Incorporation of human β-defensin-1 into immunoliposomes to facilitate targeted autophagy therapy of colon carcinoma

Abstract

Based on the discovery that human β-defensin-1 (hBD-1) triggers autophagy in colon cancer cells and inhibits proliferation, we proposed the consideration of its druggability. As a protein, its stability, targetability and bioavailability must be improved. Compared with the traditional medicinal chemistry technology, nanotechnology is more economical for increasing the druggability of hBD-1 and can be readily scaled up. Here, we propose an immunoliposome system containing hBD-1 to improve its stability and bioavailability. To enhance its targetability, anti-epidermal growth factor receptor (EGFR) antibodies were conjugated to the liposomal bilayer to produce immunoliposomes that can target EGFR, which is highly expressed in colon cancer cells. Although more studies are needed to support clinical trials and large-scale manufacturing, these immunoliposomes have great potential as therapeutics. Thus, immunoliposomes are suitable nanovesicles to improve the druggability of hBD-1; however, additional basic and translational research of these systems is warranted.

Key Words: Human β-defensin-1; Immunoliposomes; Colon cancer; SW620; Autophagy

Core Tip: Nanotechnology is a cost-effective strategy for addressing the druggable properties of proteins. We propose an immunoliposome system that improves the stability, bioavailability and targeting properties of human β-defensin-1 (hBD-1), which will support its clinical translation. Our strategy is highly effective and may lead to positive clinical trials and large-scale production. We consider immunoliposomes to be suitable nanovesicles for enhancing the drug properties of hBD-1 and warrant further basic and translational research.

INTRODUCTION

In the World Journal of Gastrointestinal Oncology, Zhao et al[1] published an important work titled, ‘Human β-defensin-1 affects the mammalian target of rapamycin pathway and autophagy in colon cancer cells through long non-coding RNA TCONS_00014506’. Cancer is one of the most common causes of death worldwide[2], with colon cancer as the third most common type[3]. New anticancer drugs are urgently needed to prevent the proliferation of colon cancer cells. Several drugs have been developed to treat colon cancer, including sildenafil, exisulind, tadalafil, vardenafil and panafil as phosphodiesterase type 5 inhibitors that target colorectal cancer cells[4], doxorubicin and etoposide, which are topoisomerase inhibitors that damage DNA to cause cell death[5], and benzenesulfonamide derivatives as cationic inhibitors affecting the acid–base balance in living cells to prevent the proliferation of tumour cells[6]. That work showed that human β-defensin-1 (hBD-1) triggers autophagy in the colon cancer cell line SW620 and inhibited its proliferation. Autophagy was assessed by flow cytometry in conjunction with acridine orange labelling to detect the induction of cellular acidity in SW620 colon cancer cells. The hBD-1 exhibited autophagy induction in this cell line[7]. The effect may result from the upregulation of the long non-coding RNA (lncRNA) TCONS_00014506, which consequently inhibits the mammalian target of rapamycin (mTOR). The mTOR inhibition may occur because lncRNAs affect their regulatory function by interacting with epigenetic enzymes[8]. The authors used bioinformatics to identify the underlying mechanisms of the hBD-1 action. The major findings of this study are summarised in Table 1[1].

Table 1 Summary of the study of Zhao et al[1].

Section	Summary
Background	Colorectal cancer has a low 5-year survival rate and a high mortality rate. The hBD-1 may function in the innate immune system, which recognises and destroys cancer cells. LncRNAs are involved in cell differentiation and growth
Methods	Cell proliferation was detected using the cell counting kit 8 method to determine the optimal drug concentration. The effect of hBD-1 on the SW620 cell proliferation was evaluated by a colony formation assay. Bioinformatics was used to screen LncRNAs for potential biological significance associated with the mTOR pathway. In addition, the expression of p-mTOR, beclin1 and LC3I/II in the SW620 cells was detected by western blot analysis
Results	The hBD-1 inhibited the SW620 cell proliferation, as shown by the reduced colony-forming ability of SW620 cells following the hBD-1 exposure. The hBD-1 decreased the expression of the p-mTOR (Ser2448) protein and increased the expression of the Beclin1 and LC3II/I proteins. In addition, a bioinformatics analysis identified seven lncRNAs associated with the mTOR pathway (two up-regulated and five down-regulated). Finally, lncRNA TCONS_00014506 was selected. Following the lncRNA TCONS_00014506 inhibition, exposure to hBD-1 inhibited p-mTOR (Ser2448) and promoted beclin1 and LC3II/I protein expression
Conclusion	In the SW620 colon cancer cells, hBD-1 increased the lncRNA TCONS_00014506 expression to suppress the mTOR pathway and promote autophagy

HBD-1: Human β-defensin-1; LC3: Light chain 3; LncRNA: Long non-coding RNA; MTOR: Mammalian target of rapamycin.

HBD-1

The study of Zhao et al[1] preliminarily revealed the anticancer potential of hBD-1. We should bear in mind that there is still an ‘enduring war’ to fight, and the clinical translation of hBD-1 as an antitumor remedy is warranted. The druggability of hBD-1 must be considered. The druggability properties include water solubility, stability, targeting and bioavailability[9]. These attributes (water solubility, stability, targetability and bioavailability) must be satisfied to produce a qualified hBD-1 pharmaceutical formulation. Fortunately, the water solubility of hBD-1 is acceptable[10]; however, as a protein, it is likely vulnerable to hydrolysis and oxidation and the transmembrane absorption behaviour is likely poor[11]. Importantly, hBD-1 may not actively target colon cancer cells in vivo because a large proportion accumulates in various epithelial regions[12-14]. Therefore, the stability, targetability and bioavailability properties must be optimised to improve its druggability.

One strategy for enhancing druggability is to perform structural modification through medicinal chemistry to enhance stability, targetability and bioavailability[15]. Nevertheless, this strategy is considered time-consuming and expensive. Moreover, clinical translation is often challenging, particularly for protein-based drugs[16]. In comparison, nanotechnology is a cost-effective strategy that can address the druggability issues of proteins. Specifically, with the application of easy-to-produce nanocarriers, the stability, targetability and bioavailability may be increased without sophisticated chemical reactions for structural modification[17]. Many commercialised anticancer products based on nanotechnology are available in the global market, such as Doxil^®[18], Abraxane^®[19] and Onivyde^®[20], indicating the potential clinical translation. Thus, we proposed the design of a nanocarrier-based formulation for hBD-1.

An immunoliposome system was proposed to achieve high stability, targetability and bioavailability. The design details are as follows: (1) To improve stability and bioavailability, hBD-1 should be accommodated in a liposomal system. The liposomal system serves as a confinement framework for hBD-1 to mitigate contact with water and oxygen, thereby reducing hydrolysis and oxidative stress[21]. This mechanism stabilises hBD-1. Furthermore, this system comprises a biocompatible, lecithin-based lipid bilayer, which is favourable for fusion with cell membranes[22], facilitates transmembrane absorption and increases the hBD-1 bioavailability. It is worth noting that hBD-1 is a cationic protein (also mentioned by Zhao et al[1]), and liposomes are negatively charged. Thus, the interaction between hBD-1 and the liposomal bilayer is predicted to be strong, which is beneficial for drug loading; (2) To improve targetability, immunoliposomes can exploit the principle of high-selectivity antigen–antibody recognition to achieve satisfactory targeting outcomes[23]. The epidermal growth factor receptor (EGFR) is an important tyrosine kinase transmembrane receptor that mediates multiple signalling cascades (Ras/mitogen activated protein kinase, phosphatidylinositol-3-kinase/protein kinase B and Jak/STAT) that regulate cell proliferation, growth and apoptosis. In contrast, the overexpression and/or mutation of the EGFR in tumour cells result in dysregulation of EGFR signalling and an increase in angiogenesis and proliferation[24,25]. The EGFR is frequently up-regulated in colon cancer cells, including cell line SW620[26]. The receptor tyrosine kinase or human epidermal growth factor (HER) receptor family (also known as ErbB or EGFR family) consists of four structurally related receptors: (1) EGFR (HER1/ErbB1); (2) EGFR 2 (ErbB2); (3) EGFR 3 (ErbB3); and (4) EGFR 4 (ErbB4)[27]. Their structure shows three domains responsible for their activity and subsequent phosphorylation of key tyrosine residues, including an extracellular ligand-binding domain, a transmembrane domain and an intracellular tyrosine kinase domain[28-32]. Anti-EGFR antibodies can selectively bind to EGFR. The EGFR antibody may be conjugated to the liposomal bilayer to produce immunoliposomes, which selectively target colon cancer cells following administration. Based on this approach, an irinotecan-loaded immunoliposome was developed and showed an excellent targeted delivery profile[33].

The abovementioned design is illustrated in Figure 1. The proposed immunoliposomes are anticipated to enhance the stability, targetability and bioavailability of hBD-1 to facilitate clinical translation. For clinical application, intravenous injection may be a preferred route of administration for the system because the anti-EGFR structure may be destroyed by gastric potential of hydrogen or digestive enzymes associated with oral administration[34].

Figure 1 Scheme for the human β-defensin-1-loaded immunoliposome design. EGFR: Epidermal growth factor receptor; HBD-1: Human β-defensin-1.

Clinical trials and large-scale manufacture of the proposed immunoliposomes require further study. On the one hand, nanotoxicology in humans must be considered during clinical trials[35]. On the other hand, strong standardised methodologies must be developed and implemented to support research practices involving the engineering and manipulation of nanomaterials[36]. Safety and reproducibility will be necessary for hBD-1 immunoliposomes to be commercially successful. This strategy is expected to achieve positive outcomes in clinical trials and large-scale manufacture because a scientific foundation has been established.

CONCLUSION

Overall, immunoliposomes are suitable nanovesicles for improving the druggability of hBD-1; however, further basic and translational studies on this system are warranted. It is suggested that journals, such as the World Journal of Gastrointestinal Oncology, should increase awareness of this topic.