Abstract
Thalidomide has demonstrated clinical activity in various malignancies affecting immunomodulatory and angiogenic pathways. The development of novel thalidomide analogs with improved efficacy and decreased toxicity is an ongoing research effort. We recently designed and synthesized a new class of compounds, consisting of both tetrafluorinated thalidomide analogues (Gu973 and Gu998) and tetrafluorobenzamides (Gu1029 and Gu992). In this study, we demonstrate the antiangiogenic properties of these newly synthesized compounds. We examined the specific antiangiogenic characteristics in vitro using rat aortic rings with carboxyamidotriazole as a positive control. In addition, further in vitro efficacy was evaluated using human umbilical vein endothelial cells (HUVEC) and PC3 cells treated with 5 and 10 μmol/L doses of each compound. All compounds were seen to reduce microvessel outgrowth in rat aortic rings as well as to inhibit HUVECs to a greater extent, at lower concentrations than previously tested thalidomide analogs. The antiangiogenic properties of the compounds were also examined in vivo in fli1:EGFP zebrafish embryos, where all compounds were seen to inhibit the extent of outgrowth of newly developing blood vessels. In addition, Gu1029 and Gu973 reduced the anti-inflammatory response in mpo:GFP zebrafish embryos, whereas Gu998 and Gu992 showed no difference. The compounds' antitumor effects were also explored in vivo using the human prostate cancer PC3 xenograft model. All four compounds were also screened in vivo in chicken embryos to investigate their teratogenic potential. This study establishes these novel thalidomide analogues as a promising immunomodulatory class with anticancer effects that warrant further development to characterize their mechanisms of action. Mol Cancer Ther; 14(10); 2228–37. ©2015 AACR.
Introduction
When thalidomide was originally made available in the 1950s, it was used as a nonaddictive, non-barbiturate sedative, and soon after as an anti-emetic, alleviating the symptoms of morning sickness in pregnant women. Shortly thereafter, it was found that thalidomide had adverse effects upon fetal development, leading to multiple severe deformities in newborns, including phocomelia (shortening of the long bones of the limbs), as well as causing miscarriages (1–3). The mechanism for these teratogenic effects has since been linked to thalidomide having antiangiogenic characteristics (4, 5). Thalidomide was removed from the market in late 1961 and never received FDA approval in the United States for the purpose of treating morning sickness. However, interest in thalidomide has resurfaced over the past 2 decades, as the drug possesses potent anti-inflammatory and antiangiogenic properties.
Thalidomide is currently FDA approved for the treatment of erythema nodosum leprosum (6, 7) and multiple myeloma (8–15). Furthermore, thalidomide has shown some activity in acute myeloid leukemia (16), metastatic renal cell carcinoma (17), high-grade gliomas (18), prostate cancer (19, 20), and Kaposi sarcoma (21). Recent studies have also shown the efficacy of 2 thalidomide analogues, lenalidomide and pomalidomide, for the treatment of select malignancies (22). When compared to thalidomide, these compounds show increased response rates and less toxicity, however, patients still suffer from related side effects, and eventually many will develop resistance to these drugs (23, 24). The development of novel thalidomide analogues that are safer and with improved activity has been an ongoing research effort. A previous collaboration between the National Cancer Institute and the Gütschow laboratory in Germany led to the synthesis of 118 N-substituted and tetrafluorinated thalidomide analogues, where 7 of these compounds (4 N-substituted and 3 tetrafluorinated) displayed antiangiogenic activity when tested in vitro and 1 also displayed antiangiogenic activity in vivo in chicken and zebrafish embryos (5, 25). These analogues were found to be active at much lower concentrations than thalidomide. On the basis of these results, a novel collection of tetrafluorinated compounds has been synthesized, composed of both tetrafluorinated thalidomide analogues and tetrafluorobenzamides. The structure of these analogues is based upon 5′-OH-thalidomide, a biologically active hydroxylated metabolite of thalidomide, which has been shown to have antiangiogenic activity in vitro (25, 26). We screened this library of novel compounds with in vitro and in vivo assays to determine their antiangiogenic and anti-inflammatory effects, evaluated their in vivo antitumor effects in the prostate cancer xenograft model, and studied effects on teratogenesis in the chicken embryo model. The current study has examined the therapeutic potential of the 4 lead tetrafluorinated thalidomide analogue compounds consisting of 2 tetrafluorobenzamides (Gu1029 and Gu992) and 2 tetrafluorinated thalidomide derivatives (Gu973 and Gu998).
Materials and Methods
Thalidomide analogues
Analogues were synthesized by Dr. Agnieszka Ambrozak in the group of Dr. Michael Gütschow at the University of Bonn (Bonn, Germany). U.S. patents of these analogues have been filed (Patent No. US 8,143,252 B2, March 27, 2012). Their chemical structures are shown in Fig. 1.
Structures of the tetrafluorobenzamides, Gu1029 and Gu992, and the tetrafluorinated thalidomide analogues Gu973 and Gu998.
Cell lines and reagents
DMSO was purchased from Sigma-Aldrich. All water used was ultra filtered by a MilliQ system (Millipore). Human umbilical vein endothelial cells (HUVEC; Clonetics) were purchased from Lonza (2007); no independent authentication was performed by the authors. Cell lines were maintained and cultured according to suppliers' protocols and used at early passages. PC3 cells were purchased from ATCC (2007) and authenticated June 2008 by IMPACT I PCR profiling (Radil).
Rat aortic ring assay
A rat aortic ring assay was performed to determine the extent of the antiangiogenicity of the 4 lead compounds on the basis of previous similar assays (5, 25, 26). Briefly, 12-well tissue culture plates were covered with 250 μL Matrigel (Becton-Dickinson) and allowed to gel for 30 to 45 minutes at 37°C and 5% CO2. Sections of thoracic aorta were removed from 8- to 10-week-old male Sprague Dawley rats. Following excision of fibroadipose tissue, the aortic sections were cut into 1-mm-long cross-sections, placed on Matrigel-coated wells, and layered with an additional 250 μL of Matrigel. These were then allowed to set, after which the cross-sectional rings were covered with endothelial cell growth media (EGM-II) and incubated under 5% CO2 at 37°C overnight. The EGM-II was composed of endothelial cell basal medium (EBM-II; Lonza), in addition to endothelial cell growth factors. The culture medium was then traded for EBM-II that was supplemented with 2% FBS, 0.25 μg/mL amphotericin B, and 10 μg/mL gentamicin. The aortic rings were treated daily with vehicle (0.5% DMSO), carboxyamidotriazole (CAI; 12 μg/mL; positive control), Gu973, Gu992, Gu998, or Gu1029, each at a dose of 50 μmol/L, for 4 days. This was replicated 4 times using aortas from 4 different rats. The area of angiogenic sprouting, reported in square pixels, was quantified using Adobe Photoshop. Data were presented as percentage of growth based on the negative control (vehicle), which was normalized to 100% growth.
Antiangiogenic and anti-inflammatory zebrafish embryo assays
Zebrafish were maintained in an approved aquarium habitat at 28°C and all necessary ethical and legal approvals were obtained for embryo work. Sexually mature males and females were crossed and the progeny were screened for the expression of GFP or EGFP. Chorions were removed at 24 hours postfertilization (hpf) using forceps. All screening and imaging was performed using a Nikon SMZ1500 stereo dissecting microscope and camera.
fli1:EGFP.
fli1:EGFP zebrafish (27) were used to assess antiangiogenic properties of the compounds. Embryos were exposed to either a vehicle control at 24 hpf or a Gu compound (5–100 μg/mL) for 24 hours. Embryos were anesthetized in 0.1% tricaine, and the length and number of intersegmental vessels were quantified as previously described (28).
mpo:GFP.
mpo:GFP zebrafish [ref. 29; also known as Tg(mpo:GFP)114] were used to assess the anti-inflammatory properties of the compounds. Adult fish were crossed, embryos were obtained and incubated until 72 hpf, whereupon they were sedated in 0.1% tricaine then given a small cut in the dorsal third of the tail fin. They were then incubated with a vehicle control (0.1% DMSO) or a compound of interest (5–100 μg/mL) in aquarium water for 24 hours. The number of fluorescent neutrophils migrating to the wound site was quantified as described previously (28).
NCI 60 cell line screening
Gu973, Gu992, Gu998, and Gu1029 were each tested in the NCI60 cell line screen at 5 different concentrations using previously published and standardized methodology (30). This screen assesses the ability of a compound to inhibit growth in 59 different human tumor cell lines.
Cell proliferation assay
The 4 lead compounds were tested for their ability to inhibit cell proliferation in vitro, using a CCK-8 assay. HUVECs and PC3 cells (prostate cancer cell line) were plated in 12-well plates with a density of 30,000 cells per well and allowed to attach overnight at 37°C in 5% CO2. The media were then removed and replaced with media containing vehicle (0.5% DMSO), Gu973, Gu992, Gu998, or Gu1029, either at 5 or 10 μmol/L for each compound.
In vivo mouse xenograft study
The in vivo efficacy of the 4 lead compounds was determined using a variation of a previously described prostate cancer xenograft mouse model (31). PC3 cells were allowed to grow in culture and then injected (∼3 million cells) into a mouse flank subcutaneously. Tumors were allowed to grow for 9 days before daily intraperitoneal injections of vehicle (5% DMSO in saline), Gu973 (1.55 mg/kg), Gu992 (2.34 mg/kg), Gu998 (0.42 mg/kg), or Gu1029 (4 mg/kg) were begun. These doses were the maximum tolerated doses determined in a previous study and based upon drug solubility (data not shown). Treatment lasted for 4 weeks, except for Gu973, which was stopped after 3 weeks due to adverse effects. All animal care was provided in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals” (National Research Council; 1996; National Academy Press). The study design and protocol were approved by the NCI Animal Care and Use Committee (Bethesda, MD).
Chicken embryo teratogenicity analysis
Fertilized white leghorn chicken embryos were incubated for 3 days in an incubator at 37°C, 5% CO2. The eggs were windowed and embryos staged according to the Hamburger–Hamilton (HH) stages of chicken embryo development (32). Embryos at HH St 18 (E3) were retained for experimentation, as at this stage, the embryo is undergoing organogenesis and rapid growth. Drug administration was carried out as previously described (5, 28). Briefly, embryonic membranes were removed and the compound of interest was applied over the embryo. After the procedure, the window was sealed with tape and returned to the incubator. All embryos were treated with 100 μL of dosing solution. Initially, compounds were applied to developing chick embryos at varying concentrations 50, 100, and 200 μg/mL (5–20 μg/embryo) to establish a dose–response relationship. These doses were tested, as they are within the ranges of previous thalidomide and thalidomide analogues screens (5, 28, 33). Once a dose–response relationship had been determined for each compound, the concentrations allowing the best survival rates were re-tested in a new cohort of embryos. Gu973, Gu998, and Gu1029 were applied at 10 μg/embryo (concentrations of 258.2, 241.1, and 266.5 μmol/L, respectively), and Gu992 was tested at 20 μg/mL (513.7 μmol/L). The compounds were given as a single dose and the development of each embryo was noted at 24 and 48 hours after drug application. Developmental defects and the survival of the embryos were recorded each day.
Statistical considerations
All results were presented as mean ± SEM. Comparisons were made with one-way ANOVA, followed by Dunnett test, with a P < 0.05 as the criterion for statistical significance. Statistical analysis was performed using GraphPad Prism.
Results
Effect of lead compounds on rat aortic angiogenesis
Rat aortic microvessel outgrowth was normalized to 100% on the basis of vehicle (0.5% DMSO) control. All lead compounds, each at 50 μmol/L, demonstrated approximately 90% inhibition of angiogenesis, comparable with CAI (Fig. 2). Gu1029 and Gu973 attenuated microvessel outgrowth to 9.02% and 6.23% of control, respectively. Gu992 and Gu998 were also effective antiangiogenic compounds, each demonstrating inhibition of outgrowth to 10.2% and 6.19% of control, respectively (P < 0.001, n = 5).
In vitro screening was conducted in the rat aortic ring (RAR) angiogenesis assay following the standard protocol. CAI was used as a positive control and shows little to no outgrowth. The Gu compounds were dosed at a concentration of 50 μmol/L. In comparison to the control, outgrowth was inhibited in every instance. ***, P < 0.001.
In vitro cancer cell proliferation
We assessed the anticancer capabilities of the compounds in the NCI60 screen, as well as the PC3 prostate cancer cell line. In the NCI60 screen, 3 compounds significantly reduced growth at a single dose of 10 μmol/L and went on to be tested at 5 different doses. All 3 compounds had a mean 50% growth inhibition (GI50) of all cell lines tested over 5 different doses. Gu973, Gu992, and Gu998 had mean GI50 values of 5.12, 36.7, and 2.12 μmol/L, respectively. Gu1029 did not induce significant growth inhibition in the initial NCI60 cell screen (at a single dose of 10 μmol/L) and thus testing of this compound in the NCI60 cell lines was not continued.
PC3 cells exhibited a decrease in cell viability with each lead compound. Gu973 and Gu998 inhibited PC3 proliferation in a dose-dependent manner (Fig. 3), where Gu973 inhibition decreased to 26.0% and 10.9% of control at 5 and 10 μmol/L, respectively, and Gu998 inhibited growth to 31.0% and 9.7% of control at 5 and 10 μmol/L, respectively (P < 0.0001, n = 8). However, at 10 μmol/L, Gu1029 and Gu992 demonstrated almost no decrease in proliferation in PC3 cells, at 87.7% and 95.5% of control (P > 0.05, n = 8), consistent with the NCI60 cell line testing at 10 μmol/L (Gu1029, 95.2%; Gu992, 93%).
In vitro testing of Gu compounds. Each compound reduced cell proliferation in HUVEC cells at 5 and 10 μmol/L. Compounds Gu1029 and Gu992 were tested in PC3 cells at 10 μmol/L and exhibited no effect, whereas Gu973 and Gu998 were tested at both 10 and 5 μmol/L causing a reduction in cell proliferation. ****, P < 0.0001; ns, not significant.
In vitro endothelial cell proliferation
We then investigated the compounds' effect on endothelial cell proliferation by using the HUVEC cell line. HUVECs showed significant reduction in cell viability when treated with each compound (n = 8). Gu973 and Gu992 both demonstrated dose-dependent inhibition of HUVEC proliferation at 5 and 10 μmol/L (Fig. 3), where Gu973 reduced proliferation to 27.2% and 9.0% of control, respectively, and Gu992 reduced to 27.1% and 10.7% of control, respectively. Gu1029 significantly reduced HUVEC cell proliferation by more than 85% at both 5 and 10 μmol/L. Gu998 was the most effective of the compounds, potently inhibiting HUVEC proliferation by more than 97% at 5 and 10 μmol/L (P < 0.0001).
Gu compounds exhibit antiangiogenic activity in zebrafish embryos
Thalidomide exhibits antiangiogenic properties in vitro (4), in vivo (33), and when assessed with in ovo chicken embryo assays (34). Moreover, antiangiogenic analogues of thalidomide cause teratogenic effects by inhibiting the growth of newly forming vessels (2, 3, 5). fli1:EGFP zebrafish, which are known to be sensitive to teratogens (5, 27, 28), were used to assess the ability of Gu compounds to affect the development of the forming vasculature. We used the formation of the intersomitic vessels (ISV) as markers of vessel outgrowth. At 24 hpf, ISVs are present in the anterior of the embryos, forming in the spine of the embryos and are yet to form in the posterior of the embryos. Compared with embryos treated with a vehicle control (0.1% DMSO, Fig. 4A and F, n = 33), treatment with Gu973 reduced the outgrowth of the ISVs at 1 μg/mL (P < 0.05, n = 9), 10 μg/mL (P < 0.01, n = 9), and at 20 μg/mL (Fig. 4B and F, P < 0.05, n = 17). Gu992 decreased outgrowth of forming vessels at all concentrations tested (Fig. 4F, 1 μg/mL, P < 0.001, n = 15; 10 μg/mL, P < 0.001, n = 20; 20 μg/mL, P < 0.001, n = 11; 50 μg/mL, Fig. 4C, P < 0.01, n = 5). Gu998 decreased outgrowth (Fig. 4F) at 1 μg/mL (P < 0.05, n = 10) and 10 μg/mL (Fig. 4D, P < 0.05, n = 7). Gu1029 decreased outgrowth at all concentrations tested (Fig. 4F, 1 μg/mL, P < 0.05, n = 10; 10 μg/mL, P > 0.05, n = 9; 20 μg/mL, P < 0.001, n = 18; 50 μg/mL, Fig. 4E, P < 0.001, n = 11); however, the extent of inhibition was not significant at 10 μg/mL. The number of sprouting ISVs was also quantified (Fig. 4G). Gu973 decreased vessel number but only significantly at 10 μg/mL (P < 0.01, n = 9) and 20 μg/mL (P < 0.05, n = 17). Gu992 inhibited sprouting significantly at 50 μg/mL (P < 0.001, n = 5). Gu998 showed a nonsignificant decrease in sprouting vessels. Gu1029 was the only compound that had no effect on the vessel number. We found that testing at higher concentrations (100 μg/mL) killed all the embryos, possibly due to toxicity, although we could not rule out that the embryos died because of systemic vessel and circulatory failure.
The effect of Gu compounds on transgenic zebrafish. Vessel length is decreased by Gu compounds. After incubation with vehicle control (0.1% DMSO) for 24 hours, embryos had normal blood vessel outgrowth (A and F). Treatment with Gu973 reduced the outgrowth at 1 and 10 μg/mL, with a slight increase at 20 μg/mL (B and F). Gu992 decreased outgrowth at all concentrations tested (F and C). Gu998 decreased outgrowth (F) at 1 and 10 μg/mL (D). At higher concentrations, all embryos were dead. Gu1029 decreased outgrowth at all concentrations tested (F); however, the extent of inhibition was not significant at 10 μg/mL. The number of sprouting vessels was also quantified (G). Gu1029 was the only compound that had no effect on the vessel number. Gu998 showed a nonsignificant decrease in sprouting vessels. Gu973 decreased vessel number but only significantly at 10 μg/mL. Gu992 inhibited sprouting significantly at 50 μg/mL. H, Gu compounds effects upon the inflammatory response in mpo:GFP zebrafish. Compared with control embryos, Gu973 and Gu1029 significantly reduce neutrophil cell migration. In contrast, Gu992 and Gu998 did not affect the inflammatory response at concentrations tested. Scale bar, 100 μm. *, P < 0.05; **, P < 0.001; ***, P < 0.001.
Gu compounds action upon the inflammatory response in zebrafish embryos
Thalidomide exhibits anti-inflammatory actions (1–3), and its analogue, lenalidomide, is known to be clinically effective in the treatment of multiple myeloma via its anti-inflammatory properties (35, 36). We therefore tested the Gu compounds to determine whether they have an effect on the inflammatory response. We tested the compounds on mpo:GFP zebrafish embryos, where neutrophils express GFP under the control of the myeloperoxidase promoter, and are recruited to the wound site following tissue injury/damage which allows the study of the inflammatory response (28, 29). Treatment with Gu1029 caused a reduction in neutrophil migration to the wound site in a concentration-dependent manner (Fig. 4H), where the response at 10 μg/mL (n = 13), 15 μg/mL (n = 8), 20 μg/mL (n = 13), 40 μg/mL (n = 8), 50 μg/mL (n = 9), and 100 μg/mL (n = 7) were all significant (P < 0.001). Gu973 showed a significant inhibition of neutrophil numbers and migration at 2.5 μg/mL (P < 0.01, n = 5), 5 μg/mL (P < 0.05, n = 5), and 6.25 μg/mL (P < 0.01, n = 8) compared with embryos with a tail fin cut but without a drug. Gu992 (5 and 10 μg/mL, n = 16 and n = 8, respectively) and Gu998 (5 μg/mL, n = 10) gave no significant response, and neutrophil behavior was similar to the controls.
In vivo efficacy of Gu compounds in the prostate cancer xenograft model
Finally, the behavior of the compounds in mouse models of cancer was evaluated. Mice (n = 29) were divided into 5 groups and treated daily for 4 weeks with a vehicle control or 1 of the 4 lead compounds. Before day 21 of the study, no compound was observed to have a statistically significant decrease in tumor volume size compared with vehicle control (Fig. 5). However, from day 21 and continuing through the end of the study (day 39), Gu1029 showed statistically significant decreases in tumor volume size (compared to vehicle) at each time point (P < 0.05). By day 36 of the study, tumor growth inhibition was 48.19% compared with control. Treatment lasted 4 weeks for all compounds except Gu973, which was stopped after 3 weeks due to animals experiencing abdominal swelling, with moderate weight loss of approximately 10% of baseline body weight. The weight loss of mice treated with Gu973 was significant but stable over the course of the experiment and supplementation with transgel did not reverse condition.
In vivo efficacy of Gu compounds. A, tumor volume in mice inoculated with 3 million PC3 cells (n = 29) was monitored over time. All compounds were compared with vehicle (5% DMSO in saline). Significance was determined by both Student t and Mann–Whitney tests, with results being similar in both instances. B, weight loss of treated mice. Weight loss of Gu973-treated mice was significant but stable. Supplementation with transgel did not abrogate the weight loss. Severe weight loss after the treatment period was seen in some mice with larger tumors.
Given there was no statistical response from Gu992 and Gu998, these in vivo results indicate that Gu1029 is a potent compound at inhibiting prostate tumor growth.
Teratogenicity of lead compounds in the developing chicken embryo
Screening of these compounds during chicken embryo development was conducted to assess their potential for teratogenesis (Fig. 6). The results are indicative of the concentration at which embryos survived the treatment and exhibited defects.
Application of Gu compounds at HH St 18 (E2.5) causes developmental defects in the chicken embryo. Examples of phenotypical outcomes with application of Gu compounds at HH St 18. A, application of Gu1029 induces hemorrhaging in the embryo (black arrowhead; A) and necrosis in the CAM in ovo (white arrowhead; A; n = 10/10). B, Gu998 causes hemorrhaging in the limb of the treated embryo (arrowhead; B; n = 2/10). The embryo also has growth retardation (n = 5/10). C, application of Gu992 causes retardation of limb growth (arrowhead; C; n = 1/12). D, Gu973 causes hemorrhaging through the body of the embryo (n = 3/10). The limbs in particular are affected (arrowhead; D). Scale bar, 1000 μm. E, application of compounds Gu1029 (n = 10), Gu992 (n = 12), Gu973 (n = 10), and Gu998 (n = 11) induces variable defects in the developing chick embryo as well as having adverse effects on the vasculature within the CAM and causing premature embryonic death.
After establishing a concentration at which embryos could survive the treatment (266.5 μmol/L, 10 μg/embryo), 10 embryos were treated with Gu1029, of which 8 survived the treatment. All embryos at 24 hours had hemorrhaging (Fig. 6A). Of the 8 surviving embryos, 3 of these had twisted spines and 6 were developmentally delayed. Gu973 administration caused defects at 258.2 μmol/L (10 μg/embryo) where 10 embryos were treated at this concentration. Two of the 8 surviving embryos presented with microopthalmia, 5 of 8 were developmentally delayed, and 3 of 8 embryos exhibited hemorrhaging. Gu992 exposure was tested at 513.77 μmol/L (20 μg/embryo) in 12 embryos, of which 11 survived the treatment. Three embryos had twisted spines, 2 had growth retardation, and 1 embryo had a limb reduction injury. Gu998 was administered over developing chicken embryos at 241.1 μmol/L (10 μg/embryo) in 11 embryos, 7 of which survived the treatment. Gu998 caused a range of complications including 2 limb defects, microopthalmia, 3 embryos with spinal defects, and 2 embryos that were underdeveloped.
Defects were seen following drug exposure in every instance (Fig. 6E), and every treatment caused hemorrhaging in the bodies and heads of the embryos, as well as constriction of the vasculature and necrosis of the chorioallantoic membranes (CAM) surrounding the embryos. These data indicate all 4 compounds are teratogenic to the developing embryo yet the severity of the injuries caused varies amongst them.
Discussion
These results show that the 4 lead tetrafluorinated thalidomide analogues tested demonstrated varying degrees of antiangiogenic, anti-inflammatory, antitumor, and teratogenic characteristics. Gu973 displayed only moderate efficacy in all assays, as well as causing unacceptable abdominal swelling, pain, and weight loss in week 3 of the mouse xenograft assay. Gu998 was the most effective compound in the in vitro tests, yet it showed no significant tumor volume reduction in the in vivo efficacy study. Gu992 was seen to have similar effects as Gu998 in most assays excluding the in vitro cell proliferation, where Gu992 demonstrated low potency in the NCI60 cell screen and minimal activity in inhibiting PC3 cell proliferation. Gu1029 exhibited antitumor effects in the prostate xenograft study, significant activity against HUVEC proliferation and in antiangiogenic assays but displayed no potency against cancer cell lines. Previous studies have found a number of compounds to also be inactive in the NCI60 screen at the concentrations tested, which include thalidomide, lenalidomide, aminolevulinic acid, and levamisole (37). Because the Gu compounds were not cytotoxic in this screen but did inhibit proliferation in endothelial cells, the results are indicative of the specificity of the antiangiogenic action of the compounds. However, the lack of response from the NCI60 screen may also be attributed to the fact that the efficacy of at least some of the drugs depends on their effect on the immune system or components of the tumor microenvironment (38), which would not be detectable in a cell line screen such as the NCI60 (37).
The antiangiogenic properties of these compounds compared favorably with tetrafluorinated thalidomide analogues previously reported by Ng and colleagues (31) and Lepper and colleagues (39). Multiple cell types are involved in the formation of the microtubules from the rat aortic rings, and the order in which they form the sprouts emulates the succession of formation in humans, making this assay an excellent indicator of the effect of drugs on angiogenesis (40). Analyzing microvessel growth in the rat aortic ring assay, all compounds performed equally well or better at 50 μmol/L than the previously synthesized N-substituted class of thalidomide analogues at the same dose. In addition, each compound tested here showed greater HUVEC lattice network inhibition at lower concentrations (5 μmol/L) than similar thalidomide analogues tested previously at 12.5 μmol/L (25). When comparing results from the in vitro PC3 cell proliferation assay, Gu992 and Gu1029 had no effect at 10 μmol/L. However, Gu973 and Gu998 at 5 μmol/L were both more effective at inhibiting PC3 cells than all previously tested compounds at 12.5 μmol/L (25).
The tetrafluorinated Gu compounds were designed around the structures of CPS thalidomide analogues previously found to be efficacious in prostate cancer xenograft models and antiangiogenic (25, 31). Several studies have shown tetrafluorinated compounds to exhibit increased bioactivity compared with nonfluorinated compounds (25). The increased activity of Gu973 and Gu998 in vitro compared with the CPS compounds (25) may be due to the combination of additional fluorine substituents as well as a more appropriate substituent at the phthalimide nitrogen, i.e., a dialkyl barbituric acid (Fig. 1). It has already been shown that nonfluorinated CPS compounds display less antiangiogenic activity than their tetrafluorinated counterparts (25). Compounds Gu1029 and Gu992 also possess better antiangiogenic activity than CPS compounds in vitro but less so than Gu973 and Gu998. Scission of one phthaloyl CN bond of the tetrafluorophthalimide structure (Gu973 and Gu998) and the loss of one CO unit would likely result in increased degrees of conformational freedom of the tetrafluorobenzamide structure (Gu1029 and Gu992). This may be a contributing factor toward explaining a reduced bioactivity of the tetrafluorobenzamides in the in vitro assays. Similarly, a replacement of the methylene bridge by CO in the phthaloyl ring has been shown to increase the potency of thalidomide analogues in vitro. Perhaps retaining this structural element of thalidomide is a requirement for improved antiangiogenic activity; structure–activity relationship studies are currently ongoing in our laboratory. Other thalidomide analogues reported in the literature have also demonstrated varying degrees of antiangiogenic and/or antitumor properties, for example, lenalidomide and pomalidomide. Thus, it should be noted that structural differences between thalidomide analogues and thalidomide itself may affect binding to distinct target protein(s) such as cereblon and associated downstream factors, hence subsequently modifying their mechanisms of action.
Despite these promising in vitro findings, only Gu1029 performed as well in the human prostate cancer xenograft assay as previously tested tetrafluorinated analogues (31). When examined by Ng and colleagues, (31), the 3 chosen compounds (CPS45, CPS49, CPS11) previously synthesized and described demonstrated reductions in tumor size of 51%, 68%, and 90%, respectively. In the current study, Gu1029 was the only compound that significantly inhibited prostate tumor growth. Further studies comparing the actions of Gu1029 with previously synthesized CPS compounds would be useful to determine the tolerability and in vivo efficacy of these compounds and may allow for the synthesis of new compounds with more potent properties.
Given the teratogenic nature of thalidomide, the lead compounds in this study were tested for any effects upon embryonic development using the chicken embryo assay. The chicken embryo is a well-established system to study teratogenesis and the effects of drugs upon development (2, 3, 5, 28, 33). Gu998 and Gu992 were shown to be teratogenic, causing limb, spinal, and head defects as well as causing a high death rate. Embryos treated with Gu973 and Gu1029 did not produce limb defects; however, surviving embryos did show hemorrhaging, growth retardation, and necrosis in the surrounding CAM as well as twisted spines (Gu1029) and microopthalmia (Gu973) at equivalent concentrations as Gu998. Full interpretation of this data would require further study in higher species. In addition, these studies were conducted to assess for the potential for teratogenic effects and further testing would be required to establish the effects of these compounds at lower concentrations. However, these compounds are potent and act outside the activity window of the parent molecule thalidomide in the chicken embryo, which needs to be applied much earlier in development to induce developmental defects (34). The concentrations of compounds used in our in vitro studies are comparable with those used in previous work by other groups (25, 41, 42). The concentrations used in the chicken embryo in vivo testing are lower (Gu973, Gu998, Gu1029) or equivalent (Gu992) to concentrations previously used to assess the teratogenicity of thalidomide, and its analogue lenalidomide at doses that induce maximal anti-inflammatory responses in zebrafish embryos and TNFα analysis in RAW cells (28). The compounds were applied once directly over the body of the embryo, and the chicken embryo was left to bathe in the solutions; however, how much of the drug is able to enter the embryo is unclear. Given that this amount is likely small, but the concentrations applied are lower or equivalent to concentrations of thalidomide and lenalidomide that induce teratogenesis from previous studies (28, 33), the likelihood of these compounds possessing teratogenic abilities is highly probable.
We also assessed the antiangiogenic and anti-inflammatory effects of these compounds on development of the vasculature in fli1:EGFP and mpo:GFP zebrafish embryos. We found that all the compounds reduced the extent of outgrowth of the blood vessels, although only Gu992 significantly reduced the number of forming blood vessels. Gu973 and Gu1029 had a significant decrease in the number of neutrophils migrating to the area of the cut and Gu992 and Gu998 did not. However, at high concentrations, embryos were not able to survive the treatments. This indicates the toxicity of these compounds in the zebrafish embryo.
In summary, this study demonstrated that Gu973, Gu992, Gu998, and Gu1029 possess significant in vitro antiangiogenic effects with potential antitumor activity. Of these compounds, Gu998 proved to be the most effective in the greatest number of experimental designs. However, Gu1029 displayed the most potential in the human prostate cancer xenograft assay, as well as exhibiting anti-inflammatory properties. Given its potency in both angiogenic and inflammatory assays, Gu1029 appears to be a prime candidate molecule for further screening as a potential anticancer compound. Studies are currently underway to determine the downstream targets and effector molecules of Gu1029. Preclinical assays which evaluate the efficacy of Gu1029 in the treatment of multiple myeloma would be useful to assess the activity in comparison to the currently used analogues lenalidomide and pomalidomide. Future studies on the compound's in vivo efficacy are also warranted, including optimization of dose and schedule, improved delivery and formulation, to decipher whether Gu structural analogues could be more effective or safer versions of thalidomide.
Disclosure of Potential Conflicts of Interest
E.R. Gardner, A. Ambrozak, M. Gütschow, and W.D. Figg have ownership interests on a patent on the novel compounds assessed in this study [Patent No. US 8,143,252 B2 (Mar. 27, 2012)]. N. Vargesson has been a consultant to lawyers representing alleged thalidomide victims. No potential conflicts of interest were disclosed by the other authors.
Disclaimer
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. Government.
Authors' Contributions
Conception and design: S.L. Beedie, E.R. Gardner, M. Gütschow, C.H. Chau, N. Vargesson, W.D. Figg
Development of methodology: S. Pisle, E.R. Gardner, M. Gütschow, N. Vargesson, W.D. Figg
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Pisle, C. Mahony, S. Barnett, A. Ambrozak, N. Vargesson
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S.L. Beedie, S. Pisle, C.H. Chau, N. Vargesson, W.D. Figg
Writing, review, and/or revision of the manuscript: S.L. Beedie, C.J. Peer, S. Pisle, E.R. Gardner, M. Gütschow, C.H. Chau, N. Vargesson, W.D. Figg
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Ambrozak, M. Gütschow, N. Vargesson
Study supervision: N. Vargesson, W.D. Figg
Grant Support
This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research and in part by a Wellcome Trust-NIH PhD Studentship (ref: 098252/Z/12/Z) to S.L. Beedie, W.D. Figg, and N. Vargesson.
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.
Acknowledgments
The authors thank Scott McMenemy for carrying out preliminary, early studies looking at effects of Gu compounds upon chicken embryology, as well as Charles D. Crowe, Jeffrey E. Roth, and Adam C. Rolt for critical comments on the article. fli1:EGFP zebrafish were obtained from the Zebrafish International Research Center (27). mpo:GFP zebrafish [also termed Tg(MPO:GFP)114] zebrafish were obtained from Dr. Stephen Renshaw, University of Sheffield (Sheffield, South Yorkshire, UK; ref. 29).
- Received April 21, 2015.
- Revision received July 8, 2015.
- Accepted July 14, 2015.
- ©2015 American Association for Cancer Research.
References
Volume 14, Issue 10
