Cell-penetrating apoptotic peptide/p53 DNA nanocomplex as

adjuvant therapy for drug-resistant breast cancer*

Presented by Spencer Thomas

Feb 9, 2016
BMS 494 | Missouri State University

*Wang H, Wang H, Liang J, et al. Mol Pharm. 2014;11(10):3352-60.

Purpose
To investigate

the in vitro and in vivo efficacy of chimerically

modified Smac peptides (AVPIR8), co-delivered with p53 pDNA,
on apoptosis and tumor suppression in metastatic and metastatic
drug-resistant breast cancer lines, as adjuvant therapy for
antineoplastic (doxorubicin) chemotherapy

ABBREVIATIONS: AVPI (Ala-Val-Pro-Ile Smac tetrapeptide); R8 (octa-arginine aa moiety); CPP (cell penetrating
peptide); MDR (multi-drug resistance); Pgp (ABC transporter P-glycoprotein); MT (mitochondrial); aa (amino acid)

Overview

Immortalization of cancer cells by suppressing or

evading cell-programmed death (apoptosis) is
hallmark of cancer

Multi-drug resistant (MDR) phenotypes are cause

of 30-70% of first-line chemotherapy failure
Resistant to DOX

(MDR-breast cancer)

Proapoptotic proteins, including Smac (AVPI)

tetrapeptide mimetics, demonstrate ability to
overcome intrinsic apoptotic-signalling
suppression
Previously, clinical efficacy of AVPI was limited due to
its low permeability

To overcome this, Wang et al. chimerically

modified AVPI tetrapeptide (AVPIR 8) with CPP
further allowing its use as vehicle for gene
delivery

Apoptosis is
A tightly

regulated process that maintains the

homeostatic cellular balance, protecting against:
Genomic instability (DNA damage, replicative
senescence)
Environmental (pathogen, PAMPs/DAMPs)
Intracellular stress (ROS, nutrient deficiency, genotoxic
drugs, UV or -radiation)

Extrinsic

and intrinsic pathways are

independently activated but converge on
activation of Casp-3/7 effector caspases
Domagoj V, and Fairbrother WJ. Clin Cancer Res. 2007

Extrinsic: paracrine/autocrine activation of Death receptors (FAS, TNF, TLR, TRAIL)

Intrinsic: self-regulatory signals of DNA damage, stress, or senescence that induce MT
permeabilization (directly or indirectly)

Chemotherapy and radiotherapy exert cytotoxicity

primarily through intrinsic apoptosis signalling
Chemotherapy (i.e., Doxorubicin)

Induce MT permeabilization through

indirect mechanisms
DNA damage (p53), Inhibition of apoptosis
(IAP) proteins, ROS-mediated

Radiotherapy (gamma, X-ray)

Generally believed to direct depolarize MT

membrane permeabilization
Release of Cytochrome c, Smac, and other
pro-apoptotic factors

Zhu X, Wang K, Zhang K, et al. Int J Mol Sci. 2013

AVPI: Smac derivatives or mimetics w/ conserved Nterminus 4-aa sequence

Novel approach to combat MDR and apoptotic

evasion is to reactivate apoptosis pathways
IAPs upregulated, Casp suppression, Smac inhibition

Smac protein is a 239 aa polypeptide

55 aa N-terminus encoding MT-targeting sequence is
cleaved upon maturation
Directly binds to IAPs, thus Smac is pro-apoptotic

Smac cut down to 4-aa tetra peptides (AVPI) have

shown in vitro and in vivo apoptotic activity, as well
as are currently in > 20 phase 1-2 clinical trials
AVPI effective at inducing chemo- and radiotherapy

Major drawback is AVPI peptides are poorly

permeability
Dean EJ, Ranson M, Blackhall F, et al. Cancer Treat Rev. 2007.

Chimeric AVPIR8/p53 nanocomplex

AVPI +

R8 moiety (CPP)

AVPIR8

+p53 DNA

AVPIR8/p53 nanocomplex

Chimeric

AVPI derivatives were developed utilizing a C-terminus octa-arginine (R)

sequence (R8)
AVPI-R8 or AVPIR8
Polyarginines (+++++) are considered cell-penetrating sequences

AVPI-R8

exhibits cationic polymer-like characteristic that is employed in gene delivery

vehicles
Arginine has natural affinity got DNA
Escapes MDR P-gp through endosomal uptake and proton sponge mediated endosomal rupture

Methods
Synthesizing

AVPIR8-pDNA Nanocomplexes

Solid Phase Synthesis AVPIR8

Preparation of AVPIR8/pDNA Nanocomplex
Physical

characterization

Agarose Gel Electrophoresis

Particle Size and Zeta Potential
In

Vitro experiments

Transfection
Cytotoxicity (MTT) assay in MCF-7/ADR cells
Apoptosis assay (PI-Flow cytometry)
In

Vivo experiments

Drug-resistant breast tumor model

Balb/c mice were xenografted with MCF-7/ADR (multidrug-resistant breast cancer cell model cells)

Physicochemical Characterization of AVPIR8 / p53 DNA nanocomplexes via mobility-shift assay

(1a), Zeta potential/Particle size (1b), and TEM (1c)

Fig. 1

Transfection efficiency in human MCF-7 and MCF-7/ADR (MDR) cells

Optimal transfection efficiency assayed using

luciferase reporter gene demonstrates 30%
w/w ratio AVPIR8 / pDNA (2a)
W-blot analysis of p53 expression after
AVPIR8/p53 complex transfection
demonstrates p53 expression is restored in
MDR breast cancer model (MCF-7/ADR) (2b)

Fig. 2

In vitro Cell Viability in response to doxorubicin (DOX)

Combination of doxorubicin + AVPIR8 + p53 demonstrate statistically significant greater response in

MDR-breast cancer (3a) and breast cancer model (3b), which is not observed in HUVEC control cells
with intact p53 (3c)

MCF-7: breast cancer model

MCF-7/ADR: MDR-breast cancer model
HUVEC: Human-umbilical endothelial cells

Fig. 3

Quantified Flow Cytometry

Casp-3 (effector caspase) activity in response to DOX

Flow Cytometry Analysis: Annexin/PI

Fig. 4

In vivo anti-MDR efficacy of DOX AVPIR8/p53 in

MDR-breast cancer Balb/c mice
W-blot analysis of P-gp expression in xenograft tumors confirms P-gp
role in MDR (MCF-7/ADR) breast cancer (5a)
DOX + AVPIR8/p53 inhibits tumor growth as demonstrated by tumor
volume and weight measurements (5b,d) and survival rate (5c)

Fig. 5

Off-site toxicity and effect of DOX AVPIR8/p53

on body weight (6a) and organ-specific damage
(6b)

Fig. 6

Conclusion
In

order to overcome MDR breast cancer, codelivery of cell-penetrating peptide/p53 DNA

nanocomplex was developed as adjuvant therapy by re-sensitizing MDR-cancer cells to apoptosis
Smac inhibits IAPs, thus activating Casp-3/7 executioner caspases
Restoration of p53 apoptosis feedback by AVPIR 8-mediated p53 gene delivery

AVPI

Modified with an R 8 polycationic moiety sequence, AVPIR 8, demonstrated:

High transfection capacity of low-permeable Smac-AVPI tetrapeptide

Effective use as vehicle for p53 pDNA delivery
Synergistic enhancement of DOX in non-MDR and MDR breast cancer models
Coadministration

of AVPIR 8/p53 demonstrated full arrest of xenografted MDR-breast cancer

tumors with reduced DOX LD 50

Animal studies using combination of antineoplastic (doxyrubicin) and AVPI-R 8 enabled full arrest
of tumor growth combined with decreased LD 50 thus decreasing off-site toxicity of non-malignant
cancer cells
Cancer cell lines have demonstrated evasion of Smac mimetic-induced apoptosis by up-regulation
of IAPs, which although initially degraded, rebounds and is refractory to subsequent degradation

Works Cited
1.

Zhu X, Wang K, Zhang K, et al. Ziyuglycoside II inhibits the growth of human breast carcinoma MDA-MB-435 cells via cell cycle arrest and induction
of apoptosis through the mitochondria dependent pathway. Int J Mol Sci. 2013;14(9):18041-55.

2.

Domagoj V, Wayne J. Fairbrother Clin Cancer Res. 2007;13:5995-6000

3.

Qin S, Yang C, Li S, Xu C, Zhao Y, Ren H. Smac: Its role in apoptosis induction and use in lung cancer diagnosis and treatment. Cancer Lett.
2012;318(1):9-13.

4.

James D, Parone PA, Terradillos O, Lucken-ardjomande S, Montessuit S, Martinou JC. Mechanisms of mitochondrial outer membrane
permeabilization. Novartis Found Symp. 2007;287:170-6.

5.

Nachmias B, Ashhab Y, Ben-yehuda D. The inhibitor of apoptosis protein family (IAPs): an emerging therapeutic target in cancer. Semin Cancer Biol.
2004;14(4):231-43.

6.

Dean EJ, Ranson M, Blackhall F, Holt SV, Dive C. Novel therapeutic targets in lung cancer: Inhibitor of apoptosis proteins from laboratory to clinic.
Cancer Treat Rev. 2007;33(2):203-12.

7.

Shiozaki EN, Shi Y. Caspases, IAPs and Smac/DIABLO: mechanisms from structural biology. Trends Biochem Sci. 2004;29(9):486-94.

8.

Fulda S, Debatin KM. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene. 2006;25(34):4798-811.

9.

Patra HK, Turner AP. The potential legacy of cancer nanotechnology: cellular selection. Trends Biotechnol. 2014;32(1):21-31.

10.

Giacca M, Zacchigna S. Virus-mediated gene delivery for human gene therapy. J Control Release. 2012;161(2):377-88.

11.

Dong Y, Love KT, Dorkin JR, et al. Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates. Proc Natl
Acad Sci USA. 2014

12.

Li X, Zhao Q, Qiu L. Smart ligand: aptamer-mediated targeted delivery of chemotherapeutic drugs and siRNA for cancer therapy . J Control Release.
2013;171(2):152-62.

13.

Shi Y. A conserved tetrapeptide motif: potentiating apoptosis through IAP-binding. Cell Death. 2002; 9:93-95

14.

Li H, Tsui TY, Ma W. Intracellular Delivery of Molecular Cargo Using Cell-Penetrating Peptides and the Combination Strategies . Int J Mol Sci.
2015;16(8):19518-36.

Dean EJ, Ranson M, Blackhall F, et al. Cancer Treat Rev. 2007.

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