Cancer Res Treat. 2016;48(1):355-64.

Antitumor Activity of HM781-36B, alone or in Combination with Chemotherapeutic Agents, in Colorectal Cancer Cells.

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제1저자 강미현 · 교신저자 김지현

PMID 25761479 PMC4720083 DOI 10.4143/crt.2014.260 IF 4.2 혈액종양내과

소속 (3)

Division of Hematology and Medical Oncology, Department of Internal Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea ; Biomedical Research Institute, Seoul National University Bundang Hospital, Seongnam, Korea.
Division of Hematology and Medical Oncology, Department of Internal Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea.
Department of Pathology, Seoul National University Bundang Hospital, Seongnam, Korea.

Abstract

PURPOSE HM781-36B is a novel and irreversible pan-human epidermal growth factor receptor (HER) inhibitor with TEC cytoplasmic kinase inhibition. The aim of this study is to evaluate the antitumor activity and mechanism of action for HM781-36B in CRC cell lines.

MATERIALS AND METHODS The CRC cell lines were exposed to HM781-36B and/or oxaliplatin (L-OHP), 5-fluorouracil (5-FU), SN-38. The cell viability was examined by Cell Titer-Glo luminescent cell viability assay kit. Change in the cell cycle and protein expression was determined by flow cytometry and immunoblot analysis, respectively. Synergism between 2 drugs was evaluated by the combination index.

RESULTS The addition of HM781-36B induced potent growth inhibition in both DiFi cells with EGFR overexpression and SNU-175 cells (IC50, 0.003 µM and 0.005 µM, respectively). Furthermore, HM781-36B induced G1 arrest of the cell cycle and apoptosis, and reduced the levels of HER family and downstream signaling molecules, pERK and pAKT, as well as nonreceptor/cytoplasmic tyrosine kinase, BMX. The combination of HM781-36B with 5-FU, L-OHP, or SN-38 showed an additive or synergistic effect in most CRC cells.

CONCLUSION These findings suggest the potential roles of HM781-36B as the treatment for EGFR-overexpressing colon cancer, singly or in combination with chemotherapeutic agents. The role of BMX expression as a marker of response to HM781-36B should be further explored.

Keywords

BMXColorectal neoplasmEpidermal growth factor receptor-neu receptorHM781-36B

Figures

**Fig. 1.** The growth inhibitory effect of HM781-36B on a panel of human colorectal carcinoma cell lines. The cells were treated with increasing doses of HM781-36B (0.001-10 μM) for 72 hours. The number of viable cells after the treatment was measured using the luminescent Cell Titer-Glo assay and expressed as percentage viable cells. Data represents the mean±standard error of the mean of three independent experiments (n=3), each of which was replicated six times.

**Fig. 2.** Analysis of the cell cycle and apoptosis in colorectal carcinoma cell lines after HM781-36B treatment. The cells were treated with the indicated concentrations of HM781-36B for 48 hours. (A) Distribution of the cell cycle was examined by propidium iodide staining and analyzed using fluorescence-activated cell sorting. The mean percentage of cells in the sub G1, G1, S, and G2/M phases of the cell cycle for duplicate independent experiments were plotted. Data represents the mean±standard error of the mean. Sample means were compared using a Student’s t test. ^*p < 0.01 compared with the control. (B) Apoptosis-related proteins were visualized by Western blotting using anti–cleaved caspase-3, anti–cleaved caspse-9, anti–poly(ADP-ribose) polymerase (PARP), and anti–Bcl-2 antibodies. Equal loading was identified by showing the total β-actin levels. The results are representative of two independent experiments (n=2).

**Fig. 3.** Basal level expression of the HER family and BMX, and fluorescence *in situ* hybridization (FISH) analysis in CRC cell lines. (A) The basal protein levels of epidermal growth factor receptor (EGFR), HER2, and BMX were evaluated in six colorectal cancer cell lines by Western blotting. (B) Hybridization of prepared cell lines with Vysis EGFR/CEP 7 FISH probe kit was performed, as described in the Materials and Methods section. The amplification of EGFR is positive in DiFi cells (left) and negative in the SNU-175 cells (right) (×100).

**Fig. 4.** Protein expression of the HER family and downstream signaling molecules in colorectal cancer cells following treatment with HM781-36B. Cells were treated with increasing concentrations of HM781-36B (0.001, 0.01, and 0.1 μM) for 48 hours. Cells were lysed, and proteins were analyzed by Western blotting with the indicated antibodies. Results are representative of two independent experiments (n=2). β-Actin was used as a loading control.

**Fig. 5.** The synergistic effect of colorectal cancer cells treated with HM781-36B in combination with chemotherapeutic drugs (oxaliplatin [L-OHP], 5-fluorouracil [5-FU], and SN-38). Synergism was assessed by the Chou and Talalay equation and the CalcuSyn software ver. 2.1 (Biosoft). Each column shows a combination index (CI) value at the 50% fraction affected. Data represents the means±standard error of the mean.

그림 출처: PubMed Central 공개 논문 (PMC4720083)

Conflict of interest statement

Conflict of interest relevant to this article was not reported.

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