A novel CXCL8 analog is effective in inhibiting the growth via cell cycle arrest and attenuating invasion of Lewis lung carcinoma

Abstract

Purpose: Lung cancer and other solid tumors contain not only tumor cells but various types of stromal cells, such as fibroblasts and endothelial cells. In addition, tumors are infiltrated by inflammatory cells (neutrophils, macrophages, and lymphocytes). Tumor cells, stromal cells, and the tumor-associated leukocytes are responsible for the production of chemokines inside the tumor and the maintenance of systemic circulating chemokine levels. CXCL8 and its receptors, CXCR1 and CXCR2, were found to play important roles in tumor proliferation, migration, survival, and growth. We have developed a novel ELR-CXC chemokine antagonist CXCL8-IP10 based on the structure of CXCL8 and IP10.

Patients and methods: We assessed the anticancer efficacies of the blockade of CXCL8-CXCR1/2 axis in the Lewis lung carcinoma (LL/2) model using CXCL8-IP10.

Results: We found that CXCL8-IP10 markedly reduced LL/2 cell anchorage-independent growth and invasion. Moreover, we demonstrated that CXCL8-IP10 could significantly suppress tumor growth and improve survival rate as well as lifespan of C57BL/6 mice inoculated with LL/2 cells.

Conclusion: Our results suggest that ELR-CXC chemokine antagonism would potentially be a useful therapeutic approach in patients with lung cancer.

Keywords: CXCL8; CXCR1; CXCR2; Lewis lung carcinoma; antagonist.

Figure 1

CXCL8-survival and CXCL8-EMT clinical analysis by the TCGA LUAD from the UCSC Cancer Genomics Browser. Notes: (A) CXCL8 expression predicts poor prognosis in lung adenocarcinoma. (B) CXCL8 level is associated with EMT marker expression in lung adenocarcinoma.

Figure 2

CXCL8-IP10 inhibit the EMT characteristics of LL/2. Notes: After treating CXCL8-IP10 200 ng/mL for 48 hrs, (A) Western blot analysis of endothelial, E-cadherin, and mesenchymal marker, N-cadherin, was conducted. (B) and (C) The panels of Western blot analysis were calculated and quantified into bar chart. Results were obtained from three experiments, and the bars represent means±SEM. *P<0.05; **P<0.01, Student’s t-test.

Figure 3

Inhibition of CXCL8-induced anoikis-resistant growth by CXCL8-IP10 in the doubling-time assay of suspended LL/2 cells. Notes: The cells were seeded in non-coated 24-well plates and cultured for 72 hrs in DMEM and were counted every 24 hrs by pipetting 10 μL/well to a hemocytometer using the dye exclusion test. (A) Cells were treated with CXCL8-IP10 200 ng/mL and compared to control (DMEM). (B) Cells were treated with CXCL8 100 ng/mL or CXCL8 100 ng/mL plus CXCL8-IP10 200 ng/mL. Results were obtained from three experiments, and the bars represent means±SEM. *P<0.05; **P<0.01, Student’s t-test.

Figure 4

Cell anchorage-independent growth decreased when treated with CXCL8-IP10. Notes: LL/2 cells were plated in non-coated 24-well plates and cultured in growth medium. After 24 hrs, the media with cells were taken from wells to the other 96-well plates, and CCK-8 solution was added about 2 hrs. The absorbance of each well was determined at 450 nm and cell growth rate was calculated. (A) Cells were treated with CXCL8-IP10 200 ng/mL and compared to control (DMEM). (B) Cells were treated with CXCL8 100 ng/mL or CXCL8 100 ng/mL plus CXCL8-IP10 200 ng/mL. Results were obtained from three experiments, and the bars represent means±SEM. *P<0.05; **P<0.01; ***P<0.001, Student’s t-test.

Figure 5

G1 Cell cycle arrest by CXCL8-IP10 on suspended LL/2 cells. Notes: The cells were starved for 16–18 hrs, and then treated with drugs for 24 hrs. The results were followed by analysis with a flow cytometer (BD Accuri™ C6). Cell cycle analysis of (A) CXCL8-IP10 200 ng/mL compared with control (growth medium) and (B) CXCL8 100 ng/mL plus CXCL8-IP10 200 ng/mL compared with CXCL8 100 ng/mL shows arrest in G1 phase. Results were obtained from at least three experiments, and the bars represent means±SEM. *P<0.05; **P<0.01; ***P<0.001, Student’s t-test.

Figure 6

The CXCL8 and MIP-2 capability of invasion was attenuated by CXCL8-IP10 in LL/2 cells. Notes: Cells were seeded onto Matrigel-coated polycarbonate filters to analyze their invasive potentials. The cells were then incubated for 24 hrs in modified Boyden chambers and the cells that invaded through the filters were stained by PI and counted under a light microscope. Representative fields of invasive cells on the transwell membrane (at 100x magnification) and the invasion rate show in two conditions. (A) cells were treated with CXCL8 100 ng/mL or CXCL8 100 ng/mL plus CXCL8-IP10 200 ng/mL and (B) cells were treated with MIP-2 50 ng/mL or MIP-2 50 ng/mL plus CXCL8-IP10 200 ng/mL. Results were obtained from five random fields in each well and were repeated three times. The bars represent means±SEM. *P<0.05; ***P<0.001, Student’s t-test.

Figure 7

In vivo anti-tumor effect of CXCL8-IP10. Notes: LL/2 cells were subcutaneously inoculated to male C57BL/6 mice (4- to 6-week old). Then, CXCL8-IP10 (500 μg/kg) or saline (control group) was administrated by intraperitoneal injection every 3 times a week. (A) The LL/2 tumor size was measured and (B) the survival curves of tumor-bearing mice were recorded after being treated with CXCL8-IP10 and control (saline). The survival rates of tumor-bearing mice were observed and recorded continuously until all mice were dead. (C) and (D) After mice were dead, tumor and lung tissue were taken out and pictures were taken as shown. Data are shown as means±SEM (n=6). *P<0.05; ***P<0.001 compared with control group, Student’s t-test.