الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Breast cancer (BC) is the most prevalent cancer among women worldwide and the
second leading cause of cancer death in the developed regions while in less developed
regions; it is the most frequent cause of cancer death in women. Although the etiology of
breast cancer is not well known, several risk factors have been suggested to have an influence
on the development of this malignant tumor. Genetic, epigenetic, hormonal and
environmental factors are contributing to the development of such disease. The treatment of
breast cancer is very complex and includes the combination of surgery and chemotherapy,
radiotherapy, hormonal therapy or biological (targeted) therapy.
Owing to the molecular heterogeneity of breast tumors; different molecular subtypes are
existing with approximately 70-75% of all breast tumors express estrogen receptor α (ERα)
and classified as ER positive BC. This molecular phenotype of the disease responds well to
endocrine therapy with tamoxifen is still the most currently used endocrine therapy for preand
postmenopausal women with this disease phenotype. Although tamoxifen enhanced the
survival rates of women with ER (+) BC for several years; one-third of these patients
eventually develop disease recurrence following their initial response. Many mechanisms
could explain acquired tamoxifen resistance including the upregulation of various tyrosine
kinase growth factors receptors and their crosstalk with ER signaling pathways.
This study was focused on the insulin like growth factor (IGF) signaling pathway and
aimed at targeting this pathway to overcome tamoxifen resistance. To achieve this aim; two
IGF1-R tyrosine kinase inhibitors were used as new promising and effective combinations
with tamoxifen; picropodophyllin (PPP) (selective IGF-1R tyrosine kinase inhibitor) and
TAE226 (dual tyrosine kinase inhibitor against IGF-1R and another tyrosine kinase called
focal adhesion kinase; FAK).
In our study, the tamoxifen sensitive ER+ MCF7 and the tamoxifen resistant but estrogen
sensitive MCF7/LCC2 cell lines were used. Cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 5% fetal bovine serum (FBS) at 37°C in a humidified
atmosphere containing 5% CO2. Cells were sub-cultured twice weekly at 70-80% confluency and
visualized using an inverted microscope. Cell viability and cytotoxicity assays using MTT
reagent were performed. Drug doses were selected according to the results of the MTT assay
within a cytotoxicity range of 50%. MCF7 and MCF7/LCC2 were cultured in 6 groups and each
experiment was triplicated. Drugs were added on day 1 as follows:
Cell line I (control group): Cells in this group received only the vehicle (DMSO) used for
the dissolution of drugs.
Cell line II (4-hydroxytamoxifen treated group): Cells in this group received 94.2 nM 4-
hydroxytamoxifen dissolved in DMSO.
Cell line III (PPP treated group): Cells in this group received 25.15 nM of PPP.
Cell line IV (TAE226 treated group): Cells in this group received 2.7 μM of TAE226.
Summary
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Cell line V (Combined treatment of 4-OHT and PPP)
Cells in this group received a combination of 4-OHT and PPP in doses equal to those received
in groups II & III.
Cell line VI (Combined treatment of 4-OHT and TAE226)
Cells in this group received a combination of 4-OHT and TAE226 in doses equal to those
received in groups II & IV.
After 4 days the cells were detached and collected for evaluating the effect of the
different treatment regimens on various parameters. To evaluate the effect of these drugs on the
cancer cells proliferation and angiogenesis, protein expression of the key cell cycle regulator
cyclin D1 (CD1) and the vascular endothelial growth factor (VEGF) was assessed by ELISA.
Our study revealed that both CD1 and VEGF were elevated in the MCF7/LCC2 cell lines as
compared to their MCF7 counterparts demonstrating that they may play a crucial role in
rendering cancer cells evading tamoxifen action. Our study also showed that all treatment
regimens significantly decreased the levels of CD1 and VEGF in the two cell lines with the
highest response achieved by the two combination regimens. Similarly; when the apoptosis was
assessed by measuring caspase-3 activity by colourimetric assay, all treatment regimens
produced a significant change in caspase-3 activity with the highest response demonstrated by
the combination regimens.
Furthermore; the phosphorylated form of both the IGF-1 receptor (IGF-1R) and FAK as
well as their downstream effector kinase; protein kinase B (AKT) was assessed by ELISA. Our
results revealed that all these phosphorylated forms were elevated in the MCF7/LCC2 compared
to the MCF7 positive control cells demonstrating their potential role in acquiring tamoxifen
resistance in these resistant cells. Our results regarding the effects of the single drug and
combination regimens on these signaling molecules were contradictory in the tamoxifen
sensitive cell line and need further investigation. On the other hand; the effects of the
combination regimens were more prominent and promising in the tamoxifen resistant cell line. In
addition; the effect of 4-OHT combination with TAE226 on phosphorylated FAK and AKT was
superior to that achieved with its combination with PPP.
In summary; the combination of 4-OHT with either PPP or TAE226 appeared to be
efficient regarding all parameters in the tamoxifen resistant cell line. Taken together, this work
provided an evidence that each of PPP and TAE226 addition to tamoxifen therapy could restore
tamoxifen sensitivity via blocking multiple deregulated processes through direct or indirect action.