Research Profile - College of Medicine and Health Science - United ...

Department of Pharmacology
IGF receptors. Drugs targeting receptor tyrosine
kinases (ErbB2/HER2 and ABL/c-kit) impact the
PI3K/Akt pathway. Consequently, the inhibition
of Akt is considered to be an attractive cancer
therapeutic target. Three isoforms of Akt
(Akt1/PKB·, Akt2/PKB‚, and Akt3/PKBÁ) are overexpressed
and activated in cancers, but the
degree of functional redundancy between them
on cancer cell survival and invasion are unclear.
The identification of the Akt isoform that is the
most promising target for cancer therapy is
unknown and will be addressed in the following
specific objectives:
a. To explore the respective roles of Akt-1, -2
and -3 in cancer cell survival, invasion, tumor
growth and metastasis, studies will be
conducted using RNA interference technology
in LNM35 human lung cancer cells and HT29
human colon cancer cells.
b. To characterize the ability of different isoforms
of Akt (Akt-1, -2 and -3) to transactivate different
subsets of target genes to orchestrate either
cell survival or invasion. This will be investigated
in colon and lung cancer cells using microarray
analysis.
using microarray analysis and confirm the results
by real-time RT-PCR. It is anticipated that this
project will provide evidence that targeting
Akt-1 and/or -2 is a promising strategy for the
treatment of colon and lung cancer.
2. Screening of potential anti-cancer
compounds using in vitro and in vivo assays
The overall goals of this part of my research are
directed towards studying the in vitro and in vivo
cytotoxic properties of the synthesized/purified
drugs (Luteolin, Thymoquinone and Frondoside
A). The effective drugs will be further
investigated in order to determine their
mechanisms of action using various molecular
biological techniques. In addition, we will
attempt to investigate the effects of these drugs
on cell survival and invasion in vitro and on
tumor growth in vivo. The effects on metastasis
in nude mice using various cancer cell lines will
also be investigated.
The effects of knockdown of Akt-1, -2 or -3 on
cellular survival will be determined by cellular
viability, DNA fragmentation, cell cycle and
TUNEL assays, caspases activity, PARP cleavage,
cytochrome-c release and expression of proand
anti- apoptotic proteins. The impact of Akt-
1, -2 or -3 silencing on invasion will be
investigated using the collagen type I and the
chick heart invasion assays, cell-cell and cellmatrix
adhesion and expression of E-Cadherin.
The effect of knock down of the Akt’s on HT29
cell line on tumour growth will be tested in
nude mice. In addition, we will assess the
development of new blood vessels (CD31
antibody), the presence of apoptotic cells
(caspase 3 activity), and proliferative activity
(ki67 antibody). We’ll test the therapeutic impact
of the Akt isoforms inhibition on metastasis
using the highly invasive and metastatic lung
cancer cell line LNM35 targeting the lymph
nodes and lungs. This cell line is stably
transfected with a vector encoding luciferase,
to allow a rapid and reliable quantification of
micro-metastases. To identify the mechanisms
underlying the effects of Akt isoform inhibition,
we will search for differentially expressed genes
65