phd thesis summary - usamv cluj · phd thesis summary molecular profiling of immune response in...
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Univeristy of Agricultural Sciences and Veterinary
Medicine Cluj-Napoca
FACULTY OF VETERINARY MEDICINE
Department of Immunology
PhD THESIS SUMMARY
Molecular profiling of immune response in malign tumors: Validation on cell cultures
and animal models
Scientific advisor PhD student
Prof. dr. Gheorghe Răpuntean ing. Almăşan Claudia
married Gherman
Cluj-Napoca
2012
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Table of contents
Abstract ........................................................................................................................... 3
1. Molecular profiling in colorectal cancer .................................................................. 5
1.1. Evaluation of the molecular mechanisms of oxaliplatin, in vitro study on colorectal cell lines. .......................................................................................................... 5
1.1.1. Evaluation of cell viability of Colo 320 by using MTT test .......................... 5
1.1.2. RT-PCR evaluation of expression levels of genes involved in apoptosis induced by oxaliplatin ................................................................................................... 7
1.2. Evaluation of the apoptotic profile induced by combining oxaliplatin treatment with 5-fluorouracil ............................................................................................................ 9
1.2.1. Evaluation of antiproliferative effects induced by oxaliplatin, 5-FU and combined treatment. ...................................................................................................... 9
1.2.2. Evaluation of gene expression pattern of the apoptotic process. ................. 10
1.2.3. Evaluation of the immunologic profile induced by the combined treatment of oxaliplatin and 5-FU ............................................................................................... 12
2. In vitro and in vivo evaluation of the antitumor and immunomodulatory effects of EGCG ........................................................................................................................... 12
2.1. In vitro evaluation of the antitumor and immunomodulator effects of RGCG on Ehrlich ascites cell cultures ............................................................................................ 12
2.1.1. In vitro evaluation of the antimor effects of EGCG on ascitic cells. .......... 13
2.2. In vivo evaluation of the antitumoral and immunomodulatory effect of EGCG 14
2.2.1. In vivo evaluation of the immunomodulatory effect of EGCG ................... 14
2.2.2. Apoptosis evaluation with Annexin V/Cy5/Calcein using the “Lab on a Chip®” fluorescence method on Agilent 2100 Bioanalyzer ...................................... 17
3. Carbon nanotubes: multifunctional biological transporters with application in medicine, testing the biological effect on in vitro or in vivo systems .......................... 19
3.1. Experiment to determine the internalization capacity of NTC on Hep2G ......... 20
3.2. Applications of carbon nanotubes in targeted transport of TNF-α with siRNA . 21
3.2.1. Chemical characterization of carboxylated carbon nanotubes .................... 22
3.2.2. siRNA internalisation in Hep2G liver cells ................................................. 23
3.2.3. Evaluation of cell survival (MTT) ............................................................... 23
4. GENERAL CONCLUSIONS ................................................................................ 27
REFERENCES ........................................................................................................... 278
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Abstract
The progress made in the last decade in the tumor immunology field has led to the
identification of new molecular mechanisms with high impact in immune response to
anticancer therapy. A new vision in this field is represented by the tumor-host
interactions, an extremely complex and dynamic process which contributes to the
elimination of the tumor cells or to the creation of an equilibrium between the elimination
rate and tumor proliferation, for in the end these events to induce tumor growth and the
migration of the malign cells.
During this process, the tumor cell selection takes place according to their capacity
to induce immune response, being selected only the cells with low immunogenity,
apoptosis resistant and ability to survive in unfavorable conditions. Tumor cells have the
ability to specifically modulate the immune system, raising their tolerance in the host
organism, which lead to immunosuppression in final stages of tumor development. Also,
some tumor phenotypes, such as colorectal cancer are validated for some inflammatory
factors. That’s why, the molecular immune response induced through characteristic
factors involved in the modulation of mRNA transcription and protein synthesis
represents an important process in tumor pathology.
The aim of this thesis is to identify and characterize the role of some genes in
tumor immunosurveillance, by modulating mechanisms involved in tumor development
and antitumor therapies.
This thesis approaches a subject of actuality, which is the international tendency to
use in vitro cell culturing as an alternative to animal in vivo studies, only the validation
step being done in in vivo systems.
The theme of this project presents new directions in developing alternative
antitumor strategies by modulating the immune system by using natural and synthetic
chemical compounds, nanomaterials or gene therapy. For the toxicity and functional
genomics studies were used in vitro and in vivo evaluation systems that are of actuality,
according to the European Legislation.
Functional genomics studies allow the evaluation of the relationship between
genome and phenotype both in normal, healthy state and disease. This can be done
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through gene expression quantification by using array technologies (microarray, PCR
array) or quantitative PCR (RT-PCR). The PCR array or microarray technologies allows
the simultaneous evaluation of high number of genes, with a global view of the altered
transcriptomic profile at some point, or give information regarding the tumor response to
treatment.
The studies presented in this thesis have been done taking into consideration the
clinical needs in complex pathologies such as cancer by using state of the art
technologies.
The antiproliferative effects at cellular and molecular levels are investigated
differently according to the molecular target: cytotoxicity, immunotoxicity, genotoxicity
or mutagenesis.
The antiproliferative and immunomodulatory actions are usually determined by
foreign compounds (xenobiotics), synthetic (drugs) or natural compounds that alter the
cellular functionality (metabolic pathways) with or without affecting the morphologic
structure. The antitumor compounds can interact with proteins, specific enzymes, or
antibodies (immunotoxicity) by affecting the viability and cellular metabolism. In this
context, this thesis proposed the in vitro and in vivo evaluation of some synthetic
cytostatic (oxaliplatin) or natural (cathechins) agents.
In this thesis were used different cellular and animal systems (Erlich ascites and
cell lines cultures) and representative antitumoral compounds, oxaliplatin and EGCG
with the capacity to modulate multiple metabolic pathways and immunomodulate the
microenvironment.
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1. Molecular profiling in colorectal cancer
1.1. Evaluation of the molecular mechanisms of oxaliplatin, in vitro study on
colorectal cell lines.
One of the most active cytostatic in colorectal cancer is oxaliplatin. This is an
alchilating agent from the platinum compounds class that inhibits the DNA synthesis by
forming inter and intra strands Pt-DNA bridges that lead to cell cycle arrest, replication
inhibition and apoptosis (Temmink şi colab., 2007).
1.1.1. Evaluation of cell viability of Colo 320 by using MTT test
The oxaliplatin effects on colorectal cell line Colo 320 induce modifications in the
cell bioactivity. Cell viability has been measured by using the MTT test, showing that
cell viability is dependent on concentration and the length of time. Cell viability is
measure in percent of control, which is considered 100%, figure 1. At low cytostatic
concentrations the cells present minimal cytotoxicity at 24, 48 and 72 hours. The
decrease in cell viability is associated with the increase of oxaliplatin at different times.
Figure 1. Cell proliferation evaluation by using MTT test (% of control) on Colo320 cell
line treated with multiple oxaliplatin doses at 24, 48, 72 hours.
-2 -1 0 1 2 30
20
40
60
80
100
120
24h
48h
72h
MTT test
log(conc, µµµµg/ml)
% o
f co
ntro
l
6
According to the obtained results in figure 1, the oxaliplatin IC50 has been
established (the concentration at which 50% of the cells are dead). The evaluation is
based on the statistical analysis using GraphPadPrism5. The results are presented in
Table 2.
Table 2.
IC 50 values of oxaliplatin at different times of incubation
Cell line Time (hours) IC 50 (µg/ml) Slope R2
Colo320
24 14.45 3.101 0.9511
48 7.858 0.6428 0.9698
72 8.356 2.624 0.9328
The evaluation of antiproliferative effect of multiple doses at 24, 48 and 72 hours
is presented in figure 2. After the administration of a double dose of 5µg/ml at time =0
and after 8 hours, no significant change has been observed when compared to the single
dose of 5µg/ml at T=0. The figure represent the data measured at 48 hours after the
administration of a double dose of 5µg/ml at T=0 and 24 hours. The administration of a
triple dose of 3.33 µg/ml oxaliplatin at T=0, 24 and 48 hours, followed by viability
evaluation at 72 hours showed a decrease in cell proliferation more efficient than single
dose of 10 µg/ml oxaliplatin.
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Figure 2. MTT results (% of control) of Colo320 treated with multiple doses of
oxaliplatin at 24, 48 and 72 hours.
1.1.2. RT-PCR evaluation of expression levels of genes involved in apoptosis
induced by oxaliplatin
The relative gene expression levels of PDGF, NFkB, p53 evaluated on Colo320
colorectal tumor cell line induced by a single dose of 10 µg/ml of oxaliplatin after 24
hours are presented in table 2. β-Actin was used as a reference gene. As presented in
figure 3, the results show that the oxaliplatin treatment induces the underexpression of
PDGF gene which is involved in tumor angiogenesis and the overexpression of p53 and
NFkB genes with a role in triggering cell death and cell proliferation inhibition (Nemoto
şi colab., 2009). The values for gene expression in oxaliplatin treated cells in comparison
with untreated cells are presented in Table 3.
MTT test
10 (2
4 h)
2X5
(24h
)
10 (4
8 h)
2X 5
(48h
)
10 (7
2h)
3X3.3
(72h
)0
20
40
60
80
100
120
140
Concentratia oxaliplatinei (µµµµg/ml)
% d
e co
ntro
l
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Table 3
Gene expression levels for PDGF, NFκB, p53
Gene Oxaliplatin (10 µg/ml)
Fold change SEM (+), (-)
β−Actin 1.000 0.433 0.302
PDGF 0.368 0.217 0.136
NFκB 1.662 0.552 0.414
p53 1.840 0.658 0.485
Figure 3. Dynamics of gene expression after oxaliplatin treatment in Colo320 cell line
In this study we demonstrated that oxaliplatin inhibits colorectal tumor cell
proliferation depending on concentration and time of administration. The
chemotherapeutic agent has sensitized the colorectal tumor cells when multiple doses
were administered, which led to a significant decrease of the viable tumor cells.
The mechanisms that govern the tumor cell response to oxaliplatin involve the
induction of apoptosis through transcriptional activation of p53 and NFkB, but also
inhibition of PDGF (Yang şi colab., 2011; Kim şi colab., 2009; Nemoto şi colab., 2009).
PDGF NfkB p530
1
2
3
Oxaliplatina (10 µg/ml)
niv
elul d
e ex
pre
sie
rela
tiv a
gen
elor
9
These results suggest a new strategy in oxaliplatin administration, multiple doses
being rather more efficient than single dose, with possible implications in patients
survival and life quality. Gene expression results for PDGF, NFkB and p53 are in
concordance with the ones presented in the literature.
1.2. Evaluation of the apoptotic profile induced by combining oxaliplatin
treatment with 5-fluorouracil
The first condition for a compound to be considered chemotherapeutic agent is to
induce alterations in tumor cell homeostazis. In the last years, the research has been
focused on understanding the molecular mechanisms that control the defects of apoptotic
processes in tumor cells, but also developing new therapies to restore these defective
processes (Rigas şi colab., 2002). Apoptosis inhibition leads to homeostasis alterations,
thus allowing the development and evolution of tumor masses. The apoptotic process is
inhibited in colorectal cancers (Bedi şi colab., 1995) which led the researchers to
develop several treatment strategies. 5-Fluorouracil it’s a pro-cytostatic frequently used
in colon cancer treatment (Rahman şi colab., 2006); once it enters the cell, it is processed
to the biologically active form 5-floro-2’-deoxiuridin-5’-trifosfat (FdUTP) that can be
incorporated in the DNA and RNA sequences and promote strand instability (Meyers şi
colab., 2003).
1.2.1. Evaluation of antiproliferative effects induced by oxaliplatin, 5-FU
and combined treatment.
The viability MTT test presented in figure 4., shows that at 24 hours after
treatment 5-FU induced a strong cytotoxic effect, while oxaliplatin is less toxic.
However, at 48 hours the effects are reversed, which suggest that for a long term
oxaliplatin is more toxic that 5-FU
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(A) (B)
Figure 4. Antiproliferative effects of oxaliplatin, 5-FU or combined at A) 24 hours and B)
48 hours post treatment
1.2.2. Evaluation of gene expression pattern of the apoptotic process.
Upon oxaliplatin treatment 50 genes have presented statistical significant changes
in the treated group when compared to untreated cells. Of these, 33 were overexpressed
and 4 underexpressed. Most of these genes are involved in inducing and maintaining the
apoptotic process. In figure 5 are presented the 33 genes that were found to be
statistically significant upon the combined treatment of oxaliplatin with 5-FU. Of these,
19 genes were overexpressed and 14 underexpressed. Among the overexpressed genes
we found genes that code for caspases which are the effector of apoptosis.
-2 -1 0 1 2 3
20
40
60
80
100
120
Oxaliplatin
Oxaliplatin+5-FU
MTT test (24h)
5-FU
log(conc)
% o
f co
ntro
l
-2 -1 0 1 2 3
20
40
60
80
100
120
Oxaliplatin
Oxaliplatin+5-FU
MTTtest (48h)
5-FU
log(conc)
% o
f co
ntro
l
11
Figure 5. PCR analysis for establishing the molecular pattern of genes involved in the
apoptotic process: red- overexpressed genes, green- underexpressed genes
Conclusions
The aim of this study was to identify the main genes involved in the apoptotic
process induced by 5-FU, oxaliplatin and combined treatment in the colorectal cancer cell
line Colo 320 (Matuo şi colab., 2009). The results prove that the two cytostatics are more
efficient in combination than alone, but there does not seem to act in a synergistically
manner.
Even if the PCR array data proves the effects of the two cytostatics at gene levels,
following studies are needed in order to identify drug resistance biomarkers and new
therapeutic targets.
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1.2.3. Evaluation of the immunologic profile induced by the combined
treatment of oxaliplatin and 5-FU
13 genes were identified to be significant at mRNA level upon treatment of
colorectal tumor cells Colo 320 with 5-FU in comparison with the control group. Of
these, 5 are overexpressed and 8 underexpressed. Upon oxaliplatin treatment, 27 genes
were identified to be overexpressed, but only 14 were statisticaly significant (p value
<0.05) and one gene of 8 was underexpressed, this gene being a citokine (Dunn şi colab.,
2002; Engel şi colab.,1997). Upon the combined treatment, 5 genes were overexpressed
and 11 underexpressed.
Conclusions
The evaluation of gene expression of some inflammatory citokines in Colo320
upon 5-FU, oxaliplatin and the two combined can lead to the identification of some
molecules involved in inflammatory processes with prognostic and diagnostic value and
could be used in the selection of the patients that could benefit of neoadjuvant therapy.
2. In vitro and in vivo evaluation of the antitumor and immunomodulatory effects
of EGCG
The aim of this study was to investigate the bioactive properties of some flavonoid
compounds (EGCG). The study was done on both in vitro and in vivo systems, with a
view to evaluate the ability of EGCG to activate the apoptotic processes and immune
response with aplicability in cancer therapy (Prodan şi colab., 2009)
2.1. In vitro evaluation of the antitumor and immunomodulator effects of RGCG
on Ehrlich ascites cell cultures
The tumor Ehrlich ascitic cells have a high proliferation rate in vitro and in vivo
systems (Bhattacharyyaet, 2003), being largely used in chemotherapeutics studies. The
literature describes the inhibitory effects of poliphenolic compounds in citokyne
synthesis as well as an association of citokines and inhibition of tumor growth (El-
Mowafy şi colab., 2010). Previous studies have showed that EGCG induces apoptosis in
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several cancer cell lines. In our study we investigated if EWGCG has antitumor effects in
vitro on tumor Ehrlich ascitic cells.
2.1.1. In vitro evaluation of the antimor effects of EGCG on tumor Ehrlich
ascitic cells.
Proliferation inhibition and decrease of cell viability induced by EGCG was
evaluated in vitro on tumor Ehrlich ascitic cells. The effects (Shimizu şi colab., 2008) at
different time points (24, 48, 72 and 96 hours) is presented in figure 6a. The results
indicate that a unique dose of 10 µM EGCG induces the decrease of cell proliferation
with a maximum at 48 hours, after which the efficiency is decreasing, according to the 72
hours results (figure 6b). The proliferation data are confirmed by the viability ones.
EGCG does not induce necrosis, but apoptosis (Shimizu şi colab., 2008; Liu şi colab.,
2008; D'Archivio şi colab., 2008; Braicu şi colab., 2009)
(A) (B)
Figure 6. EGCG effects on viability and cell. (A) EGCG inhibits the cell proliferation in a
time dependent, (B) EGCG reduces cell viability 2.1.1. Evaluating the
immunomodulatory effect of EGCG in vitro on Ehrich ascites cell cultures.
The immune response using TNF-α and IL-6 was analyzed with ELISA
techniques on Ehrlich ascites cell cultures by taking measurements at 24 and 48 hours
after treating the cultures with EGCG 10 µM. At 24 hours post treatment there were no
0 20 40 60 80 1000.0
2000000.0
4000000.0
6000000.0
8000000.0
Control
EGCG*
*
*
Cel
ls/m
l
24 48 72 960
102030405060708090
100110
Control
EGCG
**
Timp (ore)
% in
rap
ort
cu
cont
rolu
l
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significant differences in the synthesis of the two cytokines, and at 48 hours the levels of
both were low (Figure 7).
Figure 7. Quantification of IL-6 and TNF-α levels in the cell supernatant, in the presence
of EGCG (* P<0.05 compared to control, using the T-test) at 24 and 48 hours post
treatment.
2.2. In vivo evaluation of the antitumoral and immunomodulatory effect of
EGCG
In this study we evaluated the molecular profile of TNF-α and IL-6 in various organs
involved in the immune response. We also tried to observe correlations between the gene
and protein expression levels for TNF-α and IL-6, as well as the degree of tumor
proliferation and the level of apoptosis.
2.2.1. In vivo evaluation of the immunomodulatory effect of EGCG
2.2.1.1. Evaluating protein expression level of TNF-αααα and IL-6 using the
immunoenzymatic method ELISA, after EGCG treatment
Neoplastic cells present an alteration of the response to exogenous cytokines, because of
increased endogenous cytokine production, alteration of expression of membrane
receptors, alteration of second messengers (da Silva et al., 2002; Kumar et al., 2010). The
TNF-αααα
Contro
l (24
h)
EGCG (2
4h)
Contro
l (48
h)
EGCG (4
8)65
70
75
80
85
90
95
*
Conce
ntr
atia
(pg
/ml)
IL-6
Control (24
h)
EGCG
(24h
)
Control (48
h)
EGCG
(48)
0
100
200
300
*
Conc
entrat
ia (pg
/ml)
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serum concentration of the two immune mediators for the four groups of animals was
determined and is presented in figures 8 and 9.
The results obtained for the ascites group wit and without EGCG were compared to the
control group. We observed significant differences only for the 48 hour interval, when
the serum level for IL-6 was increased; this reaction induced by inoculating ascites was
counteracted by EGCG. At 72 hours post-treatment we observed a nonspecific activation
for the ascites group treated with EGCG, which may be due to the pro-oxidant effect of
EGCG manifested via the metabolites that stimulate cytokine secretion.
Figure 8. IL-6 serum levels for the ascites group, and the group with ascites and EGCG,
compared to the control group at 24, 48, 72 hours, and 6 days post-treatment
48 hours after inoculating ascites, we observed an inhibition of the TNF-α serum levels in
the tumor microenvironment. The levels of TNF-α produced by the splenic alveolar
macrophages is raised, due to EGCG induced stimulation at 48 hours post treatment; the
effect is not maintained at 72 hours, which suggests the need for multiple EGCG
administrations.
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Figure 9. TNF-α serum levels for the ascites group, and the group with ascites and
EGCG, compared to the control group at 24, 48, 72 hours, and 6 days post-treatment
2.2.1.2. Evaluation of gene expression profile for TNF-α and IL-6 using
RT-PCR technology following EGCG treatment
Among the growth factors and cytokines involved in the immune response modulation,
we evaluated TNF-α and IL-6 in spleen and thymus. The expression levels of two
molecules in the organs mentioned above at 24 and 48 hours after ascites inoculation and
EGCG treatment is presented in Figure 10 and 11.
(A) (B)
Figure 10. Variation of gene expression ratio for TNF-α in (A) spleen, (B) thymus.
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(A) (B)
Figure 11. Variation of gene expression ratio for IL-6 in (A) spleen, (B) thymus.
24 hours after inoculation with Ehrlich ascites we observed a reduction in relative
expression level for TNF-α in the thymus of the test animals, in both the EGCG treated
and untreated groups. Paradoxically, we observed the same reduction of the expression
level when normal saline solution was administered. At splenic levels, the situation was
different, since at 24 hours we observed a strong activation, although EGCG treatment
usually reduces TNF-α expression at mRNA level. IL-6 generally displays an increased
expression level for the three studied groups (serum, ascites ascites+EGCG), both at 48
and 72 hours post inoculation. In the thymus, IL-6 activation is only observed at 48
hours, the relative expression level being reverted to the control group values, or, in other
words, we can assert that there is an inhibition of the expression levels for the
ascites+EGCG.
2.2.2. Apoptosis evaluation with Annexin V/Cy5/Calcein using the “Lab on a
Chip®” fluorescence method on Agilent 2100 Bioanalyzer
This study proved the pro-apoptotic effect of EGCG, with a peak around 72 hours post
treatment, as it can be observed in Figure 12.
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Fig. 12. Apoptosis evaluation with Annexin V/Cy5/Calcein using the “Lab on a Chip®”
fluorescence method on Agilent 2100 Bioanalyzer, at 24, 48 and 72 hours, and 6 days
post-treatment for group nr. 1 – healthy mice inoculated with Ehrlich ascites, and group
nr. 2 – healthy mice inoculated with Ehrlich ascites and treated with a single dose of
EGCG.
It is a known fact that EGCG specifically causes apoptosis in tumor cells by means of
several metabolic pathways, one being the Fas path, which is the main mechanism
responsible for inducing immune mediated apoptosis; unfortunately, this effect can be
maintained only by administering multiple doses. Thus, we noticed the fact that, by using
a double dose of EGCG at a 72 hour interval the apoptotic effect is maintained, as it can
be observed in the case on group nr. 3 in Figure 13.
48h
25.90 % celule apoptotice
EGCG
72h
29.5 % celule apoptotice
0 % celule apoptotice
6 zile
(A)
(B)0 % celule apoptotice
0 % celule apoptotice
0 % celule apoptotice
19
Figure 13. Apoptosis evaluation with Annexin V/Cy5/Calcein using the “Lab on a
Chip®” fluorescence method on Agilent 2100 Bioanalyzer, at 24, 48 and 72 hours, and 6
days post-treatment for group nr. 1 – healthy mice inoculated with Ehrlich ascites, and
group nr. 2 – healthy mice inoculated with Ehrlich ascites and treated with a single dose
of EGCG, group nr. 3 – healthy mice inoculated with Ehrlich ascites and treated with a
double dose of EGCG at 72 hours, group nr. 4 – healthy mice inoculated with Ehrlich
ascites and treated with a triple dose of EGCG at 72 hours.
3. Carbon nanotubes: multifunctional biological transporters with application in
medicine, testing the biological effect on in vitro or in vivo systems
The carbon nanotubes (CNT) are important materials, with a wide range of
applications in the domain of biomedicine, cosmetics, aeronautics and electronics.
Because of their physical and chemical properties, they gained an enormous attention in
6 zile
Lot 2
Lot 2
9 zile
0 % celule apoptotice 0 % celule apoptotice
22 % celule apoptotice
0 % celule apoptotice
9 % celule apoptotice33 % celule apoptotice
Lot 4
0 % celule apoptotice0 % celule apoptotice
Lot 3
Lot 1
Lot 1
Lot 3
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biomedicine for different applications; as new transport systems for different drugs,
nucleic acids or antibodies and as a new part of the photothermic treatment for cancer.
3.1. Experiment to determine the internalization capacity of NTC on Hep2G cell
line
In this study we evaluated the internalization capacity of NTC on Hep2G cell line
(Endo et al., 2008). In figure 14 are highlighted the particularities of cellular structures as
a result of treatment with NTC at 2, 5, 24, 48 hours, each experiment was performed in
triplicate. Thus there were selected some representative images for those four selected
intervals on three types of tested nanotubes.
Figure 14. Image of electron microscopy of Hep2G cells treated with functionalized
carbon nanotubes ( S SWNTC-COOH, DWNTC-COOH, MWNTC-COOH) at 4, 24 and
48 hours
Control1:8000
Control1:12000
Imagini de microscopie electronica la 5 ore dupa tratemetul cu NTCNTC prins in vacuola
SWNTC-COOH1:15000
SWNTC-COOH1:8000
SWNTC-COOH1:15000
DWNTC-COOH1:8000
DWNTC-COOH1:12000
SWNTC-COOH1:20000
MWNTC-COOH1:8000
M-DAH71:12000MWNTC-COOH1:12000
MWNTC-COOH1:20000
4 ore dupa tratamentul cu NTC (0.25 µg/ml)
24 ore dupa tratamentul cu NTC (0.25 µg/ml)
48 ore dupa tratamentul cu NTC (0.25 µg/ml)
21
Electron microscopy highlights the abnormalities of the hepatocyte organelles.
Some of them, such as hypertrophy of the endoplasmic reticulum, are phenomena of
adaptation, increasing the activity of the microsomal oxidation system. The carbon
nanotubes seem to be present in cytosol and nucleus as cytoplasmic inclusions or
vacuoles. These vacuoles contain a granular material, probably precipitations of some
salts from the surface of the nanotubes. At 4 hours after treatment no cells in division
were observed, but at 24 and 48 hours there was observed an increased number of cells in
division, with keeping of the microvilli, this fact confirms the data obtained by MTT test
and electron microscopy.
Conclusions
By using the electron microscopy we demonstrated the penetration capacity of
NTC in cytosol level and more rarely in the nucleus. At 4 hours after treatment Hep2G
cells haven’t been observed in division, but at 24 and 48 hours the number of cells in
division was increased, with keeping the microvilli.
3.2. Applications of carbon nanotubes in targeted transport of TNF-α with
siRNA
RNA interference (RNAi) is an inhibition post-transcriptional method of gene
expression. The specialty literature suggested new mechanisms of transport to avoid the
lysosomal degradation, such as carboxylated carbon nanotubes (SWNTC-COOH). These
carboxylated carbon nanotubes are able to offer a high level of protection for the
therapeutic compounds. Thus, in this study was tested the gene inhibition capacity of a
commercial system (siPORTNeoFX, Ambion) in parallel with a conjugate product of
TNF siRNA with SWNT-COOH.
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3.2.1. Chemical characterization of carboxylated carbon nanotubes
Thermogravimetric analysis (TGA) of pristine and carboxylated nanotubes
(SWNT-COOH) has been performed for confirmation the purity of carboxylated carbon
nanotubes (Fig..15a). FT-IR spectrum (Fourier transform infrared) is presented in figure
15b and confirms the oxidation of SWNTC. By comparing these two spectra, red for
SWNT-COOH and black for SWNT, it can be observed two specific oxygen bands at
3420 cm-1. The carbonyl and carboxyl groups are visible at 1580 and 1380 cm-1. The UV-
vis spectrum clearly confirms functionalization and attachment of siRNA on the surface
of nanotubes. Electron microscopy confirms the accumulation of siRNA on the surface of
nanotubes (Figure 15d). The arrows mark the areas where siRNA is attached to SWNT-
COOH.
Figure 15. Thermogavimetric elavulation of pristine and oxidized nanotubes (SWNT-
COOH). (B) FTIR spectrum of pristine oxidized nanotubes (C) UV-vis spetrum and
evaluation with electron microscopy (D) of SWNT-COOH and of the nanoconjugate
product SWNT-COOH-siRNA
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3.2.2. siRNA internalisation in Hep2G liver cells
Previous studies described the internalization of functionalized nanotubes with
siRNA in some cellular types, but the internalization of the nanoconjugate product
SWNT-COOH-siRNA on the HepG2 cell line hasn’t been described in present. Thus, the
evaluation of transfection efficacy has been made using the fluorescence microscopy
(Figure 16). The nanoconjugate product with SWNT-COOH has the penetration capacity
of the cellular membrane; the transfection capacity is similar to the commercial
transfection agent. By comparing with the control group, is observed a high range of
internalization of siRNA in the case of both transfection complexes.
Figure 16. Representative fluorescent images (400x) for internalization of siRNA
negative control in case of siPort NeoFX and SWNT-COOH complex at 24 hours after
reverse transfection.
3.2.3. Evaluation of cell survival (MTT)
Every transfection agent can have a different performance, which was also
demonstrated in our experiments. As you can notice, in both cases of transfection with
siPORT NeoFX and SWNT-COOH, the cell survival at 24 and 48 hours was almost
unchanged, but the agent in the case of transfection complexes with siRNA negatively
24
influenced the cell proliferation. Reduction of cell survival in the case of transfection
complexes with siRNA may be caused by activation of apoptosis processes or
angiogenesis inhibition associated with gene inhibition and not because of the cytotoxic
effects (Figure 17).
Figure 17. Evaluation of antiproliferative effect on Hep2G cell line
The efficiency of gene inhibition was evaluated by analyzing the gene expression
level of β-actine and TNF-α in the transfected cells with siRNA ( TNF-α siRNA) using
siPORT NeoFX, respectively SWNT-COOH compared to the cells from the control
group. A good gene inhibition level was registered in the case of the three siRNA-s
tested. The level of gene inhibition for transfection with siPORT NeoFX for TNF- α
siRNA is 56,7. Relatively similar level of gene inhibition was noticed in the case of the
system using SWNT-COOH. SWNT-COOH has been proven to be an efficient transport
system for siRNA, as it was demonstrated by the results concerning the gene expression
level (figure 18).
MTT test
siRNA- N
eoFX
αααα
TNF-
NeoFX
siRNA-S
WNTC
-COOH
αααα
TNF-
SWNTC
-COOH
0102030405060708090
100110120
% o
f co
ntro
l
25
Figure 18. Analysis of gene expression level for TNF-α
The quantification of expression level for TNF-α from culture media was realized
by using the ELISA technique (figure 19).
Figure 19. Analysis of protein expression level
The immunofluorescent staining method confirmed RT-PCR data. ELISA data
concerning the secretion level in the culture medium of TNF-α was in concordance with
the registered data at mRNA level. The low expression level of mRNA was confirmed in
the case of transfection with TNF-α siRNA. High levels of TNF-α were observed only in
TNF-
asiR
NA-Neo
FX
NeoFX
siRNA-S
WNTC
-COOH
αααα
TNF-
SWNTC-C
OOH
1
2
3
TNFα gene
**
*
Rela
tive g
ene
exp
ress
ion
leve
l (fo
ld c
hang
e)
TNF-αααα expression level
siRNA-N
eoFX
αααα
TNF-
Neo
FX
siRNA-S
WNTC-C
OOH
αααα
TNF-
SWNTC-C
OOH
0
50
100
150
24
48
*
** *
*
*
% o
f co
ntro
l
26
the group treated with siPORT NeoFX and in the case of transfection complex
VEGFsiRNA-siPORT NeoFX.
Conclusions
The gene inhibition with SWNT-COOH represents a valid alternative for the
transfection lipid systems in the case of targeted RNAi therapy for cancer treatment. The
obtained data indicate that SWNT-COOH is a solid structure, with a good efficiency and
a low toxicity in the case of in vitro systems. This study offers perspectives of using
SWNT-COOH combined with siRNA in the targeted cancer therapy, but this data must
be validated on in vitro systems (Gannon et al., 2007; Kesharwani et al., 2012).
Supplementary studies must focus on developing new particles with a higher
biodegradation capacity that copies the properties of nanotubes for siRNA transport.
27
4. GENERAL CONCLUSIONS
1. In this study we identified a number of genes involved in the resistance to
Oxaliplatin and 5-Fluorouracil which induce apoptosis and modulate the immune
response.
2. The evaluation of the genes involved in apoptosis and in the immune response
after treatment with Oxaliplatin and 5-Fluorouracil administered either alone or
together, leads to the identification of certain molecules that might have
prognostic value in colorectal pathology.
3. We identified the antiproliferative and immunomodulatory effect of EGCG both in
in vitro and in vivo systems. EGCG treatment lead to the enhancement of
immunologic parameters evaluated both at protein and at mRNA level for TNF-α
and IL-6, but its action was only transitory, since administering multiple doses
lead to the activation of antitumor mechanisms and apoptotic processes.
4. We demonstrated on in vitro systems that SWNTC-COOH are a valid alternative
to the lipid based transfection systems used for the targeted siRNA therapy of
cancer. The in vitro studies showed that treatment with SWNTC-COOH could
lead to the activation of pro-inflammatory mechanisms, which could be also
involved in modulating tumor antiproliferative processes.
5. We observed the internalization of nanotubes in the cytosol and nucleus of the
treated cells by means of electron microscopy and also by fluorescence
microscopy, which allows for the use of these nanostructures in the targeted
transport of siRNA with minimal side effects. The FTIR analysis gave us
information regarding the pharmacokinetics of carbon nanotubes.
The use of immunology, toxicology and transcriptomic studies (cell cultures,
immunohistochemical studies, optic and electron microscopy, flow cytometry, real-time
PCR, PCR array, cytotoxicity tests) and by correlating the results with complex
bioinformatics analyses allow for the development of new therapeutic strategies for
cancer treatment.
28
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