HCT-116

Recombinant Chemokines and Supernatants Collected From HCT-116 Colorectal Cancer Cells Stimulate the Mobilization of Intracellular Ca²⁺ in NK92 Cells

It is known that there is a strong association between chemotaxis and the mobilization of intracellular calcium. To confirm such principles in NK92 cells, the mobilization of intracellular Ca²⁺ was assessed. First, Triton-X was used as a control to induce the release of intracellular calcium and EGTA to chelate this response. Next, two different concentrations of CXCL16, CCL27, and CCL28 were examined, namely 10 and 100 ng/mL, and observed that the 100 ng/mL concentration of the three chemokines induced the mobilization of (Ca²⁺); however, only the 10 ng/mL concentration of CCL28 produced a positive response. Intriguingly, the undiluted, 1:1 dilution or 1:10 dilution of supernatants collected after 24 or 48 h from HCT-116 cells also provoked the mobilization of (Ca²⁺) in NK92 cells.

To verify whether the supernatants collected from HCT-116 may utilize similar receptors to that of recombinant chemokines, desensitization experiments were performed. To gain insight into this issue, fura-2-AM-labeled NK92 cells were incubated with undiluted supernatants collected from HCT-116 cells after 24 h or 48 h incubation. The results indicate that addition of 24 h supernatants entirely desensitized the effects of the 24 h supernatants, the CXCL16 and CCL28, but there were partial desensitization of the 48 h supernatants and no effect on CCL27 (Fig. 1a). However, addition of the 48 h supernatants desensitized the calcium mobilization effects of the 48 h supernatants, 24 h supernatants, CXCL16, CCL27, and CCL28 (Fig. 1a). The reciprocal experiments were performed where recombinant chemokines were added before the addition of the supernatants. CXCL16 desensitized CXCL16 activity but not the activity of the supernatants (Fig. 1b). Similarly, CCL27 and CCL28 desensitized CCL27 and CCL28 activity, respectively, but not the supernatants with the exception where CCL28 partly desensitized the effect of 24 h supernatants (Fig. 1b). These results confirm that 24 h supernatants contain CXCL16 and CCL28, whereas 48 h supernatants contain all the three chemokines examined.

Fig. 1 Desensitization of chemokine receptors with recombinant chemokines and HCT-116 supernatants. (Elemam NM, et al., 2019)

Overexpression of MDR1 Induces Drug Resistance Phenotype in HCT-116 Cells

The MDR1 gene was transfected into HCT-116 cells and analyzed the resulting transfected cells (HCT-116/R) for the drug-resistant phenotype under a microscope. The cells overexpressing MDR1 had more intracellular vacuoles compared to the wild-type cells (HCT-116) (Fig. 2a). Next, the ability of HCT-116/R cells to reduce the drug uptake was analyzed using rhodamine 123 staining. Most of the HCT-116 cells showed intense rhodamine 123 fluorescence, whereas the fluorescence of HCT-116/R cells was weaker, which indicates that the uptake of rhodamine 123 by the MDR1-overexpressing cells was reduced (Fig. 2b). The intracellular distribution and accumulation of doxorubicin were analyzed in both cell lines and found that most of HCT-116 cells displayed prominent doxorubicin fluorescence in the nucleus, while HCT-116/R cells showed no doxorubicin fluorescence in the nucleus and diminished fluorescence in the cytoplasm (Fig. 2c). The overexpression of MDR1 in HCT-116/R cells was also confirmed by immunoblotting (Fig. 2d). Moreover, expression of HIF-1α and Nrf-2 was also upregulated. These results suggest that the elevated expression of MDR1, HIF-1α, and Nrf-2 is associated with the development of drug resistance.

The chemosensitivity of HCT-116 and HCT-116/R cells was determined to 5-FU. The half-maximal inhibitory concentration (IC₅₀) of 5-FU for HCT-116 and HCT-116/R was ~241.40 and ~702.30 µM, respectively (Fig. 2e), which indicates that the HCT-116/R cells are highly resistant to 5-FU. Therefore, the MDR1 overexpression confers drug resistance in human colorectal carcinoma (HCT-116) cells.

Fig. 2 Characterization of drug-resistant HCT-116/R cells obtained by transfection of HCT-116 cells with the MDR1 gene. (Waghela BN, et al., 2021)