A novel antimicrobial peptide M1- 8 targets the lysosomal pathway to inhibit autolysosome formation and promote apoptosis in liver cancer cells

Lysosomes, a central regulator of autophagy, play a critical role in tumour growth. Lysosomal protease cathepsin D can initiate apoptosis when released from lysosomes into the cytosol. In this study, we observed that Musca domestica cecropin (Mdc) 1– 8 (M1- 8), a small anti- tumour peptide derived from Mdc, inhibits hepatoma cell growth by blocking autophagy– lysosome fusion. This effect is likely achieved by targeting lysosomes to activate lysosomal protease D. Additionally, we examined whether lysosomal content and cathepsin D release were involved in M1- 8- induced apoptosis. After exposure to M1- 8, human hepatoma HepG2 cells rapidly co- localized with lysosomes, disrupted lysosomal integrity, caused leakage of lysosomal protease cathepsin D, caspase activation and mitochondrial membrane potential changes; and promoted cell apoptosis. Interestingly, in M1- 8- treated HepG2 cells, autophagic protein content increased and the lysosome– autophagosome fusion was inhibited, suggesting that M1- 8 can cause apoptosis through autophagy and lysosomes. This result indi-cates that a small accumulation of autophagy and autolysosome inhibition in cells can cause cell death. Taken together, these data suggest a novel insight into the regulatory mechanisms of M1- 8 in autophagy and lysosomes, which may facilitate the development of M1- 8 as a potential cancer therapeutic agent.


| INTRODUC TI ON
Liver cancer is a global health problem, representing the sixth most frequent malignancy in terms of incidence and the fourth most common cause of cancer-related deaths worldwide. The major risk factors that contribute to hepatocellular carcinoma (HCC) are hepatitis B or C virus infection, alcohol abuse, intake of the microbial metabolite aflatoxin B1 and non-alcoholic fatty liver disease (NAFLD).
Conventional chemotherapeutic agents (e.g., doxorubicin, fluoropyrimidines, platinum derivatives and irinotecan) are inefficient in HCC treatment and do not improve patient survival. 1,2 Thus, development of novel strategies and anti-tumour agents that can overcome cancer drug resistance and achieve enhanced efficacy is the major aim of current cancer research, including HCC.
Peptides are naturally produced by all organisms and exhibit a wide range of physiological, immunomodulatory and wound healing functions. 3 Over the past few years, some studies have demonstrated that novel synthetic antimicrobial peptides can effectively inhibit growth and induce apoptosis in a wide variety of human malignant cells. 4 Antimicrobial peptides (AMPs) are small molecules with effective tissue penetration, which can infiltrate the lipid bilayer membrane and cause breakdown of the transmembrane electrochemical gradient, resulting in the loss of energy for water and ion transport across the membrane, eventually promoting cell shrinkage and lysis, and thereby inhibiting the growth of cancer cells and bacteria. 4,5 The indirect mechanism involves the entry of peptides into the cell without disturbing the membrane, inhibiting protein synthesis or damaging the mitochondrial membrane, which results in the activation of the apoptotic pathway-mediated by caspases. 6 AMPs with dual antimicrobial and anticancer activities are promising therapeutic agents that can be used to combat HCC as a standalone treatment or as part of a synergistic treatment regimen. 7 However, the use of peptides has limitations, including a short plasma halflife, haemolysis and immunogenicity. 8 Rational design of antimicrobial peptides can improve their stability and safety. Furthermore, the rational design of sequences for anticancer peptides involves fragmentation, which is a type of alteration of the original sequence into shorter sequences. For example, the 10 amino acids at the Nterminus of cecropin can be used and repeated three times to create a CB1 peptide. 9 It is also expected that shorter peptides can reach the membrane phospholipid bilayer more efficiently and reduce the polypeptides cytotoxicity.
Musca domestica cecropin (Mdc) is a linear molecule containing 40 amino acids, with three α-helical structures appearing at residues 1-6, 9-21 and 27-39. Previous studies have demonstrated that Mdc has strong antibacterial and anticancer activity. [10][11][12] The peptide's structure may restrict its internal properties and contribute substantially to production costs. To obtain a more economical, more effective, simpler, less toxic, more selective or more stable anticancer peptide, we simplified Mdc appropriately by retaining the sequence related to its anticancer activity.
M1-8 (G-W-L-K-K-I-G-K) is derived from the N-terminal 1-8 amino acids of Mdc. However, whether M1-8 has an effective inhibitory effect on hepatoma cells is unclear. Furthermore, the precise inhibition mechanisms of M1-8 remain unknown. Therefore, in this study, we aimed to investigate the effects and mechanisms of M1-8 on liver cancer in vitro and in vivo. Doxorubicin hydrochloride (Dox) at over 98% purity was purchased from MP Biomedicals LLC. The apoptosis inhibitor Z-VAD was purchased from APExBIO.

| Cell culture
HepG2, L02, Chang liver and NCM460 cells were purchased from the China Center for Type Culture Collection. The cells were cultured in Dulbecco's modified eagle medium (DMEM) or 1640 medium (Gibco) containing 10% heat-inactivated fetal bovine serum (FBS) in a humidified CO 2 incubator (5% CO 2 in air) at 37°C.

| MTT Assay
The viability of HepG2, L02 and NCM460 cells treated with the peptides was determined using the MTT assay. The cells were seeded in 96-well plates at a density of 4 × 10 3 cells/well. Then, 100 μl of peptide solutions of different concentrations were introduced into the wells, followed by further incubation for 24 h. Subsequently, 20 μl of MTT (5 mg/ml) was added to each well. After culturing for 4 h,

| Cell migration assay
On the outsole of the six-well plate, four lines were evenly spaced with a marker pen at 1-cm intervals. HepG2 cells in the logarithmic growth phase were inoculated in a six-well plate and cultured to 90% confluent state. The cells were cultured in a serum-free medium for 24 h. The plates were scratched using a 10 μl pipette tip with a pattern of two lines perpendicular to each other. After 0, 24 and 48 h, the cells were observed and photographed under a microscope (Zeiss). The distance of scratches was then calculated, and the changes in migration distance were compared. 13

| Cell invasion assay
Matrigel (Corning) was carefully added to each well at 37°C for

| Cell apoptosis assay via flow cytometry
Annexin V (MultiSciences) was used to assess phosphatidylserine exposure, and propidium iodide (PI, MultiSciences) was used for cell viability assessment. The treated cells were harvested and stained using the Annexin V-FITC/PI apoptosis kit or PI according to the manufacturer's instructions. 15,16 Cell apoptosis was analysed via flow cytometry (Bio-Rad). An equal volume of saline was injected into the control mice. The length (L) and width (W) of the tumours were measured every 2 days.

| Inhibition of tumour growth in vivo
Tumour volume was calculated using the formula: The mice were sacrificed on the second day after the last administration, and the tumours were removed for immunohistochemical analysis. The tumour inhibition rate was calculated as follows:

| Laser confocal detection of drug localization
The organelles were stained using MitoTracker® Red CMXRos and LysoTracker Red according to the manufacturer's instructions. Colocalization with FITC-labelled M1-8 was observed using a laser scanning confocal microscope (NikonTi-EA1).

| Immunofluorescence staining and fluorescence microscopy
Cells were seeded on 6-mm round glass coverslips and placed at the bottom of 24-well plates. After M1-8 treatment, cells on glass coverslips were fixed with cold methanol/acetone (1:1) for 5 min, followed by incubation with 10% BSA in PBS and 0.5% Triton X-100 for 20 min. After washing, the cells were incubated with primary antibodies and immunostained with fluorescent-labelled secondary antibodies. The DNA was counterstained with DAPI for 15 min at 25°C. Fluorescence images were captured using a fluorescence microscope (Zeiss).
The intralysosomal pH was estimated using LysoTracker as per the manufacturer's instructions. 17 The fluorescence intensity was observed under a fluorescence microscope (Leica, Switzerland) and (1) The cells were fixed with 4% paraformaldehyde for 20 min.
Then, the cells were stained with Hoechst 33258 (2 μg/ml) in the dark for 15 min at 25°C. All fluorescent images were captured using a fluorescence microscope and quantified using Image J software.

| Transmission electron microscope assay
After treatment with M1-8 (400 μg/ml) for 24 h, the cells were pelleted via centrifugation and diced into blocks <1 mm 3 , which were placed in fresh fixative (1% OsO 4 ) for 2 h at 4°C. Subsequently, the blocks were dehydrated using an ascending series of alcohol solutions. The samples were stained with 2% uranyl acetate for 30 min.
The samples were then embedded in an epoxy resin overnight at 60°C. Following polymerization, the blocks were cut into 0.5-mm thick slices, stained with 1% toluidine blue and observed using a JEM1400 (Jeol) transmission electron microscope. 18

| Quantitative reverse transcription PCR (qPCR)
After administration of M1-8 or Ah for 48 h, the cells in all groups were collected to determine the mRNA level. All primers were purchased from the Invitrogen Trading Company. The primer sequences are shown in Table 1. The RNAiso Plus extraction method was used to extract total RNA from the cells (TaKaRa). Reverse transcription was performed using a PrimeScript RT Reagent Kit (TaKaRa). 16

| Isolation of lysosomes
Lysosomes were isolated using the Lysosome Enrichment kit according to the manufacturer's instructions (Thermo-Fisher Scientific, USA). Cathepsin D proteins within lysosomes were detected by Western blot. for protein level quantification; appropriate film exposures were scanned. The density of bands was determined with ImageJ and normalized to band intensity for GAPDH or β-Actin.

| Histology and immunohistochemistry assays
The excised tumours were fixed in 4% paraformaldehyde, paraffinembedded, sectioned at 5 μm thickness and prepared for H&E staining or tumour proliferation antigen P53 and Caspase-3 assays.
Immunohistochemical staining was performed according to the manufacturer's instructions. The heart, liver, spleen, lung, kidney and tumour tissues of the mice bearing orthotopic xenograft tumours were harvested at the end of the experiment. The samples were excised, fixed with 10% neutral phosphate-buffered formalin and embedded in paraffin. Continuous sections (5 μm thick) were obtained and stained with H&E for histomorphometric analysis.

| Statistical analysis
Data are presented as mean ± standard errors. Statistical significance was analysed using the unpaired two-tailed Student's t-test for at least three independent experiments using GraphPad Prism (GraphPad Software). The p-value <0.05 was considered statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001.

| The effect of M1-8 on proliferation, migration and invasion of cells
We synthesized a novel derivative of Mdc, that is, M1-8. Thereafter, we tested the inhibition of cell growth in three human cell lines

| Effect of antimicrobial peptides M1-8 on autophagy in HepG2 cells
Under fluorescence microscopy and laser confocal microscopy, the green fluorescence-labelled LC3 protein in the M1-8 group showed an aggregate spot-like distribution. In contrast, the green fluorescence in the negative control group showed a diffuse distribution.
The green fluorescence labelled LC3 protein in the M1-8 group showed an aggregate spot-like distribution, whereas the green fluorescence in the negative control group showed a diffuse distribution ( Figure 4A). The fluorescence intensity in the drug administration group was considerably stronger than that in the negative control group, indicating that M1-8 could increase LC3 II protein expression.
Laser confocal microscopy revealed the green puncta formation by LC3 of the M1-8 group ( Figure 4B).
The microstructure of HepG2 cells was observed using a transmission electron microscope (TEM) ( Figure 4C). The cell membrane of the negative control group was intact, the nucleus was regular and intact, the structure of each organelle was not abnormal, the cytoplasmic distribution was even, and no obvious vacuoles ap- indicating that the M1-8 mechanism of action is related to autophagy ( Figure 4E).

| Effects of M1-8 on autophagic flux in HepG2 cells
As shown in Figure 4D,

| In vivo anti-tumour efficacy
The results of the experiments are shown in Figure 6A.

| CON CLUS IONS
In summary, the advantages of using the derivative M1-8 from Mdc include, but are not limited to, the following: First, M1-8 improved

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Our data has not been shared previously and is not under consideration for publication elsewhere, in whole or in part.