Platelet extracts induce growth, migration and invasion in human hepatocellular carcinoma in vitro

Abstract

Background: Thrombocytopenia has been reported to be associated with small size HCCs, and thrombocytosis to be associated with large size HCCs. The aim was to examine the effects of platelets in relation to HCC cell growth.

Methods: The effects of time-expired pooled normal human platelets were examined on human HCC cell line growth and invasion.

Results: Blood platelet numbers increased with increasing HCC tumor size and portal vein invasion. Platelet extracts enhanced cell growth in 4 human HCC cell lines, as well as cell migration, medium AFP levels and decreased apoptosis. Cell invasion was significantly enhanced, using a Matrigel-coated trans-well membrane and 3D (Real-Time Imaging) invasion assay. Western blots showed that platelets caused enhanced phospho-ERK and phospho-JNK signaling and anti-apoptotic effect with increase of Bcl-xL (anti-apoptotic marker) and decrease of Bid (pro-apoptotic marker) levels. Their growth effects were blocked by a JNK inhibitor.

Conclusions: Platelets stimulated growth and invasion of several HCC cell lines in vitro, suggesting that platelets or platelet growth factors could be a potential pharmacological target.

Figure 1

Effects of platelet extracts on HCC cell line growth. (A) Comparison of serum concentrations on platelet actions. PLC/RFP/5 cells were cultured in different FBS percentage (0-5%) for 48 h in presence of PLTs or equivalent FBS concentration (control). Performing MTT assay, a significant difference in growth is detected only at 1% FBS. (B-E) Effects of platelets on the growth of different human HCC cell lines. PLC/PRF/5 (B), HepG2 (C), Hep3B (D) and Huh7 (E) cell lines were cultured in 1% FBS medium in presence of different platelets concentrations or FBS and MTT assay was assessed after 48 h. (F) Comparison of different concentrations of red cells (RBC), white cells (WBC) and platelets on PLC/PRF/5 cell growth evaluated after 48 h by MTT assay. (G) Comparison of time windows (0-24 h, 0-48 h, 48-72 h, 24-72 h and 0-72 h) for effects of two different platelet concentrations on growth evaluated after 72 h by MTT assay. The results are expressed as mean ± SD. *P < 0.05; **P < 0.001; ***P < 0.0001.

Figure 2

Effects of platelets on PLC/PRF/5 cells. (A) AFP levels in the cell culture medium of PLC/PRF/5 cell lines after treatment with different platelets concentrations or FBS. (B) Migration assay. PLC/PRF/5 cells were treated with different platelet concentrations or FBS and microscopically analyzed at the time of the scratch and after 72 h. Values were expressed as percentage of migration, 100% representing the completely closed wound. (C) Apoptosis assays. On the left are shown examples of results obtained using the Muse Annexin V kit (upper panel) or Caspase-3/7 kit (lower panel) to evaluate the percentage of apoptotic PLC/PRF/5 cells cultured whit PLTs or FBS. On the right the mean of three independent experiments is plotted in the relative graph. The results are expressed as mean ± SD. *P < 0.05; ***P < 0.0001.

Figure 3

Effects of platelets on hepatocellular carcinoma cell motility and invasion. (A) Huh7-GFP cells were assayed by Matrigel chemo-invasion with BSA 1% (control) or different dilution of platelet factors added to a lower compartment of the chamber at the indicated concentrations. (B) Tracking the migration of Huh7-GFP cells by live optical imaging. GFP-expressing Huh7 were imaged for 16 h and the behavior of motile cells was recorded. (C) The tracks of 6 representative cells (3 controls and 3 stimulated with platelet factors) are plotted inWind-rose plots with the initial position of each track (left panel).Scale bar, 100 μm. The temporal increase in the productive motility of these representative tracks from controls and stimulated Huh7-GFP cells are also plotted in the graph (right panel). (D) 3D Invasion assay of Huh7-GFP cells in the presence of platelet soluble factors. The rate of cell migration was determined at different time intervals (6, 12, 24 h). Three representative tracks for each condition (cells stimulated with platelet factors or with BSA 1% as control) are plotted in the graph. All experiments were carried out in triplicate. The results are expressed as mean ± SEM. P values were determined by Mann–Whitney one-tailed test.

Figure 4

Cell signaling changes induced by platelets. (A) Analysis of changes in p-ERK, p-JNK, p-STAT3, p-p38 and p-AKT. PLC/PRF/5 cells were treated with 3.75 × 10⁷ PLTs or FBS for different times. The changes in protein expression were evaluated using Western blot (WB) analysis. (B) The Western blot (WB) analysis confirmed the anti-apoptotic effect of platelets as showed in Figure 2C. (C) To test the significance of p-JNK increase, observed in WB analysis (A), on platelet-mediated growth induction, SP600125 (JNK inhibitor) was used, resulting in inhibition of PLT action. *P < 0.05.