br Cell Viability MTS Assay br SW cells
Cell Viability MTS Assay
SW620 cells in 96-well plates (106 cells/plate) were treated with trifluoroacetic SCH772984 (TFA) 0.1% or cathelicidin peptide (LL-37). Cell viability was measured with MTS solution (G3580, Promega), as described previously.16
Cell Migration and Invasion Assays
Cell migration and invasion assays of SW620 cells were performed using a modified Boyden chamber approach (ECM508 and ECM554, EMD Millipore), as we described previously.37 Briefly, TFA and cath-elicidin peptide (LL-37) were added to the lower chamber. Control-LV-and TUBB3-LV-infected SW620 (2.5 104) cells were seeded into the upper chamber and incubated for 7 h (for cell migration) and 24 h (for cell invasion) at 37 C. The FPRL1 antagonist WRW4 and the P2RX7 antagonist KN62 were added to both upper and lower chambers at the beginning of the cell migration experiments. Some of the SW620 cells were pretreated with control inhibitor (YI00199006, QIAGEN) and miR200c-3p inhibitor (YI04100915, QIAGEN) 24 h before the cell migration assay began. The migrated colon cancer cells through the membrane were stained and determined by absorbance at 650 nm.
The total RNA from lung and liver tissues were extracted with an RNeasy kit (74106, QIAGEN). The RNA was converted to cDNA by iScript cDNA Synthesis kit (170-8891, Bio-Rad). The mRNA
Figure 6. Cathelicidin Inhibited Colon Cancer Metastasis via P2RX7
(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 AAV admin-istration reduced human CK18 expression in lungs and liver of nude mice.
expression was determined with a Bio-Rad CFX384 PCR system, us-ing Thermo Fisher Scientific’s inventoried primers (Table 1) and iTaq Universal Probe Supermix (172-5135, Bio-Rad). Human colon cancer PCR arrays (HCRT101) were purchased from Origene. Relative mRNA expression was normalized to human 18S or mouse GAPDH mRNA expression. miRNAs were converted to cDNA by miRCURY LNA RT kit (339340, QIAGEN). PCR reactions were determined using miRCURY LNA SYBR Green PCR kit (339346, QIAGEN). Relative miRNA expression was normalized to RNU1A1 expression. Results were expressed as fold induction compared to their respective controls, as we described previously.16
Tubulin Tracker Staining
45 min, followed by Hoechst 33342 (H3570, Invitrogen), and viewed under a Zeiss AX10 confocal microscope, as described previously.16
Results were analyzed using Prism professional statistics software program (GraphPad). Unpaired Student’s t tests were used for intergroup comparisons. Quantitative results were expressed with error bars as means ± SEM. Only p values of statistically signif-icant differences (p < 0.05) are shown in the figures.
Molecular Therapy: Oncolytics
Figure 7. Cathelicidin Inhibited TUBB3 mRNA Expression in Metastasized Tumors via P2RX7
(A and B) Human keratin 20 mRNA expression (human colon cancer cell marker) in (A) lungs and (B) liver of nude mice was significantly reduced by CAMP-HA-AAV. Intraperitoneal KN62 treatment increased the presence of cytokeratin 18 protein and keratin 20 mRNA expression in the CAMP-HA-AAV-treated nude mice. (C and D) Human TUBB3 mRNA expression in (C) lungs and (D) liver of nude mice was significantly reduced by CAMP-HA-AAV. Intraperitoneal KN62 treatment increased the presence of human TUBB3 mRNA expression in the CAMP-HA-AAV-treated nude mice. (E) Diameters of subcutaneous tumors. Intravenous CAMP-HA-AAV significantly reduced subcutaneous tumor diameters in nude mice that were partially reversed by KN62 treatment.
J.W., M.C., I.K.M.L., C.O., and M.S. contributed the animal study and cell culture data. H.W.K. supervised the study, drafted the manu-script, and approved the submitted final version. All authors reviewed and approved the final version of the manuscript.
CONFLICTS OF INTEREST
The authors declare no competing interests.
This work was supported by an NIH grant (K01-DK084256) to H.W.K. and a student research fellowship from the Crohn’s and Colitis Foundation of America (287244) to M.C. The funding sponsors were not involved in the design of the study or the collec-tion, analysis, and interpretation of the data. We thank Prof. Char-alabos Pothoulakis for financial assistance and Samantha Ho for
technical assistance in the experiments. The data and materials described in this manuscript are available for sharing. Please con-tact H.W.K.