Anlotinib suppresses metastasis and multidrug resistance via dual blockade of MET/ABCB1 in colorectal carcinoma cells

Abstract

Anlotinib, a highly selective multi-targeted tyrosine kinase inhibitor (TKI) has therapeutic effects on non-small-cell lung cancer (NSCLC). In this study, the anti-tumor activity and molecular mechanism of anlotinib in metastatic colorectal cancer (mCRC) was explored. The anti-angiogenesis, anti-metastasis, anti-proliferative, and anti-multidrug resistance efficacy of anlotinib were analyzed by using in vitro and in vivo models of human CRC cells. The results indicated that anlotinib boosted chemo-sensitivity of CRC cells, and restrained its proliferation. Besides the suppression of the MET signaling pathway, anlotinib also inhibited invasion and migration of CRC cells. Furthermore, anlotinib prevented VEGF-induced angiogenesis, N-cadherin (CDH2)-induced cell migration, and reversed ATP-binding cassette subfamily B member 1 (ABCB1) -mediated CRC multidrug resistance in CRC. The CRC liver metastasis and subcutaneously implanted xenograft model testified that anlotinib could inhibit proliferation and liver metastasis in CRC cells. Such an observation suggested that a combination of anlotinib with anti-cancer drugs could attenuate angiogenesis, metastasis, proliferative, and multidrug resistance, which constitutes a novel treatment strategy for CRC patients with metastasis.

Keywords: ABCB1; MET; anlotinb; colorectal carcinoma; metastasis.

Figure 1

Anlotinib inhibits cell proliferation and induces cell apoptosis in human CRC cells. (A and B) HT-29, LoVo, HCT116 and HCT116/L cells were treated with anlotinib for 24 or 48 hours. Cell viability was measured by CCK8 kit. (C) anlotinib reduces colony formation in CRC cells by clonogenic assay. (D) Anlotinib enhances the chemosensitivity of CRC cells to oxaliplatin (OXA). (E) The percentages of apoptotic cells in the indicated cell populations are shown in the histogram. (F) Western blot assay was used to detect the expression of the apoptosis-related proteins cleaved PARP in HT-29, LoVo, HCT116 and HCT116/L cells, *P<0.05, **P<0.01.

Figure 2

Anlotinib inhibits migration in CRC cells. (A and B) Cell migration was measured by wound healing assay. The degrees to which the wounds healed in each group are shown in the histogram. (C and D) Cell invasion was measured by transwell assay. (E) The mRNA levels of metastatic markers were detected using qRT-PCR assay after 24 hours of anlotinib treatment at concentration of 0, 1.25, 2.5 and 5.0 µM. (F) N-cadherin levels were detected by IF staining after 24 hours of anlotinib treatment at 5.0 µM. N-cadherin stained red, and the nuclei, which were stained with DAPI, stained blue. *P<0.05, **P<0.01.

Figure 3

Anlotinib inhibits MET and EGFR signaling pathway in vitro. (A) MET and EGFR mRNA expressions were measured by qRT-PCR. (B) MET and EGFR protein levels were detected by IF staining after 24 hours of anlotinib treatment at 5.0 µM in HT-29, LoVo, HCT116 and HCT116/L cells. MET and EGFR protein stained red, and the nuclei, which were stained with DAPI, stained blue. (C) Western blot analysis was performed to detect EGFR and MET phosphorylation in CRC cells at concentration of 0, 1.25, 2.5 and 5.0 µM. *P<0.05, **P<0.01.

Figure 4

Anlotinib inhibits ABCB1-induced CRC cell multidrug resistance. (A) ABCB1 and ABCG2 mRNA expressions were measured by qRT-PCR. (B) EGFR protein levels were detected by IF staining after 24 hours of anlotinib treatment at 5.0 µM in HT-29, LoVo, HCT116, and HCT116/L cells. (C) Western blot analysis was performed to detect ABCB1 and ABCG2 in CRC cells at concentration of 0, 1.25, 2.5 and 5.0 µM. (D) Western blot analysis was performed to detect ABCB1-dependent signaling pathway phosphorylation in CRC cells. (E) HUVECs were preincubated with 50 ng/ml VEGF for 30 minutes, after which they were incubated with or without anlotinib at concentration of 0, 1.25, 2.5, 5.0, 10.0, and 15.0 µM for 12 hours, Western blot analysis then was performed to detect ABCB1-dependent signaling pathway phosphorylation in CRC cells.

Figure 5

Anlotinib inhibits tumor growth, metastasis, and angiogenesis in the CRC liver metastasis and subcutaneously implanted xenograft model. (A) The liver metastasis xenograft model of CRC were established, HCT116/L cells stably expressing green luciferase (-GFP) were injected into the tail veins of all mice, which were treated with an oral dose of 6 mg/kg anlotinib. (B) Liver metastasis volumes of anlotinib group was reduced compared with the NC group. (C) Hematoxylin-eosin staining of liver tumor metastasis specimens from the NC and anlotinib groups. (D) Western blot analysis was performed to detect ABCB1 and EGFR in liver metastasis tumor specimens. The subcutaneously implanted tumor model of HCT116/L cells were also established. (E and F) Tumor weights and volumes of anlotinib group were reduced compared with the NC group. (G) The percentage of apoptosis was detected by TUNEL assay, the apoptosis was decreased in the anlotinib group compared with the NC group. (H) Representative pictures of p-MET immunostaining are shown. (I) Molecular pathways involved in anlotinib inhibiting CRC liver metastases. Most notable signaling pathways are EGFR, MET, and ABCB1.