br Human CAMP mRNA expression was low in the
Human CAMP mRNA expression was low in the lungs and liver of the HA-AAV control group (threshold cycle [Ct] value, 38–40). Infection of CAMP-HA-AAVs significantly increased cathelicidin mRNA expression in the lungs and liver of the recipient mice (Fig-ure 1B). All groups carried similar intensities of HA-tagged staining in the lungs and liver, indicating equal loading of AAV particles and expression of their gene products in nude mice (Figures 1C and 1D).
The injected nude mice developed human cytokeratin 18-positive tu-mor colonies in the lungs and liver, indicating colon cancer metastasis (Figures 2A and 2B). The lung and liver tissues in the cathelicidin-overexpressing group showed much less human-specific cytokeratin 18 staining than those in the control group. Cathelicidin overexpres-sion significantly reduced human keratin-20 mRNA expression in the lungs and liver of HT-29-loaded nude mice (Figures 2C and 2D).
Figure 1. Intravenous Cathelicidin-Expressing Adeno-Associated Virus Administration Expressed Cathelicidin mRNA and Viral Marker Protein in Lungs and Liver of HT-29-Loaded Nude Mice
(A) Experimental plan. (B) Human cathelicidin mRNA expression in lungs and liver. (C and D) HA-Tag Immu-nohistochemistry of (C) lung and (D) liver tissues. AAV-infected D-AP5 were stained with HA antibody and appeared brown. The extent of AAV infection was similar among tumors from the HA-AAV and CAMP-HA-AAV groups.
Cytokeratin 18 and keratin 20 are epithelial colon cancer markers.19,20 Both approaches indicated that cathelicidin overexpression in-hibited colon cancer metastasis.
Cathelicidin Disrupted Tubulin Cytoskeleton and Inhibited Cell Migration of Colon Cancer Cells
Consistent with prior cell viability studies involving HT-29 colon cancer cells and CCD-18Co fibroblasts,16 cathelicidin peptide (LL-
37) did not affect the viability of SW620 cells (Figure 3A). LL-37 (5–10 mM) inhibited migra-tion of SW620 cells (Figure 3B), which reflected the inhibition of metastatic potential. Tumoral
tubulin expression is associated with liver metastasis of colon cancer.21 Cathelicidin-medi-ated disruption of tubulin structure in HT-29 and CCD-18Co cells suggests the potential
role of tubulin in the anti-metastatic effect of cathelicidin.16 Tubulin tracker staining demon-
strated that incubation of human advanced colon cancer SW620 cells with LL-37 (5–10 mM) disrupted the tubulin structure in a dose-dependent manner (Figure 3C). Constitutive TUBB1 mRNA expres-sion in SW620 and HT-29 cells was not affected by exposure to LL-37 (Figure 3D).
Cathelicidin Inhibited Colon Cancer Cell Migration via TUBB3 Inhibition
LL-37 (5 mM) significantly inhibited TUBB3 mRNA expression in both colon cancer cells (Figure 3E). Lentiviral overexpression of TUBB3 also led to increased colon cancer cell migration of SW620 cells, with or without exposure to LL-37 (Figure 4A). Infection of TUBB3-overexpressing lentivirus significantly increased human TUBB3 mRNA expression in SW620 cells (Figure 4B).
LL-37 Inhibited Colon Cancer Cell Migration and TUBB3 Expression via P2RX7
We used human colon cancer PCR arrays (Origene) and found that tumoral cathelicidin mRNA expression was reduced in stage II colonic tumors, but not in stage III and IV colonic tumors (Figure 4C).
The finding was consistent with a previous report.7 Cathelicidin inter-acts with two putative receptors, i.e., FPRL1 and P2RX7,22,23 which
mediate downstream effects. Normal colonic tissues and colonic tumors of all stages had positive mRNA expression of P2RX7 and FPRL1 (Figures 4D and 4E).
To identify the involvement of cathelicidin receptors in the anti-metastatic effects of cathelicidin, we pretreated the colon cancer SW620 cells with FPRL1 antagonist WRW4 and P2RX7 antagonist KN62, followed by LL-37 exposure. The LL-37-mediated inhibition of cell migration was prevented by KN62, but not by WRW4 (Fig-ure 4F). KN62 also prevented the LL-37-mediated disruption of tubulin structure in SW620 cells (Figure 5A). Exposure of SW620 cells to LL-37 (5 mM) significantly reduced TUBB3 mRNA expres-sion (Figure 5B). This reduction was reversed by short hairpin RNA (shRNA) inhibition of P2RX7 (Figure 5B). Transient transfection of P2RX7 shRNA significantly reduced P2RX7 mRNA expression in SW620 cells (Figure 5C). Also, KN62 prevented the LL-37-medi-
Figure 2. Intravenous Cathelicidin-Expressing Adeno-Associated Virus Administration Reduced the Presence of Human Colon Cancer Cells in Lungs and Liver of HT-29-Loaded Nude Mice
(A and B) Human cytokeratin-18 expression (representing human colon cancer cells) in (A) lungs and (B) liver of nude mice was identified by brown color spots (indicated by arrows). Intravenous cathelicidin expressing AAVs reduced human cytokeratin 18 expression in lungs and liver of nude mice. (C and D) Human keratin 20 mRNA expression in (C) lungs and (D) liver of nude mice was significantly reduced by CAMP-HA-AAV.