Selection and identification of a specific peptide binding to ovarian cancer cells from a phage-displayed peptide library.

OBJECTIVES: Ovarian cancer is one of the most fatal gynecological malignancies. It is emergently needed to select a novel molecular fragment as a targeting element for the future development of molecular imaging diagnosis and targeting chemotherapy to ovarian cancer. RESULTS: After five rounds of biopanning, a total of 44 positive phage clones were selected from final phage displayed peptide library. Nine consensus sequences were found based on the assay of sequencing results, then one clone of each consensus group was characterized and identified further by immunofluorescence assay. The result showed the phage clone R20 presents best targeting capacity. Then we synthesized peptide (OSP2) clone R20 displayed, it was characterized with high specificity and sensitivity binding to human ovarian cancer by a tissue chip assay. The target of OSP2 was predicted and docked as human carbonic anhydrase XII (CA12), an important protein usually deregulated in cancer. CONCLUSIONS: Taken together, OSP2 and its target indicate a novel investigation way in future to develop novel agent or drug delivery formulation for molecular imaging diagnosis and targeting chemotherapy of ovarian cancer.

Bovine extracellular vesicles contaminate human extracellular vesicles produced in cell culture conditioned medium when 'exosome-depleted serum' is utilised.

Extracellular vesicles (EVs) are important intercellular communication messengers. Half of the published studies in the field are in vitro cell culture based in which bovine serum in various concentrations and forms is used to facilitate the production of extracellular vesicles. 'Exosome depleted serum' is the type of bovine serum most widely used in the production of human EVs. Herein, we demonstrate that, despite the initial caution raised in 2014 about the persistence of bovine EVs, 'exosome depleted serum' was still used in 46% of publications on human or rodent EVs between 2015 and 2019. Using nanoparticle tracking analysis combined with detergent lysis of vesicles as well as bovine CD9 ELISA, we show that there were approximately 5.33 x 107/mL of bovine EVs remaining in the 'exosome depleted serum'. Importantly, the 'exosome depleted serum' was relatively enriched in small EVs by approximately 2.7-fold relative to the large EVs compared to that in the original serum. Specifically, the percentage of small EVs in total vesicles had increased from the original 48% in the serum before ultracentrifugation to 92% in the 'exosome depleted serum'. Furthermore, the pervasive bovine EVs carried over by the 'exosome depleted serum', even when the lowest concentration (0.5%) was used in cell culture, resulted in a significant contamination of human EVs in cell culture conditioned medium. Our findings indicate that the use 'exosome depleted serum' in cell culture-based studies may introduce artefacts into research examining the function of human and rodent EVs, in particular those involving EV miRNA. Thus, we appeal to the researchers in the EV field to seriously reconsider the practice of using 'exosome depleted serum' in the production of human and other mammalian EVs in vitro.

Screening and identification of a specific peptide binding to breast cancer cells from a phage-displayed peptide library.

OBJECTIVES: Breast cancer is a popular fatal malignant tumor for women with high of rates incidence and mortality. Development of the new approaches for breast cancer targeted diagnosis and chemotherapy is emergently needed by the current clinical practice, the important first step is finding a breast cancer specifically binding molecule or fragment as early clinical indicators. RESULTS: By a phage-displayed peptide library, a 12-mer peptide, CSB1 was screened out using MCF-7 cells as the target. The consequently results under immunofluorescence and laser scanning confocal microscope (LSCM) indicated that CSB1 bound MCF-7 cells and breast cancer tissues specifically and sensitively with high affinity. Bioinformatics analysis suggested that the peptide CSB1 targets the 5-Lipoxygenase-Activating Protein (FLAP), which has been implicated in breast cancer progression and prognosis. CONCLUSIONS: The peptide, CSB1 is of the potential as a candidate to be used for developing the new approaches of molecular imaging detection and targeting chemotherapy of breast cancer in the future.

LEF1 Enhances the Progression of Colonic Adenocarcinoma via Remodeling the Cell Motility Associated Structures.

Lymphoid enhancer-binding factor 1 (LEF1) is a key transcription factor mediating the Wnt signaling pathway. LEF1 is a regulator that is closely associated with tumor malignancy and is usually upregulated in cancers, including colonic adenocarcinoma. The underlying molecular mechanisms of LEF1 regulation for colonic adenocarcinoma progression remain unknown. To explore it, the LEF1 expression in caco2 cells was inhibited using an shRNA approach. The results showed that downregulation of LEF1 inhibited the malignancy and motility associated microstructures, such as polymerization of F-actin, ß-tubulin, and Lamin B1 in caco2 cells. LEF1 inhibition suppressed the expression of epithelial/endothelial-mesenchymal transition (EMT) relevant genes. Overall, the current results demonstrated that LEF1 plays a pivotal role in maintaining the malignancy of colonic adenocarcinoma by remodeling motility correlated microstructures and suppressing the expression of EMT-relevant genes. Our study provided evidence of the roles LEF1 played in colonic adenocarcinoma progression, and suggest LEF1 as a potential target for colonic adenocarcinoma therapy.

Development of a novel drug targeting delivery system for cervical cancer therapy.

'Targeting peptides' have demonstrated their value in diagnostic imaging and therapy and novel peptide probes specific to cervical cancer were developed. In the M13KE phage dodecapeptide (12-mer) peptide library, the phage clone S7 showed the best binding to the cancer cells as confirmed by immunofluorescence and flow cytometry assays, and was selected for continued studies. Its binding peptide, CSP3, was synthesized from the sequence of S7's 12-mer at the N-terminus of the minor coat protein pIII of this M13KE phage vector. The peptide's binding was analyzed by the same assays used for S7. It was also assessed using competitive inhibition and binding to a tissue chip. The results demonstrated that the CSP3 peptide bound to cervical carcinoma cells with high sensitivity and specificity. The positive results indicated that the peptide CSP3, conjugated with nanomaterials and chemotherapeutics, may be developed as a targeting vehicle for therapeutic drug delivery against cervical cancer, especially cervical cancer with multiple drug resistance. For this aim, we prepared a CSP3 conjugated liposome drug delivery system containing doxorubicin (DOX) and microRNA101 (miR101) expression plasmids (CSP3-Lipo-DOX-miR101), and the primary result showed that the system demonstrated significantly enhanced cytotoxicity to SiHa cells and DOX resistant SiHa cells, SiHa/ADR. Our results showed that CSP3 is a cervical cancer targeting 12aa peptide with high specificity and sensitivity, and the CSP3 conjugated drug delivery system, CSP3-Lipo-DOX-miR101 has promising potential for development as an efficient drug system for the therapy of cervical cancer.

Annexin A2 Enhances the Progression of Colorectal Cancer and Hepatocarcinoma via Cytoskeleton Structural Rearrangements.

Annexin A2 (ANXA2) is reported to be associated with cancer development. To investigate the roles ANXA2 plays during the development of cancer, the RNAi method was used to inhibit the ANXA2 expression in caco2 (human colorectal cancer cell line) and SMMC7721 (human hepatocarcinoma cell line) cells. The results showed that when the expression of ANXA2 was efficiently inhibited, the growth and motility of both cell lines were significantly decreased, and the development of the motility relevant microstructures, such as pseudopodia, filopodia, and the polymerization of microfilaments and microtubules were obviously inhibited. The cancer cell apoptosis was enhanced without obvious significance. The possible regulating pathway in the process was also predicted and discussed. Our results suggested that ANXA2 plays important roles in maintaining the malignancy of colorectal and hepatic cancer by enhancing the cell proliferation, motility, and development of the motility associated microstructures of cancer cells based on a possible complicated signal pathway.

Screening and identification of a specific peptide binding to cervical cancer cells from a phage-displayed peptide library.

OBJECTIVES: To screen and identify the probe markers specifically binding to human cervical cancer, a phage-displayed 12-mer peptide library was used for biopanning of SiHa cells. RESULTS: After four rounds of whole-cell subtraction biopanning, the phage recovery was 21-fold higher (from 3.9 × 10-5 to 8.3 × 10-4) than that of the first round, and specific phage clones were significantly enriched. 57 randomly selected phage clones were tested by ELISA, and 36 phage clones were identified as positive clones. After sequencing of positive clones, six different peptide sequences were obtained and CSP3 showed best affinity and specificity to SiHa cells via immunofluorescence assay. CONCLUSIONS: Peptide, CSP3, bound to SiHa cells specifically and sensitively. It may be a potential candidate for molecular imaging detection and targeting therapy of cervical cancer.

The further characterization of the peptide specifically binding to gastric cancer.

Targeting peptide has been considered to be useful as a small molecule probe leading to multifunctional properties for both imaging detection and targeting therapy. Thus, the identification of novel targets is urgently needed to develop innovative agents to effectively control gastric cancer metastasis and progression. Previously, we reported a novel 12-mer peptide, GP-5 (IHKDKNAPSLVP), binding to gastric carcinoma (GC) cells specifically and sensitively, and it was screened by using a phage displayed peptide library and primarily analyzed. In this study, it was further identified via fluorescence microscopy, flow cytometry, tissue chip and other methods. Our results indicated that the peptide GP-5 presents a particularly high affinity and specificity to GC cells and tissues, whereas only background detection occurred with other control cancer cells, cancer tissues or normal tissues. Taken together, all results support that the peptide GP-5 is a potential candidate to be developed as a useful molecule fragment for the imaging detection and targeting therapy of GC.

Screening of a specific peptide binding to esophageal squamous carcinoma cells from phage displayed peptide library.

To select a specifically binding peptide for imaging detection of human esophageal squamous cell carcinoma (ESCC), a phage-displayed 12-mer peptide library was used to screen the peptide that bind to ESCC cells specifically. After four rounds of bio-panning, the phage recovery rate gradually increased, and specific phage clones were effectively enriched. The 60 randomly selected phage clones were tested using cellular enzyme-linked immunosorbent assay (ELISA), and 41 phage clones were identified as positive clones with the over 2.10 ratio of absorbance higher than other clones, IRP and PBS controls. From the sequencing results of the positive clones, 14 peptide sequences were obtained and ESCP9 consensus sequence was identified as the peptide with best affinity to ESCC cells via competitive inhibition, fluorescence microscopy, and flow cytometry. The results indicate that the peptide ESCP9 can bind to ESCC cells specifically and sensitively, and it is a potential candidate to be developed as an useful molecule to the imaging detection and targeting therapy for ESCC.

A CD44 specific peptide developed by phage display for targeting gastric cancer.

OBJECTIVE: To develop a peptide probe that could be used for gastric cancer detection via binding to CD44 protein with specificity and affinity. RESULTS: A 12-mer phage peptide library was screened against immobilized CD44 protein. Bound phage counts using ELISA were performed to identify phage clones carrying the most highly selective peptide, which termed RP-1. Immunofluorescence and flow cytometry analysis indicated that the consensus peptide RP-1 could bind to CD44-positive gastric cancer cells with mean fluorescence intensities significantly higher than that of CD44-negative cells. CD44 knockdown led to decreased binding activity of RP-1 to the same cell line. Tissue array technique was used to identify the relationship (r = 0.556) between peptide binding and CD44 detection on gastric cancer tissues. Further, the hyaluronan-binding domain of CD44 was docked with RP-1 using computer modeling/docking approaches, revealing a RP-1/CD44 interaction with geometrical and energy match (-8.6 kcal/mol). CONCLUSIONS: The RP-1 peptide we screened exhibits affinity and specificity to CD44 on cells and has the potential to be used as a candidate probe for gastric cancer cell targeting.

The merged basins of signal transduction pathways in spatiotemporal cell biology.

Numerous evidences have indicated that a signal system is composed by signal pathways, each pathway is composed by sub-pathways, and the sub-pathway is composed by the original signal terminals initiated with a protein/gene. We infer the terminal signals merged signal transduction system as "signal basin". In this article, we discussed the composition and regulation of signal basins, and the relationship between the signal basin control and triple W of spatiotemporal cell biology. Finally, we evaluated the importance of the systemic regulation to gene expression by signal basins under triple W. We hope our discussion will be the beginning to cause the attention for this area from the scientists of life science.

Screening and identification of a specific peptide binding to hepatocellular carcinoma cells from a phage display peptide library.

To screen and identify the novel probe markers binding hepatocellular carcinoma specifically and sensitively, a phage-displayed 12-mer peptide library was used to make biopanning with the modified protocols on HepG2 cells. After four rounds of panning, the consensus sequences were obtained, and the PC28, a phage clone with most specific and sensitive binding to HepG2 cells, was identified as the best positive clone. The peptide probe HCSP4 (sequence SLDSTHTHAPWP) was synthesized based on the sequencing result of PC28. The specificity and sensitivity of HCSP4 were primarily analyzed using immunofluorescence, flow cytometry, and other methods. The results show that HCSP4 can bind to hepatocellular carcinoma cells with satisfactory specificity and sensitivity. It may be a promising lead candidate for molecular imaging and targeted drug delivery in the diagnosis and therapy of hepatocellular carcinoma.

Short-homology-mediated PCR-based method for gene introduction in the fission yeast Schizosaccharomyces pombe.

Schizosaccharomyces pombe is a commonly utilized model organism for studying various aspects of eukaryotic cell physiology. One reason for its widespread use as an experimental system is the ease of genetic manipulations, leveraging the natural homology-targeted repair mechanism to accurately modify the genome. We conducted a study to assess the feasibility and efficiency of directly introducing exogenous genes into the fission yeast S. pombe using Polymerase Chain Reaction (PCR) with short-homology flanking sequences. Specifically, we amplified the NatMX6 gene (which provides resistance to nourseothricin) using PCR with oligonucleotides that had short flanking regions of 20 bp, 40 bp, 60 bp and 80 bp to the target gene. By using this purified PCR product, we successfully introduced the NatMX6 gene at position 171 385 on chromosome III in S. pombe. We have made a simple modification to the transformation procedure, resulting in a significant increase in transformation efficiency by at least 5-fold. The success rate of gene integration at the target position varied between 20% and 50% depending on the length of the flanking regions. Additionally, we discovered that the addition of dimethyl sulfoxide and boiled carrier DNA increased the number of transformants by ~60- and 3-fold, respectively. Furthermore, we found that the removal of the pku70+ gene improved the transformation efficiency to ~5% and reduced the formation of small background colonies. Overall, our results demonstrate that with this modified method, even very short stretches of homologous regions (as short as 20 bp) can be used to effectively target genes at a high frequency in S. pombe. This finding greatly facilitates the introduction of exogenous genes in this organism.

Development of an efficient liposomal DOX delivery formulation for HCC therapy by targeting CK2α.

Hepatocellular carcinoma (HCC) is a digestive tract cancer with high mortality and poor prognosis, especially in China. Current chemotherapeutic drugs lead to poor prognosis, low efficacy, and high side effects due to weak targeting specificity and rapidly formed multidrug resistance (MDR). Based on the previous studies on the doxorubicin (DOX) formulation for cancer targeting therapy, we developed a novel DOX delivery formulation for the targeting chemotherapy of HCC and DOX resistant HCC. HCSP4 was previously screened and casein kinase 2α (CK2α) was predicted as its specific target on HCC cells in our lab. In the study, miR125a-5p was firstly predicted as an MDR inhibiting miRNA, and then CK2α was validated as the target of HCSP4 and miR125a-5p using CK2α-/-HepG2 cells. Based on the above, an HCC targeting and MDR inhibiting DOX delivery liposomal formulation, HCSP4/Lipo-DOX/miR125a-5p was synthesized and tested for its HCC therapeutic efficacy in vitro. The results showed that the liposomal DOX delivery formulation targeted to HCC cells specifically and sensitively, and presented the satisfied therapeutic efficacy for HCC, particularly for DOX resistant HCC. The potential therapeutic mechanism of the DOX delivery formulation was explored, and the formulation inhibited the expression of MDR-relevant genes including ATP-binding cassette subfamily B member 1 (ABCB1, also known as P-glycoprotein), ATP-binding cassette subfamily C member 5 (ABCC5), enhancer of zeste homolog 2 (EZH2), and ATPase Na+/K+ transporting subunit beta 1 (ATP1B1). Our study presents a novel targeting chemotherapeutic drug formulation for the therapy of HCC, especially for drug resistant HCC, although it is primarily and needs further study in vivo, but provided a new strategy for the development of novel anticancer drugs.

Selection and characterization of colorectal cancer cell-specific peptides.

To develop a novel peptide probe for imaging detection of colorectal cancer, a 12-mer phage display library was used to select peptides that bind specifically to the human colorectal cancer cell line Caco-2. After four rounds of panning, four phage clones that bound specifically to the Caco-2 cells were selected. The phage clone SP-2 had a particularly high affinity and specificity for Caco-2 cells. This clone was identified using a series of methods. The peptide SP-2 that was displayed on phage SP-2 exhibited a high specificity to Caco-2 cells. Thus, the peptide SP-2 could be a candidate probe for the detection of colorectal cancer.

Development of Blueberry-Derived Extracellular Nanovesicles for Immunomodulatory Therapy.

Over the past decade, there has been a significant expansion in the development of plant-derived extracellular nanovesicles (EVs) as an effective drug delivery system for precision therapy. However, the lack of effective methods for the isolation and characterization of plant EVs hampers progress in the field. To solve a challenge related to systemic separation and characterization in the plant-derived EV field, herein, we report the development of a simple 3D inner filter-based method that allows the extraction of apoplastic fluid (AF) from blueberry, facilitating EV isolation as well as effective downstream applications. Class I chitinase (PR-3) was found in blueberry-derived EVs (BENVs). As Class I chitinase is expressed in a wide range of plants, it could serve as a universal marker for plant-derived EVs. Significantly, the BENVs exhibit not only higher drug loading capacity than that reported for other EVs but also possess the ability to modulate the release of the proinflammatory cytokine IL-8 and total glutathione in response to oxidative stress. Therefore, the BENV is a promising edible multifunctional nano-bio-platform for future immunomodulatory therapies.

The roles EpCAM plays to enhance the malignancy of gastric cancer.

BACKGROUND: Gastric cancer (GC) remains a global challenge due to its high morbidity and mortality rates especially in Asia as well as poor response to treatment. As a member of the adhesion protein family and transmembrane glycoprotein, EpCAM expressed excessively in cancer cells including GC cells. The database assay showed that EpCAM is excessively expressed and easily mutated in cancers, especially in early stage of GC. METHODS: To explore the roles EpCAM plays in oncogenesis and progression of GC, the expression of EpCAM was deleted in GC cells with CRISPR/Cas9 method, and then the changes of cell proliferation, apoptosis, motility and motility associated microstructures in EpCAM-deleted GC cells (EpCAM-/-SGC7901) were detected to evaluate the rules EpCAM played. RESULTS: The results showed that EpCAM deletion caused cell proliferation, motility and the development of motility-relevant microstructures inhibited significantly, apoptotic trend and contact inhibition enhanced in EpCAM-deleted GC cells. The results of western blot suggested that EpCAM modulates the expression of epithelial/endothelial mesenchymal transition (EMT) correlated genes. All results as above indicated that EpCAM plays important roles to enhance the oncogenesis, malignancy and progression as a GC enhancer. CONCLUSIONS: Combining our results and published data together, the interaction of EpCAM with other proteins was also discussed and concluded in the discussion. Our results support that EpCAM can be considered as a novel target for the diagnosis and therapy of GC in future.

The novel insights into spatiotemporal cell biology and its schematic frame, triple W.

Numerous research results have suggested that the events occurred in a selected cell target and the fates of the cells are spatiotemporally regulated. It has been paying much attention to study the cell events with spatiotemporal proposal designation and analysis. We have been tracking and thinking the new scientific area for many years. Spatiotemporal cell biology and its schematic frame, triple W (when, where, which), are systemically introduced in this study. The triple W under pathological conditions is also discussed.

The inhibition of ABCB1/MDR1 or ABCG2/BCRP enables doxorubicin to eliminate liver cancer stem cells.

Two ATP-binding cassette transporters, ABCB1/MDR1 and ABCG2/BCRP, are considered the most critical determinants for chemoresistance in hepatocellular carcinoma. However, their roles in the chemoresistance in liver cancer stem cells remain elusive. Here we explored the role of inhibition of MDR1 or ABCG2 in sensitizing liver cancer stem cells to doxorubicin, the most frequently used chemotherapeutic agent in treating liver cancer. We show that the inhibition of MDR1 or ABCG2 in Huh7 and PLC/PRF/5 cells using either pharmacological inhibitors or RNAi resulted in the elevated level of intracellular concentration of doxorubicin and the accompanied increased apoptosis as determined by confocal microscopy, high-performance liquid chromatography, flow cytometry, and annexin V assay. Notably, the inhibition of MDR1 or ABCG2 led to the reversal of the chemoresistance, as evident from the enhanced death of the chemoresistant liver cancer stem cells in tumorsphere-forming assays. Thus, the elevation of effective intracellular concentration of doxorubicin via the inhibition of MDR1 or ABCG2 represents a promising future strategy that transforms doxorubicin from a traditional chemotherapy agent into a robust killer of liver cancer stem cells for patients undergoing transarterial chemoembolization.

The APEX1/miRNA-27a-5p axis plays key roles in progression, metastasis and targeted chemotherapy of gastric cancer.

Gastric cancer (GC) presents a challenge for conventional therapeutics due to low targeting specificity and subsequent elicitation of multiple drug resistance (MDR). As an essential enzyme for DNA repair, apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) exhibits multiple functions to affect cancer malignancy and is excessively expressed in GC. However, the roles APEX1 and its inhibitor miR-27a-5p play in modulating GC progression and MDR development remains unclear. Here, we verified APEX1 as a target of miR-27a-5p and subsequently established the APEX1-deleted SGC-7901 cell line by CRISPR/Cas9 editing. The roles of the APEX1/miR-27a-5p axis in GC progression, metastasis and doxorubicin (DOX) resistance were explored by the targeted chemotherapy facilitated by a GC-specific peptide (GP5) functionalized liposomal drug delivery formulation (GP5/Lipo/DOX/miR-27a-5p). The results showed that APEX1 deletion distinctly attenuated cell growth and metastatic properties in GC, and also sensitized GC cells to DOX. Notably, miR-27a-5p was validated as a suppressor of APEX1-dependent GC development and DOX resistance by a RAS/MEK/FOS and PTEN/AKT/SMAD2 pathway-dependent manner. The altered expression of epithelial-mesenchymal transition (EMT) signatures and signal pathway proteins in the APEX1-deleted cells implied that APEX1 potentially enhances DOX resistance of GC cells by altering the regulation of MAPK and AKT pathways, leading to compromised efficacy of chemotherapy or by initiating additional DNA damage response pathways. Taken together, these findings revealed that as a novel therapeutic target, APEX1/miR-27a-5p axis plays essential roles in modulating the GC development and MDR, and the GC targeted drug delivery formulation presents a strategic reference for the future designation of chemotherapeutics study.