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    2020-08-28


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    Figure 4. Cathelicidin-Mediated Inhibition of Colon
    Cancer Cell Migration Was P2RX7 Dependent
    (A) SW620 cells were transfected with control lenti-
    virus or TUBB3-overexpressing lentivirus, followed by
    control small interfering RNA (siRNA) or P2RX7 siRNA
    P2RX7 mRNA expression in human colon cancer
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    D
    Results were pooled from three independent experi-ments.
    (Figure S2D and S2F). Therefore, miR200c-mediated TUBB3 inhi-bition is relevant to cathelicidin-mediated inhibition of cell migra-tion. We did not determine miR200c expression in the lung and liver tissues of HT-29-loaded nude mice, because the miR200c primers could not differentiate between the human and mouse miR200c.
    Cathelicidin is a molecular target for colon cancer. 67769-47-5 Systemic infusion
    or oral administration of cathelicidin peptide is not feasible, because cathelicidin peptides degrade in body fluids or blood.31,32 A non-pep-
    tide cathelicidin mimic Ceragenin CSA13 is effective in inducing 67769-47-5 arrest and apoptosis in cultured colon cancer HCT116, HT-29, and DLD1 cells.14,33 Development of clinically applicable cathelici-din-based strategies for colon cancer metastasis needs further investigation.
    In conclusion, cathelicidin inhibits colon cancer metastasis via a P2RX7 pathway. 
    MATERIALS AND METHODS
    Cell Culture
    Human colon cancer HT-29, SW480, and SW620 cells were cultured in DMEM (10564, Thermo Fisher Scientific) containing 10% fetal calf serum (Thermo Fisher Scientific) and 1% penicillin-streptomycin (Thermo Fisher Scientific). All cultured cells were pur-chased from American Type Culture Collection (ATCC).
    Intravenous Injection of Colon Cancer Cells into Nude Mice
    HT-29 cells (1 106 cells) in Hanks’ balanced salt solution (100 mL) were injected intravenously into 8-week-old male and female nude mice (stock number 002019, Jackson Laboratory) for the determina-tion of colon cancer metastasis. The same number of cells was injected subcutaneously into the left and right flanks of the nude mice for the determination of local tumor growth. The injected nude mice were housed in the UCLA animal facility under standard conditions, as described previously.16 All animal experiments were approved by the UCLA Animal Research Committee (#2007-116).
    The human CAMP-HA-AAV and control HA-AAV were generated by Vector Laboratories, as described previously.16 HA-AAV and CAMP-HA-AAV (1 1012 genomic copies in 100 mL) were injected intravenously via tail veins into nude mice under transient isoflurane
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    anesthesia. Some of the nude mice were injected intraperitoneally with P2RX7 antagonist KN62 (5 mg/kg, Tocris) or control DMSO solution (50 mL per mouse) daily from day 21 to day 28. Lung and liver tissues were collected for analyses on day 28. Each group con-sisted of 10 mice per group in two separate experiments.
    Tail vein intravenous injection, but not orthotopic transplantation, was used because the intravenous injection model simulates how cathelicidin affects migration of CTCs to the distant organs.34,35 SW620 cells were not used in this study because intravenous injection
    of these cells fails to develop metastatic tumors in the lungs and liver of nude mic.36
    Immunohistochemistry
    Immunohistochemistry was assisted by UCLA Translational Pathol-ogy Core Laboratory (TPCL), as described previously.16 In short,  Molecular Therapy: Oncolytics
    Figure 5. Cathelicidin Inhibited TUBB3 Expression via P2RX7
    (A) Green tubulin tracker staining with blue nuclear staining in human cancer SW620 cells. SW620 cells were pretreated with DMSO (10 mL/mL) or KN62 (10 mM) for 30 min, followed by exposure to LL-37 (5 mM) for 24 h. LL-37 reduced tubulin expression in SW620 cells that was prevented by KN62. (B) SW620 cells were transiently transfected with control small interfering RNA (siRNA) or P2RX7 shRNA (1 mg/mL), followed by exposure to LL-37. TUBB3 mRNA expression in SW620 cells. (C) P2RX7 mRNA expression in the transfected SW620 cells. (D) HT-29 cells were pretreated with DMSO (10 mL/mL) or KN62 (10 mM) for 30 min, followed by exposure to LL-37. TUBB3 mRNA expression. Results were pooled from three independent experiments.
    paraffin-embedded lung and liver tissue sec-tions were stained with a rabbit monoclonal anti-LL-37 antibody (ab207758, Abcam, 1:50 dilution), mouse polyclonal anti-cytokeratin 18 antibody (sc-51583, Santa Cruz Biotech-nology, 1:50 dilution), or anti-HA-tagged anti-body (3724, Cell Signaling Technology, 1:50 dilution). Images were recorded with a Zeiss AX10 microscope.