Sticky siRNAs targeting survivin and cyclin B1 exert an antitumoral effect on melanoma subcutaneous xenografts and lung metastases

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Sticky siRNAs targeting survivin and cyclin B1 exert an antitumoral effect on
melanoma subcutaneous xenografts and lung metastases
BMC Cancer 2013, 13:338 doi:10.1186/1471-2407-13-338
Valerie Kedinger (vkedinger@polyplus-transfection.com)
Aline Meulle (meullealine@yahoo.fr)
Omar Zounib (omar.zounib@gmail.com)
Marie-Elise Bonnet (bmarieelise@yahoo.fr)
Jean-Baptiste Gossart (jeanbaptiste.gossart@yahoo.fr)
Elodie Benoit (benoitelodie@yahoo.fr)
Melanie Messmer (mmessmer047@gmail.com)
Pattabhiraman Shankaranarayanan (shankar@igbmc.fr)
Jean-Paul Behr (jpbehr@free.fr)
Patrick Erbacher (PERBACHER@polyplus-transfection.com)
Anne-Laure Bolcato-Bellemin (al_bolcato@yahoo.com)
ISSN 1471-2407
Article type Research article
Submission date 26 February 2013
Acceptance date 1 July 2013
Publication date 9 July 2013
Article URL http://www.biomedcentral.com/1471-2407/13/338
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© 2013 Kedinger et al.
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Sticky siRNAs targeting survivin and cyclin B1 exert
an antitumoral effect on melanoma subcutaneous
xenografts and lung metastases
Valerie Kedinger1*
*
Corresponding author
Email: vkedinger@polyplus-transfection.com
Aline Meulle1
Email: meullealine@yahoo.fr
Omar Zounib1
Email: omar.zounib@gmail.com
Marie-Elise Bonnet1
Email: bmarieelise@yahoo.fr
Jean-Baptiste Gossart1
Email: jeanbaptiste.gossart@yahoo.fr
Elodie Benoit1
Email: benoitelodie@yahoo.fr
Melanie Messmer2
Email: mmessmer047@gmail.com
Pattabhiraman Shankaranarayanan3
Email: shankar@igbmc.fr
Jean-Paul Behr1
Email: jpbehr@free.fr
Patrick Erbacher1
Email: PERBACHER@polyplus-transfection.com
Anne-Laure Bolcato-Bellemin4*
*
Corresponding author
Email: al_bolcato@yahoo.com
1
Polyplus-transfection SA, Bioparc, BP 90018, Boulevard Sébastien Brant,
67401 Illkirch, France
2
Current address: IBMC, 15 rue René Descartes, 67084 Strasbourg, France
3
IGBMC, 1 rue Laurent Fries, 67400 Illkirch, France
4
Current address: Quintiles, rue Jean Dominique Cassini, BP 50137, 67404
Illkirch Cedex, France

Abstract
Background
Melanoma represents one of the most aggressive and therapeutically challenging
malignancies as it often gives rise to metastases and develops resistance to classical
chemotherapeutic agents. Although diverse therapies have been generated, no major
improvement of the patient prognosis has been noticed. One promising alternative to the
conventional therapeutic approaches currently available is the inactivation of proteins
essential for survival and/or progression of melanomas by means of RNA interference.
Survivin and cyclin B1, both involved in cell survival and proliferation and frequently
deregulated in human cancers, are good candidate target genes for siRNA mediated
therapeutics.
Methods
We used our newly developed sticky siRNA-based technology delivered with linear
polyethyleneimine (PEI) to inhibit the expression of survivin and cyclin B1 both in vitro and
in vivo, and addressed the effect of this inhibition on B16-F10 murine melanoma tumor
development.
Results
We confirm that survivin and cyclin B1 downregulation through a RNA interference
mechanism induces a blockage of the cell cycle as well as impaired proliferation of B16-F10
cells in vitro. Most importantly, PEI-mediated systemic delivery of sticky siRNAs against
survivin and cyclin B1 efficiently blocks growth of established subcutaneaous B16-F10
tumors as well as formation and dissemination of melanoma lung metastases. In addition, we
highlight that inhibition of survivin expression increases the effect of doxorubicin on lung
B16-F10 metastasis growth inhibition.
Conclusion
PEI-mediated delivery of sticky siRNAs targeting genes involved in tumor progression such
as survivin and cyclin B1, either alone or in combination with chemotherapeutic drugs,
represents a promising strategy for melanoma treatment.
Keywords
Sticky siRNA, Delivery, Survivin, Cyclin B1, Tumor inhibition, Melanoma
Background
Melanoma is considered as one of the most aggressive human cancers. The majority of
melanoma-associated deaths are caused by metastases, which can occur in a wider variety of
areas than any other cancer [1]. Among target organs, the lung represents a common site of
metastasis, mostly because of its anatomic structure and high vascularization. These

characteristics make it a preferential pathway for metastatic seeding and a rich environment
for neoplastic growth [2]. Another feature commonly attributed to melanoma is its chemo-
resistance [3]. During melanoma progression, the breakdown of cell death control leading to
resistance to chemotherapeutic drugs is achieved through the combined activation of anti-
apoptotic factors, inactivation of pro-apoptotic effectors and reinforcement of survival
signals. Targeting one or more of these different factors may be a key requisite to overcome
drug resistance and thus improve clinical outcome of patients with melanoma.
Survivin, a member of the Inhibitor of Apoptosis Protein (IAP) family [4], has emerged few
years ago as a promising therapeutic target in cancers because of its overexpression in a wide
spectrum of tumors, including melanoma [5-7]. Additionally, survivin was identified as a
marker of poor prognosis in melanoma [8]. In addition to its role in apoptosis inhibition,
survivin also plays a critical role in the regulation of cell division by inducing exit from G1
checkpoint arrest and subsequent entry into S phase [9]. Finally, recent studies have involved
survivin in cell motility, which may underlie a role for this protein in promoting melanoma
metastasis [10-12]. Various approaches involving molecular inhibitors have been developed
to inhibit its expression in tumor cells [6,13-17]. Another potential therapeutic target for
cancer treatment is represented by cyclin B1, the regulatory subunit of cyclin-dependent
kinase 1 (cdk1), which plays a pivotal role in the transition of the cell cycle from G2 phase to
mitosis [18]. Altered expression of cyclin B1 has been reported in numerous cancers, where it
could contribute to chromosomal instability [19-23]. Furthermore, several studies have
demonstrated its clinical significance as a poor prognosis factor for several cancer types [24-
27], including melanoma [28], and cyclin B1 overexpression is responsible for radiotherapy
resistance in different tumors [29-31].
RNA interference represents a powerful approach for antitumor therapy by allowing in vivo
silencing of essential genes for tumor progression and provides a promising alternative to
traditional small molecule therapies. However, delivery of siRNAs still remains the most
challenging step for the development of a siRNA-based therapy. The challenge includes
efficient target gene silencing in the desired tissue while avoiding side effects such as
immune response, toxicity and off-target silencing. In this context, the cationic linear
polyethylenimine (PEI) is well known for its efficiency to transfect genes both in vitro and in
vivo as it is involved in several clinical trials for the treatment of bladder cancer
(http://clinicaltrials.gov/ct2/show/NCT00595088), pancreatic ductal adenocarcinoma
(http://clinicaltrials.gov/ct2/show/NCT01274455) and multiple myeloma
(http://clinicaltrials.gov/ct2/show/NCT01435720?term=senesco&rank=1). In this study, we
investigated its ability to deliver functional anti-tumoral siRNA. To this end, we have
developed sticky siRNAs (ssiRNAs) that mimic gene structure through reversible
concatemerization brought by sticky 3’-complementary overhangs [32]. These modified
siRNAs confer a higher stability to the complexes formed with linear PEI, thus increasing
gene silencing efficiency both in vitro and in vivo, compared with standard siRNAs. We used
this new technology to specifically target survivin and cyclin B1 in B16-F10 murine
melanoma cells. Our results show that ssiRNAs are efficient to inhibit survivin and cyclin B1
expression in vitro and that a systemic treatment with ssiRNAs targeting these two genes is
able to reduce both subcutaneous melanoma tumors and their lung metastases. Moreover,
inhibition of survivin expression increased the effect of a doxorubicin treatment on melanoma
lung metastasis. Altogether, our data are promising towards development of ssiRNAs against
survivin and cyclin B1 as a new therapeutic strategy for melanoma treatment.

Methods
Cell line
Murine melanoma B16-F10 cell line was obtained from ATCC and cultured in Dulbecco’s
modified Eagle’s medium (Eurobio, Courtaboeuf, France), supplemented with 10% fetal
bovine serum (Hyclone, Logan, UT, USA), 2 mM Glutamine (Eurobio) and 200 U/ml
penicillin / 200 µg/ml streptomycin (Eurobio).
Sticky siRNAs
IEX-HPLC-purified nucleic acids were purchased from Eurogentec (Brussels, Belgium).
Annealing was performed in annealing buffer (Eurogentec), final concentration 0.1 X for 2
min at 95°C followed by slow cooling. Sequences were as follow:
Cyclin B1 ssiRNA sense, 5’-GAGAUGUACCCUCCAGAAAdTdTdTdTdTdTdTdT-3’,
Cyclin B1 ssiRNA antisense, 5’-UUUCUGGAGGGUACAUCUCdAdAdAdAdAdAdAdA-
3’,
Survivin ssiRNA sense, 5’-CCGUCAGUGAAUUCUUGAAdTdTdTdTdTdTdTdT-3’,
Survivin ssiRNA antisense, 5’-UUCAAGAAUUCACUGACGGdAdAdAdAdAdAdAdA-3’,
Negative control ssiRNA sense, 5’-AUGUCUACUGGCAGUCCUGdTdTdTdTdTdTdTdT-
3’,
Negative control ssiRNA antisense, 5’-
CAGGACUGCCAGUAGACAUdAdAdAdAdAdAdAdA-3’.
In vitro and in vivo transfections
jetPEI® and in vivo-jetPEI® were from Polyplus-transfection (Illkirch, France). For
transfection with jetPEI® reagent, complexes were prepared as follows: for a triplicate
experiment, the required amount of ssiRNA and transfection reagent were each separately
diluted in 150 µl of NaCl 150 mM. A volume of 2.4 µl (for 75 nM of ssiRNA, N/P=6) or 3.2
µl (for 50 nM of ssiRNA, N/P=8) of jetPEI® was used per µg of siRNA. N/P ratio is defined
as the number of nitrogen residues of jetPEI® per nucleic acid phosphate. The transfection
reagent solution was added to the ssiRNA solution and left for 30 min at room temperature. A
volume of 100 µl of complexes was added to B16-F10 cells seeded in 24-well plates at
40,000 cells/well one day before, and placed in 0.5 ml of medium without serum just prior to
complexes addition. After 4 h, the medium was replaced by 1 ml of complete medium
containing 10% serum.
For in vivo delivery with in vivo-jetPEI®, complexes were prepared as follows: for 1 mouse,
the required amount of nucleic acid and PEI were separately diluted in 100 µl of 5% glucose
solution (final concentration). For injection of 0.6 mg/kg of ssiRNA, 0.16 µl of PEI were used
per µg of ssiRNA (N/P=8). For the 1 mg/kg ssiRNA injected amount, 0.25 µl of PEI were
used per µg of ssiRNA (N/P=12.5). The transfection reagent solution was added to the

ssiRNA solution and left for at least 30 min at room temperature. At this stage complexes are
stable for more than 4 h at room temperature.
Animal experiments
All animal studies were conducted in accordance to the French Animal Care guidelines and
the protocols were approved by the Direction des Services Vétérinaires. Five-weeks old
NMRI Nude female mice were obtained from Elevage Janvier (Le Genset Saint Isle, France).
For subcutaneous xenografts, B16-F10 cells (1 × 106
cells in 100 µl of culture medium
without serum) were injected subcutaneously on the right flank of animals. ssiRNA/PEI
complexes were intravenously injected through the retro-orbital sinus within 2 s. Treatment
with ssiRNA complexes started when tumors reached 50 mm3
and were performed every
other day until sacrifice of the animals. Tumors were measured at each injection, and tumor
volume was calculated as v = (π × L × l2
)/6. For lung metastasis model, B16-F10 cells (1 ×
106
cells in 300 µl of culture medium without serum) were injected intravenously through the
tail vein.
Branched DNA assay
QuantiGene assay (Panomics, Santa Clara, CA, USA) was used to quantify the amount of
mRNA in cells or lungs. Cells were lysed in 600 µl of 1 x lysis buffer and incubated for 30
min at 50 °C. Lungs were lysed in 20 ml of tissue and cell lysis solution (Tebu, Le Perray-en-
Yvelines, France), supplemented with 0.15 mg/ml of K Proteinase (Sigma-Aldrich, St Louis,
MO, USA) and incubated three times 5 min at 60 °C with 10 s vortexing. A volume of 1–60
µl of cell or lung lysate was used for branched DNA (bDNA) assay. Probe set were designed
using QuantiGene ProbeDesigner software. Target gene expression was assayed according to
manufacturer recommendations. Target expression level was normalized to corresponding
GAPDH expression from the same cell lysate.
Western blot analysis
Cells were lysed in 100 µl of RIPA buffer. Proteins were quantified with the BCA kit (Pierce,
Brebieres, France). Fifty micrograms of total protein were subjected to electrophoresis on a
10 or 15% acrylamide/bisacrylamide gel and transferred to a poly(vinylidene fluoride)
membrane (Millipore, Molsheim, France). A mouse anti-cyclin B1 monoclonal antibody
(Cell Signaling Technologies, Danvers, MA, USA) at 1/1,000, a rabbit anti-survivin
polyclonal antibody (Cell Signaling Technologies) at 1/1,000 and a mouse anti-GAPDH
monoclonal antibody (Ambion, Austin, TX) at 1/10,000 were used. Anti-rabbit or anti-mouse
secondary horseradish peroxidase-conjugated were purchased from Millipore and used at
1/10,000. Protein bands were visualized with enhanced chimioluminescence reagent (ECL,
Amersham, GE Healthcare, Velizy-Villacoublay, France).
Proliferation assay
Cell pellets were homogenized in 100 µl of 1:1 PBS/trypan blue (Eurobio) and live cells were
counted using an automatic hematocyter (TC10, BioRad, Marnes-la-Coquette, France).

For nuclei morphology analysis, cells were fixed with ice-cold methanol for 10 min, rinsed
with 1 x PBS and stained with DAPI (0.01 µg/µl) for 15 min. Cells were observed using a
Nikon inverted microscope (Nikon Eclipse TE 2000-S, Amsterdam, Netherland).
Cell cycle and apoptosis analysis
Cells were fixed in chilled 50% ethanol for 15 min at −20 °C. Pellets were incubated in 1 x
PBS with 0.1% Triton X-100 and 5% BSA for 10 min on ice, and for 30 min at 37 °C in 1 x
PBS containing 20 µg/ml of RNase A. Propidium iodide (100 µg/ml) was added for 10 min at
room temperature. Cell pellet was resuspended in 1 x PBS, 5 mM EDTA to obtain a cell
concentration lower than 5.105
cells/ml. Cells were analyzed by FACS using a Guava
apparatus from Millipore (Molsheim, France).
5’-RACE analysis
Total RNA was isolated using RNA NOW reagent (Biogentex Laboratories, Houston, TX,
USA) following instructions of the manufacturer. One µg of RNA was ligated to GeneRacer
TM
RNA Oligo (5’-CGA-CUG-GAG-CAC-GAG-GAC-ACU-GAC-AUG-GAC-UGA-AGG-
AGU-AGA-AA-3’; Life Technologies, Saint Aubin, France). Two-hundred and fifty
nanograms of Oligo were used respectively for cyclin B1 or survivin analysis. Ligated RNA
was reverse transcribed using a gene-specific primer (Table 1). To detect the cleavage
product, one or two rounds of consecutive PCR (for conditions see Additional file 1: Table
S1) were performed using primers complementary to the RNA Oligo and to cyclin B1 or
survivin gene sequence (Table 2).
Table 1 Primers used for Reverse transcription
Name Sequence
Cyclin B1 Rev (CCNBM1548R) 5′-TTC-GAC-AAC-TTC-CGT-TAG-CC-3′
Survivin Rev (810R) 5′-AGC-TCT-GGA-CTC-TGG-CCA-CCC-3′
Table 2 Primers used for PCR
Name Sequence
Cyclin B1 Rev (CCNBM975R) 5′-AGG-GCG-ACC-CAG-GCT-GAA-GT-3′
Survivin Rev (810R) 5′-AGC-TCT-GGA-CTC-TGG-CCA-CCC-3′
Survivin RevN (743R) 5′-GCC-ACC-TCC-CTG-TGG-ACT-CA-3′
Histology
After mice sacrifice, lungs were perfused with 4% paraformaldehyde, incubated for 16 h in
4% paraformaldehyde and processed for paraffin embedding. Paraffin sections (7 µm) were
generated and Hematoxylin/Eosin (H&E) stained.
Statistical analysis
All statistical analyses were performed using GraphPad prism software package (version 5).
P values were calculated according to Mann-Withney test and were considered significant
when lower than 0.05.

Results and discussion
in vitro ssiRNAs gene silencing efficiency in B16-F10 murine melanoma cells
We used previously validated survivin and cyclin B1 siRNA sequences [33,34], designed
sticky siRNAs with these sequences by adding 3’ complementary overhangs and first tested
their silencing efficiency both at the mRNA and protein levels in B16-F10 cells. A sequence
presenting no homology with mRNA databases was used as a negative control. Transfection
of B16-F10 cells with jetPEI® and different concentrations of specific ssiRNAs ranging from
25 to 100 nM was performed and all led to a significant reduction of cyclin B1 and survivin
mRNA levels. The level of inhibition was optimal at 50 nM, N/P of 8 and 75 nM, N/P of 6
(Figure 1a and b). Indeed, both concentrations induced a 70 to 80% inhibition of the two
target mRNA expression compared to control cells. This downregulation of mRNA
expression induced a diminution of the corresponding protein level for at least 48 h for both
target proteins, as analyzed by Western blot (Figure 1c).
Figure 1 Inhibition of survivin and cyclin B1 expression in B16-F10 cells. (a, b) mRNA
expression level of cyclin B1 and survivin analyzed by branched DNA 16 h after transfection
with ssiRNAs and PEI. Cyclin B1 and survivin levels are expressed relative to GAPDH. (c)
Protein level of cyclin B1 and survivin analyzed by Western blot 24, 48 and 72 h after
transfection with a negative control ssiRNA (2) or ssiRNAs against cyclin B1 or survivin (3)
(50 nM, N/P=8). Non transfected cells (1) were also loaded. GAPDH was used as a loading
control. (d) In vitro 5’-RACE analysis of RNA extracted from B16-F10 cells either
untransfected (1) or 16 h after transfection with negative control (2), cyclin B1 or survivin (3)
ssiRNAs and PEI (50 nM, N/P 8). Top part, schematic representation of the 5’-RACE
procedure.
In order to confirm that the gene silencing observed after transfection occurred through a
RNAi mechanism, we then performed rapid amplification of cDNA ends (5’-RACE) on
RNAs extracted from B16-F10 cells transfected with cyclin B1, survivin or negative control
ssiRNA. A band corresponding to the predicted cleavage product (158 bp for cyclin B1 and
330 bp for survivin) was specifically detected from RNAs extracted of both cyclin B1 and
survivin ssiRNA transfected cells (Figure 1d). Sequencing analysis of the PCR products
confirmed their specificity (data not shown).
The inhibition of cyclin B1 and survivin expression observed after ssiRNA transfections was
accompanied by a specific cellular effect, as cells presented a growth inhibition of more than
80% compared to control cells 48 h post-transfection (Figure 2a). The proliferation rate of
cells transfected with the negative control ssiRNA was also somewhat lowered compared to
control cells. This diminution can be attributed to the transfection itself, which causes a
transient blockage of the cell cycle progression. To further characterize the growth inhibition
induced by the down-regulation of both cyclin B1 and survivin, we analyzed by flow
cytometry B16-F10 DNA content 48 h after transfection of ssiRNAs (Figure 2b). Whereas
only 17.9 and 12.6% of nontransfected or negative control ssiRNA-transfected cells were
located in G2/M phase, respectively, more than 50% of cells transfected with survivin
ssiRNA were arrested in G2/M phase. Comparatively, the effect was milder after cyclin B1
inhibition, yet a significant blockage of the cells in G2/M phase was still present (Figure 2b).
Moreover, percentage of apoptotic cells was also increased following downregulation of
survivin and cyclin B1 expression compared to controls (Figure 2c). Finally, DAPI staining

of cells in which cyclin B1 or survivin expression was inhibited showed a high proportion of
cells presenting mega-nuclei (more than 50% of the cells), a characteristic of cell cycle arrest
(Figure 2d).
Figure 2 Effect of survivin and cyclin B1 inhibition on B16-F10 cell cycle progression.
(a) Proliferation assay 48 h after transfection of ssiRNAs. The proliferation rate is
represented as a percentage of cell growth relative to control untransfected cells. (b) Flow
cytometer analysis of the cell cycle distribution of untransfected B16-F10 cells or 48 h after
transfection with negative control, survivin or cyclin B1 ssiRNAs with PEI (50 nM, N/P=8).
(c) Table presenting the percentage of cells in apoptosis (subG1 population) of 3 independent
experiments determined after FACS analysis in the presence of propidium iodide 48 h after
transfection with negative control and specific ssiRNAs with PEI (50 nM, N/P=8). (d) DAPI
staining, 48 h after transfection with negative control, cyclin B1 or survivin ssiRNAs with
PEI (50 nM, N/P=8) showing the presence of mega-nuclei and chromosomal aberrations in
cells transfected with either cyclin B1 or survivin ssiRNAs.
Altogether, these results validate the ssiRNA approach to silence cyclin B1 and survivin
through a mechanism of RNA interference in vitro in B16-F10 murine melanoma cells.
Systemic treatment with ssiRNAs inhibits growth of established subcutaneous
melanoma xenografts
B16-F10 cells have the ability to form tumors when injected subcutaneously in nude mice
[35]. We took advantage of this model mimicking a primary melanoma tumor to test the
potency and specificity of PEI-mediated delivery of ssiRNAs in vivo. First, in order to
confirm a RNA interference mechanism of ssiRNAs in vivo, we performed a 5’-RACE
analysis after intra-tumoral injection of ssiRNA (0.6 mg/kg, N/P=8) complexed with in vivo-
jetPEI®. As shown in Figure 3a, we detected a band specific for the cleavage product after
injection of either cyclin B1 or survivin ssiRNAs in B16-F10 xenografts that could not be
detected in glucose or negative control ssiRNA injected tumors.
Figure 3 B16-F10 tumor xenograft growth inhibition upon systemic treatment of mice
with ssiRNAs and PEI. (a) In vivo 5’-RACE analysis of RNA extracted from subcutaneous
tumors 16 h after intra-tumoral injection of 0.6 mg/kg of negative control (2) and survivin or
cyclin B1 (3) ssiRNA and PEI (N/P=8). As a control, 5’-RACE was also performed on RNA
extracted from a tumor injected with glucose only (1). (b, c) Mice bearing tumor xenografts
were treated intravenously every other day with glucose (diamond), negative control ssiRNA
(triangle) and survivin (b) or cyclin B1 (c) ssiRNA (square) at 1 mg/kg, N/P=12.5. Tumor
growth was monitored after each ssiRNA injection and represented as a mean tumor volume
± SEM. n = 10 animals per group. P values were calculated between control (glucose treated)
group and specific ssiRNA groups; * p<0.05; **p<0.01.
We then assessed the effect of a systemic treatment of ssiRNAs delivered with in vivo-
jetPEI® on the growth of B16-F10 xenografts. Intravenous delivery of ssiRNAs at 1 mg/kg
(N/P = 12.5) was performed every other day. The mean tumor size monitored after each
injection is represented in Figure 3b for each group. We started to observe a reduction of
tumor growth after the third injection of ssiRNA against survivin (i.e., 5 days after tumor
cells injection), which persisted until the end of the experiment and reached a significant
inhibition of up to 50% compared to control mice (Figure 3b). The effect of cyclin B1
ssiRNA was less pronounced, yet still significant as it leads to 44% inhibition of tumor

growth compared to controls (Figure 3c). The difference observed between the two target
genes could be explained by the multifunctionality of survivin compared to cyclin B1, as the
first protein is implicated in apoptosis, proliferation and motility [36], whereas the second
one only plays a critical role in the control of the cell cycle progression and apoptosis [18].
The mice treated with negative control ssiRNA presented a minor, statistically nonsignificant,
reduction of tumor size. The difference between the control and negative control ssiRNA
groups could reflect the nonspecific effect of transfection on the cell cycle previously
observed in vitro (see above).
Melanoma lung metastases are reduced following systemic treatment with
ssiRNAs
Metastases, which represent the leading cause of mortality for patients with melanoma, are
the most challenging cells to target. PEI was previously shown to preferentially deliver active
nucleic acids, plasmids and siRNAs, to the lung after intravenous injection [37-40]. We
therefore wondered whether systemic treatment of antitumoral ssiRNAs delivered with PEI
could reduce the formation of melanoma lung metastases. When injected into nude mice tail
vein, B16-F10 cells rapidly give rise to lung metastases. The advantage of this model is that it
circumvents the initial step of tumor cell migration and allows us to evaluate the effect of our
treatment on the implantation of metastatic cells in the host tissue and their subsequent
proliferation. Treatment with ssiRNAs was performed as described in Figure 4a. At the end
of the experiment, lungs were excised and observed for metastatic nodules. Treatment with
cyclin B1 and survivin specific ssiRNAs led to an important reduction of the number of
metastases (black nodules) compared to controls as illustrated by representative pictures of
lungs (Figure 4b). The lung weight was also used as an indicator of metastasis progression.
We determined the mean percentage of lung over body weight in each group and observed a
significant decrease in groups treated with survivin and cyclin B1 ssiRNAs compared to
control groups (Figure 4c). Histological analysis of lung sections was performed which
further confirmed the drastic diminution of both nodule size and number in both survivin and
cyclin B1 ssiRNA treated lungs (Figure 4d).
Figure 4 Anti-metastatic therapeutic effect of a systemic treatment with ssiRNAs and
PEI. Nude mice were i.v. injected with B16-F10 cells, randomized and treated with glucose,
negative control, survivin or cyclin B1 ssiRNAs and PEI (1 mg/kg, N/P=12.5). (a) Schematic
representation of the treatment procedure. (b) Representative pictures of lungs excised from
mice of the different treated groups. (c) Overall mean lung weight relative to the body weight
of the mice. n = 8 animals per group. P values were calculated; * p<0.05; **p<0.01. (d)
Representative images of Hematoxylin-Eosin stained lung sections after treatment with
glucose, survivin or cyclin B1 ssiRNAs and PEI.
Even if representative of the metastatic grade, lung weight evaluation is not the best
quantitative method, and probably leads to an underestimation of the effect produced by the
ssiRNAs injection. Indeed, with this method only 20 and 25% reduction of lung tumor weight
were observed following cyclin B1 and survivin ssiRNA treatment, respectively. We thus
developed a quantitative assay based on MITF dosage. Indeed, as MITF is a melanocyte
specific transcription factor [41], it allows quantification of the B16-F10 nodules present in
the lungs. When MITF expression is detected in lungs, its level should be directly
proportional to the number of B16-F10 tumor cells. To verify this hypothesis, we analyzed
MITF mRNA level in lungs containing increasing number of B16-F10 tumors (Figure 5a). As
expected, no MITF mRNA was observed in lungs without B16-F10 tumors. In lungs

presenting an intermediate number of B16-F10 tumors, an intermediate level of MITF was
observed, whereas in lungs with a high level of tumor cells, a high level of MITF was
observed (Figure 5a). In addition, we validated the linearity of our MITF assay (see
Additional file 2: Figure S1).
Figure 5 Quantification of the B16-F10 lung metastases using dosage of MITF
melanocyte specific transcription factor expression. (a) Branched DNA analysis of RNA
extracted from wild-type or B16-F10 tumor-bearing lungs for determination of MITF
transcription factor mRNA expression level. (b) MITF RNA level was analyzed by branched
DNA in B16-F10 tumor-bearing lungs untreated (control) or after a systemic treatment with
survivin and cyclin B1 or negative control ssiRNA delivered with in vivo-JetPEI® (1 mg/kg,
N/P=12.5). n = 6 to 9 animals per group. P values were calculated; * p<0.05; ***p<0.001.
We then looked at the MITF mRNA level in lungs of ssiRNA-treated mice, and observed a
significant diminution after either survivin or cyclin B1 ssiRNA systemic treatment compared
to the level of MITF present in lungs of glucose or negative control ssiRNA treated mice (65
and 56% inhibition compared to negative control ssiRNA, respectively, Figure 5b). These
results are in better agreement with our macroscopic and microscopic observations, and
confirm the anti-metastatic effect of a systemic treatment of mice with ssiRNAs targeting
genes implicated in the cell cycle regulation.
Alternated treatment with doxorubicin and survivin ssiRNA induces an
additive diminution of melanoma lung metastases
The poor prognosis attributed to melanomas largely results from resistance to conventional
chemotherapy [3,42]. Doxorubicin, a topoisomerase II inhibitor, has been used for years to
treat diverse types of cancers, and it is one of the most effective anticancer drugs currently
known. However, its clinical use is limited by dose-dependent toxicity, low specificity
against cancer cells and emergence of resistance [43]. Indeed, melanoma tumors are known
to be partially refractory to doxorubicin [3], and high doses with subsequent toxic effects are
necessary to induce tumor regression. Since survivin was shown to be a key factor in chemo-
resistance, we hypothesized that a treatment with survivin ssiRNA could enhance the effect
of doxorubicin on the inhibition of melanoma lung metastasis growth. In order to answer this
question, we first established the optimal dose of doxorubicin, which would induce tumor
growth inhibition with the least toxicity. To this end, three doxorubicin treatments (at day 4, 8
and 10) at three different doses (1, 2.5 and 10 mg/kg) were performed following B16-F10
tumor cells injection. Tumor growth inhibition was evaluated by lung weight and toxicity was
determined by body weight loss during the course of the experiment. The treatment with
doxorubicin at 1 mg/kg was not toxic at all but had no effect on tumor growth either. On the
other end, the 10 mg/kg dose was very efficient on lung tumors but induced a drastic body
weight loss, almost 15% at the end of the experiment, accompanied by a general posture of
the mice which was characteristic of a high toxicity (Figure 6a and b). We thus retained the
intermediate dose of 2.5 mg/kg which had only a mild effect on tumor growth inhibition but
also shows minimal toxicity for the animals. We then evaluated the effect of an alternating
treatment of doxorubicin and survivin ssiRNA compared to a treatment with either
doxorubicin or survivin ssiRNA alone. The treatment with doxorubicin and ssiRNA was
performed every alternate day as illustrated on Figure 6c. Doxorubicin (2.5 mg/kg) and
survivin ssiRNA (1 mg/kg) had an additive effect on lung metastasis growth inhibition
(Figure 6d). Whereas doxorubicin alone at 2.5 mg/kg was poorly efficient, its combination
with survivin ssiRNA induced a significant metastasis inhibition as determined by a lung

weight decrease compared to controls, and this without producing any toxicity. A strong
diminution of the number and size of tumor nodules was observed visually (data not shown).
Moreover, dosage of MITF mRNA level showed a 87% inhibition of tumor growth (MITF
level, 0.6 × 105
+/− 0.06 × 105
) following the alternating treatment of doxorubicin and
survivin ssiRNA compared to controls (MITF level, 4.3 × 105
+/− 0.6 × 105
) which was
significantly higher than the survivin ssiRNA conditions without doxorubicin (54% inhibition
compared to control, MITF level, 1.9 × 105
+/− 0.4 × 105
).
Figure 6 Additive effect of survivin ssiRNA and doxorubicin on the treatment of lung
metastases. (a) Dose dependent effect of doxorubicin (1, 2.5 or 10 mg/kg) on lung metastasis
growth assessed by the overall mean lung weight relative to the body weight of the mice. n =
9 animals per group. (b) Body weight measurement from the beginning to the end of the
treatment was evaluated to determine the toxicity of doxorubicin treatments. (c) Schematic
representation of the treatment procedure with doxorubicin alone, survivin ssiRNA alone, or
both. (d) Overall mean lung weight relative to the body weight of the mice after an alternated
treatment of doxorubicin and survivin ssiRNA with PEI, compared to treatment with
doxorubicin or survivin ssiRNA alone. n = 9 animals per group. P values were calculated; *
p<0.05; **p<0.01.
These results are very encouraging for the development of survivin ssiRNA as a new strategy
to circumvent the inherent chemo-resistance of melanomas or to enhance the
chemosensitivity of melanomas. This combination therapy using ssiRNAs and chemotherapy
offers a novel strategy for cancer treatment and is confirmed in other cancers [44,45].
Conclusion
RNA interference is evolving as a promising strategy for cancer treatment. However, delivery
of siRNAs in vivo still remains the major issue for the development of a successful siRNA-
based therapy. Our present study highlights an emerging RNAi technology, based on the use
of sticky siRNAs delivered with PEI. These modified siRNAs, by mimicking gene structure,
enhance siRNA delivery into cells and consequently lead to a better inhibition of genes
involved in malignancies. In order to validate this new technology, we chose to design sticky
siRNAs against two well-known players of the tumor progression process, survivin and
cyclin B1 [19,46,47]. The results presented in this work demonstrate that PEI-mediated
systemic delivery of sticky siRNAs against survivin and cyclin B1 lead to an efficient
inhibition of tumor growth. Moreover, we showed in a previous study that no major
inflammation is induced by linear PEI-mediated nucleic acid delivery in vivo, as neither pro-
inflammatory cytokines nor hepatic enzymes were produced [48]. Altogether, these data are
very encouraging for the clinical development of such therapies which could represent a
promising approach for melanoma treatment.
Abbreviations
ssiRNA, sticky siRNA; PEI, polyethyleneimine; N/P, PEI amine over nucleic acid phosphate
charge ratio

Competing interests
All the authors except Pattabhiraman Shankaranarayanan are employees of the Polyplus-
transfection company, they declare no competing interests.
Authors’ contributions
VK carried out in vivo and in vitro experiments, participated in the design of the study and
drafted the manuscript, AM carried out in vivo experiments and participated in the design of
the study, OZ carried out in vivo and in vitro experiments, MEB carried out in vivo and in
vitro experiments, JBG carried out in vivo and in vitro experiments, EB carried out in vivo
experiments, MM carried out in vitro experiments, PS gave some material and critically
revised the manuscript, JPB participated in the design of the study and critically revised the
manuscript, PE conceived the study, coordinated and helped to draft the manuscript, ALBB
conceived the study, participated in its design and coordinated and helped to draft the
manuscript. All authors read and approved the final manuscript.
Acknowledgments
We thank Dr Claire Wartel-Weil and Geraldine Guerin-Peyrou for critical reading of the
manuscript. This work was supported by OSEO Innovation and Conseil Régional d’Alsace.
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Additional files
Additional_file_1 as PPTX
Additional file 1: Table S1 PCR conditions for 5’ RACE analysis.
Additional_file_2 as PPTX
Additional file 2: Figure S1 Linearity of MITF assay. Branched DNA analysis of RNA
extracted from wild-type or B16-F10 tumor-bearing lungs to determine MITF mRNA
expression level. Different volumes of lung extract (0.1; 0.5; 1 and 5 µl) were analyzed,
showing a good linearity of the assay.

a b
c Cleavage site
GeneRacer
RNA adaptor mRNA 5Ó"cleavage product
IT7Ó Rev
ITP7Ó RevN
Hkpcn"7Ó-RACE PCR product
1
cyclin B1survivin
300
500
400
2 31 2 3
17 KDa
36 KDa
36 KDa
58 KDa
d
0
0 . 5
1
1 . 5
2
2 . 5
3
3 . 5
5 0 n M , N / P = 8 7 5 n M , N / P = 6
S u
r
v
i
v
i n
/
G
A P
D
H
c o
n t r
o l c e l l s s s i R
N A n
e g a
t
i v e c o
n t r
o l s s i R
N A S u r
v i v i
n
0
0 . 1
0 . 2
0 . 3
0 . 4
0 . 5
0 . 6
0 . 7
0 . 8
0 . 9
5 0 n M , N / P = 8 7 5 n M , N / P = 6
C y
c
l
i n
B
1
/
G
A P
D
H
c o
n t r
o l c e l l s s s i R
N A n
e g a
t
i v e c o
n t r
o l s s i R
N A
c y c l i
n B 1
300
200
100
survivin
GAPDH
Cyclin B1
GAPDH
1 2 3 1 2 3 1 2 3
24 h 48 h 72 h
Figure 1

a
ssiRNA negative control
ssiRNA survivin
d
b ssiRNA negative control
ssiRNA survivin
Control cells
Control cells
ssiRNA cyclin B1
ssiRNA cyclin B1
% A p o p t o t i c
c e l l s
C o n t r o l c e l l s
7 . 0 ± 2 . 4 4
s s i R N A n e g a t i v e c o n t r o l
7 . 1 ± 3 . 1 8
s s i R N A s u r v i v i n
1 8 . 4 ± 3 . 9 5
s s i R N A c y c l i n B
1 1 4 . 2 ± 4 . 8 8
c
G1 = 77,4 %
G2/M = 17,9 %
G1 = 79,7 %
G2/M = 12,6 %
G1 = 41,4 %
G2/M = 52,9 %
G1 = 66,4 %
G2/M = 25,4 %
0
20
40
60
80
100
ssiRNA
survivin
ssiRNA
cyclin B1
ssiRNA
negative
control
Control cells
%cellgrowth/controlcells
50 nM, N/P=8 75 nM, N/P=6
Figure 2

cyclin B1survivin
300
200
100
300
500
400
1 2 31 2 3
*
**
**
0
200
400
600
800
1000
1200
1400
0 2 4 6 8 10
Tumorvolume(mm3)
Days after the first ssiRNA injection
Control
ssiRNA cyclin B1
ssiRNA negative control
a
b
**
0
500
1000
1500
2000
2500
0 2 4 6 8 10
TumorVolume(mm3)
Days after the first ssiRNA injection
Control
ssiRNA Survivin
ssiRNA Negative Control
c
Figure 3

Lungweight
(%ofbodyweight)
Control ssiRNA
survivin
ssiRNA
cyclin B1
ssiRNA
negative
control
0.6
0.8
1.0
1.2
1.4
i.v. injection of 1x106
B16-F10 cells
Sacrifice of
the mice
i.v. injections of 1 mg/kg sticky siRNA
and PEI N/P=12.5
0 3 7 9 days4 8 101 2 5 6 11
**
*
Control
ssiRNA
survivin
ssiRNA
cyclinB1
ssiRNA
negative
control
Control
ssiRNA
survivin
H&E staining
ssiRNA
cyclinB1
a c
b d
Figure 4

0
2
4
6
8
10
12
14
16
MITFlevel(x105)AU
***
*
a
b
MITFlevel(x105)AU
control ssiRNA
survivin
ssiRNA
cyclin B1
ssiRNA
negative
control
0
2
4
6
8
10
Figure 5

20
22
24
26
28
30
32
0 5 10
Bodyweight(g)
Days after cell injection
Control Doxorubicin 1 mg/kg
Doxorubicin 2.5 mg/kg Doxorubicin 10 mg/kg
i.v. injection of 1x106
B16-F10 cells
Sacrifice of
the mice
i.v. injections of 1 mg/kg sticky siRNA
and PEI N/P=12.5
0 3 7 9 days4 8 101 2 5 6 11
i.v. injections of
2.5 mg/kg doxorubicin
ns
*
**
a b
c d
Lungweight
(%ofbodyweight)
0.6
0.8
1.0
1.2
1.4
Lungweight
(%ofbodyweight)
0.5
1.0
1.5
Figure 6

Additional files provided with this submission:
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http://www.biomedcentral.com/imedia/9043992371030811/supp1.ppt
Additional file 2: 4255147589299120_add2.ppt, 111K
http://www.biomedcentral.com/imedia/7368279091030811/supp2.ppt

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