Activation of Toll-like receptors 2, 3, and 4 on human melanoma cells induces inflammatory factors

Abstract

Toll-like receptors (TLR) have been shown to be expressed on various types of cancers; however, their functional activity is not known. We examined TLR profiles of human melanoma cells and showed that TLR2, TLR3, and TLR4 were found to be highly expressed. By PCR array analysis, specific stimulation of TLR2, TLR3, and TLR4 on melanoma cells showed significant activation of the adaptor protein MyD88, as well as downstream signal transduction factors nuclear factor-kappaB and inflammatory response-related factors. Specific ligand activation of TLR2, TLR3, and TLR4 was shown to induce cell migration. Peripheral blood lymphocytes and melanoma purified RNA was shown to activate TLR3 on melanoma cells. These studies show expression and functional activity of specific TLRs on melanoma cells and as potential therapeutic targets to control tumor progression.

Figure 1

Expression of TLRs in human melanoma lines. A, expression of TLRs (TLR1–TLR10) in six representative human melanoma lines and two normal donor PBLs was analyzed by RT-PCR and gel electrophoresis. Colon cancer cell line SW480 was used as a negative control (Ctrl). B, expression of TLRs in melanoma lines, melanocytes, and healthy donor PBLs was analyzed by qRT-PCR. The X axis is the copy number of each gene over the copy number of the housekeeping gene GAPDH. Small bars, SD. ■, TLR2; □, TLR3; , TLR4. D, six melanoma cells were stained with FITC-LPS and visualized by fluorescence light microscopy. PBL from healthy individuals was used as a positive control, and PANC1 was used as a negative control. C, representative melanoma cell lines fluorescence-activated cell sorting analysis of TLR2, TLR3, TLR4, and early endosome antigen 1. Early endosome antigen 1 is endosome protein and a positive control of TLR3. PANC1 was a negative control. Left curve shaded area is the isotype control.

Figure 1

Expression of TLRs in human melanoma lines. A, expression of TLRs (TLR1–TLR10) in six representative human melanoma lines and two normal donor PBLs was analyzed by RT-PCR and gel electrophoresis. Colon cancer cell line SW480 was used as a negative control (Ctrl). B, expression of TLRs in melanoma lines, melanocytes, and healthy donor PBLs was analyzed by qRT-PCR. The X axis is the copy number of each gene over the copy number of the housekeeping gene GAPDH. Small bars, SD. ■, TLR2; □, TLR3; , TLR4. D, six melanoma cells were stained with FITC-LPS and visualized by fluorescence light microscopy. PBL from healthy individuals was used as a positive control, and PANC1 was used as a negative control. C, representative melanoma cell lines fluorescence-activated cell sorting analysis of TLR2, TLR3, TLR4, and early endosome antigen 1. Early endosome antigen 1 is endosome protein and a positive control of TLR3. PANC1 was a negative control. Left curve shaded area is the isotype control.

Figure 2

Expression of TLRs in isolated melanoma cells from tumor biopsy specimens. A, expression of MART-1, CD45, CD3-γ, cytokeratin 20, and CD34 in melanoma cells derived from 10 primary cutaneous melanoma was analyzed by RT-PCR. SC-MM is a positive control of primary melanoma cells. Melanoma lines (ME2 and ME20), normal donor PBLs (PBL1, PBL2), Huvec, and colon cancer line (SW480) were used as positive controls of each marker, respectively. B, expression of TLRs in isolated melanoma cells derived from biopsy specimens of melanomas was analyzed by qRT-PCR. SC-MM(+) is a positive control of isolated melanoma cells, ME2 is a melanoma line, and PBL5 is from a healthy individual. The first set of columns on the left shows the average for the nine biopsy-derived cells. Y axis is the gene copy number over the housekeeping gene GAPDH copy number. Small bars, SD. ■, TLR2; □, TLR3; , TLR4.

Figure 3

Heat map of quantitative PCR array analysis. Genes appear on the bottom of the heat map, and melanoma lines stimulated with ligand appear on the right side. Red, yellow, and green indicate higher, equivalent, and lower expression, respectively, in comparison with the expression in melanoma lines before stimulation.

Figure 4

Expression of MyD88 after stimulation using TLR ligands. Expression of MyD88 in two melanoma lines and one negative control cell line (PANC1) was analyzed by qRT-PCR. Each cell line stimulated by LPS, PIC, or zymosan was compared with each unstimulated cancer line. The vertical axis is the copy number of MyD88 over the copy number of the housekeeping gene GAPDH. Small bars, SD. ■, unstimulated; □, LPS; , PIC; ▤, zymosan.

Figure 5

Response to TLR ligands of three melanoma cells (ME2, ME5, and ME7) and one negative control cell line (PANC1) was assessed by a cell migration assay. The number of migrating cells in 10 randomly selected fields was counted after 24 h of incubation with and without ligands for TLR2, TLR3, and TLR4. One representative experiment of three is depicted. Small bars, SD. ■, unstimulated; □, ligand-stimulated cancer line.

Figure 6

Melanoma line response to stimulated PBL supernatant and RNA. A, expression level of TLR2, TLR3, TLR4, and MyD88 in four melanoma lines (ME1, ME2, ME5, and ME7) cultured with supernatant of PBLs from four donors (PBL1, PBL2, PBL3, and PBL4) was analyzed by qRT-PCR. ■, melanoma cells cultured with a control medium; □, melanoma cells cultured with supernatant from unstimulated PBLs; , melanoma cells cultured with supernatant from PHA stimulated PBLs. Small bars, 95% confidence intervals. The Y axis is the copy number of each gene over the copy number of the housekeeping gene GAPDH. B, expression level (mean) of TRIF mRNA in three melanoma lines (ME1, ME5, and ME7) cultured with supernatant of ■ PBLs (three donors) stimulated with PHA-L and □ ME (two melanoma cell lines ME1 and ME5) analyzed by qRT-PCR. The Y axis is the percentage of increase of TRIF mRNA expression in cells cultured with RNA compared with cells without RNA. Small bars, SD. *, actual mean increase of TRIF expression was 566 ± 497% for ME7 line incubated with melanoma cell RNA.