Effect of adipose-derived stem cell-conditioned medium on the proliferation and migration of B16 melanoma cells

Abstract

Adipose-derived stem cells (ASCs) are a population of cells derived from adipose tissue. ASCs exhibit multilineage development potential and are able to secrete various factors, which influence adjacent cells. Previous studies have reported the effectiveness of ASC-conditioned medium (ASC-CM) in wound healing, anti-melanogenesis, wrinkle improvement and hair growth. In the present study, the anticancer function of ASC-CM was investigated in vitro and in vivo. An MTT assay revealed that ASC-CM significantly decreased the proliferation of B16 melanoma cells in a time- and dose-dependent manner (P<0.01). Cell cycle analysis indicated that ASC-CM significantly increased the number of cells in G1 phase while reducing the number of cells in the S and G2/M phases (P<0.01). Furthermore, a wound migration model demonstrated that ASC-CM treatment significantly decreased the migration ability of B16 melanoma cells (P<0.01). In addition, C57BL/6 mice were administered with a single intratumoral injection of ASC-CM, daily or every other day, and a significant reduction in the volume of the tumor mass was observed compared with that of the control group (P<0.01). Thus, the findings of the present study indicated that ASC-CM has an anti-tumorigenic effect on B16 melanoma cells in vitro and in vivo, and may potentially be used to support the treatment of melanoma in the future.

Keywords: B16 melanoma cells; adipose-derived stem cell-conditioned media; adipose-derived stem cells; melanoma.

Figure 1.

ASC-CM suppresses the proliferation of B16 melanoma cells. B16 melanoma cells were incubated in DMEM supplemented with 10% FBS and ASC-CM at various concentrations. Control cells were incubated with DMEM (10% FBS) alone for 72 h. (A) Cells were treated with 10X ASC-CM for 24, 48 and 72 h prior to measuring cell viability using a CCK-8 assay. (B) Following incubation with 10X ASC-CM for 72 h, cell density was observed by phase-contrast microscopy (magnification, x100). (C) Cells were incubated with medium containing 1X, 5X and 10X ASC-CM for 72 h, prior to measuring cell viability by performing a CCK-8 assay. Values are presented as the mean ± standard deviation. **P>0.01 vs. control. ASC-CM, adipose-derived stem cell-conditioned medium; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CCK-8, Cell Counting Kit-8.

Figure 2.

ASC-CM induces cell cycle arrest in B16 cells at G1 phase. Cell cycle analysis of B16 melanoma cells incubated with ASC-CM. (A) Cells were incubated with 10X ASC-CM for 24 h and stained with propidium iodide. Cell cycle distribution was analyzed by flow cytometry. (B) Relative number of cells in the G1/S, S, and G2/M phases were compared between the control group and the ASC-CM-treated cells. A representative result from three independent experiments is shown. **P<0.01 vs. control. (C) Cells were treated with or without ASC-CM (10X) and the protein expression levels of key cell cycle regulatory proteins, including cyclin D1, p27, cyclin E and CDK2, were determined by performing an immunoblot analysis. β-actin was used as the loading control. ASC-CM, adipose-derived stem cell-conditioned medium; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; con, control; CDK, cyclin-dependent kinase.

Figure 3.

ASC-CM inhibits B16 melanoma cell migration. (A) Cells were grown to 90% confluence in 60-mm diameter culture dishes prior to scratching with a razor blade. Cells were cultured in medium containing 10% fetal bovine serum alone (control) or ASC-CM. Cells were allowed to migrate for 12 and 24 h before migration was recorded by phase contrast microscopy. (B) Quantification of cell migration by counting the number of cells that moved beyond the reference line. Values are represented as the mean ± standard deviation of three independent experiments. **P<0.01 vs. control. ASC-CM, adipose-derived stem cell-conditioned medium.

Figure 4.

ASC-CM suppresses tumor growth in a mouse xenograft model. Following subcutaneous injection of B16 melanoma cells (1×10⁶ cells) into C57BL/6 mice, an intratumoral injection of 10X ASC-CM (100 µl) was administered (A) daily or (B) every other day, for a total of five times. Tumor size was measured using calipers and tumor volume calculated as 0.5 × long axis × (short axis)². (C) Immunohistochemical analysis of Ki-67 and CD34 protein expression in the isolated tumors xenografts of control and ASC-CM-treated mice (magnification, x400). n=5 per group. **P<0.01 vs. control. ASC-CM, adipose-derived stem cell-conditioned medium; CD34, cluster of differentiation 34.