thesis doctor of philosophy duffy ciara evelyn lilly 2020 part 3

Figure 3.4 | Melittin was localised in the plasma membrane of breast cancer cells overexpressing HER2 growth factor receptors treated with honeybee venom, melittin, and RGD1-melittin.. I

Figure 3.4 | Melittin was localised in the plasma membrane of breast cancer cells overexpressing HER2 growth factor receptors treated with honeybee venom, melittin, and RGD1-melittin Immunofluorescence images of HER2-enriched breast cancer (SKBR3) cells

treated with vehicle, honeybee venom, melittin, RGD1-melittin and DEDE-melittin for 30 minutes In blue: cell nuclei, in red: anti-HER2, and in green: anti-melittin Merged images are shown The white outlines in the merged images indicate the respective regions in the zoomed images Scale bars indicate 25 µm for all images except the zoomed images, which have scale bars of 6.25 µm Representative images are shown for each treatment

3.4.4 Honeybee venom and melittin suppressed the ligand-induced phosphorylation of EGFR and HER2 in breast cancer and modulated downstream signalling pathways in a time dependent manner

The molecular mechanisms by which melittin suppresses tumour cell proliferation were next assessed Considering the highly predominant localisation of melittin in the plasma membrane in tumour cells overexpressing EGFR and HER2, the next step was to assess whether honeybee venom and melittin interfered with growth factor receptor signalling as a mechanism of reducing the proliferation and survival of breast cancer cells For this reason, the hypothesis was that both honeybee venom and melittin could disrupt RTK-associated signalling pathways in EGFR and HER2 overexpressing cells by rapidly blocking the ligand-dependent activation of these receptors in the plasma membrane of breast carcinoma cells

To assess this, Western blot analysis was conducted on SKBR3 (HER2+, EGFR+) and SUM159 (EGFR+) extracts of cells previously stimulated with epidermal growth factor (EGF) and treated with the IC50 of honeybee venom or melittin from 2.5 to 20 minutes (Figure 3.5) Importantly, the results reveal that honeybee venom and melittin down-regulated the phosphorylation of the RTKs and modulated associated PI3K/Akt and MAPK signalling pathways in a time-dependent manner

In particular, treating SKBR3 cells with honeybee venom and melittin strongly downregulated p-HER2 (Tyr1248), p-EGFR (Tyr1068), p-p44/42 MAPK (Thr202/Tyr204), and p-Akt (Ser473 and Thr308) from 5 minutes onwards (Figure 3.5, a) In SKBR3, there was also a slight decrease in total HER2, EGFR, and Akt protein only after 10 minutes of honeybee venom treatment, which could relate to membrane degradation and/or endosome-mediated receptor degradation 50

In SUM159, p-EGFR (Tyr1068) was down-regulated by honeybee venom from 10 to 20 minutes, and melittin from 15 to 20 minutes of treatment (Figure 3.5, b) Treating SUM159 with melittin also suppressed p-Akt (Thr308) at all time points, yet upregulated p-p44/42 MAPK (Thr202/Tyr204) from 10 to 20 minutes, whereas honeybee venom upregulated p-p44/42 MAPK (Thr202/Tyr204), and p-Akt (Ser473 and Thr308) from 10 to 20 minutes The anti-melittin antibody indicated an increasing amount of melittin present in the lysates of both cell lines over time, with a higher signal for the melittin treatment compared to whole honeybee venom in both cell lines

The effects of honeybee venom and melittin on JAK/STAT pathway inhibitors were also assessed in TNBC (SUM159) cells because melittin was previously shown to inhibit JAK2/STAT3 signalling in ovarian cancer 52 No modulatory effects were observed after a 60-minute treatment with the IC50 of honeybee venom or melittin with or without the stimulation of 20 ng/mL EGF for 5 minutes (Supplementary Figure 3.1)

Figure 3.5 | Honeybee venom and melittin suppressed the ligand-induced phosphorylation of EGFR and HER2 and modulated downstream signalling pathways in breast cancer in a time dependent manner Western blots showing the phosphorylation

kinetics of the receptor tyrosine kinases (EGFR and HER2), downstream signalling pathways (MAPK

and Akt), and total melittin after treatment with honeybee venom or melittin in (a) SKBR3 and (b) SUM159 breast cancer cells α-Tubulin was used as the loading control.

α-Total AKTα-p-EGFR Tyr 1068α-Total EGFRα-p-44/42 MAPK Erk1/2Thr 202 / Tyr 204α-p-AKT Ser 473α-p-AKT Thr 308

α-Total MELITTINα-TUBULINα-p-HER2 Tyr 1248

α-Total MELITTINα-TUBULINα-Total AKT

60 kDa60 kDa60 kDa175 kDa185 kDa185 kDa

175 kDa

50 kDa2.85 kDa44, 42 kDa

60 kDa60 kDa60 kDa175 kDa175 kDa

Minutes of treatment 2.5 5 10 15 20 2.5 5 10 15 20

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

Considering TNBC and HER2-enriched breast carcinoma cells are highly dependent on the activation of EGFR and HER2 for signalling and growth, bioluminescence resonance energy

transfer (BRET) experiments were performed to determine whether melittin directly

interfered with the binding of EGF to EGFR leading to the observed suppressed growth factor receptor phosphorylation The NanoLuc reporter has been extensively shown to be useful in monitoring receptor-ligand binding in real time BRET assays 43,44 In this experiment, NanoLuc was used as the bioluminescent donor molecule, and genetically fused to EGFR 41,42

Kinetic and saturation BRET experiments were used to monitor the proximity of EGFR with the fluorescently tagged acceptor molecules TAMRA-EGF (positive control), FITC-melittin, and the inactive FITC-DEDE-melittin (negative control) The experiments were conducted with HEK293FT cells previously transfected with NanoLuc-EGFR fusion plasmid Transfer of energy from the bioluminescent donor to the fluorescent acceptor occurs over distances less than 10 nm and is indicative of interactions between the tagged molecules of interest 40

NanoLuc-First, cell viability was assessed by treating HEK293FT cells with melittin and DEDE-melittin to obtain IC50 values for the subsequent experiments (Supplementary Figure 3.2) Then, a range of concentrations of each peptide was selected including the IC50

FITC-of FITC-melittin, with the corresponding molar concentrations FITC-of FITC-DEDE-melittin The kinetics of the interaction of the fluorescent peptides with NanoLuc-EGFR were assessed with BRET using a range of peptide concentrations over time (Figure 3.6, a) The BRET signal increased in a dose-dependent manner for TAMRA-EGF, FITC-melittin and FITC-DEDE-melittin Interestingly, FITC-DEDE-melittin displayed much higher BRET ratios

than FITC-melittin at the same concentrations, as well as reaching maximal BRET ratios at each dose very rapidly (Figure 3.6, a, right) In all cases, higher concentrations of peptide correlated with a higher BRET ratio As an additional negative control, a non-specific peptide designed against the Engrailed 1 (EN1) transcription factor 47 (FITC-EN1-mutant) exhibited similar BRET ratios and kinetics to the FITC-DEDE-melittin, supporting the specificity of the interaction of active forms of melittin with EGFR (Supplementary Figure 3.3) Further experiments were required to ascertain the specificity of the binding interactions of each peptide with EGFR

To determine this specificity of melittin binding to EGFR at the EGF binding site, saturation BRET assays were conducted to assess the competition of the endogenous ligand EGF with each of the peptides binding to NanoLuc-EGFR The BRET signal of TAMRA-EGF with NanoLuc-EGFR was saturable and significantly reduced from 2.5 nM and all higher concentrations of TAMRA-EGF when it was combined with 1 µM EGF (Figure 3.6, b, left; two-way ANOVA, F 7,28 = 38, p<0.0001) confirming that TAMRA-EGF was binding to the EGF ligand binding site in the absence of endogenous EGF In contrast, the BRET signal of FITC-melittin and FITC-DEDE-melittin were not saturable and were not significantly different with or without 1 µM EGF (Figure 3.6, b, middle and right; F 7,32 = 0.004, p>0.999, and F 7,32 = 0.025, p>0.999, respectively), suggesting that neither melittin nor DEDE-melittin bound to EGFR at the EGF binding site The BRET ratio of the interaction of FITC-melittin and FITC-DEDE-melittin was higher than with TAMRA-EGF, likely due to the much higher concentrations of fluorescent peptide used which were required to reach concentrations above the IC5o

Figure 3.6 | Melittin interacted with EGFR in the plasma membrane in a time and concentration dependent manner (a) Bioluminescence resonance energy transfer (BRET)

kinetic analysis of TAMRA-EGF, FITC-melittin and FITC-DEDE-melittin interaction with EGFR in HEK293FT cells The peptides were added after the cells were equilibrated in the reader

NanoLuc-with the NanoLuc-substrate furimazine for 5 minutes (b) BRET saturation binding analysis of

increasing concentrations of TAMRA-EGF, FITC-melittin and FITC-DEDE-melittin in HEK293FT cells transfected with NanoLuc-EGFR in the presence or absence of unlabelled EGF (1 µM; to define

non-specific binding) Data are expressed as raw BRET ratios with mean ± SEM (c) Illustration of

the proposed BRET kinetics (top) and saturation (bottom) analysis for TAMRA-EGF (left), melittin (middle) and FITC-DEDE-melittin (right) with NanoLuc-EGFR BRET signals were recorded when the fluorescent acceptor molecules were within 10 nm of NanoLuc-EGFR.

FITC-An illustration to explain the BRET kinetics and saturation results is proposed (Figure 3.6, c) This illustration demonstrates that the kinetics results (top panel) for TAMRA-EGF and FITC-melittin were similar because increasing concentrations of both fluorescent acceptor molecules came into close proximity with NanoLuc-EGFR until the recording saturated for each concentration of peptide used This is most likely because TAMRA-EGF bound directly to NanoLuc-EGFR, and melittin inserted into and moved laterally in the plasma membrane 53 to interact with NanoLuc-EGFR in a fluid manner within a distance of 10 nm In contrast, FITC-DEDE-melittin was not expected to enter the cell membrane due to the previously observed lack of cell reactivity to this mutant peptide, and so the same BRET levels were recorded across the hour as the peptide came within 10 nm of NanoLuc-EGFR in the extracellular fluid in a concentration dependent manner

For the saturation results (Figure 3.6, c, bottom panel), there was a significantly reduced BRET signal when TAMRA-EGF was combined with unlabelled EGF, because the latter bound to and thus blocked the EGF binding site of NanoLuc-EGFR, which interfered with the interaction between TAMRA-EGF and NanoLuc-EGFR In contrast, there was no change in the BRET signal for both FITC-melittin and FITC-DEDE-melittin with or without unlabelled EGF, because the latter did not block the capacity of these fluorescent acceptor molecules to interact with NanoLuc-EGFR Overall, the BRET data indicates that melittin may be positioned at a distance within 10 nm from the RTKs without interfering with the endogenous growth factor binding site

3.4.6 Melittin indirectly interfered with RTK phosphorylation

Considering melittin did not bind to EGFR at the endogenous EGF binding site, it was still unclear whether melittin bound directly to EGFR elsewhere on the receptor in order to interfere with receptor phosphorylation To assess this, a co-immunoprecipitation experiment was conducted between biotin-melittin and NanoLuc-EGFR (Figure 3.7)

HEK293FT cells were either untransfected (wild-type) or transfected with Empty Vector or NanoLuc-EGFR fusion plasmid for 24 hours The cells were then treated for 10 minutes with either vehicle, biotin-melittin or biotin-DEDE-melittin using the IC50 of biotin-melittin (3.512 µM), and the co-immunoprecipitation conducted with streptavidin coated beads to pull down biotinylated melittin Considering no band was detected in the lane where the cells were transfected with NanoLuc-EGFR and treated with biotin-melittin (as indicated by the grey arrow in Figure 3.7), these results indicate that melittin does not bind directly to EGFR, at least by 10 minutes of treatment In summary, it is possible that melittin interacts with the plasma membrane broadly, indirectly interfering with the phosphorylation of growth factor RTKs such as EGFR and HER2 in breast cancer cells

Figure 3.7 | Melittin indirectly interfered with growth factor RTK phosphorylation A

co-immunoprecipitation assay was conducted to assess the association of melittin with EGFR HEK293FT cells were treated with media (wild-type) or transfected with Empty Vector or NanoLuc-EGFR fusion plasmid for 24 hours, and then treated with vehicle, biotin-melittin or biotin-DEDE-melittin for 10 minutes Cell extracts were collected and subjected to co-immunoprecipitation using streptavidin coated Dynabeads, followed by Western blot with an anti-EGFR antibody Wild-type and Empty Vector transfected cells, and vehicle and biotin-DEDE-melittin, served as controls In the case that melittin bound directly to EGFR, a band would have been detected in the lane where the cells were transfected with NanoLuc-EGFR and treated with biotin-melittin (as indicated by the grey arrow).

3.5 Discussion

As discovered in Chapter 2, further investigation was required into the molecular mechanisms underpinning the interaction of melittin with plasma membrane receptors and downstream signalling pathways, and whether the breast cancer specificity of melittin could be enhanced The aim of this chapter was to alter the sequence of melittin to enhance breast cancer cell selectivity and assess whether honeybee venom and melittin suppressed cancer signalling pathways by interfering with the phosphorylation of EGFR and HER2

The results revealed that altering the amino acid sequence of melittin drastically altered the anti-cancer activity Changing the positive charge of the C-terminus of melittin by grafting a DEDE sequence completely abolished the cell death and apoptosis-inducing capacity of melittin Mutagenesis of this sequence with another positively charged α-helical sequence known to have cell penetration capacity 10 (the SV40-melittin) rescued the cytotoxic effect of DEDE-melittin to levels similar to the parental melittin Thus, the positively charged residues at the C-terminus of melittin are necessary for the functional activity of melittin 1,3,9, enabling membrane binding and subsequent pore formation and cell lysis in breast cancer In the future, the C-terminus of melittin could be replaced with uncharged residues rather than positively charged domains or DEDE, to assess how this affects the anti-cancer activity of melittin Cleaved caspase-3 immunoblotting could also be conducted with the SV40-melittin peptide to assess whether rescuing the positive charge of melittin also rescued the apoptosis inducing capacity of SV40-melittin to the same extent as parental melittin

Importantly, the selectivity of melittin towards breast cancer cells was significantly enhanced with the engineering of an N-terminal RGD peptide Previous studies have shown that RGD motifs (comprising the Arg-Gly-Asp sequence) can be engineered into therapeutic peptides in order to target αvβ3 and αvβ6 integrins overexpressed in tumours and associated vasculature 18,20 Here, melittin was N-terminally linked with RGD1, a sequence derived from

pro-TGF-β, which has very high binding affinity for αvβ6 integrins overexpressed in TNBC cells 18 Consistently, the results showed that RGD1-melittin significantly enhanced the selectivity of melittin to TNBC cells compared to normal cells, and induced apoptosis to the same extent as melittin This targeting method may be superior to previous studies using disintegrin and uPA-cleavable linkers 22–24 because RGD1-melittin possessed enhanced cancer selectivity, is relatively cheap and easy to synthesise due to the short length, and melittin did not require release at the plasma membrane via a uPA-cleavable linker

The melittin-specific monoclonal antibody showed that melittin was localised predominantly in the plasma membrane of cells overexpressing EGFR and HER2 receptors RGD1-melittin was more membrane associated than melittin particularly in the SUM159 breast cancer cells, however TGF-β has also been shown to promote HER2-driven cancer by increasing metastasis, invasion and migration 54 Therefore, in the future, cell viability assays could be conducted to assess whether RGD1-melittin has enhanced selectivity for HER2-enriched breast cancer cells compared to wild-type melittin, and immunoblotting used to detect the levels of αvβ6 integrins in both SUM159 and SKBR3 cell lines

Both HER2-enriched and EGFR expressing TNBC tumours are highly dependent on PI3K/Akt oncogenic signalling for proliferation and survival 25 The results revealed that honeybee venom and melittin suppressed the ligand-induced phosphorylation of EGFR and HER2, dynamically modulating downstream signalling pathways in breast cancer cells In the HER2-enriched breast cancer cells (SKBR3), both honeybee venom and melittin potently suppressed the phosphorylation of HER2, EGFR, MAPK, and Akt from 5 minutes treatment onwards Interestingly, a previous study in human hepatocellular carcinoma cells demonstrated that 8 ng/µL melittin supressed the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) 55, another plasma membrane receptor from a different

RTK family, and a study in Lewis lung carcinoma cells showed that bee venom from Apis

mellifera linguistica (the Italian honeybee) down regulated the expression of VEGF and

VEGFR-2, but not of VEGFR-1 39 However, neither of these studies provided an explanation for how honeybee venom or melittin interfered with VEGF and VEGFR-2 expression A reduction in total HER2 and EGFR receptor expression was also observed for SKBR3 cells after 10 minutes of honeybee venom treatment, which may have been due to membrane disruption, endocytoses and/or receptor degradation 50,51,56

Similarly, in TNBC cells (SUM159), both honeybee venom and melittin potently suppressed the phosphorylation of EGFR (and phospho-Akt for melittin treatment) in a time dependent manner Interestingly, the phosphorylation of MAPK was upregulated by both honeybee venom and melittin from 10 minutes of treatment in SUM159 This may be due to the release of a negative regulatory feedback loop that triggers ERK signalling to protect the cells from apoptotic cell death 57,58 In SUM159, Akt phosphorylation may have also been upregulated at 10 minutes treatment with honeybee venom due to cross-talk between the PI3K-mTOR and Ras-ERK pathways 59 In breast cancer, previous reports have shown that melittin suppressed the phosphorylation of Akt and ERK in TNBC cells (MDA-MB-231), but increased the phosphorylation of ERK in luminal breast cancer (MCF7) cells 38

Interestingly, Tu et al also found that the ERK pathway was phosphorylated in human

melanoma cells as early as five minutes after treatment with honeybee venom, while phosphorylated Akt was significantly reduced 57 As activation of the Akt pathway has been associated with resistance to chemotherapeutics, particularly in claudin-low breast cancers 60, melittin could be combined with chemotherapies to suppress these negative feedback loops and prevent adaptive resistance to therapy 61

The BRET results suggested that after insertion into the phospholipid bilayer, melittin interacts with RTKs in the plasma membrane and may suppress RTK phosphorylation by indirectly interfering with homo- and hetero-dimerisation of growth factor receptors such

as EGFR and HER2 Melittin interacted with EGFR in the plasma membrane at a distance within 10 nm without binding directly to EGFR Interestingly, FITC-melittin had much lower BRET ratios compared to FITC-DEDE-melittin at the same peptide concentrations, which could be due to the N-terminus of melittin being internalised into the plasma membrane containing the FITC tag, melittin inducing internalisation of EGFR, and/or melittin being taken up into the cells by endocytosis For the latter explanation, melittin was shown to enter cancer cells via receptor endocytosis following sub-cytotoxic treatment, subsequently triggering enlargement of the lysosomal compartments and cytosolic translocation of cathepsin B 50 This occurred particularly through interaction with the heat shock cognate 70 kDa (Hsc70) protein chaperone which could aid in the cell entry and cytolytic activity of melittin In the future, a control NanoLuc receptor other than NanoLuc-EGFR could be used in order to further improve our understanding of the specific interaction of melittin and DEDE-melittin with EGFR and omit any background BRET signals Furthermore, BRET trafficking assays could be used to determine whether EGFR is internalised into the cell after melittin treatment 62

In summary, this chapter demonstrated that melittin can be targeted to breast cancer cells overexpressing αvβ6 integrins using an RGD motif, and the positive charge of melittin is necessary for membrane interaction and breast cancer cell lysis Furthermore, both honeybee venom and melittin suppressed the phosphorylation of EGFR and HER2 in a time dependent manner, by indirectly interfering with the homo- or heterodimerisation of RTKs in the plasma membrane Some of the most effective treatments for breast cancer now involve combination therapy, where chemotherapies or small molecules are delivered simultaneously to improve the therapeutic effect The transmembrane pores formed by melittin may facilitate the delivery and/or internalisation of chemotherapy into cancer cells 53, and so the next step is to combine melittin with chemotherapies to assess whether

there is a synergistic interaction in vitro and in vivo, which will be investigated in Chapter 4

3.6 Supplementary figures

Supplementary Figure 3.1 Relating to Figure 3.5 | Honeybee venom and melittin did not modulate the levels of JAK/STAT pathway inhibitors after a 60-minute treatment in TNBC cells Western blot for the detection of JAK/STAT pathway inhibitors in SUM159 cells

after a 60-minute treatment with vehicle, or the IC50 of honeybee venom (5.58 ng/µL) or melittin (4.24 ng/µL), with or without the stimulation of 20 ng/mL EGF for 5 minutes α-Tubulin was used

as the loading control

Supplementary Figure 3.2 Relating to Figure 3.6 | HEK293FT cells treated with melittin and FITC-DEDE-melittin Cell viability dose-response curves of HEK293FT cells

FITC-treated with FITC-melittin and FITC-DEDE-melittin for 24 hours to obtain IC50 values for the BRET experiments Viability was assessed using the CellTiter-Glo 2.0 assay Data were normalised to the average luminescence of the vehicle condition (untreated, considered 100% viability) Data are

presented as mean ± SEM

-1.0 -0.5 0.0 0.5 1.0 1.5 2.00

log [peptide] (µM)

HEK293FT peptide treatment

FITC-melittin IC50 = 3.61 µMFITC-DEDE-melittin IC50 > 20 µM

Supplementary Figure 3.3 Relating to Figure 3.6 | A non-specific peptide designed against the Engrailed 1 (EN1) transcription factor 47 exhibited similar BRET kinetics to FITC-DEDE-melittin Kinetic analysis of FITC-EN1-mutant interaction with NanoLuc-EGFR by

bioluminescence resonance energy transfer (BRET) in HEK293FT cells FITC-EN1-mutant was added after the cells were equilibrated in the reader with the NanoLuc-substrate furimazine for 5 minutes

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4.1 Introduction

Chapter 3 revealed that melittin can be targeted to breast cancer cells and is localised in the plasma membrane of tumour cells overexpressing EGFR and HER2, subsequently suppressing the phosphorylation of these growth factor receptors Further investigation is still required into whether the combination of melittin with breast cancer chemotherapy is beneficial in treating TNBC cells This is especially important considering melittin forms transmembrane pores with an inner diameter of ~4.4 nm 1–3, which could be sufficient to allow the passive entrance of chemotherapeutics into cancer cells The focus of Chapter 4 is therefore to assess whether honeybee venom and melittin could synergise with breast cancer chemotherapies, leading to more potent breast cancer cell death and therapeutic efficacy

The effects of combining melittin with docetaxel will be determined by cell viability in vitro, and tumour growth, proliferation and induction of apoptosis in vivo Most cancer

treatments are now administered in combination, where multiple drugs are administered concurrently to increase the effect of the treatment 4 A synergistic effect is achieved when the effect is greater than the sum of the individual effects of the two agents combined 5,6 By achieving a synergistic effect on cell death, the potential side effects and induction of drug resistance can be reduced 4,6

4.1.1 The mechanism of action of docetaxel and cisplatin

Many chemotherapy agents are currently used clinically for the treatment of cancer, including docetaxel and paclitaxel (which stabilise microtubule assembly), cisplatin and carboplatin (platinum based alkylating agents) and doxorubicin (a topoisomerase inhibitor) 7,8 Docetaxel is a semisynthetic taxane compound and potent inhibitor of cell replication 9 Docetaxel was originally prepared from a noncytotoxic precursor extracted

from the needles of the European yew plant (Taxus baccata L) 10 The first clinical trials using docetaxel were in 1990, and it is now one of the most commonly prescribed

chemotherapies for TNBC, and routinely used for the treatment of metastatic breast cancer 11–14 Docetaxel exhibits anti-cancer effects through the promotion and stabilisation of microtubule assembly inhibiting cell proliferation in the S-phase, therefore resulting in apoptosis 15

Cisplatin, or cisplatinum, is also a well-known chemotherapy commonly used in the treatment of many cancers including breast, cervical, ovarian, prostate, testicular, head and neck, lung, and bladder cancer 16–18 Cisplatin triggers cellular apoptosis by forming adducts with DNA, inhibiting DNA polymerisation and thus inducing DNA damage 16 Like docetaxel, cisplatin has also been used effectively in the clinical treatment of cancer patients in combination with other drugs such as cyclophosphamide for the treatment of small cell lung cancer 19, and with cetuximab in the treatment of metastatic TNBC 20,21

4.1.2 Limitations of current chemotherapy treatment

Unfortunately, there are some serious limitations of modern chemotherapy treatment Firstly, a lack of tumour cell selectivity, causing damaging side effects in normal cells 22 Many chemotherapeutic agents target cells replicating faster than the normal cell cycle, and therefore damage healthy cells with a high rate of proliferation Secondly, many chemotherapy drugs face limited tumour cell penetration, limiting their pharmacological effect after systemic administration 22 Thirdly, chemotherapy has a propensity to induce drug resistance in tumour cells, as these cells develop new survival pathways for growth and replication, increasing patient mortality 20,23

Resistance to chemotherapies is devastating considering it can often be followed by metastatic disease and mortality 24 Even for docetaxel treatment, the occurrence of taxane resistance in TNBC is a matter of concern where mechanisms such as the overexpression of transcription factors have been proposed to be responsible 24–26 Some mechanisms by

which cancer cells develop drug resistance include apoptotic pathway defects and expression of drug transporters 25,27 Furthermore, cancer stem cells or tumour-initiating cells possess an intrinsic resistance to conventional chemotherapies, and are able to self-renew and regenerate the heterogeneity of tumours, leading to metastasis and cancer recurrence 24 Drug resistance is also an issue for cisplatin The DNA damage-mediated apoptotic signals induced by cisplatin can be attenuated by reduced drug uptake, increased drug inactivation, and increased DNA adduct repair 17 These occur at the molecular level, and include mechanisms such as the overexpression of HER2 and activation of the PI3K/Akt pathway 17

over-4.1.3 Combining honeybee venom and melittin with chemotherapy

Previous studies have examined the effects of the interaction of honeybee venom or melittin with chemotherapy or immunotherapy for the treatment of cancer cells Melittin potentiated the cytotoxicity of the DNA damaging drug bleomycin A2 in murine leukemia cells (L1210) 28.Sub-lethal doses of honeybee venom were also used alone or in combination with bleomycin in human cervical carcinoma cells (HeLa) and Chinese hamster lung fibroblasts cells (V79), and there was a dose-dependent decrease in cell survival 29 These researchers concluded that honeybee venom prevented the repair of damaged DNA after bleomycin treatment through the induction of apoptosis, necrosis and lysis Furthermore, natural killer cell based immunotherapy cells (NK-92MI) were pre-treated with honeybee venom, and then co-cultured with lung cancer cells, which enhanced the anti-cancer effect of the immunotherapy through the inactivation of NF-κB 30.

In terms of the combination with either docetaxel or cisplatin, additive and/or weak synergistic effects were reported between honeybee venom and cisplatin in human cervical

carcinoma (HeLa) and human laryngeal carcinoma (HEp-2) cells in vitro 31 This combination was successful in postponing and reducing the development of tumour cell

resistance to cisplatin The cisplatin-resistant sublines of HeLa and HEp-2 cells (HeLa CK and CK2, respectively) were also assessed in terms of cell viability after honeybee venom treatment The HeLa CK cells were more sensitive to honeybee venom compared to HeLa cells, yet CK2 cells were more resistant to honeybee venom compared to HEp-2 cells 31 This may have been due to genetic and biological variation between the cell types tested In another study, human lung cancer cell lines (A549 and NCI-H460) were treated with honeybee venom combined with docetaxel and cisplatin, and the combination resulted in increased cell death and overexpression of death receptor 3 32 The lethal effects of cisplatin were also potentiated by non-lethal doses of honeybee venom in human ovarian cancer (A2780cp) cells 33 Further, the synergistic effects of combining melittin with cisplatin were confirmed by metabolomic profiling in human ovarian cancer cell lines (both cisplatin-sensitive A2780, and cisplatin resistant A2780CR) 34 Interestingly, the metabolic effects of melittin and cisplatin in combination were very different from that of each agent alone in these cell lines These beneficial combinations of honeybee venom and melittin with anti-cancer compounds such as docetaxel and cisplatin show great potential for a synergistic interaction in the treatment of breast cancer The combination of honeybee venom or melittin with chemotherapeutic agents could also mitigate the upregulation of oncogenic signalling and the subsequent development of drug resistance

4.1.4 Immune system modulation by melittin

The immune checkpoint protein programmed death ligand-1 (PD-L1) reduces the functionality of activated T cells in the immune system PD-L1 is commonly overexpressed on tumour cells, and drugs that reduce PD-L1 levels in cancers are beneficial for reducing adaptive immune resistance 35 Previous reports have shown that melittin selectively reduced the number of M2-like tumour-associated macrophages in the tumour microenvironment in a lung carcinoma mouse model 36 Researchers have also specifically

targeted M2-like tumour-associated macrophages in an in vivo Lewis lung carcinoma model

without affecting other leukocytes such as T cells and dendritic cells 37 They achieved this by conjugating melittin to dKLA, a pro-apoptotic peptide that induces mitochondrial death after cell membrane penetration 37 Therefore, the effects of the combination of melittin and docetaxel on PD-L1 will be assessed

4.2 Aim

While many cancer treatments are now administered in combination, the co-treatment of honeybee or melittin with chemotherapy agents is still under-researched, particularly in breast cancer Assessing these drug combinations is important considering melittin forms transmembrane pores which may enable the passive entrance of chemotherapeutics into cancer cells or target different mechanisms of cancer proliferation The aim of this chapter was therefore to assess whether there is a synergistic interaction when aggressive TNBC cells are co-exposed to honeybee venom or melittin with the chemotherapies docetaxel and

cisplatin in vitro, and to melittin and docetaxel in vivo.

4.3 Methods

4.3.1 Chemical reagents and antibodies

Melittin was synthesised and purified by China Peptides Corporation (Shanghai, China), suspended in phosphate buffered saline (PBS) and stored at -80 ºC CellTiter-Glo 2.0 from the Luminescent Cell Viability Assay was obtained from Promega Docetaxel was obtained from LC Laboratories BD Matrigel Matrix High Concentration was obtained from BD Bioscience D-Luciferin was obtained from Cayman Chemical Acrymount IHC mounting media was obtained from StatLab The anti-Ki-67 mouse monoclonal antibody was obtained from Cell Signaling Technology The anti-PD-L1 antibody [PDL1/2746] was obtained from Abcam The In Situ Cell Death Detection Kit for TUNEL was obtained from Roche TWEEN 80 and Hoechst were obtained from Sigma-Aldrich Unless stated otherwise, the vehicle (negative control) was cell media

4.3.2 Bee venom collection

The honeybee venom used in these experiments was collected in Perth, Australia as described in Section 2.3.2

4.3.3 Cell culture

T11 (murine p53- claudin-low breast cancer) cells were obtained from the University of North Carolina at Chapel Hill and maintained in RPMI 1640 medium with 10% FBS The cells were incubated at 37 °C and 5% CO2 and supplemented with 1% Antibiotic-Antimycotic The media used was manufactured by the Harry Perkins Institute of Medical Research to ATCC specifications

4.3.4 Cell viability assays

Cell viability was determined as described in Section 2.3.4

4.3.5 Production of the primary monoclonal antibody against melittin

The mouse monoclonal anti-melittin antibody was developed at the Monoclonal Antibody Facility at the Harry Perkins Institute of Medical Research as described in Section 2.3.5

4.3.6 Analysis of combined drug effects

T11 cells were plated in 96-well culture plates at a density of 3000 cells/well and incubated at 37 °C and 5% CO2 for 24 hours Honeybee venom or melittin were combined with docetaxel or cisplatin and administered at the concentrations indicated in a non-constant ratio for 24 hours Cell viability was assessed using CellTiter-Glo 2.0 as described in Section 2.3.4 The combined effect of honeybee venom or melittin with docetaxel or cisplatin was assessed by the median dose effect method using CompuSyn software (ComboSyn, Version 1.0) as described in Section 4.3.10 5

4.3.7 Animal model and treatments

All animal experiments were performed in accordance with protocols approved by the Animal Ethics Committee of the University of Western Australia T11 cells were lentivirally transduced with the ZsGreen-luciferase construct and sorted three times to achieve an enrichment greater than 99% luciferase positive cells To simulate an advanced model of claudin-low breast cancer, 250,000 luciferase-positive T11 cells were suspended in serum free media and BD Matrigel Matrix High Concentration in a 1:1 ratio to a total volume of 100 µL and injected subcutaneously into the flanks of five week old BALB/cJ females (Animal Resources Centre, WA, Australia) using a 26G needle Melittin was suspended in Milli-Q water + 5% dextrose Docetaxel (in powder) was suspended in 25% TWEEN 80, and 75% of a mixture of 15.25:84.75 (v/v) solution of absolute ethanol and purified water, respectively, and stored at -20 °C Immediately before the treatments, docetaxel was freshly diluted in Milli-Q water + 5% dextrose at the required final concentration Three days after

the generation of T11 tumours (~50 mm3), mice were randomised into four groups (n=12 mice/group) The treatments were injected intratumourally on days 3, 5, 7, 9, 11, 13 and 15 post-inoculation of T11 cells, with vehicle (Milli-Q water + 5% dextrose), melittin (5 mg/Kg), docetaxel (7 mg/Kg), or a combination of melittin (5 mg/Kg) and docetaxel (7 mg/Kg) Animals were monitored for tumour size every two days and volumes calculated by the modified ellipsoid formula (Volume = width2 x length/2) Animals were humanely sacrificed when the tumours reached 800 mm3

4.3.8 Immunohistochemical analysis of the tumours

Staining and imaging of the tumour samples were performed similar to that previously described 38 Tumour tissues were fixed in 4% paraformaldehyde, washed three times in PBS and left in 70% ethanol Tumours were embedded in paraffin and 5 µm sections were prepared For haematoxylin/eosin staining, slides were dewaxed, hydrated using a decreasing solution bank of ethanol, stained with Gill’s haematoxylin, dehydrated using 70% ethanol, stained with eosin, further dehydrated using 100% ethanol, cleared using toluene and mounted in coverslips using Acrymount IHC mounting media (StatLab) Tumour cell apoptosis was determined in tissue sections by TUNEL assay (In Situ Cell Death Detection Kit; Roche)

4.3.9 Bioluminescence imaging

To accurately track changes in in vivo tumour growth with the treatments, bioluminescence

analysis was performed using the Caliper IVIS Lumina II imaging system at the Centre of Microscopy, Characterisation and Analysis at the University of Western Australia The analyses were conducted every two days after the generation of tumours Mice were injected intraperitoneally with 200 µL of D-Luciferin (Cayman Chemical) at the final concentration of 150 mg/Kg dissolved in PBS prior to being anesthetized at 4% isoflurane Once anaesthetized, mice were placed inside the pre-warmed chamber of the bioluminescence

imager and imaged under 2% isoflurane 7–12 minutes after injection of D-Luciferin, until bioluminescence signal intensity had reached steady state The optimal time of imaging was previously determined by using a reduced number of mice and plotting the curves of bioluminescence signal over 40 minutes

4.3.10 Statistical analysis

All data were derived from multiple experiments conducted at least in triplicate Statistical analyses were performed with GraphPad Prism (GraphPad Software Inc., Version 8), Office Excel 365 (Microsoft, Version 16) and SPSS Predictive Analytics Software (IBM, Version 26) For the cell viability assays, data were normalised to the average luminescence of the vehicle condition (untreated), which were considered 100% viability The IC50s were derived in GraphPad Prism The Combination Index (CI) was determined by the median dose effect method using CompuSyn software (ComboSyn, Version 1.0), which is based on the effect of a combination between two agents (where CI<1 is synergistic, CI>1 is antagonistic, and CI=1 is additive) 5 The CI equation for two drugs (D)1 and (D)2 is:

CI = (D)1/(Dx)1 + (D)2/(Dx)2

(Dx)1 = the dose of (D)1 alone that inhibits a system x% (Dx)2 = the dose of (D)2 alone that inhibits a system x%

(D)1 + (D)2 = dose of (D)1 and (D)2 in combination that inhibit a system x%

For the tumour volume, immunohistochemistry and immunofluorescence, statistical significance was determined using one-way ANOVAs with the Tukey HSD post hoc test correcting for multiple comparisons For the quantification of melittin in the immunohistochemistry, the percentage of melittin positive cells were normalised to vehicle control (considered as 0%) for all treatment groups F values are presented as F df,N For all tests, differences were considered significant at p<0.05 (*), p<0.01 (**) and p<0.001 (***).