The inhibitory effect of a novel polypeptide fraction from Arca subcrenata on cancer-related inflammation in human cervical cancer HeLa cells

Abstract

Inflammation is known to be closely associated with the development of cancer. The study was launched in human cervical cancer HeLa cells to investigate the antitumor and anti-inflammatory effects of P2, a marine polypeptide fraction from an important fishery resource Arca subcrenata. The basic research showed that P2 could suppress the production of nitric oxide in LPS-induced RAW264.7 macrophage cells as well as the secretion of inflammatory cytokines IL-6 and TNF- α in human cervical cancer HeLa cells. For the molecular mechanisms, P2 was shown to downregulate the gene expression of proinflammatory cytokines IL-6 and IL-8 and to inhibit the COX-2 and iNOS-related pathways in HeLa cells. In consequence, P2 might inhibit tumor development by blocking the interaction between tumor microenvironment and proinflammatory mediators. All findings indicate that P2 possesses the potential to be developed as a novel agent for cancer therapy.

Figure 1

(a) Effects of P2 and LPS on the cell viability of RAW264.7 cells. RAW264.7 cells were treated by P2 and LPS at different concentrations for 24 h and 48 h. Cell viability was determined by MTT assay. The results are representative of three independent experiments and expressed as mean ± SD. Differences were defined as significant at P < 0.05. (b) Effects of P2 on NO production in LPS-stimulated RAW264.7 cells. Cells were stimulated with LPS for 2 h followed by treatment with P2 (15–150 μg/mL) for 48 h. Nitrite in the medium was measured using Griess reagent. ***P < 0.001 compared with NC (negative control); ^# P < 0.05 compared with LPS-treatment only. The results are representative of three independent experiments and expressed as mean ± SD. Differences were defined as significant at P < 0.05.

Figure 2

Effect of P2 on proinflammatory cytokine secretion in HeLa cells. Levels of IL-6 and TNF-α secreted by HeLa cells after 48 h incubation were assayed by human IL-6 and TNF-α ELISA kits. *P < 0.05 and **P < 0.01 compared with normal control (NC). The results are representative of three independent experiments and expressed as mean ± SD. Differences were defined as significant at P < 0.05.

Figure 3

(a) Effect of P2 on immune-related gene expression in HeLa cells. mRNA expression of proinflammatory cytokines in HeLa cells treated with P2 as described in Section 2.6 was analyzed by RT-PCR. GAPDH was used as an internal control. Results are representative of three independent experiments. (b) Quantification of transcript levels by RT-PCR. The primer pair for GAPDH was used as the reference gene. **P < 0.01 and ***P < 0.001 compared with NC. The results are representative of three independent experiments and expressed as mean ± SD. Differences were defined as significant at P < 0.05.

Figure 4

(a) Effects of P2 on immune-related proteins expression in HeLa cells. Following treatment with P2 (1.33, 4, 12 μg/mL), whole lysates were collected and analyzed by western blotting with antibodies specific for COX-2 and iNOS. GAPDH was used as an internal control. Results are representative of three independent experiments. (b) Quantification of expression levels by western blotting. The inhibition of COX-2 and iNOS were 78.67% and 69.65% for the high concentration, respectively, after treatment with P2. GAPDH was used as an internal control. *P < 0.05 and **P < 0.01 compared with NC. The results are representative of three independent experiments and expressed as mean ± SD. Differences were defined as significant at P < 0.05.