الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Hepatocellular carcinoma is a common malignant tumor with an insidious nature and
dreadful outcome. On a global scale, HCC is always near the top of the list of cancer incidence
and ranks among the leading causes of cancer related mortality. Nearly 600,000 new cases of
HCC are detected annually, worldwide. Mostly, HCC develops on top of underlying liver
diseases such as viral infection, alcohol, nonalcoholic fatty liver and metabolic and genetic
diseases. In Egypt, a remarkable elevation in the incidence of HCC occurs because of the
improvement of screening and diagnostic tools and the increased survival of cirrhotic patients
allowing more time for development of liver cancer. Despite recent improvements in the
treatment of HCC, the overall survival rate is still disappointing, with a 5-year survival rate
being only 10 % globally, as most of the cases are detected in the advanced stages in which
curative therapy for liver cancer is useless.
The pathogenesis of HCC is complicated resulting mainly from deregulation of various
signaling pathways and the advanced knowledge of its molecular basis opens opportunities for
targeted therapy. The front line systemic targeted therapy for late stages of HCC, is sorafenib;
a multikinase inhibitor of VEGFR, PDGFR, RAF-1, B-RAF and c-KIT, causing a slight
survival improvement. Therefore, various trials are still ongoing in search for agents that can
be used alone or in combination with the standard HCC therapy to improve the response in
patients with advanced disease .
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In this regards, the current study aimed at evaluating the potential antitumor effect of
thioridazine, an antipsychotic drug belonging to the phenothiazine drug group, alone and
assessing the beneficial outcome of its combination with sorafenib in HepG2 cell line.
In the present study, the cytotoxic effect, of either sorafenib or thioridazine, was
evaluated by using the MTT assay. The IC50 of both drugs were equivalent to 15.58μM and
22.35μM, respectively, following 72 hours of incubation. The combination of sorafenib and
thioridazine was performed in a fixed ratio of 2:1 respectively, and the CI and the DRI were
evaluated. The CI was 0.9 indicating a synergistic effect of this combination. Moreover, the
co-administration of sorafenib with thioridazine reduced their doses to 2.6 and 1.88 folds,
respectively, thus reducing their potential toxicity. Following the treatment period, apoptosis,
angiogenesis, inflammation, metastasis and key mediators in the signaling pathways were
assessed by ELISA and flow cytometric techniques.
Both RAS/MAPK/ERK and PI3K/AKT/mTOR are the main pathways activated in
liver cancer. In the RAS/MAPK/ERK pathway, sorafenib alone and in combination with
thioridazine reduced the activity of ERK with more significant reduction observed in the
combination group, whereas thioridazine had no effect on the activity of ERK, compared to
the control group. On the other hand, the activity of AKT, in the PI3K/AKT/mTOR pathway,
declined in both the thioridazine and the combination groups with more pronounced decrease
in the combination group, while the activity of AKT in the sorafenib group was not
significantly different from the control group. Surprisingly, all treatment groups showed
attenuation of the activity of mTOR and p70S6K, compared to the control group, with a marked
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reduction detected in the combination group. These results indicate that the combination of the
two drugs is more beneficial in suppressing both signaling pathways and increasing the
therapeutic efficacy of each drug .
Apoptosis, inflammation, metastasis and angiogenesis are important hallmarks of
cancer. Disruption of the various signaling pathways and mediators regulating them may
therefore, improve the therapeutic outcome.
The apoptotic effect of the different treatments, in the present work, was evaluated by
annexin V/PI double staining. The percent of apoptotic cells increased in all groups in
comparison to the control group, with marked effect observed in the combination-treated
group.
Several chemotherapeutic agents attack the DNA, in tumor cells, breaking the double
strands and killing cells by induction of apoptosis. In the present study, the DNA damage was
evaluated by the level of gamma H2AX. A high level of gamma H2AX in the thioridazinetreated
group, whereas the level of gamma H2AX in the sorafenib-treated group was not
significantly different from the control group indicating the DNA damaging effect of
thioridazine. In comparison to sorafenib and thioridazine, combining both drugs caused a more
pronounced elevation in the gamma H2AX level, indicating that sorafenib augmented the DNA
damaging effect, induced by thioridazine, via inhibiting the DNA repair system. Moreover, the
effect of the combination on the gamma H2AX level was in concordance with its apoptotic
effect as persistence of the DNA damage resulted in induction of apoptosis in cancer cells.
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One of the key regulatory pathways for inflammation in HCC is the upregulated NFκB.
In the current study, a decline in the activity of NFκB was noticed in sorafenib and thioridazinetreated
groups. The combination of both drugs caused a further decline in NFκB activity,
compared to each drug alone .
Vascular endothelial growth factor is a potent inducer of angiogenesis that is
significantly increased in several tumors. Findings of the current work revealed that either drug
alone significantly decreased the VEGF levels. The combination treatment showed a further
significant inhibition of VEGF compared to monotherapies, thus illustrating a more efficient
antiangiogenic potential .
The effect of the studied drugs on metastasis; one of the causes of the decrease in the
survival of HCC patients, was evaluated in this study by assessing the level of E-cadherin and
N-cadherin. To metastasize, tumor cells must change their phenotype from epithelial to
mesenchymal which is detected by the up-regulation of N-cadherin and the down-regulation
of E-cadherin. Both sorafenib and thioridazine monotherapies caused a significant increase in
E-cadherin and a decrease in N-cadherin, compared to the control group, thus hindering
metastasis. A more favorable therapeutic response was observed in the combination group, in
comparison to each drug, exemplifying a synergistic anti-metastatic effect.
In conclusion, the combination of sorafenib and thioridazine offers an advantage of a
more pronounced and effective therapeutic efficacy due to their distinct complementary
mechanisms.