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Targeting delivery of platelets inhibitor to prevent tumor metastasis Marzieh Geranpayehvaghei, Quanwei Shi, Baochang Zhao, Suping Li, Xu Junchao, Mohammad Taleb, Hao Qin, Yinlong Zhang, Khosro Khajeh, and Guangjun Nie Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00457 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 21, 2019
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Targeting delivery of platelets inhibitor to prevent tumor metastasis Marzieh Geranpayehvagheia#, Quanwei Shi b,c#, Baochang Zhaod#, Suping Li b,c, Junchao Xu b,c, Mohammad Taleb b,c, Hao Qin b,c, Yinlong Zhangb,c*, Khosro Khajeha*, Guangjun Nieb,c* a
Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, 14115-175, Iran b
CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China c
Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049, China d School
of Life Sciences, Shandong First Medical University & Shandong Academy of Medical Sciences, Taian 271016, PR China
#These
authors contributed equally to this work.
*Address correspondence to: Guangjun Nie,
[email protected], National Center for Nanoscience and Technology, Beijing, China Khosro Khajeh,
[email protected], Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran Yinlong Zhang,
[email protected], National Center for Nanoscience and Technology, Beijing, China
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Abstract Since activated platelets have a high affinity to tumor cells, and this can protect these cells from environmental stress and immune attacks. Therefore, preventing platelet-tumor cell interaction can lead to the elimination of circulating tumor cells via natural killer cells and finally metastasis inhibition. It is also shown that CREKA (Cys-Arg-Glu-Lys-Ala), a tumor-homing pentapeptide, targets fibrin-fibronectin complexes that are found on tumor stroma and the vessel walls. In this study, we linked CREKA to Ticagrelor, a reversible antagonist of the P2Y12 receptor on platelets. In vitro experiments indicated that CREKA-Ticagrelor could inhibit the platelet-induced migration of tumor cells with an invasive phenotype and also could prevent tumor-platelet interaction. In vivo anti-tumor and anti-metastasis results of this drug showed that CREKATicagrelor could specifically target the tumor tissues within 24 hours post intravenous injection and suppress lung metastasis. Meanwhile, by having this anti-platelet drug targeted, its side effects were minimized and bleeding risk was decreased. Thus, CREKA-Ticagrelor offers an efficient anti-metastatic agent. Key words: Platelets inhibitor, CREKA-Ticagrelor, Tumor metastasis
Introduction Metastasis is responsible for about 90% of the cancer-related deaths 1-3. Thus, searching efficient methods to prevent metastasis is urgently needed in clinical practice. During the metastatic cascade, tumor cells get detached from the primary lesion and then enter the blood stream, through which the migration occurs. Finally, they are transported to distant tissues or organs, where the tumor cell is trapped and cell proliferation happens 2, 4. Platelets are generally famous for their role in coagulation and maintaining hemostasis when the blood vessels suffer from a physical injury 5. However, some evidences of their involvement in inflammation, cancer progression, and also metastasis have been observed
6, 7.
Among all their
characteristics, platelets have two features that promote cancer progression: first, they release a plethora of agents such as lipids, proteins, and nucleic acids which are essential for tumor development. Second, there are various receptors expressed on their surface, which can bond to tumor cells and promote their metastasis
7, 8.
Integrin is one of the receptors expressed on the 2
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platelet surface that facilitates their attachment to the tumor cells. β1 and β3 integrins are specifically involved in cancer cell-platelet interactions and therefore, responsible for cancer metastasis 9. When cancer cells come into the blood stream they are protected by platelets via different mechanisms: platelets form thrombus around the tumor cells and protect them from the shear stress in the circulation. Platelets also help to protect the cancer cells against the attack of Natural Killer (NK) cells. Moreover, cancer cells’ adhesion to both endothelial cells and leukocytes is also facilitated by the platelets, which is of importance for the establishment of metastatic niche 10-12. In addition, platelets are also able to promote cancer cell proliferation. All of these suggest that using therapies based on antiplatelet drugs to hinder the bioactive components’ signaling can be helpful for cancer treatment
13,
14.
However, systemic
administration of platelets inhibitors has the risk to lead to severe bleeding complications. To address this issue, in the current study, we developed a strategy to targeted delivery of ticagrelor, a reversible antagonist of the P2Y12 receptor on platelets, specifically to the tumor site for metastasis prevention
15, 16.
Briefly, a small linear tumor-homing pentapeptide, CREKA
(Cysteine–Arginine–Glutamic acid–Lysine–Alanine), which can target to the fibrin-fibronectin complexes on vessel walls and tumor stroma
17-20,
was used to conjugate with Ticagrelor,
designated as CREKA-Ticagrelor. We explored the influence of CREKA-Ticagrelor on the migration of tumor cells in vitro and also tested its in vivo anti-tumor and anti-metastasis effects on 4T1 breast cancer tumor mouse models. The results indicated that CREKA- Ticagrelor could efficiently suppress tumor lung metastasis and simultaneously did not bring an obvious bleeding threat.
Results and Discussion Synthesis and characterization of Ticagrelor-CREKA Apart from its specificity binding ability to tumor fibrin, CREKA’s production is very compatible with solid-phase synthesis compared to other tumor homing peptides
21.
Here,
CREKA was generated using solid-phase synthesis method. In this process all amino acids Ntermini and side chains were covered by protecting groups. A supporting substrate was used at the beginning to start the reaction with. Fmoc was used to protect the N-termini and it was removed using a mild base. Each amino acid was attached to the previous one after having the 3 ACS Paragon Plus Environment
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protection group removed and this way the linear peptide was synthesized as expected. To finally conjugate the synthesized peptide with Ticagrelor, oxalic acid was added to link with the peptide. As the last step, Ticagrelor was attached to the other end of oxalic acid to form the final product Ticagrelor-CREKA (Figure 1A). The mass spectrometry result indicated the molecular weight of Ticagrelor-CREKA was 1211.6 Da (Figure 1B), which was consistent with the theoretical calculation. Ticagrelor-CREKA was purified by high-performance liquid chromatography (HPLC) and achieved a purity of more than 90% (Figure 1C).
Figure 1. Design and synthesis of CREKA-Ticagrelor and its characterization. (A) Schematic illustration of CREKA-Ticagrelor. (B) Mass spectrometry analysis of the crude product was performed to verify whether the conjugation was successful. The peak at 1211.6 was consistent with the expected molecular weight of 1211.6. (C) The product was purified and collected using HPLC.
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Functional characterization of Ticagrelor-CREKA Ticagrelor, as a P2Y12 antagonist, has been previously shown to dose dependently being able to inhibit platelet aggregation by enhancing adenosine concentration 22, 23. To investigate the effect of CREKA on Ticagrelor’s functionality, platelet aggregation assay was performed. In this experiment free CREKA, Ticagrelor, and CREKA-Ticagrelor were used. As illustrated in Figure 2A, CREKA’s addition to Ticagrelor did not make an evident difference in increasing or decreasing Ticagrelor’s inhibition effect on platelet aggregation. While free CREKA did not show any effect on ADP-induced platelet aggregation, free Ticagrelor and CREKA-Ticagrelor had almost the same activity at different concentrations. Activated platelets have shown a high capability to bind to tumor cells and promote metastasis and cell proliferation 12. This is due to their escape from the immune system elimination that is natural killer cells
14, 24.
In this project we hypothesized that tumor-platelet interaction could be
blocked by using Ticagrelor as the antagonist of P2Y12 receptor. Adhesion of platelets to cancer cells in the presence and absence of Ticagrelor-CREKA was next detected using a confocal microscope. Tumor cells were co-incubated with the DiL-labeled platelets as the control. When CREKA-Ticagrelor was introduced to the incubation system, the platelet-tumor cell interaction was greatly blocked (Figure 2B). The result indicated that platelets alone had the ability to attach to the tumor cells. However, the adhesion of platelet-tumor cell could be efficiently prevented by CREKA-Ticagrelor.
Figure 2. Examination of platelet aggregation and Ticagrelor’s ability to prevent the adherence of the platelets to the tumor cells. (A) CREKA’s effect on Ticagrelor’s activity. Platelet aggregation assay was carried out after treatment with Ticagrelor, CREKA- Ticagrelor, 5 ACS Paragon Plus Environment
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and CREKA. (B) Ticagrelor’s effect on preventing the platelets from being attached to tumor cells. DiL-labeled platelets were observed using a confocal microscope.
In vitro anti-migration assay It was already shown that Ticagrelor can improve wound healing between 84 to 97% depending on the time
25.
To examine its effect after being attached to a targeting peptide, monolayers of
4T1 cells were scratch-wounded and they were incubated in RPMI 40 medium with or without platelets, and were observed 18 h and 24 h after the scratch. The distance between the edges was measured using ImageJ software and wound healing was calculated by comparing the results in different time points. CREKA-Ticagrelor had the ability to block platelets and prevent them from being attached to the tumor cells and therefore stop their migration. To evaluate the results, a positive control (platelets) and a negative control was used. To keep consistent with the literatures, although platelets stimulated cell invasion and migration, using CREKA-Ticagrelor could significantly inhibit this cancer cells characteristic. As shown in Figures 3A and 3B in the platelet group, the cells migrated faster to re-populate the scratch area and 24 hours after the scratch about 65% of the area was refilled. However, the control group demonstrated the slowest migration with covering only 15% of the area, followed by the platelet + CREKA-Ticagrelor group with the result of 30% of repopulation after 24 h. CREKA-Ticagrelor could significantly inhibit platelet-induced cell migration compared to the only platelet group at all the time points. We also investigated the ability of CREKA-Ticagrelor in inhibiting the platelet functionality, by observing the tumor cells and their transition into mesenchymal-like cells in vitro. Mouse 4T1 tumor cells were co-cultured with different components including: saline, platelets, and platelets with CREKA-Ticagrelor. They were kept at 37ºC for 24 h and finally tumor cell morphology was recorded using light microscopy. Tumor cells of the group with platelets had the appearance similar to fibroblasts and were spindle-shaped which was close to mesenchymal cells (Figure 3C). To further analyze the possible mechanism of platelets in tumor migration, the expression levels of proteins that are involved in epithelial–mesenchymal transition (EMT) e.g. E-cadherin and vimentin, was investigated. E-cadherin downregulation and vimentin upregulation promote EMT 26.
We observed a significant decrease of E-cadherin expression and increase of vimentin 6 ACS Paragon Plus Environment
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expression in 4T1 cells in platelet treated groups, which are the indicators of metastasis 37. These alterations in E-cadherin and vimentin induced by platelets were reversed by CREKA-Ticagrelor incubation (Figure 3D, 3E, 3F). All these results indicated that the migration and invasion characteristic of tumor cells was mediated by platelets and could be suppressed by CREKATicagrelor treatment.
Figure 3. Anti-migration effect of Ticagrelor. (A) Wound healing assay was performed to evaluate CREKA- Ticagrelor’s effect to stop the migration of 4T1 cancer cells in the presence of platelets. (B) Quantification of wound healing assay. (C) Polygonal to spindle-shaped changes of 4T1 cells as the indicator of CREKA-Ticagrelor inhibition of platelet induced invasive type of these cancer cells. (D) Western Blot analysis. Expression of E-cadherin and Vimentin as indicators of EMT was measured in three groups including saline (negative group), platelets (positive group), and platelet + CREKA-Ticagrelor. β -actin is used as a loading control. The results are representative of three independent experiments. (E) Quantification of E-cadherin in D. **p
