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. 2009 Apr 1;15(7):2380-6.
doi: 10.1158/1078-0432.CCR-08-2387. Epub 2009 Mar 17.

Small-molecule antagonists for CXCR2 and CXCR1 inhibit human melanoma growth by decreasing tumor cell proliferation, survival, and angiogenesis

Seema Singh  1 , Anguraj Sadanandam, Kalyan C Nannuru, Michelle L Varney, Rosemary Mayer-Ezell, Richard Bond, Rakesh K Singh
Affiliations

Small-molecule antagonists for CXCR2 and CXCR1 inhibit human melanoma growth by decreasing tumor cell proliferation, survival, and angiogenesis

Seema Singh et al. Clin Cancer Res. .

Abstract

Purpose: Melanoma, the most aggressive form of skin cancer, accounts for 75% of all skin cancer-related deaths and current therapeutic strategies are not effective in advanced disease. In the current study, we have investigated the efficacy of orally active small-molecule antagonist targeting CXCR2/CXCR1.

Experimental design: Human A375SM melanoma cells were treated with SCH-479833 or SCH-527123, and their effect on proliferation, motility, and invasion was evaluated in vitro. We examined the downstream signaling events in the cells following treatment with antagonists. For in vivo studies, A375SM cells were implanted subcutaneously into athymic nude mice followed by administration of SCH-479833, SCH-527123, or hydroxypropyl-beta-cyclodextrin (20%) orally for 21 days and their effect on tumor growth and angiogenesis was evaluated.

Results: Our data show that SCH-479833 or SCH-527123 inhibited the melanoma cell proliferation, chemotaxis, and invasive potential in vitro. Treatment of melanoma cells with SCH-479833 or SCH-527123 also inhibited tumor growth. Histologic and histochemical analyses showed significant (P < 0.05) decreases in tumor cell proliferation and microvessel density in tumors. Moreover, we observed a significant increase in melanoma cell apoptosis in SCH-479833- or SCH-527123-treated animals compared with controls.

Conclusion: Together, these studies show that selectively targeting CXCR2/CXCR1 with orally active small-molecule inhibitors is a promising therapeutic approach for inhibiting melanoma growth and angiogenesis.

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Figures

Fig. 1
Fig. 1
SCH-479833 and SCH-527123 inhibit melanoma growth. Eight-week-old mice were injected with highly metastatic human melanoma A375SM cells subcutaneously.Twenty-four hours after injection, mice were orally fed with SCH-479833 or SCH-527123 antagonist (100 mg/kg in 0.2 mL of 20% HPβCD) once a day for 21d. Control mice were fed with 0.2 mL vehicle (20% HPβCD) alone. Growth curve of A375SM cells in athymic nude mice treated with vehicle, SCH-479833, and SCH-527123. Average ± SE tumor volume.
Fig. 2
Fig. 2
SCH-479833 and SCH-527123 inhibit proliferation and survival in human melanoma tumors. Immunohistochemical staining for PCNA (A) andTUNEL (C) were analyzed based on DAB staining as described in Materials and Methods. Control (left), SCH-479833 (middle), and SCH-527123 (right). Pictures at ± 200. Proliferating (B) and apoptotic (D) cells were counted in 10 arbitrarily selected fields at ×200 magnification in a double-blinded manner and expressed as average ± SE number of cells per field view. *, P < 0.05, significantly different from control HPβCD-treated animals.
Fig. 3
Fig. 3
SCH-479833 and SCH-527123 inhibit microvessel density in human melanoma tumors. Immunohistochemical staining for microvessel using anti-GS-IB4 was analyzed as described in Materials and Methods. Control (left), SCH-479833 (middle), and SCH-527123 (right). Representative pictures at ×200.
Fig. 4
Fig. 4
SCH-479833 and SCH-527123 inhibit melanoma cell motility and invasion. Cells were plated on noncoated or Matrigel-coated membranes for motility (A and B) and invasion (C and D) assays and incubated for overnight. Serum-free medium containing 10 ng/mL CXCL-8 and SCH-479833 or SCH-527123 (10 ng/mL) or HPβCD was added to the lower chamber.The cells that did not migrate through the Matrigel and/or pores in the membrane were removed, and cells on the other side of the membrane were stained and photographed at ×200 magnification. Cells were counted in 10 random fields (×200) and expressed as the average number of cells per field of view. Number ± SE of migrated cells. Representative of three experiments done in triplicate. *, P < 0.05,×significantly different from control CXCL-8-treated cells.
Fig. 5
Fig. 5
SCH-479833 and SCH-527123 inhibit A375SM cell proliferation and ERK1/2 signaling. A375SM cells (1,000 per well) in a 96-well plate were cultured in medium with or without serum (1.25%) and SCH-479833 (A) or SCH-527123 (B) in four different concentrations. Cellular proliferation was determined at 72 h by MTTassay. Mean ± SE percent inhibition of proliferation. C, A375SM cell were treated with the antagonists for 1h and then stimulated with CXCL-8 for 30 min. Cell lysates (50 μg) were fractionated by SDS-PAGE and subjected to Western blotting using pERK1/2 or ERK1/2 antibody, and GAPDH was used as a loading control.
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