br and colony formation assays
and colony formation assays, we found that combined use of AD and vemurafenib exerted a more potent eﬀect on suppression of cancer cell proliferation compared to either vemurafenib or AD treatment alone in CDK1-expressing colon cancer Acarbose (Fig. 6A and B). To examine whe-ther AD reverses the resistance of CDK1-overexpressing colon cancer cells to vemurafenib in vivo, HT29 cells with ectopic expression of CDK1 were subcutaneously injected into nude mice, and tumor-bearing mice were treated with vemurafenib, AD alone or a combination. Although vemurafenib or AD had no eﬀect or only a modest anti-proliferative eﬀect on tumors, combined vemurafenib and AD exerted a significantly synergistic eﬀect on suppressing the growth of tumor xenografts (Fig. 6C). Moreover, immunohistochemical analysis of Ki-67 prolifera-tion index also provided evidence that tumor cell proliferation was significantly inhibited by combined use of vemurafenib and AD (mean index decreased from 51.7% in the vehicle-treated group to 47.0% in the vemurafenib-treated group and 22.0% in the AD/Vemurafenib combination-treated group) (Fig. 6D). Western blot analysis of tumor xenografts showed that CDK1 activation in VR tumors was significantly inhibited by combined use of vemurafenib and AD (Fig. 6E). Con-sidering the lack of significant changes in body weight (Fig. 6F), our results demonstrate treatment eﬃcacy and safety of AD as a vemur-afenib sensitizer in colon cancer cells. r> 4. Discussion
B-Raf is frequently overexpressed in diﬀerent cancers and plays an important role in tumorigenesis [7,8]. Previous studies have demon-strated that vemurafenib exhibits significant eﬀects on suppressing melanoma cell proliferation by targeting B-Raf/B-Raf V600E. However, the mechanism by which vemurafenib suppresses cancer cell pro-liferation remains unclear, and the therapeutic eﬃcacy of vemurafenib on colon cancer is reduced compared to melanoma . Understanding the molecular mechanisms underlying drug resistance can provide clues to identify potential targets for development of anticancer drugs. A recent study demonstrated that colon cancer is unresponsive to ve-murafenib treatment due to feedback activation of EGFR . More-over, cancer cells will also be unresponsive to targeted drugs if certain “front-line proteins”, including direct performers, such as cyclins and epithelial-mesenchymal transition (EMT) factors that receive signals from upstream factors and lead to tumorigenesis and cancer develop-ment, are activated . This finding suggests that targeting “front-line proteins” may be a better strategy for overcoming tumor resistance to chemotherapeutic drugs. In this study, we uncovered the mechanism for chemoresistance of colon cancer cells to vemurafenib and aimed to resolve this problem by focusing on “front-line proteins”. Using IPA analysis, we found that cell cycle checkpoint signaling is one of the biological processes aﬀected by vemurafenib, and expression of CDK1, one of the “front-line proteins”, was significantly decreased. Further-more, CDK1 overexpression significantly increased colon cancer cell resistance to vemurafenib, indicating that CDK1 is an eﬀector of ve-murafenib and plays an important role in vemurafenib resistance in colon cancer (Figs. 1–2, Supplementary Fig. S1).
Signal transduction is dependent on specific protein-protein inter-actions in cells [46,47], and emerging evidence suggests that these protein-protein interactions are potential targets for cancer treatment [48,49]. Modified ELISA has been used to detect protein-protein in-teractions and facilitates the screening of small molecules targeting protein-protein interaction in vitro. For example, researchers identified SLCB050 as a novel anti-tumor compound that inhibits DX2-p14/ARF
Fig. 5. AD suppresses tumorigenesis of colon cancer cells in vitro and in vivo. (A) HCT116 and HT29 cells treated with DMSO or AD (10 μM) for 48 h were stained with CFSE, and the rate of cell division was analyzed by flow cytometry. (B) Comparison of cell viability in colon cancer cells with or without AD treatment by WST-1 assays. (C) Colony formation ability of colon cancer cells with or without AD treatment was examined by colony formation assays. (D) Nude mice bearing HT29-derived xenografts were orally administered AD (10 mg/kg or 50 mg/kg) or vehicle daily (n = 6 per group). Tumor curves showing that AD significantly suppressed growth of tumor xenografts. (E) Expression levels of cyclin A2, pCDK1 and CDK1 were compared between tumors from mice treated with AD and vehicle, by Western blot. (F) Body weight of nude mice during the AD treatment period. (G) Representative images of liver, kidney and lung specimens stained with hematoxylin and eosin (H&E) indicated no toxic eﬀects. Bars, SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared to the control group treated with DMSO.