The Mechanism Underlying Inhibition of Colorectal Cancer Invasion by Quxie Capsule Through CRK-RAC Signaling Pathway Regulation.

BackgroundOur previous studies showed that Quxie capsule (QX) remarkably prolongs the survival of patients with advanced colorectal cancer (CRC) and downregulates CT10 regulator of kinase (CRK) protein expression in responders. However, the underlying mechanism through which QX inhibits CRC invasion remains unclear.ObjectiveTo elucidate the mechanism through which QX suppresses CRC invasion.MethodsThe IC50 values of QX and 5-fluorouracil (5-FU) were determined by CCK-8 assay to establish drug intervention concentrations. Flow cytometry, Transwell assays, and hematoxylin-eosin (HE) staining were used to evaluate the effects of QX and 5-FU combination on the proliferation, apoptosis, cell cycle, migration, invasion, and tube formation ability of CRC cells. Modulation of the CRK-RAC signaling pathway was analyzed by qRT-PCR and western blotting assay. For in vivo validation assay, tumor-bearing nude mouse models were utilized to measure tumor volume, and HE staining, immunohistochemical assay were conducted to assess tumor proliferation and apoptosis. A sh-CRKL knockdown stable cell line was established to investigate the role of CRKL in CRC invasion through simultaneous in vitro and in vivo experiments.ResultsProteomics analysis identified Crk-like protein (CRKL) as a differentially expressed protein in CRC. In vitro experiments demonstrated that the combined treatment of QX (0.589 mg/mL, 0.597 mg/mL) with 5-FU (4.036 μg/mL, 6.417 μg/mL) could enhance the inhibition of the proliferation of CRC cells (HCT116 and SW620) and induced apoptosis (P < 0.05), while significantly downregulating the CRKL-RAC signaling pathway activity. Knocking down the CRKL gene further confirmed that CRKL deficiency inhibits cancer cell invasion; in the CRKL-deficient model, the combination therapy of QX and 5-FU further enhanced the inhibitory effect on tumor progression (P < 0.05). In vivo experiments also validated the efficacy of this combination therapy: compared with the monotherapy group, the combination of QX and 5-FU significantly inhibited tumor growth in tumor-bearing mice (P < 0.05) and was accompanied by downregulation of key proteins in the CRKL-RAC signaling pathway (P < 0.05).ConclusionQX inhibits CRC invasion by downregulating CRKL protein expression and modulating the CRK-RAC signaling pathway; this finding provides a mechanistic basis for validating the clinical efficacy of QX.