الفهرس | Only 14 pages are availabe for public view |
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 102 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. |