Wu Luo

Screening a phage display library for a novel FGF8b-binding peptide with anti-tumor effect on prostate cancer

Fibroblast growth factor 8b (FGF8b) is the major isoform of FGF8 expressed in prostate cancer and it correlates with the stage and grade of the disease. FGF8b has been considered as a potential target for prostate cancer therapy. Here we isolated 12 specific FGF8b-binding phage clones by screening a phage display heptapeptide library with FGF8b. The peptide (HSQAAVP, named as P12) corresponding to one of these clones showed high homology to the immunoglobulin-like (Ig-like) domain II(D2) of high-affinity FGF8b receptor (FGFR3c), contained 3 identical amino acids (AVP) to the authentic FGFR3 D2 sequence aa 163-169 (LLAVPAA) directly participating in ligand binding, carried the same charges as its corresponding motif (aa163-169) in FGFR3c, suggesting that P12 may have a greater potential to interrupt FGF8b binding to its receptors than other identified heptapeptides do. Functional analysis indicated that synthetic P12 peptides mediate significant inhibition of FGF8b-induced cell proliferation, arrest cell cycle at the G0/G1 phase via suppression of Cyclin D1 and PCNA, and blockade of the activations of Erk1/2 and Akt cascades in both prostate cancer cells and vascular endothelial cells. The results demonstrated that the P12 peptide acting as an FGF8b antagonist may have therapeutic potential in prostate cancer.

Identification of galectin-1 as a novel mediator for chemoresistance in chronic myeloid leukemia cells

Multidrug resistance protein-1 (MDR1) has been proven to be associated with the development of chemoresistance to imatinib (Glivec, STI571) which displays high efficacy in treatment of BCR-ABL-positive chronic myelogenous leukemia (CML). However, the possible mechanisms of MDR1 modulation in the process of the resistance development remain to be defined. Herein, galectin-1 was identified as a candidate modulator of MDR1 by proteomic analysis of a model system of leukemia cell lines with a gradual increase of MDR1 expression and drug resistance. Coincidently, alteration of galectin-1 expression triggers the change of MDR1 expression as well as the resistance to the cytotoxic drugs, suggesting that augment of MDR1 expression engages in galectin-1-mediated chemoresistance. Moreover, we provided the first data showing that NF-κB translocation induced by P38 MAPK activation was responsible for the modulation effect of galectin-1 on MDR1 in the chronic myelogenous leukemia cells. Galectin-1 might be considered as a novel target for combined modality therapy for enhancing the efficacy of CML treatment with imatinib.

Inhibition of autophagy promotes apoptosis and enhances anticancer efficacy of adriamycin via augmented ROS generation in prostate cancer cells.

The interplay between autophagy and apoptosis response to chemotherapy is still a subject of intense debate in recent years. More efforts have focused on the regulation effects of apoptosis on autophagy, whereas how autophagy affects apoptosis remains poorly understood. In this study performed on prostate cancer cells, we investigated the role of autophagy in adriamycin-induced apoptosis, as well as the mechanisms mediating the effects of autophagy on apoptosis response to adriamycin (ADM). The results show that ADM not only inhibited cell viability and enhanced apoptosis, but also promoted autophagy via PI3K/Akt(T308)/mTOR signal pathway. Inhibition of autophagy by either pharmacological inhibitor chloroquine (CQ) or RNA interference of Atg5 increased ADM-induced apoptosis and enhanced the chemosensitivity of prostate cancer cells. Moreover, blockade of autophagy augmented reactive oxygen species (ROS) generation induced by ADM. Scavenging of ROS by antioxidant N-acetyl-cysteine (NAC) reversed the strengthened effects of CQ on ADM-induced apoptosis and rescued the cells from apoptosis. The results identified ROS as a potential mediator directing the modulation effects of the protective autophagy on apoptosis response to ADM. Suppression of the protective autophagy might provide a promising strategy to increase the anticancer efficacy of agents in the treatment of prostate cancer.

A short peptide derived from the gN helix domain of FGF8b suppresses the growth of human prostate cancer cells.

Previous studies have demonstrated that fibroblast growth factor 8b (FGF8b) is up-regulated in a large proportion of prostate cancer patients and that it plays a key role in prostate carcinogenesis. In this study, we designed and synthesized a gN helix domain derived short peptide (termed 8b-13) based on the analysis of the FGF8b-FGFR structure. The synthetic peptides inhibited the proliferation of prostate cancer cell lines, including PC-3 and DU-145 cells. Further investigations indicated that 8b-13 arrested the cell cycle at the G0/G1 phase, reduced the activation of the Erk1/2, P38, and Akt cascades, and down-regulated the expression of G1/S-specific cyclinD1. The suppression of DNA synthesis and the G1 to S phase transition due to the expression of proteins related to proliferation and cell cycle progression may contribute to the inhibitory effect of 8b-13 peptides on cellular proliferation. Our results not only suggest that 8b-13 exerts an antitumor effect in prostate cancer but also confirm the essential role of the gN helix domain in mediating the activity of FGF8b.

Selection of a novel FGF23-binding peptide antagonizing the inhibitory effect of FGF23 on phosphate uptake

Fibroblast growth factor 23 (FGF23) is a bone-derived endocrine regulator of phosphate homeostasis and has been considered as a potential therapeutic target for hypophosphatemic disorders. Herein, we isolated a novel FGF23-binding peptide by screening a phage display library with FGF23180–205, the minimal epitope of FGF23 binding to the binary fibroblast growth factor receptor (FGFR)-Klotho complex. The corresponding peptide (referred to as 23-b6) showed high homology to the immunoglobulin-like (Ig-like) domain III (D3) of FGFR1c, the predominant receptor mediating the phosphaturic activity of FGF23. The 23-b6 peptide and panning target FGF23180–205 carried opposite charges and shared similar hydrophilic profiles. Functional analysis indicated that synthetic 23-b6 peptide exhibited antagonistic effect on the inhibition of phosphate uptake by FGF23 in opossum kidney cells (OK cells). The mechanisms of 23-b6 peptide impairing the bioactivity of FGF23 involved blockade of the activation of Erk cascade and up-regulation of NaPi-2a and NaPi-2c expression in OK cells. Our results demonstrate that the 23-b6 peptide is a potent FGF23 antagonist with increased effect on phosphate uptake in kidney cells and might have therapeutic potentials in hypophosphatemic disorders characterized by an abnormally high level of FGF23.

Inhibition of Proliferation of Non-small Cell Lung Cancer Cells by a bFGF Antagonist Peptide

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related mortality worldwide. Basic fibroblast growth factor (bFGF) is up-regulated in NSCLC patients and plays an important role in tumor growth. In this paper, we attempt to evaluate the therapeutic potential of bFGF binding peptide (named as P7) using as a potent bFGF antagonist via exploration of its anti-proliferation effect on NSCLC cells. Our experiments showed that P7 peptide inhibited bFGF-stimulated proliferation of NSCLC cell lines including A549, H1299, and H460. The inhibitory mechanism of P7 involved cell cycle arrest at the G0/G1phase caused by suppression of cyclin D1, blockage of the activation of Erk1/2, P38, Akt, and inhibition of bFGF internalization. Strategies using bFGF antagonist peptides with potent anti-proliferation property may have therapeutic potential in NSCLC.

P7 peptides targeting bFGF sensitize colorectal cancer cells to CPT-11

The low survival rate of patients with colorectal cancer (CRC) is mainly due to the drug resistance of tumor cells to chemotherapeutic agents. It has been reported that basic fibroblast growth factor (bFGF) is an essential factor involved in the epigenetic mechanisms of drug resistance, which provides a novel potential target for improving the sensitivity of tumor cells to chemotherapeutic agents. In this study, we first demonstrate that a novel bFGF antagonist, peptide P7, previously isolated by phage display technology, reversed bFGF-induced resistance to irinotecan hydrochloride (CPT-11), and counteracted the anti-apoptotic effects of bFGF on CPT-11-treated HT-29 cells. Further experiments indicated that the inhibition of Akt activation, the suppression of bFGF internalization, the increase in the Bax to Bcl-2 ratio and the downregulation of cytokeratin 8 (CK8) by P7 may contribute to the counteracting of the anti-apoptotic effects of bFGF, and further reversal of bFGF-induced resistance to CPT-11. Our results suggest that peptide P7 may have therapeutic potential in CRC as a sensitizer to chemotherapeutic agents by targeting bFGF.

Identification of a crucial amino acid responsible for the loss of specifying FGFR1-KLB affinity of the iodinated FGF21: LUO et al.

Previous studies suggest that specific binding to the complex consisting of fibroblast growth factor receptor‐1 (FGFR1) and the coreceptor beta‐Klotho (KLB) is the premise for human FGF19 and FGF21 activating the downstream signaling cascades, and regulating the metabolic homeostasis. However, it was found that human FGF21 loses its ability to bind to FGFR1–KLB after iodination with Na125I and chloramine T, whereas human FGF19 retained its affinity for FGFR1–KLB even after iodination. The molecular mechanisms underlying these differences remained elusive. In this study, we first demonstrated that an intramolecular disulfide bond was formed between cysteine‐102 and cysteine‐121 in FGF21, implying that the oxidation of the cysteine to cysteic acid, which may interfere with the active conformation of FGF21, did not occur during the iodination procedures, and thus ruled out the possibility of the two conserved cysteine residues mediating the loss of FGF21 binding affinity to FGFR1–KLB upon iodination. Site‐directed mutagenesis and molecular modeling were further applied to determine the residue(s) responsible for the loss of FGFR1–KLB affinity. The results showed that mutation of a single tyrosine‐207, but not the other five tyrosine residues in FGF21, to a phenylalanine retained the FGFR1–KLB affinity of FGF21 even after iodination, whereas replacing the corresponding phenylalanine residue with tyrosine in FGF19 did not alter its binding affinity to FGFR1–KLB, but decreased the receptor binding ability of the iodinated protein, suggesting that tyrosine‐207 is the crucial amino acid responsible for the loss of specifying FGFR1–KLB affinity of the iodinated FGF21.

Mechanism of bFGF-binding Peptide Reversing Adriamycin Resistance in Human Gastric Cancer Cells

Development of drug resistance is a challenging problem in cancer chemotherapy. It has been shown that basic fibroblast growth factor (bFGF) plays an important role in an epigenetic mechanism of drug resistance. We have isolated a bFGF binding peptide P7 with inhibitory activity against bFGF-induced proliferation of human gastric cancer cells by screening a phage display library. In this study, we found that P7 peptide also has efficacy of reversing bFGF-induced resistance to Adriamycin (ADM) in human gastric cancer cells. Further investigations with SGC-7901 cells revealed that inhibition of Akt activation triggered by bFGF, and reversal of bFGF-induced up-regulation of Bcl-2 and XIAP and down-regulation of Bax, contribute to P7 peptide counteracting the anti-apoptotic effect of bFGF, and further reversing bFGF-induced resistance to ADM. The results suggested that the bFGF-binding peptide may have therapeutic potential of drug resistance in gastric cancer.

A bFGF Antagonist Peptide with Anti-angiogenesis Properties in Non-small Cell Lung Cancer Cells

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related mortality worldwide. Basic fibroblast growth factor (bFGF) is up-regulated in NSCLC patients and plays an important role in tumor growth and angiogenesis. Therefore, it is regarded as a potential therapeutic target of NSCLC. We have previously obtained a high-affinity bFGF antagonist peptide (named P7) with the potential of suppressing angiogenesis stimulated by bFGF from the phage display random heptapeptide library. Herein, we further revealed the underlying anti-angiogenic mechanisms of P7 peptides in human NSCLC cells. P7 not only inhibited the expressions of angiogenesis related factors including VEGF, MMP-2 and MMP-9 at both transcriptional and translational levels, but also induced significant changes in the expressions of proteins related to tumor growth and progress. Our results suggested that P7 peptides with potent anti-angiogenic activity may have therapeutic potential in NSCLC.