Inhibition of breast cancer xenografts in a mouse model and the induction of apoptosis in multiple breast cancer cell lines by lactoferricin B peptide

Abstract Breast cancer has a diverse aetiology characterized by the heterogeneous expression of hormone receptors and signalling molecules, resulting in varied sensitivity to chemotherapy. The adverse side effects of chemotherapy coupled with the development of drug resistance have prompted the exploration of natural products to combat cancer. Lactoferricin B (LfcinB) is a natural peptide derived from bovine lactoferrin that exhibits anticancer properties. LfcinB was evaluated in vitro for its inhibitory effects on cell lines representing different categories of breast cancer and in vivo for its suppressive effects on tumour xenografts in NOD‐SCID mice. The different breast cancer cell lines exhibited varied levels of sensitivity to apoptosis induced by LfcinB in the order of SKBR3>MDA‐MB‐231>MDA‐MB‐468>MCF7, while the normal breast epithelial cells MCF‐10A were not sensitive to LfcinB. The peptide also inhibited the invasion of the MDA‐MB‐231 and MDA‐MB‐468 cell lines. In the mouse xenograft model, intratumoural injections of LfcinB significantly reduced tumour growth rate and tumour size, as depicted by live imaging of the mice using in vivo imaging systems (IVIS). Harvested tumour volume and weight were significantly reduced by LfcinB treatment. LfcinB, therefore, is a promising and safe candidate that can be considered for the treatment of breast cancer.

cancers are categorized as follows: luminal A (ER high , Her2 low ), luminal B (ER low , Her2 low ), Her2-enriched (Her2 + , ER -), basal-like or triple-negative (ER -, PR -, Her2 -) and claudin-low (ER -, Claudin low , E-cadherin low ) types, most of which are associated with a poor short-term prognosis. 2 Her2-positive breast cancer is characterized by Her2 receptor overexpression and dependence on Her2 pathway for survival. The prognosis of Her2-positive breast cancers improved dramatically following the use of monoclonal antibodies such as trastuzumab and kinase inhibitors for therapy. 3 Luminal B type is more aggressive and has worse prognosis than luminal A type. 4 Triple-negative breast cancer (TNBC) is highly aggressive, heterogeneous and the most difficult type of breast cancer to treat, with a 10 year survival rate of 60%-70%. TNBC responds only to chemotherapy due to the absence of hormone receptors. 5,6 Antibody-drug conjugates, adjuvant and neo-adjuvant therapies have significantly increased patient outcome although toxicity, relapses and drug resistance are also reported. 3 Chemotherapy is often accompanied by the development of drug resistance facilitated by multiple resistance mechanisms leading to serious side effects. This has prompted research into the development of natural agents that might help overcome these issues. Many studies have focused on anticancer peptides to explore their potential to kill cancer cells, but most were found to be highly toxic.
Bovine lactoferrin is an iron-binding glycoprotein present in cow milk with antimicrobial, anticancer, and immunomodulatory properties. 7 Administration of lactoferrin in vivo is safe and well-tolerated, and is effective against some cancers. 8 Lactoferricin B (LfcinB) is a 25-amino acid peptide released from lactoferrin by acid-pepsin hydrolysis. Similar to other cationic antimicrobial peptides, LfcinB exhibits antibacterial and antifungal properties. 9,10 as well as anticancer properties. Interference with cell cycle, induction of apoptosis, inhibition of cell migration and immunomodulation are proposed to be the mechanisms of action of lactoferrin and its peptide derivatives against cancer cells. 8 LfcinB and a core peptide derived from Lfcin have been found to induce apoptosis of leukaemic T cells, B lymphoma cells and a gastric cancer cell line. [10][11][12][13] LfcinB also exerted an inhibitory effect against neuroblastoma cells and B lymphoma cells both in vitro and in vivo. 15,16 In MDA-MB-435 cells, LfcinB-induced DNA fragmentation was enhanced by C6 ceramide. 17 Several peptides derived from LfcinB have also been shown to exert anticancer effects. [17][18][19] While these reports confirm the anticancer effects of LfcinB, a focused exploration of the ability of LfcinB to inhibit diverse breast cancer subtypes has yet to be undertaken. The present study found that the inhibitory effects of LfcinB peptide on cell lines representing different categories of breast cancer were varied. LfcinB also had antitumorigenic effect in an in vivo xenograft model of breast cancer in immunodeficient mice. Scientific, Inc), and maintained in a humidified incubator at 37˚C with 5% CO 2 . The MCF-10A cell line was cultured in DMEM/F12 supplemented with 5% horse serum, 0.02% epidermal growth factor, 0.05% hydrocortisone, 0.1% insulin, 1% non-essential amino acids, 1% glutamine and 1% penicillin-streptomycin and maintained at 37˚C with 5% CO 2 . All the components were purchased from Thermo Fisher Scientific, Inc.

| Peptide
LfcinB peptide (FKCRRWQWRMKKLGAPSITCVRRAF) was synthesized by Mimotopes Pty. Ltd. The peptide was dissolved in incomplete DMEM to a working concentration of 1 mg/ml.

| Apoptosis assay
The assay was carried out using the Annexin V-FITC early apoptosis detection kit from Cell Signaling Technology, Inc., following

| Cell invasion assay
Boyden chambers (8 μM pore size) were pre-coated with fibronectin and placed in 24-well companion plates. In the upper chamber, 2 × 10 4 cells were placed in serum-free medium containing various concentrations of LfcinB peptide. Complete DMEM (600 µl) was placed in the lower chamber. After 12 h of incubation, the cells that had invaded into the fibronectin-coated lower surface of the membrane were fixed with methanol, stained with 1% crystal violet and counted using Nikon Eclipse Ti-S inverted microscope at 200X magnification.

| In vivo imaging systems (IVIS)
For live imaging, the mice were anaesthetized by isoflurane (3% induction dose and 1.5%-2% maintenance dose via a precision vaporizer) and injected with 100 µl of diluted XenoLight D-Luciferin-K + salt, a bioluminescent substrate (PerkinElmer, Inc) intraperitoneally (i.p.) at a concentration of 150 mg/kg bodyweight. The mice were placed in the imaging chamber for fluorescent imaging using IVIS ® Spectrum (PerkinElmer, Inc) and imaged ventrally.

| Histological analysis
The harvested tumours were fixed in 10% formalin and embedded in paraffin for histological analysis. The tissue blocks were cut into 4 µm sections using a microtome (RM2255; Leica Biosystems, Inc). Slides were prepared and stained with haematoxylin and eosin (H&E), and images were captured using Nikon Eclipse 80i microscope at 200X and 400X magnifications.

| Statistical analysis
Data were analysed using a Student's t test. Tumour growth curves were compared using a one-way analysis of variance (ANOVA) followed by post hoc Tukey's honest significant difference (HSD).
Differences between treatments were considered significant at the P < .5 probability level.

| Varied sensitivity of different breast cancer cell lines to LfcinB-induced apoptosis
The different breast cancer cell lines exhibited varying degrees of   The normal MCF-10A cells were non-invasive (data not shown).

| Impairment of tumour growth by LfcinB
The tumour growth rate was slower in mice that received intratumoural injections of LfcinB peptide compared to control mice treated with saline ( Figure 4A). The difference in size was apparent from day 7 post-injection, and over time, the gap between the treated and control groups (5 mice/group) became wider. By day 16, while tumour size in the control group reached ~1,000 mm 3 , that of the treatment group remained <500 mm 3 . Throughout the experimental period, the bodyweight of the mice was found to be stable in all the groups ( Figure 4B).
Imaging of live mice using the IVIS ® Spectrum revealed that tumour size and density increased over time in the control group, as depicted by an increasingly intense red colour compared to the LfcinB-treated groups that exhibited a decrease in tumour size, as well as in density. The LfcinB-treated mice had smaller tumours with lower density, as indicated by the shift towards blue colour coupled with a decrease in red colour by day 16 ( Figure 5).
Excised tumour volume and weight exhibited the same decreasing trend. The control group had a mean tumour volume of 1,272 mm 3 , whereas the treatment groups had mean volumes of 319 and 291 mm 3 , respectively ( Figure 6A). Similarly, tumour weight decreased from a mean of 0.81 g in the control group to ~0.23 g in the LfcinB-treated groups ( Figure 6B). Images of the excised tumours revealed a marked difference between the control and treated groups ( Figure 6C).

| Induction of apoptosis in tumours treated with LfcinB
H&E staining of tumour sections from the LfcinB-treated mice revealed an increased number of apoptotic cells compared to the control mice (Figure 7). This indicated that the intratumoural injection

| D ISCUSS I ON
The development of peptides as anticancer agents is aimed at increasing the target specificity to cancer cells while decreasing toxicity and undesirable side effects. Anticancer peptides are generally cationic and exert their toxic effects by interacting with the anionic cancer cell membrane. 21,22 LfcinB is a cationic peptide rich in basic amino acids with a net positive charge of 7.84, 23 and its selective targeting of cancer cells might be attributed to their elevated negative membrane potential. 22 The differential sensitivity of breast cancer cell lines to therapeutic agents has been reported with the antimicrobial peptide Tempoprin-1CEa, while a similar observation with bladder cancer cell lines was made with the peptide SKBR3>MDA-MB-468>MCF7>MDA-MB-231. 27 The SKBR3 cells, which lack functional p53, but have functional caspase-3 were the most sensitive to the drugs used in the present study. In contrast, the MCF7 cells which have functional p53, but not functional caspase-3, were relatively resistant. While the MCF7 cells are responsive to hormone therapy and are associated with the most favourable prognosis, they are relatively resistant to other drugs as well as LfcinB. 28,29 Smaller peptide derivatives of LfcinB have been synthesized and have exhibited anticancer properties. A study using the core peptide A variety of mechanisms have been proposed to underlie the anticancer effects of lactoferrin, LfcinB and its derivatives. A common mechanism proposed by many is the induction of apoptosis, membrane disruption and cell cycle arrest in cancer cells treated with lactoferrin or its derivatives. 35,38 Human lactoferrin was found to arrest the cell cycle at the G1 to S transition phase in MDA-MB-231 cells by inhibiting cyclin-dependent kinases. 36 In another study, lactoferrin was reported to induce cell cycle arrest and inhibit the mTOR signalling pathway, thereby inducing stress, but not apoptosis of breast cancer cell lines. 37 Both lactoferrin and LfcinB were found to induce apoptosis by modulating the expression of pro-apoptotic and anti-apoptotic proteins and caspases in different cancer cell lines, as well as in implanted tumours in animal models. 13,15,16,31,[34][35][36] The induction of reactive oxygen species (ROS) F I G U R E 5 LfcinB reduces tumour size and density as depicted by live imaging of mice by IVIS. Photographs depicting tumour size and density taken using the in vivo imaging systems (IVIS, Perkin Elmer) to detect the luminescence of MDA-MB-231-GFP-luc cells in the tumours. Images were taken on the day of first injection (day 0) followed by day 10 and day 16 post-treatment F I G U R E 6 LfcinB reduces excised tumour volume and weight. Graphs depicting (A) the average volume and (B) the average weight of excised tumours from mice that received intratumoural injections of saline or LfcinB (4 or 5 mg/mouse). *The result is significant at P < .05. (C) Photographs of tumours excised from the different groups of mice was implicated by some studies in the apoptotic pathway induced by LfcinB. 11,12,40 Mitochondrial death pathways and caspases have been proposed to be involved in apoptosis induced by LfcinB. 14,31,41 JNK signalling pathway, which is implicated in mitochondriamediated apoptosis, was induced by LfcinB in oral cancer cells. 42 Anti-angiogenic properties have also been attributed to LfcinB as evident by its anti-VEGF effect and decreased expression of matrix metalloproteases (MMPs). 43 Both lactoferrin and lactoferricin have tumour-suppressing functions. The expression of lactoferrin cDNA effectively reduced tumours derived from breast cancer as well as cervical cancer. 44,45 The direct tumour-suppressive effects of LfcinB have been observed in in vivo studies using animal models. [39][40][41][42] In melanoma and colon carcinoma tumours, disruption of cell membrane, lysis and haemorrhagic necrosis was observed. 46 Administration of LfcinB dendriplexes intravenously suppressed the growth of A431 and B16-F10 tumour xenografts in mice and prevent tumour metastasis. 47,49 The intratumoural injection of a 9-mer peptide, LTX-302, which is a derivative of LfcinB, caused tumour necrosis and tumour infiltration by inflammatory cells followed by complete regression of tumours in a T lymphocyte-dependent manner in immunocompetent BALB/c mouse tumour model. 48 LTX-315, an oncolytic peptide derived from LfcinB, was shown to exert an additive effect when used along with chemotherapy as a combination therapy against triple-negative breast cancers in a mouse model. 50 It was also able to modulate immune response and reprogram the tumour microenvironment, and is being evaluated in clinical trials. 51 Our study has shown that LfcinB is effective against different breast cancer cell lines including triple-negative types and is capable of inducing apoptosis, and inhibiting cell invasion. Intratumoural administration of LfcinB caused significant reduction in tumour burden in vivo. Furthermore, it specifically targeted cancer cells without affecting the normal cells. These properties make LfcinB a promising natural product to be safely considered as a candidate for combination therapy against breast cancer. It can help minimize the dose of chemotherapeutic drugs thereby mitigating the side effects and reduce the overall cost of cancer treatment by serving as a cheaper alternative. Further studies are to be undertaken to evaluate this peptide for treatment of human breast cancer.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated and analysed during this study are included in this article. Further details are available upon request.