CXCR1/2 antagonism with CXCL8/Interleukin-8 analogue CXCL8(3-72)K11R/G31P restricts lung cancer growth by inhibiting tumor cell proliferation and suppressing angiogenesis - PubMed (original) (raw)
CXCR1/2 antagonism with CXCL8/Interleukin-8 analogue CXCL8(3-72)K11R/G31P restricts lung cancer growth by inhibiting tumor cell proliferation and suppressing angiogenesis
Muhammad Noman Khan et al. Oncotarget. 2015.
Abstract
CXCR1 and CXCR2 together with cognate chemokines are significantly upregulated in a number of cancers, where they act as key regulators of tumor cell proliferation, metastasis, and angiogenesis. We have previously reported a mutant protein of CXCL8/Interleukin-8, CXCL8(3-72)K11R/G31P (G31P), which can act as a selective antagonist towards CXCR1/2 with therapeutic efficacy in both inflammatory diseases and malignancies. In this study, we investigated the effect of this ELR-CXC chemokine antagonist G31P on human non-small cell lung cancer cells and lung tumor progression in an orthotopic xenograft model. We report increased mRNA levels of CXCR1 and CXCR2 in human lung cancer tissues compared to normal counterparts. Expression levels of CXCR1/2 cognate ligands was determined by ELISA. CXCR1/2 receptor antagonism via G31P leads to decreased H460 and A549 cell proliferation and migration in a dose-dependent manner. G31P also enhanced apoptosis in lung cancer cells as determined by elevated levels of cleaved PARP, Caspase-8, and Bax, together with a reduced expression of the anti-apoptotic protein Bcl-2. In an in vivo orthotopic xenograft mouse model of human lung cancer, G31P treatment suppressed tumor growth, metastasis, and angiogenesis. At the molecular level, G31P treatment was correlated with decreased expression of VEGF and NFкB-p65, in addition to reduced phosphorylation of ERK1/2 and AKT. Our results suggest that G31P blockage of CXCR1 and CXCR2 can inhibit human lung cancer cell growth and metastasis, which offers potential therapeutic opportunities.
Keywords: CXCR antagonist; ELR-CXC chemokine; G31P; lung cancer.
Conflict of interest statement
CONFLICT OF INTERESTS
Competing financial interests: The authors declare no competing financial interests.
Figures
Figure 1. Expression of CXCR1 and CXCR2 receptors in human lung cancer tissue and cell lines
A. CXCR1 and CXCR2 mRNA was detected in a panel of non-small cell lung cancer cell lines as indicated. All five cell lines show CXCR1 and CXCR2 mRNA expression. B. CXCL1, 6, and 8 chemokines were detected from conditioned media of H460, A549, and H358 through ELISA. Data are summarized from 3 independent experiments; error bars represent SEM (standard error of the mean). C. CXCR1 and CXCR2 mRNA expression was quantified through PCR in human lung cancer tissue comparing to adjacent non-cancerous tissue from same patients (n = 8). CXCR1 and CXCR2 mRNA was expressed more in cancer tissue than non-cancerous counterpart. Results represent mean ± SEM (*, p < 0.05). D. protein expression and quantification histogram represent the presence of CXCR2 receptor in non-cancerous and cancer tissues of human samples, (*, p < 0.05). E. immunohistochemistry results of CXCR2 expression in normal and cancer tissues of human lung samples. Scale bar = 200 μm.
Figure 2. CXCR1/2 antagonism by G31P inhibits NSCLC cell proliferation
A. cells were treated with G31P (at concentrations of 0, 1, 10, 50, and 100 ng/ml) for 48 h. Cell proliferation was measured by CCK8 assay at 450 nm. G31P at 100 ng/ml showed significant inhibition of growth, (*, p < 0.05). B. cells treated with CXCR1/2 siRNA or control reagents were assessed for proliferation with or without G31P. G31P and siCXCR1/2 showed similar reduction but with no additive effect (*, p < 0.05). C. validation of G31P effect on H460 and A549 cell proliferation by Ki-67 nuclear stain through immunofluorescence. Ki-67 protein expression (red fluorescence) was detected significantly lower in G31P treated cells when compared with control for both cell lines, scale bar = 100 μm. D. graph represents percentages of area with positive Ki-67 stain (mean ± SEM) from three independent experiments (*, p < 0.05). E. cell cycle analysis of G31P-treated H460 cells shows reduction of cells in S and G2/M phases. F. graph represents percentages of cells in S phase after G31P treatment. All error bars represent standard error of the mean (SEM), and * indicates p < 0.05. All data were summarized from at least 3 independent experiments.
Figure 3. G31P restricts migratory and chemokinetic capacities of H460 and A549 cells
A. migration of H460 and A549 cells was assessed by wound healing assay and migration rate (%) was calculated and demonstrated in B. The data depicted represent the relative migration rate of H460 and A549 as quantified by measuring the distance covered by cells during 24 h. C and D. impact of G31P on chemotaxis of H460 and A549 cells, as determined using modified Boyden chamber microchemotaxis assays. CXCR1/2 and mock siRNA transfected cells were loaded into upper chamber and CXCL8/IL-8 (20 ng/ml) and G31P (100 ng/ml) were filled into lower chambers. Representative images were shown in C. Histograms in D show that G31P and siCXCR1/2 treatment inhibit lung cancer cell migration. Graph represents mean ± SEM from three independent experiments. Scale bar = 100 μm, and (*, p < 0.05).
Figure 4. CXCR1 and CXCR2 antagonism induces apoptosis in NSCLC cells
A. H460 and A549 cells were treated with the indicated concentrations of G31P and assessed for uptake of dye Hoechst 33342. B. quantification data from A show percentages of apoptotic cells. Data were summarized from 3 independent experiments and error bars represent SEM. C. flow cytometric analysis of H460 cells treated with indicated concentrations of G31P by staining with Hoechst 33342. Results show a peak of intact cells on the left of each track and a second peak of apoptotic cells. D. impact of G31P on expression of the indicated apoptosis-associated proteins in H460 cells. G31P treatment augmented expression of cleaved PARP and Caspase-8, as well as Bax, but modestly reduced expression of the anti-apoptotic protein Bcl-2, α-Tubulin was used as loading control.
Figure 5. G31P inhibits H460 xenograft growth and metastasis in mouse model
A and B. weights and volume of the xenografts from each mouse were assessed and plotted respectively. Tumors from the G31P-treated mice were significantly smaller than those from the saline-treated mice (p < 0.01). C. impact of G31P on expression of the indicated apoptosis-associated proteins in xenograft tumors. G31P treatment increased expression of cleaved PARP, Caspase-8, and Bax, while mildly decreased amounts of Bcl-2. α-Tubulin was used as loading control. D. gross fluorescent pictures were taken of GFP-expressing H460 cell tumors resected from saline- (control) and G31P-treated mice, 3 from each are shown as examples. G31P effects on apoptosis and necrosis of tumor were confirmed by serial sections stained with H & E and TUNEL. GFP fluorescent micrograph was also taken to differentiate live and necrotic part of tissue. H & E staining confirmed more nucleated cells/necrosis ratio in control group than G31P group, whereas TUNEL staining of tumor serial section micrographs exhibits more apoptosis in G31P treated tumor. Scale bar = 100 μm. E. representative fluorescent images (open chest and abdomen cavities) of GFP-expressing H460 tumors in saline (control) and G31P treated mice (n = 8 in each group). The tumor volume and extent of metastasis were substantially decreased in the G31P-treated mice. Error bars represent standard error of the mean.
Figure 6. G31P suppresses angiogenesis in xenograft tumor
A. gross photographs of H460 tumors growing in nude mice. Right side chart shows quantification data in which microvessel length and tumor areas were measured to calculate microvessel densities (MVD) using the formula: density = microvessel length/xenograft area. The graph represents the summarized MVD data with mean ± SEM from 6 mice in each group. B. tissue sections from control and G31P-treated xenografts were analyzed by immunohistochemistry for PECAM-1/CD31, VEGF and NFкB-p65 expression, scale bar = 100 μm. Right side charts show quantification data calculated as integral optical density (IOD) of PECAM-1/CD31, VEGF and NFкB-p65. Reduced expression was evident with G31P. C. immunoblottings for VEGF and NFкB-p65 in resected tumors from control and G31P-treated mice show reduced VEGF and p65 levels. GAPDH was used as loading control. All error bars represent standard error of the mean, and * indicates p < 0.05.
Figure 7. G31P treatment decreases the levels of pAKT and pERK1/2 in lung cancer cells and xenografts
A. H460 and A549 cells were treated with G31P (concentrations of 0, 10, 50 and 100 ng/ml) for one h before stimulated with IL-8 (20 ng/ml) for 30 minutes. Results showed that G31P treatment with increasing concentrations suppressed the IL-8 induced upregulation of pERK1/2 and pAKT in both cell lines. B. similarly in xenografts decreased phosphorylation of ERK1/2 and AKT is evident. α-Tubulin was used as loading control, against which normalized intensity was shown for pAKT and pERK1/2.
References
- Rahib L, Smith BD, Aizenberg R, Rosenzweig AB, Fleshman JM, Matrisian LM. Projecting cancer incidence and death to 2030. The unexpected burden of thyroid. liver, and pancreas cancer in the United States. Cancer Res. 2014;74:2913–2921. - PubMed
- She J, Yang P, Hong Q, Bai C. Lung cancer in China: Challenges and interventions. Chest. 2013;143:1117–26. - PubMed
- Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61:69–90. - PubMed
- Wang JM, Deng X, Gong W, Su S. Chemokines and their role in tumor growth and metastasis. J Immunol Methods. 1998;220:1–17. - PubMed
- Vandercappellen J, Van Damme J, Struyf S. The role of CXC chemokines and their receptors in cancer. Cancer Lett. 2008;267:226–44. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical
Research Materials
Miscellaneous