Cancer Letters

Cancer Letters

Volume 293, Issue 2, 28 July 2010, Pages 240-253
Cancer Letters

Lytic peptide-mediated sensitization of TRAIL-resistant prostate cancer cells to death receptor agonists

https://doi.org/10.1016/j.canlet.2010.01.012Get rights and content

Abstract

Tumor Necrosis Factor-α Related Apoptosis Inducing Ligand (TRAIL) and agonistic antibodies to death receptors (DR) 4 and 5 have attracted significant attention in recent years due to their ability to selectively induce apoptosis in malignant cells while demonstrating little cytotoxicity in normal cells. Although these candidates are promising in cancer therapy, a number of tumor cells are resistant to TRAIL-mediated apoptosis. We describe the use of a cationic amphipathic lytic peptide, KLA (single letter sequence HHHHHKLAKLAKKLAKLAKC), for the chemosensitization of TRAIL-resistant LNCaP and PC3-PSMA human prostate cancer cells to DR agonistic antibodies. ‘Single-agent’ treatment with DR agonistic antibodies did not result in loss of viability of these cells confirming the resistance of these cells. However, the combination treatment of KLA followed by DR agonists resulted in greater cell death compared to the individual treatments acting alone, indicating synergistic action between the two components of the combination treatment. The combination of lytic peptide and DR agonists resulted in a significant increase in activated caspase-3 cleavage and cytochrome-C protein levels in cells, indicating a role for the caspase-mediated apoptotic pathway. In addition, KLA treatment also resulted in increased localization of DR5 and lipid rafts in LNCaP cells. Our results demonstrate, for the first time, that lytic peptides can be employed for sensitizing TRAIL-resistant prostate cancer cells to DR-mediated apoptosis resulting in novel combination treatments for the ablation of advanced cancer cells.

Introduction

Prostate cancer is the most commonly diagnosed malignancy and is the second-leading cause of cancer death in men; approximately 30,000 deaths occur every year in the United States due to the disease [1]. Localized prostate cancer is generally treated with surgery (radical prostatectomy), radiation therapy, or cryotherapy [2]. However, disease relapse after surgery is a common occurrence, mainly due to the outgrowth of minimal residual disease [2]. Currently, patients with recurrent, locally advanced, or metastatic prostate cancer are treated by androgen deprivation alone or in combination with local therapy. Although most patients initially respond to androgen deprivation, a large fraction of patients suffer disease progression and develop androgen-independent, hormone-refractory prostate cancer [1]. Novel therapeutic candidates and strategies with increased efficacy and/or reduced side effects are therefore an urgent medical need in prostate cancer therapy.

Tumor Necrosis Factor-α Related Apoptosis Inducing Ligand (TRAIL) is a member of the Tumor Necrosis Factor (TNF) super-family of cytokines that engages the cellular apoptotic machinery upon specific binding to death receptors (DR) 4 and 5 [3]. TRAIL has attracted significant attention in recent years due to its ability to selectively induce apoptosis in transformed (malignant) cells while demonstrating little cytotoxicity in normal cells [4], [5], [6], [7], [8], [9]. Although TRAIL and agonistic antibodies to death receptors 4 and 5 are promising candidates for cancer therapy, many tumor cells are resistant to TRAIL-mediated apoptosis. Dysfunction of the Fas-Associated Death Domain (FADD)/improper assembly of the Death-Inducing Signaling Complex (DISC), loss of caspase-8 activity [10], [11], [12], constitutively active Akt/protein kinase B [13], and over-expression of anti-apoptotic proteins such as c-FLIP [14], [15] and Bcl-2 [16] are among the main mechanisms responsible for cancer cell resistance to DR-mediated apoptosis. As a result, therapeutic strategies that enhance cancer cell sensitivity to DR-mediated apoptosis have been explored. For example, DNA-damaging radiotherapy [17], [18] and genotoxins [19], [20] activate p53-mediated death receptor transcription leading to up-regulation of death receptors on the surface of tumor cells. Subsequent treatment with TRAIL [19] or agonistic antibodies to DR4/DR5 [21] results in increased apoptosis of tumor cells indicating synergy between the two components of the combination treatment (i.e. genotoxins and death receptor agonists). Pathways independent of p53 have also been implicated in sensitizing tumor cells to DR-mediated apoptosis [22], [23].

Cationic amphipathic lytic peptides are ubiquitous in nature and form a part of the host-defense mechanism in invertebrates and vertebrates, including humans. Anti-microbial peptides belong to this class of host-defense peptides and play a role in host-defense by lysing pathogen membranes [24], [25]. The model amphipathic peptide KLA (single letter amino acid sequence: KLAKLAKKLAKLAK) was developed using de novo design and has been demonstrated to possess potent anti-microbial activity [26]. In addition, the peptide, when conjugated to cell-surface receptor-targeting peptides or antibodies, induced apoptosis in cancer cells [27], [28], [29], [30] and fat tissue [31], presumably due to its ability to lyse mitochondrial membranes and induce subsequent mitochondrial damage.

In the present work, we describe cationic amphipathic lytic peptide induced sensitization of LNCaP human prostate cancer cells to death receptor (DR) mediated apoptosis. Treatment with agonistic antibodies to DR4 and DR5 resulted in negligible loss of viability in these cells indicating that the cells were resistant to death receptor agonists. However, pre-incubation of LNCaP and PC3-PSMA cells with the lytic peptide followed by subsequent treatment with DR agonists resulted in greater cell death compared to the individual treatments acting alone, indicating synergistic action between the two components of the combination treatment. Mechanistic investigation indicated that the anti-apoptotic protein c-FLIP was down regulated in response to KLA treatment. Increased levels of cytochrome-C and cleaved caspase-8 and caspase-3, indicated the involvement of the caspase-mediated apoptosis pathway. KLA treatment also moderately increased the localization of DR5 in lipid raft regions in LNCaP cells. Our results indicate that lytic peptide-based stimulation of death receptor-mediated apoptosis might have implications for designing new combination treatments for the ablation of cancer cells.

Section snippets

Cell culture

The LNCaP human prostate cancer cell line was purchased from ATCC. The PC3-PSMA human prostate cancer cell line [32] was a generous gift from Dr. Michel Sadelain of the Memorial Sloan Cancer Center, New York, NY. Cells were grown in RPMI-1640 (ATCC) + 10% FBS (Invitrogen Corp.) + 1% penicillin/streptomycin (Invitrogen Corp.) at 37 °C in a 5% CO2 incubator. All chemicals were purchased from Sigma (St. Louis, MO) unless otherwise specified. The cationic amphipathic lytic peptide, KLA, (single letter

Results

We first investigated the susceptibility of LNCaP and PC3-PSMA cells to the apoptotic activity of DR agonists. Fig. 1a shows that the LNCaP cell viability remained invariant when cells were treated with different concentrations (0–1000 ng/mL) of DR5-Ab; similar results were observed with DR4-Ab as seen from the Live/Dead® staining results (Fig. 1b). PC3-PSMA cells were also largely resistant to the agonistic antibodies (Fig. 1c). Taken together, these results indicate that the LNCaP and PC3-PSMA

Discussion

Various chemotherapeutic strategies have been investigated for the ablation of advanced prostate cancer cells. Of these, targeted biological therapies represent an emerging class of therapeutics that can potentially reduce non-specific cytotoxicity in collateral organs such as the urethra and bladder, especially in cases of residual disease and secondary metastasis [6], [8], [41], [42]. Tumor Necrosis Factor-α Related Apoptosis Inducing Ligand (TRAIL) and agonistic antibodies to death receptor

Conclusions

In the present investigation, we employed the cationic amphipathic lytic peptide KLA in order to sensitize TRAIL-resistant LNCaP and PC3-PSMA prostate cancer cells to the apoptotic action of DR agonistic antibodies. The combination treatment (KLA followed by death receptor agonists) induced synergistic cell death when compared to the efficacies of the individual treatments (i.e. DR agonists and KLA) acting alone. Mechanistic investigation indicated that the lytic peptide did not induce changes

Acknowledgements

We thank Gertrude Gunset and Dr. Michel Sadelain at the Memorial Sloan Cancer Center, New York, NY for PC3-PSMA cells. We also thank Dr. Roger Johnson and Professor Deirdre Meldrum, Dean Ira A. Fulton School of Engineering and Director of the Center for Ecogenomics at the Biodesign Institute at ASU for access to the confocal microscope. This work was supported NIH (5R21CA131891-02) and start-up funds from ASU to KR and an NSF NIRT (DMI 0303950) grant and a NASA/NIAC grant to MLY. The authors

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