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pH-sensitive cytotoxicity of a cell penetrating peptide fused with a histidine-glutamate co-oligopeptide

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pH-sensitive cytotoxicity of a cell penetrating peptide fused with a histidine-glutamate co-oligopeptide

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PH-SENSITIVE CYTOTOXICITY OF A CELL PENETRATING
PEPTIDE FUSED WITH A HISTIDINE-GLUTAMATE
CO-OLIGOPEPTIDE

by
XINRU QIU

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

May 2015

Copyright2015 Xinru Qiu

i

TABLE OF CONTENTS
Acknowledgments iii

List of Figures and Table v

Abbreviations vi

Abstract vii

Chapter 1: Introduction 1

1.1 Characteristics of Cell penetrating peptides 1

1.2 Specific objective 4
1.3 Principles underlying the cytotoxicity assays used in the study 6
Chapter 2: Materials and Methods 8

2.1 Materials 8
2.2 Cell Line 9
2.3 Preparation of HE-MAP Fusion Peptides 9
2.3.1 Expression and purification of recombinant proteins 9

2.3.2 Bacterial lysate and GSH agarose affinity purification 10
2.3.3 Nickel- nitriloacetic acid (Ni-NTA) agarose affinity purification 10
2.3.4 Dialysis of the fusion peptide 11
2.3.5 Peptide concentration 11
2.3.6 Peptide handling 12
2.3.7 Spectrophotometric Determination of Peptide Concentration 13

ii

2.3.8 SDS-PAGE 14
2.3.9 Coomassie Blue Staining 14
2.4 Cytotoxicity Assay 16
2.4.1 MTT assay 16
2.4.2 LDH leakage assay 17
Chapter 3. Results 19

3.1 Production of recombinant proteins 19

3.2 Production of recombinant peptides 21
3.3 Dialysis and concentration of (HE)8–MAP and (HE)9–MAP
fusion peptides. 23
3.4 MTT Assay for (HE)8-MAP fusion peptide 25
3.5 LDH Leakage Assay for (HE)9 – MAP fusion peptide 27
Chapter 4. Discussion 29

References 34

iii

Acknowledgments
First of all I am grateful to work in Dr. Wei-Chiang Shen’s lab for my graduate
study. My appreciation goes to my advisor Dr. Jennica L. Zaro and my co-advisor Dr.
Wei-Chiang Shen. Dr. Jennica L. Zaro has influenced me in every aspect of my life, her
encouragement, patience and valuable research guidance gives me great support in the
graduate study. And my co-advisor Dr. Wei-Chiang Shen has been a great scientific
mentor for me during my graduate study. Dr. Wei-Chiang Shen has provided me with so
much help and guidance with my graduate studies and career development. I sincerely
appreciate all of his great advice and support. My appreciation also goes to Daisy Shen,
for her thoughtfulness and caring.
I also want to give deeply thank my Master thesis committee member Dr. Curtis
Okamoto for his advice in my course study and thesis. I appreciate the knowledge he
shared with me, and all the discussion and guidance.
Additionally, I would like to express my thankfulness to my labmates for their
support and friendship: Likun Fei ,Hsin-Fang Lee, Yu-Sheng Chen, Tzyy-Harn Yeh,
Juntang Shao, Zoe Folchman-Wagner, Yuquian Liu, and Li Zhou, Shanshan Tong,
Krutika Jain. Also, I want to give extra thanks to Likun Fei for sharing his unpublished
data.
I also want to acknowledge my boyfriend Narisu Bai, for being caring and
supportive, talk to me and cheer me up whenever I am having a bad time.

iv

Finally, I would like to thank my parents Dr. Changbo Qiu and Dr. Meihua Li for
their unconditional love and support for letting me study in USC.

v

List of Figures and Table

Figure. 1: The Proposed Function Model of pH-Sensitive HE-MAP Fusion Peptide. 5
Figure.2: Schematic of LDH Cytotoxicity Assay Mechanism. 7
Figure.3: The Spectrophotometric Determination Regression Equation to
Determine peptide Concentration. 13
Figure.4: SDS-PAGE Analysis and Coomassie Blue Staining
of Purified Fusion Proteins. 20
Figure.5: Tricine SDS-PAGE and Coomassie Blue Staining of Purified Fusion
Peptides. 22
Figure.6: Tricine SDS-PAGE and Coomassie Blue Staining of Purified Fusion
Peptides After Dialysis and Lyophilization. 24
Figure.7: pH-Dependent Cytotoxicity of (HE)
8
–MAP Fusion Peptide. 26
Figure.8: Impact of GST-HE-MAP in HeLa Cells at 4 Different pH Conditions. 32
Table 1: pH-Sensitive LDH Release in Cells Treated With
(HE)
9
–MAP Fusion Peptide. 28

vi

Abbreviations
CPP: Cell penetrating peptide
MAP: Model amphipathic peptide
EDTA: Ethylenediaminetetraacetic Acid
FBS: Fetal Bovine Serum
DS: Dosing Solution
ddH2O: double distilled water
LDH: lactate dehydrogenase
TB: Terrific broth
GSH: glutathione
Ni-NTA: Nickel nitrilotriacetic acid
PMSF: Phenylmethylsulfonyl fluoride
IPTG: Isopropyl β-D-1-thiogalactopyranoside
PBS: Phosphate buffered saline
SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis
MWCO: Molecular weight cutoff
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
SFM: Serum free medium
AMP: Antimicrobial peptide

vii

Abstract
With the growth of research and biotechnology, more proteins and peptides have
been developed as potential therapeutics. Due to their high cell surface binding and
internalization, cell penetrating peptides (CPPs) have been considered as potential
carriers for efficient intracellular drug delivery. Some amphipathic CPPs such as Model
Amphipathic Peptide (MAP) also have lytic activity, similar to antimicrobial peptides.
This cytotoxic activity could potentially be utilized as a therapeutic strategy for cancer.
However, the lack of specificity of CPPs for tumor versus normal cells is a major hurdle
for further use of CPPs such as MAP in anticancer therapy.
In order to overcome the non-specificity problem, attaching a pH-sensitive
masking peptide sequence to the CPPs becomes a solution. Histidine–glutamic acid (HE)
copolymer sequence has been shown to block the binding and internalization of CPPs at
normal pH 7.4, while both binding and uptake are recovered in the mildly acidic pH of
6.5-7. The purpose of this dissertation study was to determine if the lytic activity of MAP
could also be masked by HE at normal physiological pH, and reactivated in the mildly
acidic conditions seen in the tumor microenvironment.
In this dissertation, cytotoxicity and lytic assays in HeLa cells confirmed that the
HE-MAP construct exhibited cytotoxicity and lytic activity at pH 6.5, but was inactive at
normal physiological pH. Therefore, HE-MAP has the potential to be utilized as a highly
pH-sensitive cytotoxic agent for treatment of cancer.

1

Chapter 1: Introduction
1.1 Characteristics of Cell penetrating peptides
Due to the better understanding of the human body, the improvement of diagnostic
devices and rapid development of medical research, we currently have surgical
intervention, radiation and chemotherapy drugs to treat cancers. For most cancer
treatments, chemotherapeutic drugs are now commonly used because they cause less
body damage and are more easily administered compared to surgical intervention and
radiation. Unfortunately, the downside of chemotherapy drugs is that they do not only
eradicate cancer cells, they also kill normal cells and cause severe toxicity to the patient.
Recent reviews give a perspective on the use of nanoconstructs as indispensable tools in
cancer research (Ferrari, 2005).
Cell-penetrating peptides (CPPS) are peptides usually containing 15-30 amino
acids which can enter most cell types in an energy-dependent or independent
pathway(Jarver and Langel, 2006)
,
(Richard et al., 2003). CPPs are also known as protein
transduction domains (PTDs) or membrane transduction peptides (MTPs), frequently
used terminology to describe those peptides with the ability to cross plasma
membranes(Patel et al., 2007).
CPPs are classified into two categories including cationic or amphipathic. The
cationic CPPs are short strands of arginine- and lysine- rich peptides, which rely on their

2

cationic nature for efficient intracellular accumulation(Mo et al., 2012). This class
includes the protein derived nuclear transcription activator Tat-(47-57)
(YGRKKRRQRRR)(Vives et al., 1997), HIV-1 Rev-(34-50) (TRQARRNRRRR
WRERQR) both encoded by HIV-1, the Drosophila Antennapedia homeoprotein, Antp
(43-58) (RQIKIYFQNRRMKWKK)(Patel et al., 2007), and synthetic small
oligoarginine/oligolysine, (R)
n
/(K)
n
(n=6-9)(Futaki, 2002).
As for amphipathic CPPs, they contain both hydrophobic and hydrophilic parts and
have the characteristic that all the polar residues align to one face while the nonpolar
residues align on the opposite side. Thus, amphipathic CPPs can first interact with the
plasma membrane with their cationic hydrophilic part, and then penetrate the
hydrophobic interior of the plasma membrane. Also, some amphipathic CPPs exhibit an
α-helical structure including the model amphipathic peptide (MAP)
(KLALKLALKALKAALKLA) and its derivatives, transportan
(GWTLNSAGYLLGKINLKALAALAKKIL) derived from a fusion of a wasp venom
peptide and a neuropeptide(Fernandez-Carneado et al., 2004), MPG
(GALFLGWLGAAGSTMGAPKKKRKV), derived from a fusion of HIV-1 gp41 protein
and SV40 large T antigen(Simeoni et al., 2003).
CPPs have the ability to widely distributed throughout most tissues, and have been
shown to translocate through cell membranes in most mammal cell types(Patel et al.,
2009). Some CPPs also have toxic effects resulting from membrane perturbation at
higher peptide concentrations(Saar et al., 2005). Due to the membrane-disrupting activity,

3

cationic amphipathic peptides like MAP often possess antiviral(Reddy et al., 2004),
antimicrobial(Powers and Hancock, 2003) and antitumoral(Leuschner and Hansel, 2004)
effects.
The translocation mechanisms of CPPs are being explored, however their
mechanism for tranclocation across biological cell membranes still remains debated.
Some reports state that the uptake of Tat and penetratin is mainly endocytotic(Koppelhus
et al., 2002), also some report explains that the observation of the peptides Tat and (Arg)
9

internalization might simply be cell-fixation artefacts(Richard et al., 2003). The CPP used
in this study, MAP, has been shown in our laboratory to efficiently translocate into the
cytosolic and/or nuclear compartment of cells via an endocytic mechanism(Kenien et al.,
2012).
CPPs have the potential for being drug carriers, but there is still no clinical
breakthrough for CPPs in the recent years(Tung and Weissleder, 2003) . The major
barrier of CPPs is their in vivo lack of specificity, mainly due to their cationic nature.
Studies have shown that non-specific binding and penetrating cell membranes by CPPs
can be reduced through masking their cationic charges(Fei et al., 2011). This masking
effect has been utilized in many designs to reduce non-specific binding to the plasma
membrane(Kuai et al., 2010). A potential way to mask the CPPs to inactivate its
non-specific targeting is to take advantage of the mildly acidic environment of the target
sites, like inside endosomes or the acidic microenvironment of the tumor cells.

4

1.2 Specific objective
In this study, in order to reduce the normal cell association at neutral pH, an
HE-repeat oligopeptide was attached to MAP through a pentaglycine linker (“HE- MAP”)
and expressed in E. coli as a glutathione S-transferase (GST) fusion protein. This CPP
nanoconstruct exhibits a highly pH-sensitive cell association in the mildly acidic
region(Fei et al., 2014; Sun et al., 2014; Zaro et al., 2012), and has been shown to
specifically accumulate near the tumor site in an in vivo xenograft mouse model of breast
cancer(Fei et al., 2014).
Previous studies have shown that some amphipathic CPPs, such as MAP, exhibit
cytotoxicity via membrane destabilization(El-Andaloussi et al., 2007). The toxicity has
been shown to rely on both the cationic and hydrophobic nature of the CPP
sequence(Munyendo et al., 2012). In this study, the central hypothesis we are testing is if
the lytic activity of MAP can be targeted to the mildly acidic region using our
HE-masking sequence. To this end, two fusion peptides with different lengths of
HE-repeats (n=8, 9) were evaluated. (HE)
8
-MAP was evaluated in the cytotoxicity assay,
due to its low expression level, there was not a way to express it in enough amount to
conduct the LDH assay, so only the MTT assay data will be shown in the dissertation.
And as for the (HE)
9
-MAP, due to its HE repeat masking effect, MTT assay did not show
significant toxicity in both pH 6.5 and pH 7.4, so the MTT assay data will not be shown
in the dissertation.

5

Fig. 1. The Proposed Function Model of pH-Sensitive HE-MAP Fusion Peptide. At the
surface of normal cells (pH 7.4), the fusion peptide will be in its inactive form, and will
not bind to the cell surface. At the surface of tumor cells with the mildly acidic pH 6.5-7,
the fusion peptide will be unmasked revealing the membrane-permeable CPP for
subsequent binding and subsequent LDH leakage.

6

1.3 Principles underlying the cytotoxicity assays used in the study
A. MTT assay
The principle of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium
bromide) assay is that for most viable cells, mitochondrial activity is constant, so an
increase or decrease in the amount of viable cells is linearly related to the cell
mitochondrial activity. The mitochondrial activity of the cells is reflected by the
conversion of the tetrazolium salt MTT into formazan crystals, which is then solubilised
by isopropanol in this study. Therefore, the increase or decrease in viable cell number can
be detected by measuring formazan concentration reflected in optical density (OD) using
a plate reader at 560nm(van Meerloo et al., 2011). In this study the assay is generally
used to measure the in vitro cytotoxic effects of HE-MAP in the HeLa tumor cell line.
B. LDH leakage assay
LDH is a cytosolic enzyme present in many different cell types. Plasma membrane
damage releases LDH into the cell culture media. LDH activity was determined as
NADH oxidation or INT reduction over a defined time period, the extracellular LDH in
the medium can be quantified by the coupled enzymatic reaction that LDH catalyzes the
conversion of lactate to pyruvate via NAD+ reduction to NADH. And then diaphorase
uses NADH to reduce a tetrazolium salt (INT) to a red formazan product. Then the
product is assayed using a plate reader at 490/680nm(Decker and Lohmann-Matthes,
1988). The level of formazan formation reflects the total amount of LDH released into

7

the medium, which shows the efficiency of HE-MAP fusion peptide perturbation.
Also, in order to avoid the reduction of the effective peptide concentration, serum
was not included in the exposure medium in this study.

Fig.2. Schematic of LDH Cytotoxicity Assay Mechanism. Applied from Pierce LDH
Cytotoxicity Assay Kit instructions.

8

Chapter 2: Materials and Methods
2.1 Materials
The HeLa cell line was purchased from ATCC (Manassas, VA), RPMI 1640 media,
fetal bovine serum (FBS), and ampicillin were from Mediatech (Manassas, VA), RPMI
1640 powder, L-glutamine, penicillin-streptomycin, and trypsin-EDTA were from
Invitrogen (Carlsbad, CA), thrombin was from GE Healthcare Life Sciences (Piscataway,
NJ). Competent E. coli (DH5α and JM109) was from ZYMO Research (Irvine, CA).
PageRulerTM prestained protein ladder, SpectraTM multicolor low range protein ladder,
glutathione (GSH) agarose, and HisPurTM nickel nitrilotriacetic acid (Ni-NTA) resin
were from Thermo Fisher Scientific (Waltham, MA). Isopropyl
β-D-1-thiogalactopyranoside (IPTG), yeast extract, and sodium lauroyl sarcosine
(sarkosyl) were from Amresco (Solon, OH). 0.22 μm sterile syringe filter with
polyethersulfone (PES) membrane, 0.22 μm sterile Stericup® filter unit (PES, 500 mL),
phenylmethylsulfonyl fluoride (PMSF), and acrylamide/bis-acylamide (19:1) was from
EMD Millipore (Billerica, MA). Reduced glutathione was from Alfa Aesar (Ward Hill,
MA). Acrylamide/bis-acylamide (29:1) was from JT Baker (Central Valley, PA).
MicrosepTM centrifugal device, MWCO at 1 kDa, was from PALL (Port Washington,
NY). MTT was from Sigma-Aldrich (St. Louis, MO). LDH assay kit was from Thermo
Scientific (Rockford, lL), and other chemicals were from Sigma-Aldrich (St. Louis, MO).

9

2.2 Cell Line
HeLa cells, which are derived from human cervix carcinoma cells, were used in the
pH-sensitive cytotoxicity of HE-MAP fusion peptides. HeLa cells were cultured in
RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 50
IU/mL penicillin G sodium salt at 37 °C with 5% CO
2
in the air. Cells were replenished
with fresh complete medium the day before cells were confluent and ready for all cellular
assays.
2.3 Preparation of HE-MAP Fusion Peptides

2.3.1 Expression and purification of recombinant proteins
The fusion protein was prepared to contain GST, an 8- or 9-mer HE-oligopeptide
sequence ((HE)
8
or

(HE)
9
), a short pentaglycine (G
5
) linker, and MAP (“GST-HE-MAP”).
The plasmids with correct insertions were transformed into Escherichia coli expression
strain BL21. For expression of recombinant proteins, bacteria were incubated in terrific
broth (TB) media with 60 mg/mL ampicillin at 37 °C with 300 rpm shaking speed until
the OD
600

of the media reached 2.5 -3.0. IPTG was added into TB media to a final
concentration of 0.2 mM. After 3-4 h of additional incubation, the bacteria were collected
and stored at -80°C. Expression of the GST-fusion proteins was monitored by sodium

10

dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) followed by
Coomassie blue staining.
2.3.2 Bacterial lysate and GSH agarose affinity purification
The recombinant GST-fusion protein was purified from crude extracts using
glutathione (GSH) (which recognizes GST). The bacterial pellets were resuspended in
phosphate buffered saline (PBS for GST agarose), pH 7.4, and lysozyme was added to
reach a final concentration of 0.25 mg/mL. After 30 min incubation on ice, PMSF was
added to 1 mM and Triton X-100 was added to a final concentration of 1% (v/v). The
bacteria were lysed by sonication (Misonix Ultrasonic Liquid Processors S-4000,
Misonix, Farmingdale, NY) on ice at amplitude 10 for 4-5 min total working time at a 10
s on/ 15 s off working cycle. The lysate was centrifuged at 15,000 g for 30 min at 4°C.
The supernatant was loaded on GSH agarose column pre-balanced with PBS. The column
was washed with 1% Triton X-100 in PBS and then PBS alone. Thrombin was loaded on
to the GSH agarose for 16 h room temperature incubation.

2.3.3 Nickel- nitriloacetic acid (Ni-NTA) agarose affinity purification
After the thrombin cut off the GST of the recombinant protein GST-HE-MAP, the
HE-MAP peptides left on the GSH agarose beads were eluted with PBS, then load the
HE-MAP containing PBS onto the Nickel- nitriloacetic acid (Ni-NTA) agarose, which

11

was pre-balanced with PBS (for Ni-NTA agarose). Finally, the HE-MAP fusion peptide
was eluted with the elution buffer (250 mM imidazole, pH 8.0 for Ni-NTA agarose).
During purification, HE-MAP fusion peptides were monitored by SDS-PAGE with
Coomassie blue staining.
2.3.4 Dialysis of the fusion peptide
After the HE-MAP peptides are eluted from the Ni-NTA agarose, the solution was
poured into a presoaked Spectra/Por molecularporous membrane tubing (MWCO 3500Da)
for dialysis, and dialyzed three times in 2 L of 50mM ammonium bicarbonate for 4 h
each time at 4 °C.
2.3.5 Peptide concentration
To concentrate the purified HE-MAP solution, lyophilization, or freeze-drying, was
used. This method of drying significantly reduces loss of activity or other damage to
peptides. First, the peptide solutions were rapidly frozen over a dry ice and acetone
mixture, and then the pre-frozen sample was loaded onto a sample valve on the drying
chamber for lyophilization (Labconco, Kansas City, MO). The mass of the dried fusion
peptide is measured by using an automatic analytical balance.

12

2.3.6 Peptide handling
In cytotoxicity assays, frozen aliquots of peptides were used, and the aliquots were
prepared as follows. The amount of HE-MAP peptide was weighed, dissolved in 1mL
PBS, and then stored at -20°C. The peptide solution was monitored regularly by
SDS-PAGE and no degradation was detected. The concentration of the solutions was
verified by measuring the absorbance of the tyrosine residue in the peptide sequence at
280 nm and comparing it to a tyrosine standard curve (Figure 3).

13

2.3.7 Spectrophotometric Determination of Peptide Concentration
Preparation of the solution Tyrosine, use it as the standard solution.

Fig.3. The Spectrophotometric Determination Regression Equation to Determine peptide
Concentration. The concentration of standard stock solution Tyrosine was 1 mM, and it
was serially diluted with PBS to 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8mM. The
absorbance at 280nm was measured by using UV spectrophotometry, and a standard
regression equation was used to calculate HE-MAP peptide concentration in the samples.

y
=
1.1916x
-‐
0.0016

R²
=
0.99965
-‐0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0
0.2
0.4
0.6
0.8
1
1.2

Absorbance

at
280nm
Peptide
Concentration
(mM)
Spectrophotometric
Determination
of

Peptide
Concentration

14

2.3.8 SDS-PAGE
The sample of GST-HE-MAP fusion protein was monitored by Tris-Glycine SDS-
PAGE to examine the production efficiency. GST-HE-MAP samples were diluted 5-fold
and mixed with 5X protein reducing loading dye, and then boiled for 10 min. The
GST-HE-MAP samples were later loaded onto a polyacrylamide gel with 4% stacking 13%
separating polyacrylamide gel.
The sample of HE-MAP was monitored by Tricine-SDS-PAGE to examine the
degree of purity. HE-MAP samples were diluted 3-fold and mixed with 4X peptide
reducing loading dye. Then the HE-MAP samples are loaded onto 4% stacking, 10%
spacer, 16%/6 M urea separating polyacrylamide gel.
2.3.9 Coomassie Blue Staining
For GST-HE-MAP fusion protein monitoring, after the Tris-Glycine SDS-PAGE
process, the gel was immersed in the Coomassie Blue R-250 reagent for 30 minutes with
gently shaking. Later, the stained gel was washed in De-stain Buffer (40% methanol,
10%acetic acid mix with H
2
O), and kept gently shaking for 3 h to remove excess
Coomassie Blue reagent. De-stain Buffer was replaced with water after 3 h of washing,
and the gel was kept soaking in water overnight. Afterward, the gel was transferred to a
drying frame to dry out the gel for storage and documentation purposes.
For HE-MAP fusion peptide purity examination, after the Tricine-SDS-PAGE

15

process, the gel was immersed in the fixing buffer (50% methanol, 10% acetic acid
100mM ammonium acetate) for 30 minutes with gently shaking. Then immersed in the
Coomassie Blue R-250 reagent for 30 minutes also with gentle shaking. After Coomassie
Blue staining, the stained gel was washed in Tricine-SDS-PAGE De-stain Buffer (10%
acetic acid mix with H
2
O), and kept gently shaking for 3 h to remove excess Coomassie
Blue reagent. The De-stain Buffer was then replaced with water overnight. Finally, the
gel was transferred to a drying frame put in the fume hood to dry out the gel for storage
and documentation purposes.

16

2.4 Cytotoxicity Assay
2.4.1 MTT assay
A 1mg/mL MTT stock solution was prepared by adding 12 mL of sterile phenol
red-free, serum-free medium to one 1 mg vial of MTT. The solution was mixed by
vortexing until dissolved.
In the MTT assay, 2×10
4
HeLa cells were seeded in a 96-well flat-bottom plate
and grown to confluence. The medium was replenished the day before experiments. For
the cytotoxicity assays, the culture medium was replaced with 100 μL serum-free
medium at pH 6.5 or 7.4, and cells were incubated at 37°C, 5% CO
2
for 10 minutes,
followed by the addition of 100 μL dosing solution (DS) containing the HE-MAP
peptides at pH 6.5 or 7.4. The 96-well flat-bottom plate was sealed by parafilm and the
cultured cells were treated with DS for 4 h at 37°C, 5% CO
2
.
After 4 h, the DS was removed, each well was washed with 100μL PBS, and the
cells were incubated with the MTT solution at 37°C, 5% CO
2
for 1 h. The MTT solution
was then removed, and the MTT formazan was dissolved by 100 μL isopropanol per well
with gentle shaking for 5 minutes. The absorbance at 560nm was measured using a
spectrophotometer to determine the amount of viable cells.
The % Cytotoxicity was calculated as the MAP-treated sample group divided by
the control group (PBS-treated) and multiplied by 100:

17

%=
ℎ
ℎ
× 100
2.4.2 LDH leakage assay
The LDH Reaction Mixture was prepared by first dissolving one vial of the
Substrate Mix (lyophilizate) with 11.4 mL of ultrapure water in a 15 mL conical tube.
The Substrate Mix was then combined with 0.6mL of Assay Buffer to make the final
LDH Reaction Mixture.
For the LDH leakage assay, 2×10
4
HeLa cells were seeded in a 96-well
flat-bottom plate and grown to reach confluence. The culture medium was changed the
day before the experiments. The medium was removed, then 100 μL serum-free medium
per well was added and cells were incubated at 37°C, 5% CO
2
for 10 minutes, followed
by the addition of 100 μL DS containing the HE-MAP peptides per well. Cultured cells in
parafilm-sealed plates were incubated for 4 h with DS to induce release of LDH.
After 4 h, 50μL of each sample medium was transfered to a 96-well flat-bottom
plate, followed by the addition of 50μL of Reaction Mixture to each sample well and
mixed. The plate was incubated at room temperature for 30 minutes while protected from
light, after which time reactions were stopped by adding Stop Solution.
The absorbance at 490nm and 680nm was measured using a plate-reading
spectrophotometer to determine LDH release. The 680nm absorbance value
(background) was first subtracted from each of the the 490nm absorbance values, and the %

18

Cytotoxicity was calculated using the following equation,
%=
!"#$"%&' !"#$!#% !"# !"#$!"#$!!"#$%&$'#() !"# !"#$%$#&
!"#$%&% !"# !"#$%$#&!!"#$%&$'#() !"# !"#$%$#&
× 100
where “Compound treated LDH activity” was the MAP-treated samples, “Spontaneous
LDH Activity” was the PBS-treated (control samples), and “Maximum LDH activity”
was the lysis buffer-treated samples.

19

Chapter 3. Results
3.1 Production of recombinant proteins
A dsDNA fragment encoding the HE-MAP peptide was cloned into the
pGEX-4T-1 vector. The fusion proteins of GST-(HE)
n
-MAP (n=8,9,10) were
expressed in E.coli BL21 strain respectively. As shown in Fig.4, the SDS-PAGE with
Coomassie blue staining showed all three fusion proteins were purified with GSH
agarose to >90% purity. Between the GST-(HE)
8
-MAP, GST-(HE)
9
-MAP and
GST-(HE)
10
-MAP fusion proteins, GST-(HE)
8
-MAP has the lowest expression level
followed by GST-(HE)
9
-MAP, and the highest was GST-(HE)
10
-MAP.

20

Fig.4. SDS-PAGE Analysis and Coomassie Blue Staining of Purified Fusion Proteins.
GST-fusion proteins were purified from bacteria by affinity chromatography
according to the procedures described in the methods chapter. Lane M: Molecular
weight markers. Lane 1: GST-(HE)
8
-MAP, expected MW = 30.74kDa. Lane 2:
GST-(HE)
9
-MAP , expected MW = 31.02kDa. Lane 3: GST-(HE)
10
-MAP , expected
MW = 31.30kDa.

21

3.2 Production of recombinant peptides
The fusion proteins were then incubated with thrombin for 16 h to cut GST
from GST-HE-MAP fusion protein. As shown in Fig.5, HE-MAP fusion peptides
could be purified by Ni-NTA chromatography using the histidine residues as a
recognition site, resulting in >90% purity determined by SDS-PAGE followed by
Coomassie blue staining. Thus, the inclusion of oligohistidine in the sequence for the
fusion peptides has the advantage in the purification methods for the purification of
fusion peptides. Among the (HE)
8
-MAP, (HE)
9
-MAP and (HE)
10
-MAP fusion
peptides, (HE)
8
-MAP has the lowest expression level around 0.7 mg/L, followed by
(HE)
9
-MAP with expression level at 2.5 mg/L, and the highest expression level of
(HE)
10
-MAP was around 5.0 mg/L. As for (HE)
10
-MAP, because concentration at 668
μM showed no significant cytotoxicity through MTT assay both at pH 6.5 and pH 7.4,
so the cytotoxicity data for (HE)
10
-MAP will not be displayed in this dissertation.

22

Fig.5. Tricine SDS-PAGE and Coomassie Blue Staining of Purified Fusion Peptides.
Fusion peptides were by affinity chromatography according to the procedures
described in the methods chapter. Lane M: Molecular weight markers. Lane 1:
(HE)
8
-MAP, expected MW=4600Da. Lane 2: (HE)
9
-MAP, expected MW=4870Da.
Lane 3: (HE)
10
-MAP, expected MW= 5132Da. Note: The peptides run at an
anomalous molecular weight due to the high amount of charge in the constructs.

23

3.3 Dialysis and concentration of (HE)
8
–MAP and (HE)
9
–MAP fusion
peptides.
Fusion peptides are dialyzed three times in 2 L of 50mM ammonium bicarbonate
for 4 h at 4 °C, followed by lyophilization concentrate the HE-MAP solutions to 1 mL.
As seen from Fig.6, the peptides were highly concentrated after lyophilization. After all
purification and concentration procedures, the final concentrations of (HE)
8
–MAP and
(HE)
9
–MAP fusion peptides were around 0.3 mg/mL, and 6 mg/mL, respectively.

24

Fig.6. Tricine SDS-PAGE and Coomassie Blue Staining of Purified Fusion Peptides
After Dialysis and Lyophilization. (A) (HE)
8
–MAP fusion peptide, (B) (HE)
9
–MAP
fusion peptide. Fusion peptides were dialyzed in 50mM ammonium bicarbonate and
concentrated by lyophilization, as described in the methods chapter. (A) M: Molecular
weight markers. Lane 1: (HE)
8
-MAP before dialysis. Lane 2: (HE)
8
-MAP after dialysis.
Lane 3: (HE)
8
-MAP after lyophilization. (B) M: Molecular weight markers. Lane 1:
(HE)
9
-MAP before dialysis. Lane 2: (HE)
9
-MAP after dialysis. Lane 3: (HE)
9
-MAP after
lyophilization.

25

3.4 MTT Assay for (HE)
8
-MAP fusion peptide
Cytotoxicity of HE-MAP in HeLa cells was determined by measuring
mitochondria activity in treated cells using the MTT assay. HeLa cells grown to
confluence in 96-well plates were treated with 110 μM of (HE)
8
– MAP in SFM medium
at pH 6.5 or 7.4. As seen in Fig.7, (HE)
8
–MAP-treated HeLa cells had 45% viability at
pH 7.4 compared to the non-treated control, cells treated at mildly acidic pH of 6.5 had
24% viability. Therefore, the (HE)
8
–MAP construct showed pH-dependent cytotoxicity in
the mildly acidic pH-range.

26

Fig.7. pH-Dependent Cytotoxicity of (HE)8–MAP Fusion Peptide. Mitochondria activity
in HeLa cells after 4 h of treatment with 110 μM (HE)
8
–MAP fusion peptide in SFM
medium at pH 6.5 or 7.4. Bars represent average of duplicate wells, with error bars
indicating Standard Error.

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

pH
6.5
pH
7.4

Cell
Viability
MTT
Assay
for
(HE)
8
-‐MAP

pH
6.5

pH
7.4

27

3.5 LDH Leakage Assay for (HE)
9
– MAP fusion peptide
An LDH leakage assay was used to measure the acute membrane disturbance
caused by treatment with MAP-peptides. HeLa cells grown to confluence in 96-well
plates were treated with sterile PBS alone or in the presence 50 μM, 300 μM 615 μM
and1.23 mM (HE)
9
–MAP fusion peptide, or 50 μM cMAP at pH 6.5 or 7.4. After 4 h, the
LDH release was determine as described in the Methods Chapter.
As seen in Table 1, when (HE)
9
–MAP concentration was as low as 50 μM and
300 μM, no significance LDH leakage was detedted at both pH 6.5 and pH 7.4. But when
with higher concentration as 615 μM and 1.23 mM, neither 615 μM nor 1.23 mM (HE)
9
–
MAP fusion peptide affected membrane integrity at pH 7.4, while both concentrations of
(HE)
9
– MAP fusion peptide induced LDH leakage at pH 6.5. For 615 μM (HE)
9
–MAP
fusion peptide at pH 6.5, approximately 10% LDH leakage in HeLa cells was observed,
compared 2.07% leakage at pH 7.4. At 1.23mM (HE)
9
–MAP fusion peptide, >20% LDH
leakage was observed at pH 6.5, while only 4.09% LDH leakage was induced at pH 7.4.
For non-pH sensitive 50 μM cMAP, which does not contain the HE-masking sequence,
the leakage was approximately 16% at both pH 6.5 and 7.4.

28

Table 1. pH-Sensitive LDH Release in Cells Treated With (HE)9–MAP Fusion Peptide.
LDH leakage was determined in HeLa cells after 4 h of treatment with (HE)
9
– MAP
fusion peptide or cMAP at different concentrations and different pH in PBS. Data were
expressed as mean ± SD of triplicates.

％ Cytotoxicity

pH 6.5 pH 7.4
50μM cMAP 14.69±0.01 16.67±0.02
50μM (HE)
9
–MAP Undetectable 2,04±0.04
300μM (HE)
9
–MAP 11.97±0.04 7.43±0.03
615μM (HE)
9
–MAP 10.15±0.01 2.07±0.02
1.23 mM (HE)
9
–MAP 26.45±0.02 4.09±0.02

29

Chapter 4. Discussion
CPPs rely on their cationic nature to efficiently bind to the cell surface and
translocate into the cytosolic and/or nuclear compartment of the cells, which is relevant
for drug delivery. But the lack of specificity impedes CPPs’ application in vivo. However,
it is stated that tumor tissues’ pH is 0.4-0.8 unit lower than that of normal tissues, which
provides a great opportunity for achieving targeted delivery to tumor sites without
harming normal cells(Gerweck and Seetharaman, 1996). So in this dissertation, a series
of recombinant fusion peptides containing an HE oligopeptide sequence and an
amphipathic CPP (i.e. MAP) are engineered to target the mildly acidic tumor
microenvironment. The main goal is to obtain a peptide construct with pH-sensitive
bioactivity for treatment of cancer.
According to the undetectable cytotoxicity of (HE)
9
-MAP, (HE)
10
-MAP in the
MTT assay and Fig.7 the MTT assay of (HE)
8
-MAP, the results showed that the (HE)
8

attachment to MAP has much higher toxicity compare to (HE)
9
-MAP and (HE)
10
-MAP,
which is an evidence showed that HE oligopeptide sequence is reducing MAP
cytotoxicity. With more HE repeat, the more MAP cytotoxicity will be reduced. To
further prove this point, I referenced the data from Likun Fei’s dissertation showed in
Fig.8, which demonstrated that at pH 6.0, 6.5, 7.0, or 7.5, the GST-(HE)
8
-MAP fusion
protein has the highest cell binding and cell uptake efficiency, followed by
GST-(HE)
10
-MAP, and the GST-(HE)
12
-MAP has the lowest cell binding and cell uptake

30

efficiency.
There are also many previous researches stated that α-helical antimicrobial
peptides (AMP) conduct anticancer activity via apoptosis and/or membrane lysis manner.
For example, AMP cecropins isolated from the hemolymph of Hyalophora cecropia, its
family contain cecropin A (KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK)
and cecropin B (KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL)(Hung et al.,
1999). Cecropin A and B are able to lyse different types of human cancer cells at peptide
concentrations that are not harmful to normal eukaryotic cells(Chen et al., 1997). And
AMP magainins, which are isolated from the skin of the African clawed frog Xenopus
laevis(Zasloff, 1987). Magainins comprised of 21 to 27 amino acids that create an
α-helical secondary structure with separate cationic and hydrophobic faces. Magainin 2
(GIGKFLHSAKKFGKAFVGEIMNS) and its more potent synthetic analogues
(magainins A, B, and G) induce the rapid lysis of hematopoietic and solid tumor cell lines
with concentrations at 5-10 fold lower than its concentrations that are lytic for normal
human peripheral blood lymphocytes or neutrophils(Cruciani et al., 1991) (Jacob and
Zasloff, 1994). Also, there are reports stated that magainins can translocate to the
cytosolic of cancer cells then trigger the mitochondrial pathway of apoptosis due to the
researchers found magainins have been form channels in the membranes of isolated rat
liver mitochondria(Westerhoff et al., 1989). Therefore, it is not clear whether magainins
kill human cancer cells primarily through membrane lysis and/or apoptosis(Hoskin and
Ramamoorthy, 2008).

31

Therefore, to evaluate whether MAP conduct membrane lysis manner to the
tumor cells, and to prove that the HE-MAP membrane lysis behavior is pH-sensitive, the
LDH leakage assay was performed shown in Table.1.
From the Table.1, with the results of cMAP as the positive control, showed that
MAP does have the ability for efficiently disrupt cell membrane and induce LDH leakage.
Also comparing the cytotoxicity between pH 6.5 and pH 7.4, both at concentration 615
μM and 1.23 mM, the cytotoxicity for them both much lower than the pH 7.4. Also, from
Likun Fei’s data, Fig. 8 showed that all three fusion proteins, GST-(HE)
8
-MAP
GST-(HE)
10
-MAP and GST-(HE)
12
-MAP have much lower cell binding and cell uptake at
neutral pH comparing to pH below 6.5. So the above results demonstrated thet the
negatively charged glutamate residues in HE oligopeptide sequence can interact with
positively charged lysine residues in MAP at neutral pH in order to mask MAP
non-specific targeting for normal cells.
When comparing Fig. 7 and Table. 1, it is obvious that (HE)
8
-MAP has much better
pH sensitivity at low concentration compare to (HE)
9
-MAP, which demonstrates that the
MAP in (HE)
9
-MAP may not be fully exposed in mild acidic environment. For
(HE)
9
-MAP, the concentration should be above 1mM to achieve >20% LDH leakage.

32

Fig.8. Impact of GST-HE-MAP in HeLa Cells at 4 Different pH Conditions. (A) Total
cell surface binding of different HE repeats of GST-HE-MAP in HeLa cells at 4 different
pH conditions. (B) Total cell uptake of different HE repeat of GST-HE-MAP in HeLa
cells at 4 different pH conditions. HeLa cells were treated with different concentration
fusion proteins at pH 6.0, 6.5, 7.0, or 7.5 for 1 h at 37 °C. Data were expressed as mean ±
SD of triplicates.

33

The figures shown were from Likun Fei’s Dissertation Cell penetrating peptide-based
drug delivery system for targeting mildly acidic pH.

34

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Abstract (if available)

Abstract With the growth of research and biotechnology, more proteins and peptides have been developed as potential therapeutics. Due to their high cell surface binding and internalization, cell penetrating peptides (CPPs) have been considered as potential carriers for efficient intracellular drug delivery. Some amphipathic CPPs such as Model Amphipathic Peptide (MAP) also have lytic activity, similar to antimicrobial peptides. This cytotoxic activity could potentially be utilized as a therapeutic strategy for cancer. However, the lack of specificity of CPPs for tumor versus normal cells is a major hurdle for further use of CPPs such as MAP in anticancer therapy. ❧ In order to overcome the non-specificity problem, attaching a pH-sensitive masking peptide sequence to the CPPs becomes a solution. Histidine–glutamic acid (HE) copolymer sequence has been shown to block the binding and internalization of CPPs at normal pH 7.4, while both binding and uptake are recovered in the mildly acidic pH of 6.5-7. The purpose of this dissertation study was to determine if the lytic activity of MAP could also be masked by HE at normal physiological pH, and reactivated in the mildly acidic conditions seen in the tumor microenvironment. ❧ In this dissertation, cytotoxicity and lytic assays in HeLa cells confirmed that the HE-MAP construct exhibited cytotoxicity and lytic activity at pH 6.5, but was inactive at normal physiological pH. Therefore, HE-MAP has the potential to be utilized as a highly pH-sensitive cytotoxic agent for treatment of cancer.

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Asset Metadata

Creator Qiu, Xinru (author)

Core Title pH-sensitive cytotoxicity of a cell penetrating peptide fused with a histidine-glutamate co-oligopeptide

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 04/20/2015

Defense Date 04/01/2015

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag acidic tumor microenvironment,cell penetrating peptide,cytotoxicity,OAI-PMH Harvest,pH-sensitive

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Shen, Wei-Chiang (committee chair), Okamoto, Curtis Toshio (committee member), Zaro, Jennica (committee member)

Creator Email xinruqiu@usc.edu

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