الفهرس 
Aim of the workOnly 14 pages are availabe for public view
 |  | from | 230 |  |  |
| | | from | 230 | | |
Abstract
Summary
Hepatocellular carcinoma is the major type of primary liver malignancy
with high rates of mortality worldwide. Hepatocellular carcinoma tumors have
poor prognosis and are usually diagnosed at late stages when potentially
curative therapeutic interventions become inapplicable. Most HCC tumors are
inherently resistant to chemotherapy and despite the tremendous advances in
cancer chemotherapy, their treatment remains quite challenging.
Recently, targeted therapy gained considerable attention in cancer
chemotherapy in order to achieve specificity towards cancer cells and to limit
toxicities. This opened the door for tyrosine kinase inhibitors as potentially
curative chemotherapeutic agents. Sorafenib is an orally active multikinase
inhibitor with multiple anti-angiogenic and apoptotic activities. Since 2008,
sorafenib became the first and only pharmacological agent clinically approved
for the treatment of advanced HCC. However, despite the success achieved by
sorafenib in clinical trials, overall survival was limited suggesting the presence
of MDR mechanisms. Thus, the search for new agents acting in synergy with
sorafenib became highly essential.
Lately, natural compounds including dietary phytochemicals have
demonstrated promising chemopreventive potentials in many cancers including
HCC. Among these promising phytochemicals is I3C. Indole-3-carbinol was
extensively studied for its selective cytotoxic potentials towards cancer cells as
well as its abilities to chemosensitize several cancer cell lines to
chemotherapeutic agents.
Accordingly, the current study was conducted to investigate the potential
chemosensitizing effects of the phytochemical, I3C, on sorafenib cytotoxicity in
HCC cell line, HepG2. Also, the putative mechanisms underlying this
modulation were explored in terms of their effects on apoptotic and angiogenic
machineries as well as on tumor invasiveness. Light was shed on the
contribution of NOX-1, a member of the NOX family of enzymes, in I3C
modulatory effects as well as on clusterin which has been reported to be
involved in HCC metastasis, chemoresistance and apoptosis. To fulfill these
goals, sorafenib was used at concentrations ranged from 0.3125 to 20 μM in the
cytotoxicity assay. While, I3C was used at concentrations ranged from 12.5 to
800 μM. from fitted survival curves, IC5 of I3C was selected to study the
modulatory effects exerted by the phytochemical on sorafenib cytotoxicity in
HepG2 cells. In the mechanistic studies, cells were divided into four different
treatment groups as follows:
Control group: the group was treated with vehicle only (0.3% DMSO in
RMPI-1640 medium).
Indole-3-carbinol group: the group was treated with 113 μM I3C.
Sorafenib group: the group was treated with 2.3 μM sorafenib.
Combination group: the group was co-treated with 113 μM I3C and 2.3 μM
sorafenib.
The following parameters were investigated:
The effect of sorafenib and/or I3C was assessed on the following parameters in
HepG2 cell line:
I. Cellular proliferation of HepG2 cells using SRB assay.
II. Cell cycle distribution using flowcytometric DNA ploidy analysis.
III. Apoptosis markers by:
Active caspase-3 level using ELISA kit.
Caspase-8 enzymatic activity colorimetrically.
IV. Proliferation markers by:
Measuring the gene expression of EGFR by qRT-PCR.
Measuring the level of p-Akt using ELISA kit.
Summary and Conclusions
150
Measuring the level of p-ERK using ELISA kit.
V. Angiogenesis markers by:
Measuring the gene expression of VEGF by qRT-PCR.
Measuring the level of HIF-1α using ELISA kit.
VI. Invasiveness and metastasis markers by:
Measuring the gene expression of E-cadherin by qRT-PCR.
Measuring the level of snail using ELISA kit.
Measuring the level of clusterin using ELISA kit.
VII. Involvement of NOX enzymes by:
Measuring the level of NOX-1 using ELISA kit.
The findings of the present investigation can be summarized as follows:
I. Both I3C and sorafenib inhibited the growth of HepG2 cells in
a concentration dependent manner where the IC50 of both drugs were 197 and
2.3 μM, respectively. from fitted survival curves the IC5 of I3C was calculated
and found to be 113 μM. Co-treatment of HepG2 with I3C and sorafenib
significantly enhanced the cytotoxicity of sorafenib where the IC50 dropped
from 2.3 μM to 0.91 μM.
II. Treatment of HepG2 cells with 113 μM I3C induced a significant cell cycle
arrest at the S phase whereas sorafenib arrested the cells at the G1/G0 phase.
When HepG2 cells were treated with both drugs a prominent G1/G0 phase block
occurred.
III. Treatment of HepG2 cells with I3C, sorafenib or their combination
enhanced apoptosis significantly by increasing caspase-3 as well as caspase-8
activities. Moreover, the combination achieved a significant upregulation in
caspases activities compared to the individual treatments.
IV. Treatment of HepG2 cells with I3C, sorafenib or their combination
significantly downregulated the gene expression of EGFR as well as the levels
of p-Akt and p-ERK indicating the anti-proliferative effects of both drugs.
Summary and Conclusions
151
However, it should be noted that while the combination significantly reduced
the level of p-ERK compared to the sorafenib-only treated group, it failed to
achieve such reduction in EGFR expression or p-Akt level.
V. Treatment of HepG2 cells with I3C, sorafenib or their combination
significantly downregulated the level of HIF-1α. Regarding the expression level
of VEGF, both sorafenib and combination groups significantly downregulated
its expression. Indeed, the anti-angiogenic potentials of both drugs were
manifested in the combination group where the level of HIF-1α as well as the
gene expression of VEGF were significantly downregulated compared to the
sorafenib-only treated group.
VI. Indole-3-carbinol could suppress metastasis and invasiveness markers in
combination with sorafenib where the levels of snail and clusterin were
significantly reduced while the epithelial phenotype marker, E-cadherin, was
significantly augmented.
VII. The level of NOX-1 was significantly augmented by I3C. Sorafenib failed
to achieve any significant elevation in the level of NOX-1, however, in
combination the effects of I3C became more pronounced and NOX-1 level
increased significantly compared to the control and the sorafenib-only treated
groups.
In conclusion, to our knowledge and until the time of writing this thesis,
this is the first study to show that I3C, at a subtoxic concentration, enhances the
cytotoxic activities of sorafenib in HepG2 cancer cells and subsequently, may
shed some light on overcoming sorafenib resistance in HCC cells. The
modulatory activities of I3C could be, at least, partially attributed to increased
activity of caspases, decreased expression of p-ERK, HIF-1α and VEGF,
elevated expression of E-cadherin and downregulated levels of snail and
clusterin as well as increasing NOX-1 expression (Graphical abstract).
Summary and Conclusions
152
Graphical abstract: Schematic diagram for the modulatory effects exerted by
I3C to enhance sorafenib cytotoxicity in HepG2 cells.