Elsevier

Fitoterapia

Volume 112, July 2016, Pages 183-190
Fitoterapia

Enhanced anti-tumor activity of trichosanthin after combination with a human-derived cell-penetrating peptide, and a possible mechanism of activity

https://doi.org/10.1016/j.fitote.2016.03.019Get rights and content

Abstract

Trichosanthin (TCS), a type I ribosome-inactivating protein (RIP-I) and renowned Chinese traditional medicine, displays a broad spectrum of biological and pharmacological properties. Particularly, its anti-tumor activity has received a great deal of attention. However, the cellular mechanism for TCS uptake varies with different tumor cell lines, leading to discrepancies in its reported ability to penetrate cells. In this study, HBD, a human derived cell-penetrating peptide (CPP), was used to improve the delivery of TCS into several types of tumor cells, including HeLa cells. Recombinant TCS (rTCS) with or without the fused HBD peptide was expressed in Escherichia coli cells and successfully purified by Ni-NTA affinity chromatography. The cellular uptake efficiency of FITC-labelled-rTCS-HBD was observed in HeLa cells and compared with the uptake efficiency of non-HBD conjugated rTCS under the same conditions using laser confocal microscopy. Moreover, the IC50 value of rTCS-HBD in the tested tumor cells was much lower than that of rTCS, indicating that HBD could efficiently deliver the rTCS into tumor cells. When compared with rTCS, rTCS-HBD induced higher rates of apoptosis in HeLa cells as analyzed by flow cytometry. Furthermore, the apoptotic events observed in HeLa cells incubated with HBD-fused rTCS included activation of Caspase-9, decrease in the Bcl-2/Bax ratio, and cleavage of PARP. These results strongly suggest the participation of mitochondria in apoptosis. This report illustrates one possible method for achieving the efficient transport of TCS into cells using a CPP as a vector, and increases the likelihood that TCS can be used in the clinic.

Introduction

Cancer is one of the deadliest diseases in humans, and its incidence and mortality rate continue to rise. As a result, it is critical that anti-tumor agents demonstrate greater efficacy in treating this disease. Trichosanthin (TCS) is a protein extracted from the root tubers of T. kirilowii Maxim, and a traditional Chinese medicine that has been used as an abortifacient for centuries in China [1], [2]. TCS belongs to a family of structurally and functionally related toxin proteins, collectively referred to as ribosome-inactivating proteins (RIPs), which can be classified into three types [3], [4]. TCS is a type I ribosome-inactivating protein (RIP-I) with a molecular weight of 27 kDa, and consists of a single chain containing 289 amino acids. Full-length TCS is processed to its mature form containing 247 amino acids, without putative secretory signal peptide and carboxyl-terminal sequence [5]. TCS inactivates ribosomes by removing a single adenine residue from position 4324 in the 28S rRNA subunit of eukaryotic ribosomes [6].

Numerous patients die from the effects chemotherapeutic drugs in addition to the cancer itself. This is mainly attributed to the high doses of drugs administered, and the fact that chemotherapeutic agents usually kill normal cells in addition to cancer cells, and suppress the immune system. A previous study showed that TCS is transported across the cell membrane by low density lipoprotein receptor-related protein 1 (LRP 1) [7], and this results in its differential permeability in different cell lines [7], [8], [9], [10], [11], [12], [13]. Cells with a high concentration of LRP1 on their outer membrane (e.g., trophoblasts and choriocarcinoma cells) are more sensitive to the effects of TCS [7]. However, cells lacking LRP-1 show poor internalization of TCS, which limits its potency. Therefore, efficient internalization of TCS into tumor cells is vital for achieving the desired therapeutic effect. Cell-penetrating peptides (CPPs) have been utilized for several years to carry macromolecules into cells. As recombinant RIPs and CPPs have been proven effective as transport agents [14], [15], CPPs might be used to improve the transport and therapeutic efficacy of TCS.

The c-terminus of human extracellular superoxide dismutase (hC-SOD3, hereafter referred to as HBD), is a human-derived CPP with 27 amino acids (GPGLWERQAREHSERKKRRR ESECKAA), and was previously described by our group [16]. That study demonstrated that HBD was capable of transporting molecules such as apoptin into living cells both in vitro and in vivo without producing a toxic effect. Additionally, HBD has demonstrated its ability to enhance the anti-tumor effects of MAP30, another type I ribosome-inactivating protein [14]. Based on such findings, we predicted that integrating the c-terminus of TCS into HBD might facilitate the transport of TCS across cell membranes and enhance its toxicity to tumor cells.

In this study, we fused HBD with the C-terminus of recombinant TCS (rTCS) to improve the translocation efficiency of TCS. The recombinant fusion proteins with and without HBD were expressed and purified. The biological activities of both rTCS and HBD-conjugated rTCS were monitored using a N-glycosidase activity assay performed with a TNT® T7/SP6 Coupled Reticulocyte Lysate System. The cell penetration ability of HBD-conjugated rTCS was investigated in HeLa cells by confocal laser scanning microscopy, and its cytotoxicity was measured using the MTT assay. Apoptotic events in HeLa cells were detected by flow cytometry, and western blot studies were conducted to explore possible mechanisms for any improved anti-tumor activity.

Section snippets

Materials

Plasmids pET-28a, pET-28a-HBD, pET-28b, and Escherichia coli strains DH5α and BL21 (DE3) were maintained in our laboratory. FITC was obtained from Sigma (St. Louis, MO, USA). RPMI 1640 growth medium and FBS were purchased from HyClone (Logan, UT, USA). BCA protein assay kits were supplied by Shanghai Sangon Biotech (Shanghai, China). HeLa, A549, 95D, and SMMC-7721 cells were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). RNasin® was

Construction of expression vectors

To obtain the rTCS gene, genomic DNA was extracted from the seedling leaves of T. kirilowii Maxim by using improved cetrimonium bromide (CTAB) methods (Fig. 1A). Next, adequately purified DNA and the appropriate primers were used to clone rTCS using PCR methodology because of the absence of introns. The PCR-derived amplicons had a length of 741 bp, and the control amplicon was created without a template (Fig. 1B). rTCS containing Nde I and BamH I cleavage sites and with or without the HBD gene

Discussion

Numerous strategies have been developed for the pharmaceutical application of rTCS and other RIPs, such as constructing novel compounds by combining RIPs with monoclonal antibodies [20], [21], [22], growth factors [23], [24], gold nanoparticles [25] or cell-penetrating peptides [15]. These strategies may enhance the efficacy of RIPs both in vitro and in vivo, and thus broaden their application in medical treatment.

In this study, we designed a novel fusion protein with anti-tumor activity and

Conflict of interest statement

The authors declare having no conflicts of interest regarding the conduct or outcomes of this study.

Acknowledgment

This work was sponsored by the National Natural Science Foundation of China (grant no. 81571795).

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