In this context, OR-DLD1 and OR-RKO cells co-cultured in the presence and absence of NK cells were subjected to RT-PCR and Western blot analyses to assess the expression levels of both WBSCR22 mRNA and protein, respectively. cell lines DLD1 and RKO were exposed to long term oxaliplatin treatment to establish oxaliplatin-resistant cell lines (see materials and methods). (A) Resistant DLD1 and (B) RKO, respectively with their parental cell lines, were incubated in the presence of oxaliplatin, followed by clonogenic assay to assess their survival. Data are presented as mean SD from at least three independent experiments. **P<0.01, resistant vs parental. NK cell co-culture reduces growth of OR colorectal cancer cell lines in vitro Next, to assess the effect of NK cells on these established OR-DLD1 and OR-RKO cells, a co-culture system of employed using previously reported protocol [13]. Viability and proliferation of the OR-DLD1 and OR-RKO cells, in the presence or absence of NK cell co-culture, were then assessed by clonogenic assay and CCK8 kit, respectively. As shown in Figure 2A and ?and2B,2B, both viability and proliferation of OR-DLD1 and OR-RKO cells were significantly reduced when they are co-cultured with NK cells, compared to the control. Moreover, wound healing assay and transwell invasion assay were also used to assess migration and invasion capacities of the OR-DLD1 and ORRKO cells. Similarly, both migration and invasion were significantly inhibited when these two cell lines were co-cultured with NK cells (Figure 3A and ?and3B3B). Open in a separate window Figure 2 NK cell co-culture reduces viability and proliferation of oxaliplatin-resistant colorectal cancer cell lines. Oxaliplatin-resistant (OR)-DLD1 and OR-RKO cells were subjected to (A) viability and (B) proliferation assays, in the absence (NK cell -) or presence (NK cell +) of co-cultured natural killer cells, respectively. Data are presented as mean SD from at least three independent experiments. **P<0.01, *P<0.05, NK cell - vs NK cell +. Open in a separate window Figure 3 NK cell co-culture reduces migration and invasion capacities of oxaliplatin-resistant colorectal cancer cell lines. Oxaliplatin-resistant (OR)-DLD1 and OR-RKO cells were Sulfo-NHS-LC-Biotin subjected to (A) wound healing and (B) transwell invasion assays, in the absence (NK cell -) or presence (NK cell +) of co-cultured natural killer cells, respectively. Data are presented as mean SD from at least three independent experiments. **P<0.01, *P<0.05, NK cell - vs NK cell +. NK cell co-culture reduces growth of OR colorectal cancer Mouse monoclonal to KARS cell lines in vivo To further investigate whether NK cells could exert inhibitory effect on OR-DLD1 and OR-RKO cells in vivo, we established xenograft tumor model in nude mice, by co-injecting them with NK cells. Growth of xenograft tumors were measured on day 7, 14 and 21 after injection, and the sizes of tumors in the NK cell co-injected group were significantly bigger than those without co-injected NK cells (Figure 4A and ?and4B).4B). At the end of day 21, tumors were extracted and their weight was measured, and we found the weight of tumors in the NK cell co-injected mice were also significantly heavier than those without co-injected NK cells (Figure 4C and ?and4D4D). Open in a separate window Figure 4 NK cell co-culture reduces migration and invasion capacities of oxaliplatin-resistant colorectal cancer cell lines. A and B. Oxaliplatin-resistant (OR)-DLD1 and OR-RKO cells were injected into mice (n=8 each group), without (NK cell Sulfo-NHS-LC-Biotin -) or with co-injection of natural killer cells Sulfo-NHS-LC-Biotin (NK cell +), respectively, followed by growth curve evaluation on day 7, 14 and 21 after injection. C and D. At the end of day 21, xenograft tumors from all mice were extracted and weighed. Data are presented as mean SD, n=8 each group. **P<0.01, *P<0.05, NK cell - vs NK cell +. NK cell co-culture reduces WBSCR22 expression in oxaliplatin-resistant colorectal cancer cell lines Since WBSCR22 was very recently reported to confer oxaliplatin resistance in human colorectal cancer [14], we wondered whether NK cells could regulate its expression in the OR-DLD1 and OR-RKO cells. In this context, OR-DLD1 and OR-RKO cells co-cultured in the presence and absence of NK cells were subjected to.