Targeting Smoothened Sensitizes Gastric Cancer to Chemotherapy in Experimental Models

Abstract

BACKGROUND The Hedgehog pathway receptor smoothened (SMO) has critical roles in tumor progression. However, whether SMO is a key factor regulating gastric cancer chemotherapy resistance is unknown. MATERIAL AND METHODS We investigated the potential functions of SMO in inducing gastric cancer paclitaxel resistance in clinical samples, gastric cancer cell lines (424GC and AGS), and subcutaneous syngeneic mouse models. RESULTS We found high SMO expression in paclitaxel-resistant gastric cancer clinical samples. Paclitaxel gastric cancer cells had higher SMO expression than in drug-sensitive cells. Upregulating SMO expression induced paclitaxel resistance in gastric cells lines via enhancing cell proliferation and inhibiting apoptosis. The combination of IPI-926, an inhibitor of SMO, with paclitaxel decreased cell viability of paclitaxel-resistant gastric cancer cells in vitro and controlled tumor growth in animal models. CONCLUSIONS The Hedgehog pathway receptor SMO is an important regulator of gastric cancer paclitaxel resistance and could be a target for sensitizing paclitaxel-resistant tumors.

Figure 1

Expression of SMO protein in the tumor tissue of gastric cancer patients. (A) Representative images of IHC-stained human gastric cancer tissues with high SMO expression and low SMO expression, respectively. (B) The stage-IV patients tended to have high SMO expression, while the stage III patients tended to have low SMO expression. (C) The relationship between paclitaxel resistance and SMO expression level in gastric cancer patients. The patients who were resistant to paclitaxel treatment tended to have high SMO expression, but the patients who were sensitive to the paclitaxel treatment tended to have low SMO expression.

Figure 2

Expression of SMO in paclitaxel-resistant gastric cancer cell lines. (A) With the treatment of paclitaxel, the cell viability of paclitaxel-resistant gastric cancer cell line 424GC-R was much higher than in the wild-type 424GC cell line. (B) The cell viability of paclitaxel-resistant gastric cell line AGS-R was higher than in the wild-type AGS cell line under increasing concentrations of paclitaxel treatment. (C) The qPCR assay indicated that paclitaxel-resistant gastric cancer cell line 424GC-R and AGS-R had increased levels of SMO mRNA compared with the wide-type cell lines. (D) Western blotting assay indicated that the SMO protein expression level in the paclitaxel-resistant 424GC-R and AGS-R cell lines were higher than that of the wild-type cell lines (424GC and AGS).

Figure 3

SMO overexpression induced gastric cell line resistance to paclitaxel treatment. (A) The Western blotting assay of 424GC cells with different SMO levels. (B) Cell viability curves showed that the SMO overexpression in the 424GC cell line was highly resistant to paclitaxel treatment. (C) The BrdU incorporation of the SMO overexpression 424GC was high. (D) The cells were treated with 100 nM paclitaxel for 24 h. The activated caspase-3 level was low in the SMO-overexpression 424GC cell line, even under paclitaxel treatment.

Figure 4

SMO inhibitor reversed paclitaxel resistance in gastric cancer models. (A) The cell viability curves indicated that the 424GC-R cell line was resistant to high-dose paclitaxel (1000 nM) treatment, but when combined with IPI-929, a smaller dose of paclitaxel (100 nM) achieved significant inhibition of cell viability. (B) Tumor growth of the 424GC-R subcutaneous mouse models treated with paclitaxel + IPI-929 was much slower than in the mice treated with paclitaxel alone. (C) BrdU incorporation of the tumor tissue from the mouse models treated with paclitaxel + IPI-929 was much less than that of the mice treated with paclitaxel alone. (D) The level of activated caspase-3 was greatly increased in the tumor tissue from the mice treated with paclitaxel + IPI-929 compared with the mice treated with paclitaxel alone.