Hypoxia promotes the proliferation of cervical carcinoma cells through stimulating the secretion of IL-8

Abstract

To explore whether hypoxia and interleukin 8 (IL-8) regulate the viability and apoptosis of cervical carcinomas cells and the possible mechanism. We evaluated the expression of hypoxia inducible factor-1α (HIF-1α), IL-8 and its receptors (CXCR1 and CXCR2) in cervical cancer and cervicitis tissues by immunohistochemistry. Then the effects of hypoxia and IL-8 on the viability and apoptosis of HeLa and SiHa cells were detected by the SRB and apoptosis assays. Here we observed that the expression of HIF-1α, IL-8 and CXCR1 in cervical cancer tissues was significantly higher than that in cervicitis tissues. Hypoxic condition stimulated the secretion of IL-8 and the expression of CXCR1 and CXCR2 on HeLa and SiHa cells. Recombinant human IL-8 enhanced the viability and reduced the apoptosis in HeLa and SiHa cells. HeLa and SiHa cells cultured in 1% oxygen showed the increased viability and apoptosis, and the former effect could be partly reversed by anti-human IL-8 neutralizing antibody. This data suggested that IL-8 secreted by cervical carcinomas cells induced by hypoxia can stimulate the viability of cervical carcinomas cells in an autocrine dependent manner, and contribute to the pathogenesis of cervical cancer.

Keywords: Hypoxia; IL-8; apoptosis; cervical carcer cells; viabilty.

Figure 1

Cervical cancer cells highly express IL-8 and it receptors in cervical cancer tissues. The expression of HIF-1α, IL-8, CXCR1 and CXCR2 in the cervical tissues from cervical cancer (n=18) and cervicitis patients (n=10) was analyzed respectively by immunohistochemistry. Original magnification: ×400.

Figure 2

Hypoxia induces the secretion of IL-8 and the expression of it receptors on HeLa and SiHa cells. We cultured HeLa and SiHa cells under hypoxic condition (1% oxygen) or normoxic condition (21% oxygen) for 24h (for ELISA assay) or 8h (for flow cytometry assay), and then detected the secretion level of IL-8 in the supernatant by ELISA (A) and the expression of CXCR1 and CXCR2 by flow cytometry (B-D). Normal HeLa/SiHa: HeLa/SiHa cells cultured under normoxic condition; Hypoxic HeLa/SiHa: HeLa/SiHa cells cultured under hypoxic condition. Results are highly reproducible in three independent experiments. Data are mean±SD. *P<0.05, **P<0.01 or ***P<0.001 compared to the normoxic condition control.

Figure 3

Recombinant human IL-8 promotes the proliferation and reduces the apoptosis of HeLa and SiHa cells. HeLa and SiHa cells (7×10³ cell/well for proliferation assay; 1×10⁵ cell/well for apoptosis assay) were treated respectively with recombinant human IL-8 (rhIL-8) (1, 10, 100 ng/ml for proliferation assay; only 10 ng/ml for apoptosis assay) for 48h, with vehicle or mouse isotype as controls. Then Sulforhodamine B (SRB) proliferation assay (A, B) and annexin V-FITC apoptosis detection assay (C-E) were conducted to analyze proliferation and apoptosis of HeLa and SiHa cells, respectively. *P<0.05, **P<0.01 or ***P<0.001 compared to the vehicle control.

Figure 4

The stimulatory effect of hypoxia on the proliferation not apoptosis of cervical cancer cells is dependent on IL-8 signals. We treated HeLa and SiHa cells under the normoxic condition or under the hypoxic condition for 8h with anti-human IL-8 neutralizing antibody (α-IL-8) (0.04, 2, 10 ug/ml for proliferation assay; only 2 ug/ml for apoptosis assay), and then cultured these cells under the normoxic condition for 48h, with mouse isotype (Sino-America Co. Ltd) as control. Then SRB proliferation assay (A, B) and apoptosis assay (C, D) were performed to analyze the proliferation and apoptosis of HeLa and SiHa cells. Data are mean±SD. NS: no statistically difference.

Figure 5

Schematic roles of hypoxia in regulating biological behavior of cervical cancer cells. Under hypoxia conditions, cervical carcinoma cells secrete high level of IL-8 and express more CXCR1 and CXCR2. The increased IL-8/CXCR1/CXCR2 signals induced by hypoxia, on the one hand, may stimulate the proliferation, restrict the apoptosis and promote the migration and metastasis of cervical cancer cells in an autocrine-dependent manner; On the other hand, enhance the angiogenesis of vascular endothelial cells (VEC) in a paracrine-dependent manner. In addition, hypoxia promotes the apoptosis of cervical cancer cells through other signals. These effects finally contribute to growth and development of cervical cancer.