thesis doctor of philosophy duffy ciara evelyn lilly 2020 part 1

The anti-cancer properties of both honeybee venom and melittin, and selectivity towards TNBC and HER2-enriched breast cancers, were assessed by investigating the cellular response in a p

Honeybee venom and melittin for the treatment of HER2-enriched and

triple-negative breast cancer

Ciara Evelyn Lilly Duffy

Bachelor of Science (Honours)

This thesis is presented for the degree of Doctor of Philosophy of The University of Western Australia

School of Human Sciences

Breast cancer is the most commonly occurring cancer in women worldwide There are currently no effective targeted therapies available for triple-negative breast cancer (TNBC), which is aggressive, proliferative and associated with poor prognosis The epidermal growth factor receptor (EGFR) is commonly overexpressed in TNBCs, and is important for cell proliferation and survival, conferring oncogenic signalling often dependent on the PI3K/Akt pathway downstream To date, blocking EGFR signalling in TNBC has demonstrated limited clinical efficacy and the development of drug resistance Although therapies targeting human epidermal growth factor receptor 2 (HER2) in HER2-enriched breast tumours have dramatically improved median survival in the metastatic setting, resistance is almost inevitable for this subtype as well Clearly, the discovery of more effective and selective therapeutic strategies for these cancers is an area of prime importance in clinical oncology

Venom from the European honeybee (Apis mellifera) and melittin, the major peptide in the

venom, possess anti-cancer activity particularly by disrupting the plasma membrane However, the precise molecular mechanisms explaining the anti-cancer activity of these agents against malignant breast cells are unknown The objective of this PhD was to determine the therapeutic potential of honeybee venom and melittin to target and kill aggressive breast cancer cells The first aim was to determine the molecular mechanisms of breast cancer cell death due to honeybee venom and melittin The second aim was to enhance the breast cancer selectivity of melittin and assess the interaction of melittin with growth factor receptors overexpressed in breast cancer

cells The final aim was to combine melittin with docetaxel in vivo to assess whether there is a

synergistic interaction The anti-cancer properties of both honeybee venom and melittin, and selectivity towards TNBC and HER2-enriched breast cancers, were assessed by investigating the cellular response in a panel of breast cancer and normal-like cell lines

The results revealed that honeybee venom and melittin significantly and selectively reduce the viability of TNBC and HER2-enriched breast cancer cells and induce cell death through membrane disruption and caspase 3-mediated apoptosis Melittin modulates oncogenic Akt and MAPK signalling in breast cancer cells by suppressing the activation of EGFR and HER2 Finally,

melittin is synergistic with docetaxel both in vitro and in vivo, reducing cancer cell proliferation

and consequent tumour growth in an aggressive TNBC allograft model Overall, this PhD demonstrates that honeybee venom and melittin potently and rapidly reduce breast cancer cell viability and can be used with chemotherapies or other small molecules to treat highly aggressive

subtypes of breast cancer

Table of contents

CHAPTER 1: General introduction and literature review 1

1.1 The intrinsic molecular subtypes of breast cancer 3

1.2 Limitations of current treatments for TNBC 4

1.3 Honeybee venom and melittin 5

1.4 The anti-cancer properties of bee venom and melittin 7

1.5 Targeting melittin, combination with chemotherapy and the use of nanotechnology to deliver melittin to cancer cells 8

1.6 Purpose and significance of this research 10

1.7 Aims and location in thesis 10

1.8 References 12

CHAPTER 2: Honeybee venom and melittin induce selective, potent, and rapid breast cancer cell death 19

2.1 Introduction 21

2.1.1 Honeybees and bumblebees 21

2.1.2 Composition of honeybee venom compared to bumblebee venom 23

2.1.3 The application of bee venom and melittin in cancer 24

2.1.4 The cancer cell selectivity of honeybee venom and melittin 25

2.2 Aim 27

2.3 Methods 28

2.3.1 Chemical reagents and antibodies 28

2.3.2 Bee venom collection 28

2.3.3 Cell culture 30

2.3.4 Cell viability assays 30

2.3.5 Production of the primary monoclonal antibody against melittin 31

2.3.6 Enzyme-linked immunosorbent assay (ELISA) 31

2.3.7 Anti-melittin antibody competition experiments 32

2.3.8 Western blot for the detection of Cleaved Caspase-3 32

2.3.9 Flow cytometry 33

2.3.10 Live cell microscopy 34

2.3.11 Scanning electron microscopy 34

2.4.4 Honeybee venom and melittin induced caspase 3-mediated apoptosis, rapid cell death, and membrane disruption in breast cancer cells 43

2.4.5 Honeybee venom and melittin induced membrane disruption and nuclear condensation in breast cancer cells 45

2.5 Discussion 47

2.6 References 52

CHAPTER 3: Enhancing the breast cancer selectivity of melittin with RGD, and assessing the mechanisms of the suppression of EGFR and HER2 phosphorylation by melittin 60

3.1 Introduction 62

3.1.1 Altering the charge of melittin to modulate the lytic effect 62

3.1.2 Increasing the cancer selectivity of melittin 63

3.1.3 Suppressing the phosphorylation of EGFR and HER2, and modulating downstream oncogenic signalling pathways in breast cancer cells 65

3.1.4 Utilising bioluminescence resonance energy transfer to determine the interaction of melittin with growth factor receptors 66

3.2 Aim 67

3.3 Methods 68

3.3.1 Peptide modelling 68

3.3.2 Chemical reagents and antibodies 68

3.3.3 Bee venom collection 69

3.3.4 Cell culture 69

3.3.5 Cell viability assays 69

3.3.6 Production of the primary monoclonal antibody against melittin 69

3.3.7 Enzyme-linked immunosorbent assay (ELISA) 70

3.4.5 Melittin interacted with EGFR in the plasma membrane in a time and concentration dependent manner 86

3.4.6 Melittin indirectly interfered with RTK phosphorylation 89

4.1.1 The mechanism of action of docetaxel and cisplatin 108

4.1.2 Limitations of current chemotherapy treatment 109

4.1.3 Combining honeybee venom and melittin with chemotherapy 110

4.1.4 Immune system modulation by melittin 111

4.2 Aim 112

4.3 Methods 113

4.3.1 Chemical reagents and antibodies 113

4.3.2 Bee venom collection 113

4.3.4 Cell viability assays 113

4.3.5 Production of the primary monoclonal antibody against melittin 114

4.3.6 Analysis of combined drug effects 114

4.3.7 Animal model and treatments 114

4.3.8 Immunohistochemical analysis of the tumours 115

CHAPTER 5: General discussion and future directions 133

5.1 Summary of results and significance 135

GLM Generalised linear model

IC50 50% inhibitory concentration

PLA2 Phospholipase A2

RGD Arginine-glycine-aspartic acid motif

uPA Urokinase plasminogen activator

Acknowledgements

Thank you to everyone who has been a part of my PhD This has been an incredible journey and a transformational time of my life To my supervisors, Associate Professor Pilar Blancafort, Professor Boris Baer, Dr Anabel Sorolla, and Professor Killugudi Swaminathan Iyer; I am tremendously thankful for all that you have taught me, and all of the opportunities you gave me Thank you for enabling me the freedom to pursue my own scientific ideas and avenues of research

I would like to thank everyone who I have had the pleasure of working alongside in the Blancafort Laboratory at the Harry Perkins Institute of Medical Research In particular, I would like to acknowledge Anabel Sorolla and Edina Wang; I have learnt a great deal from both of you Thank you to Colette Moses, for your constant guidance and support throughout my research journey Thank you to the Centre for Integrative Bee Research (CIBER) team, especially Tiffane Bates, for providing training and access to the honeybees at the UWA apiary I would also like to thank my wonderful mentors; Mark Cregan, Miriam Borthwick, Kevin Pfleger, Matt Oldakowski, Intan Oldakowska, Carolyn Williams and Charlie Bass Thank you for your invaluable training, advice and encouragement

Thank you to my international research collaborators; Professor Jane Stout (Trinity College Dublin) and Professor Mark Brown (Royal Holloway University of London) I would like to sincerely thank all of the following researchers and/or beekeepers who helped me to access samples of different bee populations in Ireland and England; Saorla Kavanagh, Keith Pierce, Marcus Phelan, Susie Bioletti, David Morris, Terry Meakin, Martina Halpin, Edward Hill, Fabio Manfredini and Callum Martin Thank you to those who assisted in the venom transport across the world, including Sylvia Mathiasen, Siobhán McNamee and Rob Prouse Thank you to Alison Boyce and Steve Portugalfor assisting me with accessing and setting up

This research was supported by an Australian Government Research Training Program (RTP) Scholarship, and a PhD Top Up Scholarship from the Cancer Council of Western Australia Technical assistance was kindly provided by Kathleen Davern for the development of the anti-melittin antibody (Chapter 2); Diwei Ho, in assisting with setting up the scanning electron microscopy experiment (Chapter 2); Emily Golden, in assisting with the peptide modelling (Chapter 3); Elizabeth Johnstone, in helping set up the BRET assay (Chapter 3);

and Anabel Sorolla and Edina Wang, for assisting with the in vivo experiment (Chapter 4)

I would also like to thank Paul Rigby (CMCA) and Kevin Li (FACS Facility) at the Harry Perkins Institute of Medical Research

Lastly, thank you to all of my friends for your unwavering encouragement, to my beautiful family, and to Quentin, for your unconditional support throughout this journey Mum and Dad, I truly cannot thank you enough It is to you I dedicate my thesis

Chapter 1

General introduction and literature review

1.1 The intrinsic molecular subtypes of breast cancer

Breast cancer is a devastating disease caused by a combination of environmental and genetic factors that trigger damage to the cellular DNA 1, is the second most common cancer in the world, and the most commonly occurring cancer in women 2,3 In Australia, one in eight women will be diagnosed with breast cancer Genome-wide profiling has identified several intrinsic molecular subtypes of breast cancer using patient samples to correlate the characteristics of the tumours to clinical outcome These molecular subtypes include luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, basal-like, claudin-low, and normal-like 4–6 These subtypes differ greatly in terms of their transcriptional profiles, receptor expression, unique DNA methylation patterns, response to therapy and patient survival

Targeted therapies have yet to be established with clinical success for basal-like and claudin-low tumours, which are immunohistochemically characterised as triple-negative breast cancer (TNBC), lacking the expression of estrogen and progesterone receptors, as well as HER2 6 These TNBCs are particularly aggressive and this, coupled with the lack of targeted therapy options, leads to poor outcomes and disease-specific survival probability 7,8 TNBC accounts for 10–17% of breast cancer cases Approximately 50% of TNBCs overexpress the epidermal growth factor receptor (EGFR) 9 Another aggressive breast cancer subtype, the HER2-enriched tumours which accounts for 10% of breast cancer cases 10, overexpress HER2, another growth factor receptor tyrosine kinase (RTK) from the same family as EGFR RTKs are key regulators in the development and progression of multiple cancers, conferring oncogenic signalling often dependent on the phosphoinositide-3-kinase/protein kinase B/the mammalian target of rapamycin (PI3K/Akt/mTOR) pathway

downstream, which causes reduced apoptosis and increased cellular proliferation 9,11 Anti-HER2 and anti-EGFR therapies have been trialled in the clinic for targeting these cancers, which generally bind to the growth factor receptors on cancer cells and prevent endogenous growth factors binding in order to halt cell proliferation 12–14 However, in contrast to HER2-targeted treatments such as Trastuzumab, blocking EGFR signalling in TNBC with standard therapies has demonstrated limited clinical efficacy due to a lack of dependence on the EGFR pathway and the importance of collateral pathways 15,16 Even those breast cancers that are sensitive to EGFR therapy tend to develop resistance to EGFR inhibitors Although HER2-targeted therapies have dramatically improved median survival in the metastatic setting, resistance is almost inevitable for this subtype as well, with 80% of patients treated with docetaxel, trastuzumab, and pertuzumab relapsing at 5 years 14,17 Clearly, the discovery of more effective and selective therapeutic strategies for TNBC and HER2-enriched breast cancers is a priority area in clinical oncology

The only treatment options for patients with TNBC are based on conventional surgery such as mastectomy, as well as aggressive chemotherapy and radiation 9 Surgery and radiation can cause damaging side effects that last long after treatment has ended, including lymphedema, pain and numbness, musculoskeletal symptoms, and skin problems There are serious limitations to chemotherapy as well, including limited tumour cell penetration reducing the pharmacological effect, a lack of cancer cell specificity causing damaging side effects, and the development of drug resistance of cancer cells increasing patient mortality 18 The intrinsic propensity of these cancers to disseminate and resist chemotherapies, combined with the substantial side effects and

collateral tissue damage from current treatments, have generated broader and holistic research interests over recent years to develop alternative treatments that are more specific and less harmful for cancer patients The development of new, targeted treatments is therefore required to manage these aggressive breast cancers and overcome the damaging side effects of current clinical treatments 8

The European honeybee (Apis mellifera) has been the source of a number of products

used medicinally by humans such as honey, propolis and venom for thousands of years 19 Honeybee venom has been used in clinical trials for the treatment of Parkinson’s disease, rheumatoid arthritis and pain 20–22 The non-water components make up around 12% of honeybee venom and include numerous peptides, enzymes, amines, phospholipids, pheromones, sugars and amino acids 23 Half of the dry weight of honeybee venom corresponds to melittin which forms pores in cell membranes causing cell lysis 24–26 Melittin can also activate cellular phospholipase A2 (PLA2), by inducing prolonged activation of phospholipid hydrolysis at high levels until the cell dissolves itself in detergent lipids from its own membranes, however the precise

mechanism of this process is not clear 27,28

Melittin is a positively charged, amphipathic, 26 amino acid peptide with a molecular weight of 2.85 kDa 24–26 Each melittin molecule forms the shape of a bent rod and is comprised of two independent alpha-helical segments, separated by the helix-breaking glycine at position 12 and proline at position 14 29 It is hydrophobic at the N-terminus and hydrophilic at the C-terminus, with entirely polar residues in positions 21–26, and has a net charge of +5 to +6 24,30 The domain of lysine and arginine amino acids at the C-terminus (K21RKR24) are positive at neutral pH 31 Melittin was modelled

using PEP-FOLD3 and the image generated using PyMOL 32 (Figure 1.0)

Melittin is known to interact with cell membranes by initially adopting an orientation parallel to the membrane Then at sufficiently high concentrations, melittin moves laterally, orients perpendicular and undergoes oligomerisation into toroidal transmembrane pores 33 This in turn disrupts the membrane potential and causes cell lysis 34 Melittin peptides associate with the phospholipid head group region of the membrane bilayer, causing a continuous bend of phospholipids so that the pore is lined by both the lipid head groups and the cell lytic peptide 25,35,36

Figure 1.0 | Melittin is a short peptide with two alpha-helical chains, forming the shape of a bent rod (a) The primary amino acid sequence of melittin, from N-terminus to

C-terminus (b) The tertiary structure of melittin, modelled using PEP-FOLD3 and the image

generated using PyMOL Melittin is modelled from N-terminus (left) to C-terminus (right)

The pore formation of melittin in cell membranes has been dynamically modelled using all-atom and course-grained molecular dynamics simulations, showing that multiple melittin peptides come together in the membrane to form the transmembrane pore 26,37,38 These simulations revealed that melittin forms defects in

GIGAVLKVLTTGLPALISWIKRKRQQ

the membrane that develop into small transmembrane pores with an estimated diameter of ∼1.5 nm and a lifetime of the order of tens of milliseconds 39 These pores then act as sites of membrane rupture, leading to larger pores with an inner diameter of ~4.4 nm that may enable the internalization of additional small molecules with cytotoxic activities 39–41 The mechanism by which melittin exerts its effect in cell membranes is dependent on conditions such as peptide concentration, pH, and the composition of the target membrane (including the presence of cholesterol or proportion of positively charged lipids) 42, and this will be discussed further in Chapter 2

1.4 The anti-cancer properties of bee venom and melittin

Interest in the anti-cancer properties of honeybee venom and melittin have recently increased The first report of the anti-cancer effects of honeybee venom demonstrated that the venom delayed tumour appearance in colchicine-induced tumours in plant models 43 Since then, honeybee venom and melittin have consequently been explored

in a range of cancers, demonstrating anti-tumoural effects in vitro in melanoma 44, ovarian cancer 45, cervical cancer 46, glioblastoma 47, leukemia 48, hepatocellular carcinoma 49, and in vivo in xenograft tumour mouse models of non-small-cell lung

cancer 50 and pancreatic cancer 51 Honeybee venom and melittin have also been shown

to reduce the viability of breast cancer cells in vitro 52–54 and in vivo in murine

transplantable mammary carcinoma models 55,56 The findings from all of these studies will be covered in detail in Section 2.1 Overall, a limited number of breast cancer cell lines have been investigated, with no examination of breast cancer subtype selectivity, and the molecular mechanisms triggering cancer cell death remain poorly understood

Importantly, both honeybee venom and melittin exhibited higher cytotoxic potency to

cancer cells compared to normal cells for melanoma, leukemia, pancreatic cancer and lung cancer cells 44,48,51,57 suggesting a useful therapeutic window could exist Honeybee venom and melittin inhibited pro-oncogenic processes in cancer by blocking the PI3K/Akt/mTOR axis in breast cancer cells 53, mitogen-activated protein kinase (MAPK) in hepatocyte and melanoma cells 58,59, Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) in ovarian cancer cells 45, and nuclear factor-kappa B (NF-κB) signalling in lung cancer cells 60 Further analysis is required into the effects of honeybee venom and melittin in aggressive breast cancer cells, in terms of potential cancer cell selectivity compared to normal cells, and the molecular interaction with oncogenic signalling pathways and overexpressed membrane receptors which these cancers rely on for growth and proliferation

and the use of nanotechnology to deliver melittin to cancer cells

In the discovery of anti-cancer compounds, there are multiple methods of targeting and delivering small peptides to the tumour and tumour-associated vasculature, although this is difficult to successfully achieve Some studies have shown benefits of conjugating melittin to molecules that target characteristics overexpressed in cancer cells 61–63, which will be covered in detail in Section 3.1

Most state of the art cancer treatments are also now administered in combination, where multiple drugs are used to increase the effect of the treatment 64 Previous studies have examined the effects of honeybee venom or melittin in combination with chemotherapy on cancer cells Honeybee venom has been combined with cisplatin in cervical and laryngeal carcinoma cells 65, with docetaxel in lung cancer cells 60, and

with immunotherapy to kill lung cancer cells in vitro 66 The details of these

combination treatments will be covered in Section 4.1 Further research is required into the combination of honeybee venom and melittin with chemotherapy drugs currently used for the treatment of breast cancer in the clinic, and whether a synergistic combination can be achieved

Nanoparticles have also been used to deliver melittin to cancer cells in vitro and in vivo Targeted nanoparticles delivered melittin to syngeneic B16-F10 melanoma in immunocompetent C57BL/6 mice, xenograft MDA-MB-435 breast cancerin athymic nude (NCr-nu/nu) mice, and precancerous lesions in K14-HPV16 transgenic mice with squamous dysplasia and carcinoma 67 However, the effects of these melittin loaded nanoparticles were about five fold less effective than free melittin Melittin has also been joined to an alpha-helical peptide via a linker, and the peptide self-assembled into a ~20 nm lipid nanoparticle by interacting with the phospholipids 68 This nanoparticle model successfully reduced tumour growth of B16-F10 melanoma cells

both in vitro, and in vivo in C57BL/6 mice Another group developed a melittin peptide delivery system to kill colon, breast and ovarian cancer cells in vitro 69 Melittin has previously been electrostatically bound to a peptide vector of nanodiamonds and PEGylated polyglutamic acid, which was toxic to luminal MCF7

breast cancer cells in vitro 70 Oligopeptide–alginate nanoparticles were also used to

deliver melittin to human colorectal adenocarcinoma cells in vitro 71

Recently, Yu et al., developed nanoparticles containing melittin attached to an

amphipathic α-helical peptide (namely α-melittin-NPs) which were delivered to liver sinusoidal endothelial cells, activating the immunologic environment and reaching 80% survival rate for mice with spontaneous liver metastatic tumours 72 However, there are major limitations to the application of delivering melittin to cancer cells using nanoparticles such as reduced therapeutic efficacy, lack of cancer specificity,

unexpected cytotoxicity, enzymatic degradation, and inactivation by serum components 31,61,69,73; overcoming these limitations will be discussed in Chapter 5

1.6 Purpose and significance of this research

The purpose of my PhD research was to quantify the effectiveness of honeybee venom and melittin in treating TNBC and HER2-enriched breast cancer cells, with a particular focus on cancer selectivity, and to expand our understanding of the molecular mechanisms triggering breast cancer cell death My project addresses a significant problem considering there is currently no targeted therapy available for these aggressive breast cancers The findings of my work could potentiate the use of melittin as a small peptide for the treatment of the most aggressive subtypes of breast cancer, either alone or in combination with chemotherapeutic drugs

1.7 Aims and location in thesis

The specific project aims of my PhD were to:

1 Determine the viability of breast cancer cells compared to normal cells treated with honeybee venom, melittin, or bumblebee venom, and investigate the molecular

mechanisms of breast cancer cell death (Chapter 2)

2 Develop a targeted melittin peptide to enhance cancer cell selectivity, and test the hypothesis that honeybee venom and melittin suppress cancer signalling pathways by

interfering with the phosphorylation of EGFR and HER2 (Chapter 3)

3 Assess whether there is a synergistic interaction when aggressive TNBC cells are

co-exposed to honeybee venom or melittin with docetaxel treatment in vitro and in vivo

(Chapter 4)

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human cervical and laryngeal carcinoma cells and their drug resistant sublines

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Chapter 2

Honeybee venom and melittin induce selective,

2.1 Introduction

Chapter 1 reviewed the literature relevant to the experimental work described in this thesis, including the molecular subtypes of breast cancer, the use of honeybee venom and melittin in cancer treatment, characteristics and mechanisms of action of melittin, and a general scope of targeting peptides and combination of melittin with chemotherapy The focus of Chapter 2 is to understand the potency and selectivity of honeybee venom and melittin to breast cancer cells The introduction of this chapter reviews the composition and anti-tumour activity of both honeybee and bumblebee venom, and their anti-cancer properties

The European honeybee (Apis mellifera) and European buff-tailed or large earth bumblebee (Bombus terrestris) are bee species commonly used for pollination of crops in fields and

greenhouses 1,2 In Australia, the European honeybee was introduced over 180 years ago, yet the only bumblebees in Australia are feral bees, accidentally introduced in Tasmania in 1992 3 For honeybees and bumblebees, the colony consists of a queen (female), workers (females), and drones (males) The female bees possess a stinging apparatus, while the drones do not; rather, the drones have an endophallus for mating with a queen bee 4,5 The queen honeybees use their stinging apparatus to kill other queens reared in the colony, either before they complete pupation or in a combat to the death to become the new queen of the colony 6 Alternatively, worker bees use their sting to protect the colony from predators

Honeybees and bumblebees belong to the Apidae family, and while these species diverged about 100 million years ago, their venoms appear to be similar with 70% homology between the toxins in the venoms 7,8 These toxins share biochemical activity, which may be explained by the high level of conservation between the genetic architecture of the genomes of the two species 7 For comparative purposes, a photograph of a honeybee and bumblebee are

Figure 2.0 | The European honeybee (Apis mellifera, top) and European buff-tailed or large earth bumblebee (Bombus terrestris, bottom) The photographs are provided courtesy

of the United States Geological Survey (USGS) Native Bee Inventory and Monitoring Lab (https://www.flickr.com/photos/usgsbiml).

2.1.2 Composition of honeybee venom compared to bumblebee venom

In my PhD, honeybee venom from different populations of bees in different geographical locations, and bumblebee venom, were collected to determine whether the effects of the venoms on breast cancer cells differed across bee species and locations To date, there are no reports comparing the anti-cancer properties of the venoms from different honeybee populations The venom of honeybees and bumblebees contain highly complex mixtures of peptides, enzymes and biogenic amines The major components of honeybee venom are melittin and secretory phospholipase A2 (PLA2) 9,10 Melittin constitutes 50% of honeybee venom by dry weight PLA2 is an enzyme that catalytically hydrolyses and digests cell membrane components, and constitutes 10–12% of honeybee venom by dry weight 11,12 Both honeybee venom and bumblebee venom contain two types of PLA2 (PLA2-1 and PLA2-2) 8,13

In contrast to honeybee venom, melittin is not present in bumblebee venom, and the major components of bumblebee venom are bombolitin, PLA2, and serine protease 8,14 Bombolitin

has been discovered in other bumblebee species as well (including Megabombus pennsylvanicus, Bombus lapidaries, and Bombus ignites), and constitutes about 25% of

bumblebee venom by dry weight 15–17 Bombolitin from Bombus terrestris shares 39%

nucleotide sequence similarity with melittin 8 However, on the protein level both bombolitin and melittin share similar amphiphilic secondary structures in their α-helical conformation, and are reported to possess similar antimicrobial activity against human pathogenic bacteria 15 Further homologs between the venoms of honeybees and bumblebees are hyaluronidase (75% similarity), acid phosphatase (67% similarity), and α-Glucosidase (89% similarity) 8 Interestingly, bumblebee venom contains no homologs to the honeybee venom antimicrobial peptides apidaecin and mast cell degranulating peptide, or the neurotoxic peptides apamin and tertiapin 18–20 Bumblebee venom contains a larger number of serine proteases compared to honeybee venom Thus, bumblebee venom will be used to

2.1.3 The application of bee venom and melittin in cancer

As described in Chapter 1, honeybee venom has been shown to elicit anti-tumour effects in numerous types of cancer Both honeybee venom and melittin reduced ovarian cancer cell viability by inducing the expression of death receptors and suppressing the STAT3 pathway 21 Honeybee venom also inhibited the cell growth of non-small-cell lung cancer (NSCLC) by decreasing NF-κB activity and increasing death receptor 3 expression 22, reduced the viability of human glioblastoma cells by suppressing matrix metalloprotease-2 expression and inducing apoptosis 23, and induced apoptosis in HeLa cervical cancer cells 24

The anti-tumour properties of melittin have also been demonstrated in various models of cancer Melittin sensitised human hepatocellular carcinoma cells to TRAIL-induced apoptosis by activating Ca2+/calmodulin-dependent protein kinase 25, and induced

apoptosis in hypoxic head, neck and oesophageal squamous cell carcinoma cells in vitro and

in xenograft mouse models, making these cells more sensitive to radiation treatment 26,27 Melittin also inhibited tumour growth in a Lewis lung carcinoma mouse model by regulating the gene expression of tumour associated macrophages 28 and inhibiting tumour angiogenesis and metastasis 29, induced apoptosis in NCI-H441 NSCLC cells by inhibiting miR-183 expression and activating caspase-2, and reduced tumour growth in a NSCLC xenograft mouse model 30 Oxidative stress and cytogenetic damage were induced by melittin in human peripheral blood lymphocytes, with the modulation of genes associated with DNA damage and apoptosis 31

In breast cancer, honeybee venom reduced the in vitro proliferation and induced apoptosis

in hormone receptor positive MCF7 breast cancer cells by inducing cytochrome c release, and increasing the levels of caspase-9 and poly (ADP-ribose) polymerase (PARP) 32 The viability, invasion and migration of MDA-MB-231 (estrogen receptor negative) and MCF7