Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity

Lu, Qiang; Yang, Xu-Zhong; Fu, Long-Yun; Lu, Yv-Ting; Lü, Yanhua; Zhao, Jian; Wang, Fujun

doi:10.1016/j.pep.2015.03.008

Cited by 23 publications

(35 citation statements)

References 38 publications

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“…The cellular uptake of our FITC‐labeled recombinant proteins demonstrates that HBP, acting as a novel effective human‐derived CPP, promoted the transport of more MAP30 molecules into HeLa cells than free MAP30, which was scarcely observed inside the cells (Figure C and D). This result is consistent with the aforementioned findings (Figure ) and our previous work . Consequently, MAP30‐HBP was more cytotoxic than free MAP30 in various tumor cell lines (Figure ), and their average difference is approximately 100‐fold, according to the reported IC 50 values (Table ).…”

Section: Discussionsupporting

confidence: 92%

The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery

Luo

Cao

et al. 2016

J. Pept. Sci.

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Cell-penetrating peptides (CPPs) have been shown to be potential drug carriers for cancer therapy. The inherently low immunogenicity and cytotoxicity of human-derived CPPs make them more suitable for intracellular drug delivery compared to other delivery vehicles. In this work, the protein transduction ability of a novel CPP (termed HBP) derived from the heparin-binding domain of HB-EGF was evaluated. Our data shows, for the first time, that HBP possesses similar properties to typical CPPs and is a potent drug delivery vector for improving the antitumor activity of impermeable MAP30. The intrinsic bioactivities of recombinant MAP30-HBP were well preserved compared to those of free MAP30. Furthermore, HBP conjugated to the C-terminus of MAP30 promoted the cellular uptake of recombinant MAP30-HBP. Moreover, the fusion of HBP to MAP30 gave rise to significantly enhanced cytotoxic effects in all of the tumor cell lines tested. In HeLa cells, this cytotoxicity was mainly caused by the induction of cell apoptosis. Further investigation revealed that HBP enhanced MAP30-induced apoptosis through the activation of the mitochondrial- and death receptor-mediated signaling pathways. In addition, the MAP30-HBP fusion protein caused more HeLa cells to become arrested in S phase compared to MAP30 alone. These results highlight the MAP30-HBP fusion protein as a promising drug candidate for cancer therapy and demonstrate HBP, a novel CPP derived from human HB-EGF, as a new potential vector for antitumor drug delivery. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

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Section: Discussionsupporting

confidence: 92%

The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery

Luo

Cao

et al. 2016

J. Pept. Sci.

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“…It has been reported that MAP 30 has antitumor properties through the induction of apoptosis . To explore whether CAT can enhance the antitumor effect of MAP 30 by promoting its apoptotic activity, apoptosis induced by MAP 30‐CAT recombinant protein was analyzed with an Annexin V‐FITC apoptosis detection kit and FACS analysis.…”

Section: Resultsmentioning

confidence: 99%

Screening and characterization of a novel high‐efficiency tumor‐homing cell‐penetrating peptide from the buffalo cathelicidin family

Cao

Fu³

et al. 2019

Journal of Peptide Science

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Targeted delivery of antitumor drugs is especially important for tumor therapy. Cell‐penetrating peptides (CPPs) have been shown to be very effective drug carriers for tumor therapy. However, most CPPs lack tumor cell specificity. Here, we identified a highly efficient CPP, CAT, from the newly identified buffalo‐derived cathelicidin family, which exhibits a preferential binding capacity for multiple tumor cell lines and delivers carried drug molecules into cells. CAT showed an approximately threefold to sixfold higher translocation efficiency than some reported cell‐penetrating antimicrobial peptides, including the well‐known classical CPP TAT. Moreover, the delivery efficiency of CAT was greater in a variety of tested tumor cells than in normal cells, especially for the human hepatoma cell line SMMC‐7721, for which delivery was 7 times more efficient than the normal human embryonic lung cell line MRC‐5, according to fluorescent labeling experiment results. CAT was conjugated to the Momordica charantia‐derived type‐I ribosome‐inactivating protein MAP 30, and the cytotoxicity of the MAP 30‐CAT fusion protein in the tumor cell line SMMC‐7721 was significantly enhanced compared with that of the unconjugated MAP 30. The IC50 value of MAP 30‐CAT was approximately 83 times lower than the IC50 value of the original MAP 30. Interestingly, the IC50 value of MAP 30 alone for MRC‐5 was approximately twofold higher than the value for SMMC‐7721, showing a small difference. However, when MAP 30 was conjugated to CAT, the difference in IC50 values between the two cell lines was significantly increased by 38‐fold. The results of the flow cytometric detection of apoptosis revealed that the increase in cytotoxicity after CAT conjugation was mainly caused by the increased induction of apoptosis by the fusion protein. These results suggest that CAT, as a novel tumor‐homing CPP, has great potential in drug delivery applications in vivo and will be beneficial to the development of tumor therapeutics.

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“…Therefore, development of novel and effective therapeutic agents is highly desirable. Although previous studies have identified that MAP30 possesses antitumor effects in various tumor cells, including glioblastoma cells, the underlying molecular mechanism of MAP30 on glioblastoma cells has not yet been elucidated (4,6,(12)(13)(14)16,21,22). The results of the present study demonstrated that MAP30 displayed significant inhibition of proliferation, invasion and migration on U87 and U251 cell lines in a dose-and time-dependent manner.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, previous studies fused MAP30 with human-derived cell penetrating peptide or poly (ethylene glycol) to increase the uptake efficiency, cytotoxic activity and in vivo half-life and decrease immunogenicity of MAP30 (12,44). Even more importantly, RIPs may exhibit little or no detectable adverse effects on normal cells as they may recognize features on membranes dominant to tumor cells (4,17,(45)(46)(47).…”

Section: Ser9mentioning

confidence: 99%

“…Previous studies indicated that MAP30 exhibits a variety of anti-infection, anti-diabetic, antiviral and antitumor bioactive effects (4)(5)(6)(7)(8)(9)(10). The antitumor ability of MAP30 has been the subject of previous studies (11)(12)(13)(14)(15). However, certain researchers have already noted the effects of MAP30 on the inhibition of U87 cells in screening for anticancer effects (3,7,16,17).…”

Section: Introductionmentioning

confidence: 99%

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MAP30 promotes apoptosis of U251 and U87 cells by suppressing the LGR5 and Wnt/β-catenin signaling pathway, and enhancing Smac expression

et al. 2018

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Abstract. Significant antitumor activity of Momordica anti-human immunodeficiency virus protein of 30 kDa (MAP30) purified from Momordica charantia has been the subject of previous research. However, the effective mechanism of MAP30 on malignant glioma cells has not yet been clarified. The aim of the present study was to investigate the effects and mechanism of MAP30 on U87 and U251 cell lines. A Cell Counting Kit-8 assay, wound healing assay and Transwell assay were used to detect the effects on U87 and U251 cells treated with different concentrations of MAP30 (0.5, 1, 2, 4, 8 and 16 µM) over different periods of time. Proliferation, migration and invasion of each cell line were markedly inhibited by MAP30 in a dose-and time-dependent manner. Flow cytometry and fluorescence staining demonstrated that apoptosis increased and the cell cycle was arrested in S-phase in the two investigated cell lines following MAP30 treatment. Western blot analysis demonstrated that leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5) expression and key proteins in the Wnt/β-catenin signaling pathway were apparently decreased, whereas second mitochondria-derived activator of caspase (Smac) protein expression significantly increased with MAP30 treatment in the same manner. These results suggest that MAP30 markedly induces apoptosis in U87 and U251 cell lines by suppressing LGR5 and the Wnt/β-catenin signaling pathway, and enhancing Smac expression in a dose-and time-dependent manner.

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Recombinant expression and purification of a MAP30-cell penetrating peptide fusion protein with higher anti-tumor bioactivity

Cited by 23 publications

References 38 publications

The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery

The heparin-binding domain of HB-EGF as an efficient cell-penetrating peptide for drug delivery

Screening and characterization of a novel high‐efficiency tumor‐homing cell‐penetrating peptide from the buffalo cathelicidin family

MAP30 promotes apoptosis of U251 and U87 cells by suppressing the LGR5 and Wnt/β-catenin signaling pathway, and enhancing Smac expression

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