P-selectin-mediated platelet activation promotes adhesion of non-small cell lung carcinoma cells on vascular endothelial cells under flow

Abstract

Lung cancer is a severe disease threatening human health worldwide. Distant hematogenous metastasis results in poor prognosis and death of lung cancer patients. In the present study, we investigated the effect of circulatory platelets (PLTs) on hematogenous metastasis of non-small cell lung carcinoma (NSCLC). Laser scanning confocal microscopy was employed to assay the expression of P-selectin in lung cancer tissue, paracancerous tissue and distant tissue, respectively. Meanwhile, fluorescence-activated cell sorting (FACS) was used to determine P-selectin activation in peripheral blood. Purified PLTs were co-cultured with A549 cells and human vascular endothelial cells (HuvECs). Subsequently, the formation of PLT-lung cancer cell complexes and their effects on rolling and adhesion of A549 on the surface of vascular endothelium were assayed. Integrin α3, α5, β1, ICAM-1 and VCAM-1 mRNAs and proteins were measured by reverse RT-PCR and western blot analysis, respectively. The expression of P-selectin in lung adenocarcinoma tissue was significantly stronger compared to that in paracancerous and distant tissues. P-selectin activation in peripheral blood in lung adenocarcinoma was markedly enhanced. The rolling rate of A549 on HuvECs was significantly slowed down after co-culture of activated PLTs and A549 cells. The mRNA and protein levels of integrin α3, α5, β1, ICAM-1 and VCAM-1 were significantly increased after the co-culture. In conclusion, the PLT-lung cancer cell complexes protected the lung cancer cells from mechanical injury under blood flow. Furthermore, up-regulated integrin α3, α5, β1 and endothelial cell adhesion molecules ICAM-1 and VCAM-1 promoted the adhesion of A549 on vascular endothelial cells, which may be responsible for hematogenous metastasis of lung cancer.

Figure 1

Expression of P-selectin in different lung cancer tissue. Expression of P-selectin in lung adenocarcinoma tissue was stronger than that in lung squamous tissue; meanwhile, the expression of P-selectin in the cancer tissue was significantly increased compared with that in the paracancerous tissue and distant normal tissue, respectively.

Figure 2

Correlation between blood platelet count (BPC) and pathological types in 168 patients with primary lung cancer based on the BPC in the peripheral blood prior to chemotherapy, radiotherapy or surgery. BPC was classified into decreased (<100×10⁹/l), normal (100–300×10⁹/l) and increased (>300×10⁹/l) states. We found that the BPC was decreased, was normal and was increased in 3.57% (6/168), 69.05% (116/168) and 27.38% (46/168) of the 168 lung cancer patients, respectively. Meanwhile, the PLT count was increased in 22.64% (12/53) of squamous cell carcinoma, in 37.09% (23/62) of adenocarcinoma, in 22.70% (10/44) of small-cell carcinoma and in 14.20% (1/7) of other pathological types, respectively.

Figure 3

Activation of P-selectin in peripheral blood of NSCLC patients (mean ± SD). Expression of P-selectin in lung adenocarcinoma was significantly enhanced compared with that in the healthy control. However, there were no significant differences between squamous cell carcinoma and adenocarcinoma. ^**P<0.01 as compared with the control.

Figure 4

SEM assay of PLT adhesion. Few lung cancer cells assembled with PLTs in the control group. (A) After siRNA targeting PSGL-1 or usage of specific P-selectin antibody in A549, little PLT aggregation was noted. (B) However, activated PLTs promoted interactions between cancer cells and endothelial cells and obvious aggregation phenomenon was observed. A: 1, A549; 2, A549 + inactive PLTs; 3, A549 (siRNA targeting PSGL-1) + activated PLTs; 4, A549 + activated PLTs + P-selectin antibody. (B) A549 + activated PLTs. The arrows in the figures represented PLT-lung cancer cell complexes.

Figure 5

Adhesion of A549 cells on the surface of HuvECs compared with the control. The adhesion efficiency reached 65% and the rolling rate of A549 was decreased by 60% after co-culture. There were significant differences between the two co-cultures. This suggests that the co-culture of activated PLTs and A549 cells significantly attenuated the rolling rate of A549 on HuvECs, which promoted the interaction between A549 and HuvECs. (A) Adhesion between endothelial cells and lung adenocarcinoma cells after interaction with PLTs. (B) Rolling rate of lung adenocarcinoma cells. 1, HuvECs+A549; 2, HuvECs+A549 + inactivated PLTs; 3, HuvECs+A549 + activated PLTs; 4, HuvECs+A549 (siRNA targeting PSGL-1) + activated PLTs; 5, HuvECs+A549 + activated PLTs + P-selectin antibody. ^*P<0.05, ^**P<0.01 as compared with HuvECs+A549.

Figure 6

mRNA expression of integrin α3, integrin α5, integrin β1, ICAM-1 and VCAM-1 (±SD, n=3). mRNA levels of integrin α3, integrin α5, integrin β1, ICAM-1 and VCAM-1 after the co-culture of HuvECs, A549 and activated PLTs were significantly increased compared with that after the co-culture of HuvECs and A549. 1, HuvECs+A549; 2, HuvECs+A549 + inactivated PLTs; 3, HuvECs+A549 + activated PLTs; 4, HuvECs+A549 (siRNA targeting PSGL-1) + activated PLTs; 5, HuvECs+A549 + activated PLTs + P-selectin antibody. ^*P<0.05 as compared with HuvECs+A549.

Figure 7

Protein expression of integrin α3, integrin α5, integrin β1, ICAM-1 and VCAM-1 (mean ± SD, n=3). The protein levels of integrin α3, integrin α5, integrin β1, ICAM-1 and VCAM-1 after the co-culture of HuvECs, A549 and activated PLTs were significantly up-regulated compared with that after the co-culture of HuvECs and A549. 1, HuvECs+A549; 2, HuvECs+A549 + inactivated PLTs; 3, HuvECs+A549 + activated PLTs; 4, HuvECs+A549 (siRNA targeting PSGL-1) + activated PLTs; 5, HuvECs+A549 + activated PLTs + P-selectin antibody. ^*P<0.05, ^**P<0.01 as compared with HuvECs+A549.