Background
Platelet activity has been known for a long time to be altered in the presence of cancer, with venous thrombosis being recognized in association with occult malignancy [
1,
2]. In addition to the effects of cancer on platelet actions in blood clotting, platelets have been recognized to be involved in cancer development, progression and metastasis [
3‐
8]. Platelet levels have been shown to impact prognosis in several cancers, including those of the ovary, kidney, colon, lung and pancreas [
9‐
14]. Furthermore, whereas hepatocellular carcinoma (HCC) most typically arises on the basis of cirrhosis, with its frequently associated splenomegaly and thrombocytopenia, normal or elevated platelet levels are frequently seen in large size HCCs [
15‐
17]. We recently found that platelet extracts can stimulate HCC cell line growth in vitro, which was associated with a decrease in apoptosis [
18]. We now extend those observations, by examining the effects of platelet extracts on the effects of apoptosis-inducing HCC treatment agents and report that platelet extracts can antagonize growth inhibition mediated by Sorafenib or Regorafenib.
Methods
Cells and materials
PLC/PRF/5, Hep3B and HepG2 cells were obtained from the ATCC and were cultured as previously described [
19].
Recombinant human EGF was purchased from PeproTech (Rocky Hill, NJ, USA), mouse recombinant IGF-I from Calbiochem (San Diego, CA, USA) and serotonin from Sigma-Aldrich (Saint Louis, MO, USA), all the growth factors were dissolved in water.
Platelet lysates
The platelet samples were collected from healthy volunteers. The study protocol was approved by the institutional review boards of the University of Bari and “Saverio de Bellis” Institute of Castellana G. (BA), Italy. Additionally, written informed consent was obtained from participants for the use of their blood in this study.
The platelet-rich plasma was obtained using an automated hemapheresis procedure in a local blood transfusion center. The platelets obtained from different volunteers were mixed and then divided into aliquots. Each aliquot was subjected to several freeze-thaw cycles to disrupt their membranes and release the growth factors stored in the granules (human Platelet Lysate, hPL).
Growth assay
Proliferation assay was performed as recently described [
19,
20]. The cells were cultured in 1% FBS medium containing different hPL concentrations (2.5 - 3.75 × 10
7) or equivalent percentage of FBS in presence of 1 μM (HepG2 cell line) or 2.5 μM (Hep3B and PLC/RFP/5) of Sorafenib or Regorafenib. In the same growth condition HCC cell lines were cultured in presence of EGF 10, 25 mg/ml, IGF-I 50, 100 mg/ml and serotonin 1, 10 μM with or without Sorafenib 1 μM.
AFP measurement
Medium AFP levels were measured using an automated system (UniCel Integrated Workstations DxC 660i, Beckman Coulter, Fullerton, CA, USA) by a chemoluminescent immunometric method. Sample measurements over the calibration range were automatically re-analyzed according to manufacture’s instructions.
Migration assay
A scratch assay was performed as previously described [
19,
20]. Briefly, a wound was generated with a pipette tip, after rinsing, medium containing different concentrations of hPL or equivalent percentage of FBS and 2.5 μM Sorafenib or Regorafenib was added. Photographs were taken of each well immediately and after 24 h and 48 h. The values were expressed as percentage of migration, with 100% being when the wound was completely closed. The results were representative of three independent experiments.
Invasion assay
Cell invasion assays were performed using Matrigel (BD Transduction, San Jose, CA, USA)-coated Transwells (8 μm pore PET membrane, Millipore, Billerica, MA, USA) as previously described (19). Briefly, 2.5 μM Sorafenib or Regorafenib treated cells were suspended in low serum medium. Medium containing different hPL or FBS concentrations was added to the bottom wells. After incubation of 24 h, the invading cells were fixed and stained. The images were acquired and analyzed counting the cells with Image J Software (National Institute of Health, USA). Values obtained were expressed as fold increase of invading cells, setting the cell counts of control cells as one. Results were representative of three independent experiments.
Apoptosis assays
Annexin V
The Muse Annexin V/Dead Cell Assay Kit (Millipore, Darmstadt, Germany) for quantitative analysis of live, early/late apoptotic and dead cells was used with a Muse Cell Analyzer (Millipore). Briefly, the assay utilizes Annexin V to detect PS on the external membrane of apoptotic cells. A dead cell marker (7-AAD) is also used. PLC/PRF/5 cell line, including positive and negative controls, were cultured in 1% FBS medium supplemented with a volume of hPL corresponding to 3.75 × 107 platelets/ml or with an equivalent percentage of serum (control cells) for 48 h. The cells were then processed as described in the user’s guide.
Caspase-3/7 quantitative measurements
The Muse Caspase-3/7 kit (Millipore) permits simultaneous evaluation of apoptotic status based on Caspase-3 and -7 activation and cellular plasma membrane permeabilization (cell death). The assay provides relative percentage of cells that are live, early/late apoptotic or dead. Cells were cultured as described above and processed according to the user’s guide.
Western blots
We analyzed the MAPK signaling and anti-apoptosis markers in Hep3B cells treated with 2.5 μM Sorafenib or Regorafenib and hPL by Western blot, as previously described [
19,
20]. In brief, cells were washed twice with cold PBS and then lysed in RIPA buffer (Sigma-Aldrich, Milan; Italy). After quantization of protein concentration, equal amount of protein (50 μg) were resolved on SDS–PAGE and transferred to polyvinyldifluoride (PVDF) filters. The blots were blocked with 5% (w/v) nonfat dry milk for 2 h at room temperature and then probed with primary antibody overnight at 4°C.
The primary antibodies were directed against the following proteins: ERK and phospho-ERK (P-ERK), JNK and phospho-JNK (P-JNK), p38 and phospho-p38 (P-p38), STAT3 and phospho-STAT3 (Tyr705, Ser727) (P-STAT3), AKT and phospho-AKT (P-AKT), survivin, Bcl-xL, Bax, Bim and β-actin (Cell Signaling, Beverly, MA, USA). After three washes, incubation was followed by the reaction with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The immunoreactive bands were visualized and analyzed using the enhanced chemiluminescence detection reagents (Cell Signaling), according to the manufacturer’s instructions, and chemiluminescence detection system (ChemiDoc XRS apparatus and software, Bio-Rad).
Statistical analysis
GraphPad Prism 5.0 software (La Jolla, CA, USA) was used for all statistical analysis. Mann–Whitney nonparametric test was employed to assess the statistical significance of differences between two groups. For multiple comparisons was used one-way Anova test followed by appropriate post-test. P-values of <0.05 were considered statistically significant. All experiments were done in triplicate and data are presented as mean ± standard deviation (SD).
Discussion
We report here for the first time, the antagonizing effects of platelet extracts on growth inhibition in several HCC cell lines, that was mediated by Sorafenib or Regorafenib. Both agents were similarly antagonized by hPL. Furthermore, the previously demonstrated inhibition of AFP secretion by these drugs, was also antagonized. A main consequence of each drug is a decrease in phospho-ERK levels, secondary to Raf inhibition. hPL antagonized this early consequence of the drug action, without change in ERK levels. There was also an early and strong antagonism of the previously noted inhibitory effects of drug on phospho-p38 levels [
20], and similarly for the p38 downstream target, phospho-STAT3 (Tyr and Ser). These are important molecules in mediating cell proliferation and play a role in the induction of anti-apoptosis mediators. Both Sorafenib and Regorafenib are known to increase apoptosis in treated cells. We found that this apoptosis-induction was antagonized by addition of hPL to cells that were treated with each of these two agents, as measured by both annexin V and caspase 3/7 activation.
Consistent with our findings of increased phospho-STAT3 levels, we also found an increase in the levels of anti-apoptotic Bcl-xL and survivin and a decrease in the levels of pro-apoptotic Bim and Bax, consequent to hPL action.
Due to the important role of platelets in the metastasis mechanisms of many tumors [
8,
21], we evaluated hPL for a possible role in stimulating cell migration or invasion. We founds that the extracts also antagonized drug-mediated inhibition of HCC cell migration and invasion on Matrigel-treated membranes. In other systems, the targeting of platelets or experimental decrease in their numbers has been shown to enhance cancer chemotherapy [
22,
23].
Platelets are the source of multiple growth factors, cytokines and inflammatory mediators [
6].
Included among them are EGF, IGF-I, fibroblast growth factor (FGF), platelet derived growth factor (PDGF) and serotonin, the modulation of each having been shown to alter cancer chemotherapy sensitivity or resistance [
24‐
30]. Preliminary data, obtained with several growth factors included in hPL, revealed interesting results using EGF and IGF-I. Both these factors were able to antagonized Sorafenib in a proliferation assay, in particular when used in combination. This growth induction was more evident than that observed in absence of drug, suggesting a specific interference of these growth factors with the inhibitory action of Sorafenib.
Interestingly, the clinical insulin modulator and diabetes drug, metformin [
26] and the serotonin modulator Fluoxetine/Prozac that is used in depression treatment [
29,
30], each alter chemotherapy sensitivity in cancer cells. Multiple pathways have been found to be involved in Sorafenib-mediated growth inhibition, especially apoptosis and autophagy [
19,
31,
32] as well as others [
33‐
37] and several cytokines, or cytokine modulators that are produced by platelets can modulate Sorafenib activity [
38]. Since Sorafenib effects have been clinically modest, several approaches are under way to enhance its actions, either on its downstream targets, or by adding inhibitors of parallel pathways in combination therapies [
39]. Given the large number of candidate factors in platelets, the identification of those responsible for drug resistance is just beginning. However, FGF, IGF1 and serotonin would seem to be promising possibilities.
The recent finding that platelet inhibitors reduce hepatitis B associated experimental HCC [
40] has led to new interest in the use of aspirin and other platelet inhibitors in HCC prevention, as in colon cancer prevention [
41]. Thrombocytosis has been shown to be a negative prognostic factor for renal, breast, ovary, pancreas and colon cancers. Therefore, the results from this paper might be applicable to those tumor types, especially to renal cancer, since Sorafenib is also FDA-approved for treatment of renal cancer.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BIC involved in interpretation of clinical data, conception and design of the translational research study in vitro and wrote the first draft of the manuscript; RD, MGR and CL participated equally at the design, execution and interpretation of the experiments; NC performed Western blot experiments; GG participated in blood collection, isolation and count of platelets; CM and AC provided overall supervision for conducting the study and involved in manuscript revision and presentation. All authors read and approved the final manuscript.