1,3-Bis(3,5-dichlorophenyl) urea compound 'COH-SR4' inhibits proliferation and activates apoptosis in melanoma

Abstract

The current clinical interventions in malignant melanomas are met with poor response to therapy due to dynamic regulation of multiple melanoma signaling pathways consequent to administration of single target agents. In this context of limited response to single target agents, novel candidate molecules capable of effectively inducing tumor inhibition along with targeting multiple critical nodes of melanoma signaling assume translational significance. In this regard, we investigated the anti-cancer effects of a novel dichlorophenyl urea compound called COH-SR4 in melanoma. The SR4 treatment decreased the survival and inhibited the clonogenic potential of melanomas along with inducing apoptosis in vitro cultures. SR4 treatments lead to inhibition of GST activity along with causing G2/M phase cell cycle arrest. Oral administration of 4 mg/kg SR4 leads to effective inhibition of tumor burdens in both syngeneic and nude mouse models of melanoma. The SR4 treatment was well tolerated and no overt toxicity was observed. The histopathological examination of resected tumor sections revealed decreased blood vessels, decrease in the levels of angiogenesis marker, CD31, and proliferation marker, Ki67, along with an increase in pAMPK levels. Western blot analyses of resected tumor lysates revealed increased PARP cleavage, Bim, pAMPK along with decreased pAkt, vimentin, fibronectin, CDK4 and cyclin B1. Thus, SR4 represents a novel candidate for the further development of mono and combinatorial therapies to effectively target aggressive and therapeutically refractory melanomas.

Figure 1. Anti-proliferative and pro-apoptotic effects of SR4 in Melanoma

The chemical structure of 1,3-bis(3,5-dichlorophenyl)urea compound also called COH-SR4 (panel A). Drug sensitivity assays were performed by MTT assay using SR4 at 96 h post-treatment to determine IC₅₀. Values are presented as mean ± standard deviation from two separate determinations with eight replicates each (n= 16) (panel B). Colony-forming assay was performed and the colonies were counted using Innotech Alpha Imager HP as detailed in Materials and Methods. * p < 0.001 compared with control ( n=3, panel C). For TUNEL apoptosis assay, cells were grown on coverslips and treated with 10 μM SR4 for 24 h. TUNEL assay was performed using Promega fluorescence detection kit and examined using Zeiss LSM 510 META laser scanning fluorescence microscope with filters 520 and 620 nm. Photographs taken at identical exposure at × 400 magnification are presented. Apoptotic cells showed green fluorescence (panel D).

Figure 2. Effect of SR4 on cell cycle progression in melanoma

GST activity towards 1-chloro 2,4-dinitro benzene (CDNB) and its inhibition by SR4 was performed in 28000g crude supernatant prepared from B16-F0, Hs600T and A2058 cells. Human liver purified GST was used as a control (inset). The inhibitory effect of SR4 on GST was studied at a fixed concentration of GSH and CDNB (1 mM each) and varying concentrations of inhibitor. The enzymes were pre-incubated with the inhibitor for 5 min at 37 °C prior to the addition of the substrates (panel A). Depletion of GSTπ by siRNA and its effects on cell survival by MTT assay: GSTπ siRNA or a scrambled control was transfected into melanoma cell lines using Lipofectamine 2000 (Invitrogen). After knock-down of GSTπ by siRNA, the level of GSTπ was detected by Western blot analyses. Membranes were stripped and reprobed for β-actin as a loading control. Results were quantified by scanning densitometry: C, control siRNA; T, GSTπ siRNA (panel B). MTT assay in GSTπ siRNA transfected cells were performed 96 h after SR4 treatment. The values are presented as mean ± SD from two separate determinations with eight replicates each (n = 16) (panel C). GSTπ-depletion itself caused decreased in cell growth( panel C inset), * p<0.01 compared to control. Inhibitory effect of SR4 on cell cycle distribution was determined by fluorescence activated cell sorting (FACS) analysis (panel D). The experiment was repeated three times and similar results were obtained.

Figure 3. Measurement of serum levels of SR4

The serum levels of SR4 in control and SR4 treated mice was examined by LC-MS/MS analyses. The C57 B mice were treated with 0.1 mg/mice (4 mg/kg b.w.) of SR4 on alternate day by oral gavage for 8 weeks. On the final day of treatment blood was collected within 2 h of final dosage. The samples were processed and analyzed for serum levels of SR4 as described in the methods section. The panels represent multiple reaction monitoring (MRM) mode chromatograms of 6D-SR4 standard 5 pg/uL (panel A), control serum (panel B) and SR4 treated mice serum (panel C). The bar diagram represents the quantification (mean ± SD) of SR4 in mice serum (n = 3); ND, not detectable (panel D).

Figure 4. Effect of oral administration of SR4 on melanoma progression in mice

The C57B mice and Hsd: Athymic nude nu/nu mice were obtained from Harlan, Indianapolis, IN. In each model, ten 10-weeks-old mice were divided into two groups of 5 animals (treated with corn oil (vehicle), and SR4 compound 4 mg / kg b.w.). All animals were injected with 2 × 10⁶ melanoma cells suspensions in 100 μl of PBS, subcutaneously into one flank of each mouse. Treatment was started 10 days after the implantation to see palpable tumor growth. Treatment consisted of 0.1 mg of SR4/mice in 200 μl corn oil by oral gavage alternate day. Control groups were treated with 200 μl corn oil by oral gavage alternate day. Animals were examined daily for signs of tumor growth and body weights were recorded (panel A). Photographs of animals were taken at day 1, day 10, day 14, day 18, day 20, day 30, day 40, and day 60 after subcutaneous injection, are shown for all groups (supplementary Figures 2 and 3). Weights and photographs of tumors were also taken at day 20 (for syngeneic model), and at day 51 (for xenograft model). Photographs of tumors were also taken at day 20 (for syngeneic model), and at day 51 (for xenograft model) (panel B). Tumors were measured in two dimensions using calipers and time-course analysis of tumor regression was performed during the study (panel C).

Figure 5. Histopathologic analyses of tumor sections after SR4 treatment

Control and SR4 treated B16-F0 and A2058 melanoma bearing mice tumor sections were used for histopathologic analyses. Immuno-histochemistry analyses for Ki-67, CD31, and pAMPK expression from tumors in mice of control and SR4-treated groups. Statistical significance of difference was determined by two-tailed Student’s t test. p < 0.001, SR4-treated compared with control. Immuno-reactivity is evident as a dark brown stain, whereas non-reactive areas display only the background color. Sections were counterstained with Hematoxylin (blue). Percent staining was determined by measuring positive immuno-reactivity per unit area. Arrows represent the area for positive staining for an antigen (panel A- Histopathology of resected B16-F0 syngeneic mouse melanoma tumors; panel B- Histopathology of resected A2058 human melanoma tumors). The intensity of antigen staining was quantified by digital image analysis. Bars represent mean ± S.E. (n = 5); * p<0.001 compared with control

Figure 6. Effect of SR4 on signaling proteins in vivo models of melanoma

Western-blot analyses of signaling proteins in tumor tissue lysates in control and SR4 treated experimental groups (panel A- Western blot of lysates from resected B16-F0 syngeneic mouse melanoma tumors from C57 B mice; panel B- Western blot of resected A2058 human melanoma tumors from nu/nu nude mice xenograft model). The bar diagrams represent the fold change in the levels of proteins as compared to controls as determined by densitometry. Dotted line represents no significant change as observed with control.