Efficient delivery of anti-miR-210 using Tachyplesin, a cell penetrating peptide, for glioblastoma treatment

Abstract

The levels of microRNAs (miRNAs) are altered in various diseases including glioblastoma (GBM) and this alteration may have widespread effects on various hallmarks of cancer cells. MiR210 is overexpressed in GBM and functions as an oncogenic miRNA. Anti-miR210 therapy holds great promise but its efficient delivery remains a major challenge. Our work here explores a novel role of Tachyplesin (Tpl), a cell-penetrating antimicrobial peptide, as a nanocarrier for anti-miR210. Tpl electrostatically interacts with anti-miR210 at 1:25 and 1:50 (anti-miR:Tpl) weight ratios to form a complex and efficiently delivers anti-miR210 inside GBM cells cultured as 2D and 3D spheroid model. Treatment of GBM cells with the complex significantly inhibited miR210 levels (~90%), proliferation, migration and spheroid formation ability and induced apoptosis as evident by increased levels of caspase 3/7 and ROS. GBM cells pre-treated with anti-miR210:Tpl complex were also found to be sensitive to TMZ mediated action. Uptake of the complex in GBM cells induced the levels of miR210 targeted tumor suppressor genes, NeuroD2 and HIF3A. Overall, our work reveals a novel and efficient miRNA delivery ability of Tpl in glioma cells, holding a great promise for treatment of GBM and potentially for other cancers.

Introduction

Glioblastoma (GBM) is the most malignant grade IV tumor of the central nervous system (Parsons et al., 2008). It is a fatal brain tumor with an annual incidence of 5 in 100,000 people (Omuro and DeAngelis, 2013). The current treatment modality involves surgical resection along with radiation therapy and rounds of temozolomide (TMZ)-chemotherapy, however the outcome remains extremely poor, with the median survival period of around 15 months (Combs et al., 2011). A very aggressive growth rate, diffused boundaries, invasive behaviour and development of drug resistance make it even more resistant to the available therapies (Zhu et al., 2009).

Recent studies suggest microRNA (miRNA) based therapies as a promising option for GBM treatment. miRNAs are small non-coding RNAs that interact with a wide range of target transcripts most commonly at the 3′ UTR region and bring about their degradation or translational repression (Wu et al., 2019, Nikaki et al., 2012). Aberrant levels of miRNAs have been reported in various pathological conditions including cancer. Specific miRNAs have been shown to play an oncogenic or tumor suppressive role in various cancers (Reddy, 2015). It is widely agreed that downregulation of oncogenic miRNAs or upregulation of tumor suppressor miRNAs has immense potential for cancer treatment. miRNAs may provide more comprehensive benefits than other targets with limited activities since one miRNA can control the expression of wide range of targets and thus oncogenic pathways (Lawler and Chiocca, 2009). While the potential of miRNAs as therapeutic targets is indeed exciting, the delivery of miRNAs remain major obstacle due to their charge and biological instability (Mondal et al., 2019, Chen et al., 2015, Kwekkeboom et al., 2014). Therefore, search for a safe, highly effective delivery system for miRNA-based therapy continues.

Antimicrobial peptides (AMPs) have a broad spectrum action against gram positive and gram negative bacteria, viruses, fungi and parasites (Lakshmaiah Narayana and Chen, 2015). Recently a number of reports have shown that cationic antimicrobial peptides exhibit potent cytotoxicity towards cancer cells without harming normal cells (Smolarczyk et al., 2009, Liu et al., 2015). This selectivity of peptides towards cancer cells is mainly due to the presence of negatively charged cancer cell membrane leading to electrostatic interaction between cationic antimicrobial peptide and cancer cells (Smolarczyk et al., 2009, Gaspar et al., 2013). AMPs mainly act by membranolytic mechanism leading to membrane destabilization and cell lysis (Fadnes et al., 2009). Therefore, it is possible that the cell penetrating antimicrobial peptides with an efficient cellular uptake by cancer cells may result in efficient and selective delivery of anti-cancer therapeutics in different cancer types. In our previous reports we have successfully shown that marine AMP, Tpl, derived from horseshoe crab can act as a cell penetrating peptide (CPP) with efficient cargo delivery ability (Jain et al., 2015). Thus, employing CPPs for miRNA delivery in cancer cells seems to be a promising new approach. The cationic nature of the CPPs allows them to electrostatically bind to the phosphate backbone of nucleic acids to form a stable complex and protect the nucleic acid from degradation (Suh et al., 2013).

CPPs have the ability to cross the cell membrane and can increase transport efficiency of different cargo molecules across the blood brain barrier (BBB). Though quantitative studies are still lacking. The ability of CPPs to pass through BBB is dependent on various aspects like multiple time regression (MTR), unidirectional influx rate, brain to serum activity etc. MTR analysis of different peptides disclose divergent influx through BBB, though influx properties cannot be correlated with their cell penetrating ability. That may be because of differences in cellular influx mechanism and secondary structures (Stalmans et al., 2015).

There are many reports claiming the blood brain permeability of Tpl. The work has demonstrated that Tpl, Protegrin or peptides derived from them act as excellent delivery vehicles for transfer of various cargoes across BBB (Mouchet et al., 2003).

Our study mainly focuses on developing a peptide based nanocarrier system for the delivery of anti-miRNAs for inhibiting oncogenic miRNAs. We specially aim to target an established oncogenic miRNA- miR210 which is known to be highly expressed in various cancers such as breast cancer, prostate cancer and GBM (Giannakakis et al., 2008, Grosso et al., 2013, Gee et al., 2010). We have recently shown high levels of miR210 in GBM patients as compared to normal brain cells in both TCGA-GBM patient dataset and Indian GBM patients (Agrawal et al., 2014, Agrawal et al., 2018, Lai et al., 2015). miR210 levels increase with tumor grade and has been correlated with poor prognosis of GBM patients. miR210 plays an oncogenic role in GBM by promoting cell proliferation, migration, invasion, and inhibition of apoptosis (Agrawal et al., 2014, Agrawal et al., 2018).

Here, we have screened a number of CPPs which includes, Tat2 which is TAT dimer (Jain et al., 2015) and random coil in nature, Tpl, derived from marine origin which is cationic and β hairpin peptide (Nakamura et al., 1988) and CyLoP-1 (Ponnappan et al., 2017) snake toxin derived and is random coil in nature, LN and LDP which are α-helical in nature and are spider toxin derived (Ponnappan and Chugh, 2017), for their membrane translocation abilities in GBM cells. Our work demonstrates that Tpl, a known antimicrobial peptide, shows excellent cell penetrating ability in GBM cells and is also known to effectively cross BBB (Mouchet et al., 2003). Further, we show that Tpl forms a complex with anti-miRNA and delivers it efficiently into GBM cells with higher transfection efficiency compared to lipofectamine reagent. Further, anti-miR210:Tpl complex brings about a significant reduction in miR210 levels with a concomitant increase in the levels of its target genes. We also show that treatment of GBM cells with anti-miR210:Tpl complex brings about significant inhibition of cell proliferation, spheroid formation, migration and induces apoptosis in GBM cells. Further, anti-miR210:Tpl complex also sensitizes GBM cells to chemodrug, TMZ, treatment. Our findings demonstrate that efficient delivery of anti-miR210 molecule with CPP may hold great promise for treatment of GBM and other solid tumours with high miR210 levels.