The Mechanism Study and Target Modifications on the AMP

Temporin-PEa Triggering TNF-ɑ Necroptosis Pathway in Lung

Cancer Cell Death

Jiewen Zheng

School of Pharmacy, Queen’s University Belfast, Belfast, U.K.

Keywords: Temporin-Pea, Lung Cancer, TNF, Necroptosis Pathway, Antimicrobial Peptides, Target Modifications.

Abstract: NSCLC is the most typical lung cancer with one of the highest lethal rates in the world. However, the drug

resistance and poor prognosis make the conventional therapies less effective. A previous study has reported

that a novel AMP temporin-PEa shows anticancer activity on NSCLC, which provides more potential for lung

cancer treatment. This study investigates the antiproliferation mechanism of temporin-PEa, as well as the

therapeutic effect of this AMP, and two other modified versions in both in vitro and in vivo conditions. The

experiments will utilize NSCLC cell lines A549 and NCI-H157, and HMEC-1 cell line, and Xenograft mice

models. Antiproliferation activity is measured by MTT assay. Cytotoxicity is detected by LDH assay, and

haemolytic activity is detected using horse blood. The TNF-α necroptosis will be measured by Annexin V-

FITC/PI assay and western blot. ROS over-generation will be measured by ROS assay kit with H2DCFDA

ROS probe. The results of this study will provide important information for the exploration of the anticancer

mechanism of AMPs and target modifications improving drug therapeutic values. Further studies should

mainly focus on exploiting the specific target of temporin-PEa for further anticancer drug development, and

in vivo drug delivery system design, which could help the drug overcome various biological barriers.

1 INTRODUCTION

Non-small-cell lung cancer (NSCLC) accounts for

85-90% of all lung cancers, and is one of the leading

causes of cancer death in the world (R.L. Siegel, K.D.

Miller, A. Jemal, Cancer Statistics, 2017). However,

the poor prognosis and resistance to conventional

chemotherapy and radiotherapy have greatly

motivated the development of novel efficacious

therapeutics in treating this lethal disease (Liu, Pei,

Yang, Li, Amin, Liu, Buchan, Cho 2017).

Antimicrobial peptides (AMPs), which are

commonly believed to show strong antimicrobial

activity against a broad spectrum of microorganisms,

have been reported to represent cell-line-dependent

anticancer activity (Tornesello, Borrelli, Buonaguro,

Buonaguro, Tornesello 2020). Compared to normal

cells, cancer cell membranes exhibit more anionic,

according to the changes of the tumor

microenvironment, which might lead to

dysregulation of phospholipid transporters (Ran,

Downes, Thorpe 2002). Therefore, the electrostatic

attraction between the anionic phospholipids of

cancer cell membranes and the cationic peptides

would play a vital role in the peptide-membrane

interaction (Hoskin, Ramamoorthy 2008).

A recently discovered AMP temporin-PEa,

generated through modification on a natural-derived

peptide temporin-PE, has been reported to represent

strong antiproliferation activity on lung cancer cells

and low cytotoxicity on mammalian cells (Sang, Wu,

Xi, Ma, Wang, Zhou, Burrows, Chen 2018). This

enables temporin-PEa to become a proper candidate

with high therapeutic effects for further drug

development. However, the molecular mechanisms

by which this peptide induces tumor cell death still

remain poorly understood.

Phosphatidylserine (PS), accounting for 3-9% of

the total amount of phospholipids, is negatively

charged and normally exists on the inner leaflet of the

cell membrane, and could be transferred to the outer

membrane according to apoptosis or mutations in

cancer cells (Birge et al 2016, Shklyar, Levy-Adam,

Mishnaevski, Kurant 2013). Based on the literature,

the outer membrane exposure of PS could be

mediated by Tumour Necrosis Factor (TNF), which

Zheng, J.

The Mechanism Study and Target Modiﬁcations on the AMP Temporin-PEa Triggering TNF- Necroptosis Pathway in Lung Cancer Cell Death.

DOI: 10.5220/0011390000003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 1221-1229

ISBN: 978-989-758-595-1

1221

is significantly important for cellular homeostasis,

and is a master regulator in cell necroptosis (Parvy,

Yu, Dostalova, Kondo, Kurjan, Bulet, Lemaitre,

Vidal, Cordero 2019, Kalliolias, Ivashkiv 2016).

Necroptosis is a regulated cell death program without

caspase activation, and it is mainly mediated through

Receptor-Interacting Protein Kinase 1/3 (RIPK1/3)

and Mixed Lineage Kinase Domain-Like

(MLKL)(Gong et al 2019). Necroptosis plays a

pivotal role in oncogenesis, cancer metastasis, and

cancer immunity (Seehawer 2018). In the necroptosis

signaling pathway, the interaction of TNF-α and

TNFR would induce a downstream signaling cascade,

followed by energy depletion due to reactive oxygen

species (ROS) over-generation, and ultimately leads

to cell death (Figure 1) (Christofferson, Yuan 2010,

Vanden Berghe 2014).

Figure 1: Schematic of TNF-α-induced necroptosis

pathway and ROS accumulation.

TRADD: TNF receptor-associated death domain;

FADD: Fas-associated cell death domain.

Therefore, I hypothesize that temporin-PEa

exhibits certain antiproliferation activity on NSCLC

via the TNF-α-induced necroptosis pathway and ROS

over-generation. In this study, the molecular

mechanisms of temporin-PEa inducing NSCLC cell

death are investigated. Also, the bioactivities of

temporin-PEa and two other modified peptides are

studied, in both in vitro and in vivo occasions.

2 MATERIALS AND METHODS

2.1 Peptides Design and Synthesis

In order to improve the cell-penetrating activity and

better investigate the intracellular mechanism of

temporin-PEa, two analogues are designed. The RGD

peptide (Arg-Gly-Asp) and the TAT

(GRKKRRQRRR) peptide have been reported to

effectively deliver drugs as the cell-penetrating

peptide (CPP) in treating lung cancers, as described

previously (Duan et al 2017, Diao et al 2012).

Therefore, RGD is added to the N-terminus of the

template peptide, generating the novel peptide RGD-

temporin-PEa (RGD-PEa). Meanwhile, the other

analogue is designed by linking the TAT sequence to

the C-terminus of the original peptide, namely TAT-

PEa. The specific sequences are listed in Table 1.

Table 1: Anticancer peptide sequences.

Peptide Sequence

Temporin-PEa FLYIVAKLLSGLL-NH

RGD-PEa RGD- FLYIVAKLLSGLL-NH

PEa-TAT FLYIVAKLLSGLL-

GRKKRRQRRR-NH

*-NH

: C-terminal amidation

Temporin-PEa and the other modified peptides

will be synthesized using a Tribute 2-channel

automated peptide synthesizer through solid-phase

peptide synthesis. Briefly, the process will employ

resin with rink amide and standard Fmoc protection

chemistry, and will be performed in the PS4 peptide

synthesizer. Then the crude peptides will be purified

by the RP HPLC and then characterized by MALDI-

TOF MS.

2.2 Cell Lines, Cell Culture, and

Chemicals

Human NSCLC cell lines A549, NCI-H157 will be

obtained and cultured in the medium of DMEM/F12

with 10% FBS and 1% penicillin-streptomycin.

Human microvascular epithelial cell line HMEC-1

will be cultured in a full-growth MCDB-131 medium

with 10 mM L-Glutamine as well as 10 ng/mL of

epidermal growth factor. The specific cell lines are

shown in Table 2.

Table 2: Cell lines applied in this study.

Cell line Cell Type

NCI-H157 Human Non-small cell lung cancer cell

A549 Human Non-small cell lung cancer cell

HMEC-1 Human microvascular epithelial cell

2.3 MTT Cell Proliferation Assay

3-(4, 5-Dimethylthiazol-2-yl)-2, 5-

diphenyltetrazolium bromide (MTT) cell viability

assay is utilized to evaluate the antiproliferation

activity of temporin-PEa, and its analogues.

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Firstly, the cells with the amount of 5× 103 cells

per well will be plated in the 96-well plate and

cultured under 5% CO2 at 37°C for 24 h. The cells

will be then starved by the medium without FBS for

6 h, followed by treatment with three peptides

gradient dilutions (10-9-10-4 M) for 24 h.

Meanwhile, PBS solution will be used in the vehicle

control group, and 1% Triton X-100 will be utilized

as the positive control. After incubation for another

24 h, a total amount of 10 μL MTT with the

concentration of 5 mg/mL will be added to each well

and co-incubated for 4 h at the conventional

incubator. Finally, after removing the medium, a

certain amount of DMSO will be added to each well,

and the optical density (OD) value of each well will

be subsequently measured by a Synergy HT plate

reader at the wavelength of 490 nm. After the

experiment, the half-maximal inhibitory

concentration (IC50) values of three peptides will be

determined by GraphPad Prism Software. Also, the

Geometric Mean (GM) of each peptide inhibiting

cancer cell growth is calculated.

2.4 Lactate Dehydrogenase Release

(LDH) Assay

LDH will release from the damaged cells, and the cell

membrane integrity will be measured to estimate the

cytotoxicity of the three peptides on normal cells. The

degree of LDH release from destroyed cells after

peptide treatment will be determined by the Pierce

LDH Cytotoxicity Assay Kit. In brief, cells with a

density of 5 × 103 each well will be seeded into 96-

well plates and incubated for 24 h. The cells will be

treated with a range of doses (10-9-10-4 M) of the

three peptides or PBS solution for 24 h incubation as

sample groups and negative control group,

respectively. Also, in the positive control group, cells

will be mixed with 1% Triton X-100 and then

incubated at 37°C under 5% CO2 for 30 min to

acquire a relatively maximum LDH release. After the

incubation, the cell supernatant of each group will be

transferred to another 96-well plate, and the reaction

buffer will be added with incubation for about half an

hour at room temperature. At last, the stop solution

will be added, and the OD value will be determined

at the wavelength of 490 nm. Also, the therapeutic

index (TI) of each peptide will be calculated as the

ratio of the IC50 value for the HMEC-1 cell line to

the GM value for two NSCLC cell lines.

2.5 Haemolysis Assay

The haemolytic effect of three peptides will be

determined using defibrinated horse red blood cells.

A total volume of 100 μL peptide serial dilutions,

ranging from 1 to 128 μM, will be incubated with an

equal volume of 4% erythrocytes for 120 min at

37°C. The positive control and negative control

groups will contain the same volume of 2% Triton X-

100 and PBS solution, respectively. Then, the OD

value of each well will be measured at 550 nm.

2.6 Annexin V-PI Assay

Cancer cells (1×106) will be treated with peptide

solution at half IC50, IC50, and 2×IC50

concentrations. Z-VAD-FMK, the pan-caspase

inhibitor, followed by TNF-α, and PBS solution will

be utilized in the positive control the negative control

group, respectively. The Annexin V-FITC/PI assay

will be conducted based on the protocol, as reported

previously (Yu et al 2019). Briefly, after incubation

for 24 h, cells will be rinsed with PBS solution,

trypsinized, and resuspended in the binding buffer.

Then, the cell suspension will be incubated with

Annexin V-FITC and propidium iodide (PI) for 5 min

at room temperature in the dark. The cells will then

get analyzed for morphological changes and DNA

content by flow cytometry with a ZE5 Cell Analyzer.

2.7 Western Blot Analysis

Harvested cells will be lysed with 400 μl of lysis

buffer. Protein concentration will be determined

according to the Bradford method. A certain amount

of protein extraction will be loaded into SDS gels to

separate the protein components. After the membrane

transfer, the blotted membrane will be blocked with

skim milk for 60 min at room temperature and

incubated with the appropriate primary antibody for

10 h at 4˚C. At last, HRP will be coupled with

appropriate secondary antibodies. After 60 min,

signals from the combined antibodies will be detected

by using the chemiluminescence kit.

2.8 ROS Accumulation Detection

The ROS assay kit will be used in this section. A total

amount of cells with a density of 5×105 per dish (10

cm) will be incubated for 24 h, and then will be

treated with the peptide solution at half IC50, IC50,

and 2×IC50 for a specific time length. Ten microliters

of the ROS probe H2DCFDA will be added to the

cells suspension after PBS-rinsing, and incubated for

The Mechanism Study and Target Modiﬁcations on the AMP Temporin-PEa Triggering TNF- Necroptosis Pathway in Lung Cancer Cell

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at 37˚C, 5% CO2 for half an hour in the dark. Then,

cells will be treated with trypsin and instantly

analyzed for DCF fluorescence intensity by flow

cytometry. Hydrogen peroxide (H2O2) dilution and

PBS will be used as the positive control and negative

control respectively.

2.9 In Vivo Antiproliferation Assay

The cell line that peptides exhibit a stronger

anticancer level will be chosen to conduct the in vivo

assay. Also, the peptide with the highest TI value and

no significant haemolytic activity will be selected for

the in vivo assay.

In vivo antiproliferation will be detected by

subcutaneous injection of a total amount of 2×106

cancer cells in the right flank of the seven-week-old

BALB/c-nu/nu nude mice, based on the Guide for the

Care and Use of Laboratory Animals published by the

US NIH. When tumor volumes reach 200 mm3, mice

will be divided into six groups, including a negative

control group treated with PBS solution, a positive

control group treated with Cisplatin (DDP), and three

doses of sample groups treated with a high, medium,

and low concentration of peptide solutions,

respectively. Moreover, mice with no tumor will be

treated as the blank group.

Mice of peptide groups will receive intratumoral

injections on a daily basis, with a volume of 20 μL of

peptide solutions with different concentrations for

two weeks. Additionally, mice in the negative control

group will be daily treated with 20 μL PBS solution,

while the mice in the positive control group will

receive the seven-day intraperitoneal injections with

200 μL DDP and then will be continuously fed until

day 14. Tumour growth inhibition will be then

detected by tumor size measurement twice a week

using a digital caliper.

2.10 Statistical Analysis

Each experiment will be repeated five times. And the

statistical significance of data acquired will be

analyzed with the student’s T-Test or one-way

ANOVA on GraphPad Prism software at (p <0.05).

3 RESULTS

3.1 Possible Results on the Mechanisms

of the Peptide Temporin-Pea

Inducing Lung Cancer Cell Death

(The Overview of Four Possible

Results is Demonstrated in Table

3).

Possible Result 1: Temporin-PEa induces lung

cancer cell death via the TNF-α necroptosis

pathway and ROS over-generation.

Based on western blot analysis, there is a

significant up-regulation of the expression level of

TNF-α, phosphorylated MLKL (pMLKL), pRIPK1,

and pRIPK3, and no significant changes in the level

of the procaspase-8 as well as the cleaved caspase-8

expression. Also, ROS accumulation is detected in

the peptide-treated cells.

Possible Result 2: Temporin-PEa induces lung

cancer cell death via the TNF-α necroptosis

pathway, but not ROS over-generation.

Based on western blot analysis, there is a

significant up-regulation of the expression level of

TNF-α, pMLKL, pRIPK1, and pRIPK3, and no

significant changes in the level of the procaspase-8 as

well as the cleaved caspase-8 expression. However,

ROS accumulation is not detected.

Possible Result 3: Temporin-PEa induces lung

cancer cell death through ROS over-generation,

but not the TNF-α necroptosis pathway.

After the treatment of the peptide, the expression

levels of TNF-α, pMLKL, pRIPK1, and pRIPK3 have

not been increased significantly, and/or there are

significant changes in the expression level of

procaspase-8 and cleaved caspase-8. However, ROS

accumulation is detected.

Table 3: Possible results on the mechanisms of the peptide temporin-PEa inducing lung cancer cell death.

Result 1 Result 2 Result 3 Result 4

TNF-α + + - -

pRIPK 1/3 + + - -

pMLKL + + - -

Procaspase 8 - - + +

Cleaved caspase 8 - - + +

ROS over-generation + - + -

Note. “+” represents a significant increase from the negative control. “-” represents there is no significant difference from the

negative control.

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Possible Result 4: Temporin-PEa induces lung

cancer cell death via other signalling pathways,

instead of the TNF-α necroptosis pathway and

ROS over-generation.

After the treatment of the peptide, the expression

level of TNF-α, phosphorylated RIPK1,

phosphorylated RIPK3, and phosphorylated MLKL

has not been increased significantly, and/or there are

significant changes in procaspase-8 and cleaved

caspase-8 expression. Also, ROS accumulation is not

detected.

3.2 Possible Results on the Therapeutic

Index (TI) of the Peptides (The

Overview of Four Possible Results

is Demonstrated in Table 4).

Possible Result 5: RGD-PEa shows the highest TI

value, while PEa-TAT and temporin-PEa show

the second and the third TI value respectively.

According to the peptide antiproliferation activity

on lung cancer cells and cytotoxicity to normal cells,

the peptide RGD-PEa exhibits the highest TI value

among the three peptides, while peptide PEa-TAT

shows the second TI value, and temporin-PEa shows

the lowest.

Possible Result 6: RGD-PEa shows the highest

TI value, while temporin-PEa and PEa-TAT show

the second and the third antiproliferation activity

respectively.

Based on the antiproliferation activity and

cytotoxicity of the peptide, RGD-PEa exhibits the

highest TI value among the three peptides, while

temporin-PEa shows the medium TI value, and PEa-

TAT shows the lowest.

Possible Result 7: PEa-TAT shows the highest

TI value, and RGD-PEa and temporin-PEa show

the second and the third TI value respectively.

Based on the antiproliferation activity and

cytotoxicity of the peptide, PEa-TAT exhibits the

highest TI value among the three peptides, while

peptide RGD-PEa shows the second TI value, and

temporin-PEa shows the lowest.

Possible Result 8: PEa-TAT shows the highest

TI value, and temporin-PEa, as well as RGD-PEa

shows the second and the third TI value

respectively.

According to the peptide antiproliferation activity

on lung cancer cells and cytotoxicity to normal cells,

the peptide PEa-TAT exhibits the highest TI value

among the three peptides, while temporin-PEa shows

the medium level of TI value, and RGD-PEa shows

the lowest.

Table 4: Possible results on the TI values of the three peptides.

Result 5 Result 6 Result 7 Result 8

RGD-PEa +++ +++ ++ +

PEa-TAT ++ + +++ +++

Temporin-PEa + ++ + ++

Note. “+” represents the peptide with the lowest TI value, “++” represents the peptide with the medium TI value, and “+++”

represents the peptide that shows the highest TI value

3.3 Possible Results on The Haemolytic

Activity of The Peptides (The

Overview of Four Possible Results

is Demonstrated in Table 5).

Possible Result 9: None of the peptides shows

significant haemolytic activity.

Compared with the negative control, all of the

three peptides display no significant haemolytic

activity on mammalian red blood cells.

Possible Result 10: RGD-PEa and temporin-PEa

both show no significant haemolytic activity, while

PEa-TAT shows significant haemolytic activity.

Compared with the negative control, the peptide

RGD-PEa and temporin-PEa both display no

significant haemolytic activity on red blood cells.

However, PEa-TAT exhibits significant haemolytic

activity.

Possible Result 11: PEa-TAT and temporin-

PEa both show no significant haemolytic activity,

while RGD-PEa shows significant haemolytic

activity.

Compared with the negative control, the peptide

RGD-PEa and temporin-PEa both display no

significant haemolytic activity. However, PEa-TAT

exhibits significant haemolytic activity.

Possible Result 12: Both PEa-TAT and RGD-

PEa show significant haemolytic activity, and only

temporin-PEa shows no significance with the

negative control.

Both of the modified peptides exhibit significant

haemolytic activity, and only the original peptide

temporin-PEa shows no significance with the

negative control group.

The Mechanism Study and Target Modiﬁcations on the AMP Temporin-PEa Triggering TNF- Necroptosis Pathway in Lung Cancer Cell

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Table 5: Possible results on the haemolytic activity of three peptides.

Result 9 Result 10 Result 11 Result 12

RGD-PEa - - + +

PEa-TAT - + - +

Temporin-PEa - ---

Note. “+” represents a significant difference in haemolytic activity from the negative control. “-” represents there is no

significant difference from the negative control.

3.4 Possible Results on the In Vivo

Antiproliferation Activity of the

Peptides (The Overview of Four

Possible Results is Shown in Table

6).

Possible Result 13: The chosen peptide represents

stronger antiproliferation activity on the NCI-

H157 cancer cell line, and induces significant lung

cancer cell death in vivo.

In the in vitro detection, the chosen peptide shows

stronger antiproliferation activity on the NCI-H157

cancer cell line, which is used to establish in vivo

models. Also, the peptide induces significant lung

cancer cell death in the animal model.

Possible Result 14: The chosen peptide

represents stronger antiproliferation activity on

the NCI-H157 cancer cell line, but does not show

significant in vivo antiproliferation activity on

lung cancer.

In the in vitro detection, the chosen peptide shows

stronger antiproliferation activity on the NCI-H157

cancer cell line, which is used to establish in vivo

models. However, the peptide does not induce

significant lung cancer cell death in the animal model.

Possible Result 15: The chosen peptide

represents stronger antiproliferation activity on

the A549 cancer cell line, and induces significant

lung cancer cell death in vivo.

In the in vitro detection, the chosen peptide shows

stronger antiproliferation activity on the A549 cancer

cell line, which is used to establish in vivo models.

Also, the peptide induces significant lung cancer cell

death in the animal model.

Possible Result 16: The chosen peptide

represents stronger antiproliferation activity on

the A549 cancer cell line, but does not show

significant in vivo antiproliferation activity on

lung cancer.

In the in vitro detection, the chosen peptide shows

stronger antiproliferation activity on the A549 cancer

cell line, which is used to establish in vivo models.

However, the peptide does not induce significant lung

cancer cell death in the animal model.

3.5 Additional Possible Results on the

Temporin-Pea and Modification

Peptides Different from Previous

Researches

Possible Results 17: Compared with temporin-

PEa, modified peptides RGD-PEa and PEa-TAT

both show lower TI values.

Temporin-PEa shows the highest TI value, while

the other two modification strategies decrease the PI

value of the original peptide.

Possible Results 18: The chosen peptide shows

no significant difference in antiproliferation

activity between the two cancer cell lines.

In the in vitro detection, the chosen peptide shows

no significant difference in the anticancer activity

between NCI-H157 and A549 cancer cell lines. In

this case, cell line NCI-H157 is used for mechanism

study and in vivo detection.

Table 6: Possible results on the in vivo antiproliferation activity of the chosen peptide.

Result 13 Result 14 Result 15 Result 16

Cell line for xenograft model NCI-H157 NCI-H157 A549 A549

In vivo antiproliferation + - + -

Note. “+” represents a significant difference in antiproliferation activity from the negative control. “-” represents there is no

significant difference from the negative control.

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4 DISCUSSION

Previous studies report that AMPs show certain

anticancer activity in a cell-line-dependent manner

(Tornesello, Borrelli, Buonaguro, Buonaguro,

Tornesello 2020). An AMP temporin-PEa has been

reported to present strong antiproliferation activity on

human lung cancer cells (Sang, Wu, Xi, Ma, Wang,

Zhou, Burrows, Chen 2018). To unravel the

molecular mechanisms of this peptide-inducing lung

cancer cell death, this study further investigates

whether temporin-PEa would trigger the TNF-α

necroptosis signaling pathway and ROS

accumulation. Also, in order to assess potential target

modification strategies to improve the therapeutic

value of the peptide, the in vitro bioactivity assays are

designed, and the peptide with no significant

haemolytic activity and relatively higher TI value is

used for xenograft animal model detection.

Possible Result 1 shows that temporin-PEa would

induce lung cancer cell death through the TNF-α

necroptosis pathway and ROS accumulation, which

fully supports the hypothesis of this study. According

to the literature, there are few studies on the

exploration of temporin-peptide inducing cancer cell

death, and the mechanisms include membrane

destruction, intracellular Ca

leakage, and inducing

apoptosis pathway (Wang et al 2013, Shaheen et al

2018). Here, temporin-PEa would trigger the TNF-α

necroptosis pathway and ROS accumulation, which

could provide a new research direction for the

mechanism study on the anticancer activity of AMPs

from the temporin family. This might also provide a

new strategy for novel anticancer drug development

based on the structure of temporin-PEa. Moreover,

further investigation should be conducted on the

specific intracellular target of the peptide for target

drug delivery system design.

ROS accumulation, which always occurs

downstream of the necroptosis pathway, can lead to

energy depletion and initiate oxidative stress-induced

cancer cell death (Moloney 2018). In Possible Result

2, ROS accumulation is not detected, which might

indicate that there are other mechanisms as the

downstream of TNF-α necroptosis pathway,

including ER (endoplasmic reticulum) stress,

mitochondria membrane disruption, and Ca

leakage

(Kim, Kim 2018, Wang, Zhou, Li, Li, Tian, Wang

2013).

The negative results of temporin-PEa not

triggering the TNF-α necroptosis pathway are

described in Possible Results 3 and 4. Possible Result

3 might indicate that lung cancer cell death is induced

by other signaling pathways, such as the apoptosis

pathway or FasL-mediated necroptosis pathway,

which might trigger ROS over-generation as well

(Otani 2018, Sauler, Bazan, Lee 2019). This could be

further verified by flow cytometry and western blot

detection. In Possible Result 4, neither TNF-α

necroptosis nor ROS over-generation is detected,

which contradicts the hypothesis and probably means

that temporin-PEa could directly break the cancer cell

membrane instead of triggering the intracellular

pathway. Based on the two results, further

investigation should be done on the molecular

mechanisms of temporin-PEa inducing lung cancer

cell death.

In order to improve the therapeutic value of the

original peptide, two modifications are conducted on

temporin-PEa. Possible Results 5 and 7 show that

both of the modified peptides have higher TI value

than temporin-PEa, which is consistent with former

studies and support the hypothesis of this study (Diao

et al 2012, Hu, Chen, Huang, Chen 2018). This would

indicate that both RGD and TAT motifs could

increase the cell penetration activity and tumor

selectivity of the peptide, and thus increase the

anticancer activity while reducing the cytotoxicity to

normal cells. RGD motif shows better efficacy in

Possible Result 5, while TAT peptide behaves better

in Possible Result 7. While in Possible Results 6 and

8, only one of the two modification strategies could

improve the therapeutic efficacy.

Possible Result 9 demonstrates that all of the three

peptides show no haemolytic activity, which enables

them to be proper candidates for further clinical

therapeutic development. Except for temporin-PEa,

PEa-TAT and RGD-PEa show significant

cytotoxicity or haemolytic activity in Possible

Results 10 and 11 respectively. The modified peptide,

which is harmful to the normal cells, cannot be

utilized for in vivo experiments or further clinical

research. Both of the modified peptides display

significant haemolytic activity in Possible Result 12,

which suggests that the novel peptide might generate

new structures that change the properties of the

original peptide and result in haemolysis. These two

modification strategies are not suitable for temporin-

PEa so that further modification approaches should

be studied. Based on the Possible Results (5-12), the

peptide with no haemolytic activity and relatively

higher TI value is chosen for in vivo detection.

Possible Results 13-16 demonstrate the in vivo

detection of the chosen peptide. The results that the

peptide shows higher anticancer activity on the NCI-

H157 cell line are listed in Possible Results 13 and

14. This is consistent with the former study, as

temporin-PEa exhibits strong anticancer activity on

The Mechanism Study and Target Modiﬁcations on the AMP Temporin-PEa Triggering TNF- Necroptosis Pathway in Lung Cancer Cell

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the NCI-H157 cell line. Possible Results 15 and 16

show that the chosen peptide has better anticancer

activity on the A549 cancer cell line, which indicates

that the peptide might behave differently in various

lung cancer cell types. Also, Possible Results 14 and

16 demonstrate that the peptide would probably show

no antiproliferation activity in the xenograft animal

models, which might due to the biological barriers

and poor drug delivery efficiency.

The Possible Result 17 contradicts the hypothesis

and the current understanding of RGD and TAT

peptides improving drug therapeutic value. This

might indicate systemic errors of the experiment

design, and these two modification strategies cannot

promote the peptide cell-penetration ability or tumor

selectivity. Thus, further studies on peptide structure-

function relationship as well as peptide modifications

should be carried out. In Possible Result 18, the

peptide shows no significant difference between

NCI-H157 and A549 cell lines, which would indicate

that the choice of the two cancer cell lines might not

be appropriate, and the antiproliferation activity of

the peptide exhibiting on other lung cancer cell lines

should be further exploited.

5 CONCLUSION

In summary, this study investigates the molecular

mechanisms of the AMP temporin-PEa inducing lung

cancer cell death, and target modifications on this

peptide. The results of this study will test the

hypothesis that whether the peptide would induce

lung cancer cell death via the TNF-α necroptosis

pathway and ROS over-generation, and whether the

RGD and TAT motifs will enhance the therapeutic

value of the peptide.

The possible results on the anticancer mechanism

of temporin-PEa indicate that the peptide would

trigger TNF-α necroptosis or other pathways

followed by energy depletion processes. Also, the

results might suggest that there would be a signaling

network of AMPs triggering immunogenic cell death

(ICD), which involves both death-receptor signaling

pathways and the engagement of other organelles.

Additionally, the possible results on CPP modifying

temporin-PEa would provide potential peptide

modification strategies for further anticancer drug

development. However, novel drug delivery systems

should get further studied to help the peptide

overcome the biological barriers, and to reduce

cytotoxicity to normal cells.

Researchers have started to explore the anticancer

activity of AMPs in recent years, and the detailed

understanding of the intracellular mechanisms of

AMPs still remains largely unclear. Therefore, more

studies on the AMPs anticancer mechanisms should

be conducted to provide more therapeutic potentials

for peptide biologics development.

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