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. 2019 Nov 11;10(12):1620–1627. doi: 10.1021/acsmedchemlett.9b00339

Inhibitory Effects of Multivalent Polypeptides on the Proliferation and Metastasis of Breast Cancer Cells

Zhuangzhuang Zhang †, Yachao Li †, Huayu Wu †, Xiao Zhang ‡, Dan Zhong †, Yahui Wu †, Xianghui Xu ‡,*, Jun Yang §, Zhongwei Gu †,‡,*
PMCID: PMC6912875  PMID: 31857837

Abstract

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The susceptibility of peptide drugs to enzymatic degradation has limited their clinical applications. To overcome this limitation, we attached the peptide tyroserleutide (YSL) to a molecular scaffold in order to produce homogeneous monovalent, bivalent, tetravalent, and octavalent YSL dendrimers with highly ordered secondary structures. These multivalent YSL dendrimers were resistant to proteolysis and were better able to induce cytotoxicity in tumor cells in vitro as compared with monomeric peptides. These multivalent YSL dendrimers were also better able to constrain tumor cell metastasis. Compared with monovalent YSL, the multivalent YSL dendrimers displayed enhanced in vivo antitumor activity and suppressed tumor growth and metastasis in BALB/c mice bearing 4T1 tumors. These findings indicate that multivalence can significantly enhance ligand potency and represent a potential method for the development of peptide drugs with high therapeutic potential.

Keywords: Tyroserleutide, multivalence, nature products, invasion, antitumor

Peptides are widely distributed in the human body and regulate diverse biological functions.1 Peptides can also be utilized as building blocks for construction of biological materials owing to the intrinsic bioactivity and biocompatibility.2,3 Recently, peptide-based cancer therapy has attracted wide attention.4 Different from many traditional chemotherapeutics, peptides are nongenotoxic and are of low toxicity.5 Short peptides that have no more than 50 amino acid repeating units have been intensively studied owing to their biological origins and amenability to structural modification.6 For example, muramyl dipeptide from mycobacterium can inhibit tumor growth and has been utilized as a vaccine adjuvant.7,8 However, peptides suffered from rapid degradation problems. For example, dipeptides and tripeptides derived from l-amino acids are easily degraded (e.g., the half-life time of Gly-Pro-Hyp on brush-border membrane vesicles is 4 h).9 Therefore, improving peptides’ biological stability is the key to improve their therapeutic efficacy.10

Multivalent interaction is a well-accepted strategy to significantly enhance the interactions between ligands and their receptors.11 For example, the number of RGD peptides per nanoparticle was positively related with the nanoparticle’s cellular uptake,12 and the number of α-conotoxin ImI (α-ImI) per nanoparticle was directly associated with the resultant conotoxin potency.13 Liposomes functionalized using multimeric peptides have similarly been able to better mediate drug delivery to targeted tumor cells.14 Multimeric approaches have also been used in other context, as in a study wherein researchers developed multivalent macrophage-targeting peptides with both increased serum stability and avidity.15 In addition, a wide selection of scaffolds are available for peptide multimerization strategies, including polylysine dendrimers assembled from l-lysine amino acids that are easily synthesized and induce minimal cytotoxicity, making them the subject of intense research interest.16,17 To date, a variety of therapeutic dendrimers have been designed through rational molecular and supramolecular engineering approaches in order to serve as antitumor agents both in vitro and in vivo, including tryptophan-rich peptide dendrimers.18−20 However, the conjugation of multiple peptide ligands to dendrimers presents many challenges, particularly for high generation of tailor-made dendritic peptides.

Tyroserleutide (YSL), a natural peptide identified in splenic tissues, inhibits the growth and metastasis of specific cancers and has reached phase II in preclinical programs.21 YSL can inhibit the invasiveness and metastatic potential of hepatocellular carcinoma (HCC) through down-regulation of hypoxia-inducible factor 1α (HIF-1α) and transmembrane protease serine 4 (TMPRSS4) and inhibition of epithelial-mesenchymal transition (EMT), having a potential as a new antimetastasis agent for radiotherapy.22 As others have previously observed improved efficacy for multivalent RGD peptides,12 we therefore wanted to explore whether multivalent YSL peptides similarly exhibited enhanced antitumor efficacy. To that end, we synthesized monovalent, bivalent, tetravalent, and octavalent YSL peptide dendrimers (Figure 1) and explored their ability to inhibit breast cancer growth and metastasis. Interestingly, we found that the YSL-dendrimers showed enhanced stability in serum and improved inhibition of proliferation and metastasis of 4T1 cells.

Figure 1.

Figure 1

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Chemical structures of YSL and YSL-based dendrimers.

The monovalent, bivalent, tetravalent, and octavalent YSL (Figure 1) were synthesized by conjugation of YSL to l-lysine-based dendrimer backbone, which was synthesized via a divergent method (see synthetic details in the Supporting Information). Peaks at m/z 418.23, 909.47, 1892.12, and 3857.32, [M + Na]+, in MALDI-TOF mass spectra, were consistent with the predicted molecular weights of monovalent, bivalent, tetravalent, and octavalent YSL (cal. m/z 418.21, 909.41, 1892.27, and 3857.12, [M + Na]+), respectively (Figure 2A and Table S1). Phenol signals from the YSL peptide (δ = 6.75 and 7.25 ppm) in 1H NMR spectra confirmed the incorporation into the poly(l-lysine) dendrimer scaffold. RP-HPLC results showed that YSL and multivalent YSL dendrimers had high purity of more than 90% (Figures S13–S16). Taken together, multivalent YSL dendrimers were successfully prepared with high purity.

Figure 2.

Figure 2

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(A) MALDI-TOF mass spectra and (B) serum stability of YSL, bivalent YSL, tetravalent YSL, and octavalent YSL.

We next investigated the secondary structures of YSL dendrimers using circular dichroism (CD) spectroscopy (Figure S17). All YSL dendrimers showed negative maxima at 205–209 nm and positive maxima at 220–225 nm, indicating the presence of a polyproline helix (PPII).23 In contrast, the multivalent dendron backbones showed a positive maximum at 195–205 nm, exhibiting a β-sheet structure (Figure S18). The PPII elements in the multivalent YSL dendrimers thus originated from the attached YSL ligands. Taken together, these results indicated that free YSL and YSL dendrimers had similar highly ordered architectures. What’s more, the data may suggest each YSL in the dendrimer was structurally intact and free to interact with cells independently, thus allowing potential multivalent interactions.13

The stability of YSL dendrimers in serum was studied to evaluate their biological stability (Figure 2B and S19). Free YSL showed rapid degradation in serum; about 60% and 100% YSL was degraded at 4 and 12 h, respectively. In contrast, more than 80% YSL in the bivalent, tetravalent, and octavalent YSL dendrimers remained intact after 12 h incubation. The enhanced serum stability of YSL in the YSL dendrimers was probably attributed to the branched architecture of YSL dendrimers, which provided steric hindrance for the efficient contact with serum. The stability of YSL dendrimers in serum was increased with increasing of YSL number per peptide dendrimer. In addition, peptide backbones of multimerization can generate clusters, and dynamic aggregation may occur, which contributes to the resistance of the protease.24,25

However, the use of all d-amino acids has been employed to improve peptide stability.26 As for all-d-amino acid peptides, d-amino acid replacement at a specific position may decrease the activity of the peptides.27 In addition, most d-amino acid peptides have high cost due to the lack of natural sources. Cyclization is also envisioned to enhance the selective binding, uptake, potency, and stability of linear precursors; however, not all cyclization strategies and constrained geometries enhance these properties to the same extent.28 What’s more, multivalent YSL dendrimers that do not contain directional converter residues present no synthetic difficulties, there still does not appear to exist a generic strategy for the preparation of retro-inverso peptides. Although there are many limitations to multivalent dendrimers, they can thus also exhibit advantageous properties that make them well-suited to additional applications.4,29−31

Previous study established that YSL can inhibit human hepatocellular carcinoma cells with an IC50 value (concentration for 50% cell inhibition) of 3.03 mM.32 We then studied the in vitro antitumor activity of YSL and YSL dendrimers on 4T1 cancer cells by an MTT assay. No significant inhibition effect of cancer cell growth was evident in response to a 0.1 mM dose of any compound, whereas at the 0.2 mM dose, rates of cell death for monovalent, bivalent, tetravalent, and octavalent YSL were 2%, 5%, 10%, and 20%, respectively. The IC50 value for octavalent YSL when used to treat 4T1 tumor cells was 0.4 mM (Figure 3A), making this compound more efficient than YSL, and Taxol was also able to efficiently induce 4T1 tumor cell death with an IC50 of 10–3 mM (Figure S20). As the antitumor mechanism of multivalent dendrimers and Taxol may be similar according our assays, we chose Taoxl as the positive control. However, G1-Lys, G2-Lys, and G3-Lys mixing with YSL under identical conditions failed to induce any significant cytotoxicity (Figure S21). As expected, the antitumor activity of the multivalent YSL dendrimers was thus dependent upon protein structure. To further examine the impact of YSL dendrimers on normal cell viability, we analyzed the effect of a range of dendrimer concentrations on normal L929 cells (Figure S22). Next, we aim to endow the multivalent YSL dendrimers with tumor-specific toxicity by tumor-specific responsiveness strategies such as tumor microenvironment targeting.33 In addition, we have conducted a hemolysis assay and provided quantification of the resultant data (Figures S23–S24). Visual and quantitative results of hemolysis assay were presented as a hemolysis rate of under 5%, thus placing the red blood cell (RBC) adverse reaction within acceptable limits even at a high concentration of the YSL-dendrimers (3.2 mM).34

Figure 3.

Figure 3

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Antitumor activity in vitro of YSL and multivalent YSL dendrimers. (A) Cell viability assays, (B) live–dead cell staining, (C) cell cycle distribution, and (D) apoptosis analysis upon Annexin V-FITC/PI staining of 4T1 treatment with YSL, bivalent YSL, tetravalent YSL and octavalent YSL after 24 h (mean ± SD, n = 6, *p ≤ 0.001, YSL compared with octavalent YSL; #p ≤ 0.001, bivalent YSL compared with octavalent YSL; &p ≤ 0.001, tetravalent YSL compared with octavalent YSL).

We next treated cancer cells with YSL dendrimers to assess apoptotic induction, using the IC50 value for octavalent YSL as a target dose. As shown in Figure 3B, controls, as well as the monovalent, bivalent, and tetravalent YSL groups (0.4 mM) exhibited PI staining consistent with cytotoxicity and potential apoptosis. PI staining at this dose was strongest for those cells treated with octavalent YSL, which is better than positive control. Consistent with our earlier results, when cells were instead treated with a higher 0.8 mM dose of octavalent YSL or Taxol, this led to significantly increased tumor cell death in a concentration-dependent manner (Figure S25). This thus suggested that increased YSL multivalency also increased the ability of these compounds to induce cytotoxic cell death in tumor cells.

Cell cycle distribution analyses were further used to explore the mechanism whereby these dendrimers induced tumor cell death. As shown in Figure 3C, the percentage of cells in the G0/G1 phase following octavalent YSL treatment was higher than in the YSL group (44.4% vs 37.4%). In cells treated with Taxol, a similar elevation in the frequency of cells in the G0/G1 phase was observed (42.3%). This analysis confirmed that the multivalent YSL dendrimers induced cell cycle arrest at the G0/G1 phase.35 The percentage of cells in the G2/M stage following treatment with monovalent (15.3%), bivalent (15.3%), and tetravalent YSL (15.5%) were comparable, whereas cells treated with octavalent YSL exhibited reduced G2/M progression (7.1%).

Afterward, we assessed the induction of apoptotic cell death in 4T1 cells upon multivalent YSL dendrimer treatment with an Annexin V-FITC/PI detection kit (Figure 3D). Then, 56.2% and 36.9% of cells treated with octavalent YSL and Taxol were in the late stages of apoptosis as determined based upon Annexin V and PI positivity. In the monovalent, bivalent, and tetravalent YSL groups, in contrast, the percentages of cells in the late stage of apoptosis were 10.3%, 18.8%, and 20.4%, respectively. The octavalent YSL dendrimer thus induced the highest levels of apoptosis-like cytotoxicity in tumor cells.

Previous studies have indicated that YSL inhibits cancer cell metastasis and migration.22 We therefore assessed the effects of the multivalent YSL dendrimers on the migratory and invasive properties of tumor cells. To examine the direct impact of these multimers on tumor cell migration without introducing cytotoxicity artifacts, we selected the lower 0.2 mM dose. In the wound healing assay, the wound closure rates of cells treated with control, YSL, bivalent YSL, tetravalent YSL, octavalent YSL, and Taxol were 82%, 80%, 79%, 76%, 30%, and 43%, respectively. The results demonstrated that octavalent YSL dendrimers exhibited enhanced inhibition of cancer cell migration (Figure 4A,B and S26). In a Transwell-based migration assay, the frequencies of migrated cells were 82%, 68%, 39%, 10%, and 30% in the YSL, bivalent YSL, tetravalent YSL, octavalent YSL, and Taxol groups, respectively, following a 24 h treatment with 0.2 mM of the indicated compounds (Figure 4C–D). Together, these wound healing assay and migration results thus both demonstrated that octavalent multivalent YSL strongly inhibited the migration of cancer cells. The inhibitory activity of octavalent YSL was 4.5-fold higher than that of YSL (**p ≤ 0.001), 3-fold higher than that of bivalent YSL (**p ≤ 0.001), and 1.5-fold higher than that of tetravalent YSL (**p ≤ 0.001). Next, we analyzed the impact of these multivalent YSL dendrimers on cancer cell invasion with a Matrigel-based Transwell assay system. We found that, relative to untreated control cells, invasion rates in cells treated with octavalent YSL (0.2 mM) were reduced to 20%, being lower than rates for cells treated with YSL (92%), bivalent YSL (70%), tetravalent YSL (41%), and Taxol (34%) (Figure 4E,F). These observations were thus consistent with wound healing and Transwell migration assay results.

Figure 4.

Figure 4

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Impact of YSL and multivalent YSL dendrimers on in vitro tumor metastasis. (A) In vitro scratch wound healing assays, (B) wound closure was quantified by measuring the distance between the scratch edges after 36 h, (C) cell migration assays and (D) migration rates of 4T1 cells were quantified by measuring cell numbers on the bottom of the porous membrane, and (E) cell invasion assays and (F) invasion rates of 4T1 cells were quantified by measuring the number of cells on the bottom of the Matrigel-coated porous membrane (mean ± SD, n = 3, **p ≤ 0.001, *p ≤ 0.01), scale bar: 200 μm.

Tumor cell invasiveness arises from the ability of these cells to mediate dramatic cytoskeletal remodeling and matrix metalloproteinase secretion.36 Scanning electron microscopy (SEM) and cytoskeletal imaging were used to further assess the YSL-mediated inhibition of cancer cell metastasis. As shown in Figure 5A, at a low concentration of 0.2 mM, no pseudopods were observed in the tetravalent-YSL, octavalent-YSL and Taxol groups (10–5 mM) after a 24 h treatment. We further stained treated cells for F-actin using Alexa Fluor 488 Phalloidin to reveal changes in cell morphology. We found in comparison to the extension of slender actin filaments in the untreated group, multivalent YSL dendrimers and Taxol displayed disrupted F-actin arrangements (Figure 5B). Treatment with tetravalent or octavalent YSL dendrimers resulted in cell shrinkage and actin filaments shorter than YSL treatments. The observed disruption in actin consistent with the results of our migration and invasion assays.

Figure 5.

Figure 5

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Mechanistic basis for multivalent dendrimer impact on in vitro tumor metastasis. (A) SEM images and (B) fluorescent images of untreated, multivalent YSL dendrimer-treated, and Taxol-treated 4T1 cells after 24 h. Green and blue present Fluor 488 phalloidin-stained cytoskeleton channel and Hoechst 33342-stained nucleus channel.

We next sought to confirm the previously documented impact of multivalent YSL dendrimers on matrix metalloproteinase activity,37 shown in Figure S27; relative to control samples, MMP-9 activity was reduced to 75%, 70%, and 34% following treatment with bivalent, tetravalent, and octavalent YSL, respectively. These observations suggested that MMP-9 activity and subsequent matrix degradation were inhibited following treatment with the YSL dendrimers.

To explore the in vivo antitumor efficacy of these multivalent YSL dendrimers, BALB/c mice bearing 4T1 tumors were treated with YSL and YSL dendrimers (Figure 6A). Animals were treated with identical doses of the indicated dendrimers (6 doses, 10 mg/kg body weight, the YSL monomer molar concentrations of each compounds were same), with the average tumor volume in octavalent YSL-treated mice being half that observed in saline-treated mice, whereas only very limited tumor growth inhibition was observed in animals treated with monovalent and bivalent YSL. Hematoxylin and eosin (H&E) staining of the tumor tissue revealed that treatment with the YSL dendrimers resulted in marked damage to the tumor tissue after 24 days (Figure 6C). The tumor tissues of mice in tetravalent YSL, octavalent YSL, and Taxol groups exhibited significantly higher rates of cytotoxic tumor cell death relative to those in saline group. More importantly, compared to saline group, the octavalent YSL group exhibited higher rates of damage to CD31-positive blood vessels and reduced the numbers of Ki-67 positive tumor cells. As the valence of YSL dendrimers increased, the number of TUNEL-positive 4T1 tumor cells increased. What’s more, representative whole lung photographs and histopathological examination of murine lungs (Figure S28) suggested that increasing YSL dendrimer valence was associated with a reduction in the number and size of lung tumor metastatic nodules as compared with saline-treated animals. These results thus indicated that octavalent YSL potentially suppressed tumor metastasis to the lungs. As shown in Figure S29, no lesions were observed in major organs (heart, liver, spleen, and kidney) in YSL dendrimer treatment groups, and normal biochemical parameters were observed in all animals (serum creatinine and blood urea nitrogen, Figures S30–S31). No significant differences in mouse body weights (Figure 6B) also evidenced that these compounds caused negligible side effect.

Figure 6.

Figure 6

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Antitumor efficacy of YSL and multivalent YSL dendrimers in the subcutaneous 4T1 tumor-bearing mouse model: (A) tumor growth curves, (B) animal weights, (C) H&E, CD31, Ki-67, and TUNEL staining results in 4T1 tumors from treated mice (mean ± SD, n = 5, *p ≤ 0.01). Scale bar: 200 μm.

In conclusion, we have synthesized multivalent YSL dendrimers through a careful design and purification approach. The resultant multivalent YSL dendrimers had intact structures and a peptide spatial configuration similar to that of YSL. In comparison to monomeric YSL, multivalent YSL dendrimers significantly enhanced the serum stability and the ability to inhibit breast cancer metastasis and growth through cytoskeletal disruption, reducing MMP-9 expression, and cytotoxic tumor cell death. Multivalent YSL dendrimers inhibited tumor growth in 4T1 tumor-bearing mice. These multivalent peptide constructs thus represent a promising approach for the optimization of peptide drugs’ biological stability and efficacy.

Acknowledgments

The National Natural Science Foundation of China (81621003, 21674067, and 31771067), the National Key Research and Development Program of China (2017YFC1103501), the Key R&D Plan of Jiangsu Province (BE2018010-3), and the Scientific Research Foundation for Talent Introduction of Nanjing Tech University (39803130 and 39803134) all provided financial support for this work.

Glossary

ABBREVIATIONS

DMSO

dimethyl sulfoxide

CD

circular dichroism

MTT

3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide

RP-HPLC

reversed-phase high-performance liquid chromatography

L929 cells

mouse fibroblast cells

RBC

red blood cell

F-action

fibrous actin

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications Web site. Supporting Information includes The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.9b00339.

  • Full experimental methods, NMR and MALDI-TOF results of compounds, cytotoxicity, hemolysis, apoptosis, wound-healing assay, and H&E staining results (PDF)

The authors declare no competing financial interest.

Supplementary Material

ml9b00339_si_001.pdf (5.3MB, pdf)

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