbom S,
    5. Buchert M,
    6. Fasol U,
    7. et al.
    A phase I dose-escalation study of regorafenib (BAY 73–4506), an inhibitor of oncogenic, angiogenic, and stromal kinases, in patients with advanced solid tumors. Clin Cancer Res 2012;18:2658–67.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Eisen T,
    2. Joensuu H,
    3. Nathan PD,
    4. Harper PG,
    5. Wojtukiewicz MZ,
    6. Nicholson S,
    7. et al.
    Regorafenib for patients with previously untreated metastatic or unresectable renal-cell carcinoma: a single-group phase 2 trial. Lancet Oncol 2012;13:1055–62.
    OpenUrlCrossRefPubMed
  20. 20.↵
    1. Folkman J.
    Tumor angiogenesis: therapeutic implications. N Engl J Med 1971;285:1182–6.
    OpenUrlCrossRefPubMed
  21. 21.↵
    1. Liu JY,
    2. Park SH,
    3. Morisseau C,
    4. Hwang SH,
    5. Hammock BD,
    6. Weiss RH
    . Sorafenib has soluble epoxide hydrolase inhibitory activity, which contributes to its effect profile in vivo. Mol Cancer Ther 2009;8:2193–203.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    1. Morisseau C,
    2. Hammock BD
    . Impact of soluble epoxide hydrolase and epoxyeicosanoids on human health. Annu Rev Pharmacol Toxicol 2013;53:37–58.
    OpenUrlCrossRefPubMed
  23. 23.↵
    1. Spector AA,
    2. Fang X,
    3. Snyder GD,
    4. Weintraub NL
    . Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function. Prog Lipid Res 2004;43:55–90.
    OpenUrlCrossRefPubMed
  24. 24.↵
    1. Node K,
    2. Huo Y,
    3. Ruan X,
    4. Yang B,
    5. Spiecker M,
    6. Ley K,
    7. et al.
    Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids. Science 1999;285:1276–9.
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    1. Yu Z,
    2. Xu F,
    3. Huse LM,
    4. Morisseau C,
    5. Draper AJ,
    6. Newman JW,
    7. et al.
    Soluble epoxide hydrolase regulates hydrolysis of vasoactive epoxyeicosatrienoic acids. Cir Res 2000;87:992–8.
    OpenUrlAbstract/FREE Full Text
  26. 26.↵
    1. Webler AC,
    2. Michaelis UR,
    3. Popp R,
    4. Barbosa-Sicard E,
    5. Murugan A,
    6. Falck JR,
    7. et al.
    Epoxyeicosatrienoic acids are part of the VEGF-activated signaling cascade leading to angiogenesis. Am J Physiol Cell Physiol 2008;295:C1292–301.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    1. Pozzi A,
    2. Macias-Perez I,
    3. Abair T,
    4. Wei S,
    5. Su Y,
    6. Zent R,
    7. et al.
    Characterization of 5,6- and 8,9-epoxyeicosatrienoic acids (5,6- and 8,9-EET) as potent in vivo angiogenic lipids. J Biol Chem 2005;280:27138–46.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    1. Wang Y,
    2. Wei X,
    3. Xiao X,
    4. Hui R,
    5. Card JW,
    6. Carey MA,
    7. et al.
    Arachidonic acid epoxygenase metabolites stimulate endothelial cell growth and angiogenesis via mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt signaling pathways. J Pharmacol Exp Ther 2005;314:522–32.
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    1. Cheng LM,
    2. Jiang JG,
    3. Sun ZY,
    4. Chen C,
    5. Dackor RT,
    6. Zeldin DC,
    7. et al.
    The epoxyeicosatrienoic acid-stimulated phosphorylation of EGF-R involves the activation of metalloproteinases and the release of HB-EGF in cancer cells. Acta Pharmacol Sin 2010;31:211–8.
    OpenUrlCrossRefPubMed
  30. 30.↵
    1. Panigrahy D,
    2. Edin ML,
    3. Lee CR,
    4. Huang S,
    5. Bielenberg DR,
    6. Butterfield CE,
    7. et al.
    Epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice. J Clin Invest 2012;122:178–91.
    OpenUrlCrossRefPubMed
  31. 31.↵
    1. Wang D,
    2. Dubois RN
    . Epoxyeicosatrienoic acids: a double-edged sword in cardiovascular diseases and cancer. J Clin Invest 2012;122:19–22.
    OpenUrlCrossRefPubMed
  32. 32.↵
    1. Zhang G,
    2. Panigrahy D,
    3. Mahakian LM,
    4. Yang J,
    5. Liu JY,
    6. Stephen Lee KS,
    7. et al.
    Epoxy metabolites of docosahexaenoic acid (DHA) inhibit angiogenesis, tumor growth, and metastasis. Proc Natl Acad Sci U S A 2013;110:6530–5.
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    1. Inoue H,
    2. Kauffman M,
    3. Shacham S,
    4. Landesman Y,
    5. Yang J,
    6. Evans CP,
    7. et al.
    CRM1 blockade by selective inhibitors of nuclear export attenuates kidney cancer growth. J Urol 2013;189:2317–26.
    OpenUrlCrossRefPubMed
  34. 34.↵
    1. Nithipatikom K,
    2. Endsley MP,
    3. Isbell MA,
    4. Falck JR,
    5. Iwamoto Y,
    6. Hillard CJ,
    7. et al.
    2-arachidonoylglycerol: a novel inhibitor of androgen-independent prostate cancer cell invasion. Cancer Res 2004;64:8826–30.
    OpenUrlAbstract/FREE Full Text
  35. 35.↵
    1. Kim J,
    2. Carlson ME,
    3. Kuchel GA,
    4. Newman JW,
    5. Watkins BA
    . Dietary DHA reduces downstream endocannabinoid and inflammatory gene expression and epididymal fat mass while improving aspects of glucose use in muscle in C57BL/6J mice. Int J Obes 2016;40:129–37.
    OpenUrlCrossRef
  36. 36.↵
    1. Yang J,
    2. Schmelzer K,
    3. Georgi K,
    4. Hammock BD
    . Quantitative profiling method for oxylipin metabolome by liquid chromatography electrospray ionization tandem mass spectrometry. Anal Chem 2009;81:8085–93.
    OpenUrlCrossRefPubMed
  37. 37.↵
    1. Nording ML,
    2. Yang J,
    3. Georgi K,
    4. Hegedus Karbowski C,
    5. German JB,
    6. Weiss RH,
    7. et al.
    Individual variation in lipidomic profiles of healthy subjects in response to omega-3 Fatty acids. PloS One 2013;8:e76575.
    OpenUrlCrossRefPubMed
  38. 38.↵
    1. Koch AE,
    2. Polverini PJ,
    3. Kunkel SL,
    4. Harlow LA,
    5. DiPietro LA,
    6. Elner VM,
    7. et al.
    Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science 1992;258:1798–801.
    OpenUrlAbstract/FREE Full Text
  39. 39.↵
    1. Skovseth DK,
    2. Kuchler AM,
    3. Haraldsen G
    . The HUVEC/Matrigel assay: an in vivo assay of human angiogenesis suitable for drug validation. Methods Mol Biol 2007;360:253–68.
    OpenUrlPubMed
  40. 40.↵
    1. Guo D,
    2. Jia Q,
    3. Song HY,
    4. Warren RS,
    5. Donner DB
    . Vascular endothelial cell growth factor promotes tyrosine phosphorylation of mediators of signal transduction that contain SH2 domains. Association with endothelial cell proliferation. J Biol Chem 1995;270:6729–33.
    OpenUrlAbstract/FREE Full Text
  41. 41.↵
    1. Chou TC.
    Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 2010;70:440–6.
    OpenUrlAbstract/FREE Full Text
  42. 42.↵
    1. Ivanov SV,
    2. Kuzmin I,
    3. Wei MH,
    4. Pack S,
    5. Geil L,
    6. Johnson BE,
    7. et al.
    Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes. Proc Natl Acad Sci U S A 1998;95:12596–601.
    OpenUrlAbstract/FREE Full Text
  43. 43.↵
    1. Kurban G,
    2. Hudon V,
    3. Duplan E,
    4. Ohh M,
    5. Pause A
    . Characterization of a von Hippel Lindau pathway involved in extracellular matrix remodeling, cell invasion, and angiogenesis. Cancer Res 2006;66:1313–9.
    OpenUrlAbstract/FREE Full Text
  44. 44.↵
    1. Donadio JV Jr..,
    2. Bergstralh EJ,
    3. Offord KP,
    4. Spencer DC,
    5. Holley KE
    . A controlled trial of fish oil in IgA nephropathy. Mayo Nephrology Collaborative Group. N Engl J Med 1994;331:1194–9.
    OpenUrlCrossRefPubMed
  45. 45.↵
    1. Hwang SH,
    2. Wecksler AT,
    3. Zhang G,
    4. Morisseau C,
    5. Nguyen LV,
    6. Fu SH,
    7. et al.
    Synthesis and biological evaluation of sorafenib- and regorafenib-like sEH inhibitors. Bioorg Med Chem Lett 2013;23:3732–7.
    OpenUrlCrossRefPubMed
  46. 46.↵
    1. Schebb NH,
    2. Ostermann AI,
    3. Yang J,
    4. Hammock BD,
    5. Hahn A,
    6. Schuchardt JP
    . Comparison of the effects of long-chain omega-3 fatty acid supplementation on plasma levels of free and esterified oxylipins. Prostaglandins Other Lipid Mediat 2014;113–115:21–9.
    OpenUrl
  47. 47.↵
    1. Morisseau C,
    2. Inceoglu B,
    3. Schmelzer K,
    4. Tsai HJ,
    5. Jinks SL,
    6. Hegedus CM,
    7. et al.
    Naturally occurring monoepoxides of eicosapentaenoic acid and docosahexaenoic acid are bioactive antihyperalgesic lipids. J Lipid Res 2010;51:3481–90.
    OpenUrlAbstract/FREE Full Text
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Molecular Cancer Therapeutics: 15 (5)
May 2016
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Addition of DHA Synergistically Enhances the Efficacy of Regorafenib for Kidney Cancer Therapy
Jeffrey Kim, Arzu Ulu, Debin Wan, Jun Yang, Bruce D Hammock and Robert H. Weiss
Mol Cancer Ther May 1 2016 (15) (5) 890-898; DOI: 10.1158/1535-7163.MCT-15-0847

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Addition of DHA Synergistically Enhances the Efficacy of Regorafenib for Kidney Cancer Therapy
Jeffrey Kim, Arzu Ulu, Debin Wan, Jun Yang, Bruce D Hammock and Robert H. Weiss
Mol Cancer Ther May 1 2016 (15) (5) 890-898; DOI: 10.1158/1535-7163.MCT-15-0847
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