الفهرس 
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Abstract
Colorectal cancer is a significant cause of mortality worldwide. c-kit stimulation and overexpression is one of the causes of colorectal cancer. Imatinib is a well-known tyrosine kinase inhibitor which has been approved by the FDA to be used in the treatment of CML and GIST, primarily through its inhibitory actions on Bcr-Abl and c-kit tyrosine kinase receptors.
However, imatinib monotherapy resulted in rapid development of resistant cells. Besides, severe neutropenia in almost 45% of imatinib-treated patients resulting in dose interruptions and thus, compromised patient outcome limited the clinical usefulness of imatinib as a monotherapy.
The current study is the first in-vitro trial to investigate the modulatory effect of sodium selenite on imatinib cytotoxicity in human colorectal cancer cells HCT116. The possible modulatory mechanisms were explored by studying the cell cycle perturbation, effects on apoptotic and autophagic machineries, effects on reduced thiols as well as nitric oxide content.
Imatinib and selenite were used at concentration range from 10 to 50 μM. Human colorectal tumour (HCT116) cells were divided into four treated groups. The first group served as the control, in which cells are treated with vehicle (0.1 % DMSO in RPMI-1640 medium). The second and third groups of cells were treated with either imatinib or selenite while the last group was co-treated with imatinib and selenite.
The following parameters were investigated:
Expression of c-kit tyrosine kinase receptors in HCT116 cells using immunocytochemistry.
Cytotoxic activity of imatinib, selenite and their combination using SRB assay.
Percentage distribution of different phases of the cell cycle using flowcytometric analysis.
Determination of effects on apoptotic machinery by measuring caspase-3 activity and visualization of DNA fragmentation.
Determination of effects on autophagic machinery by detecting Beclin-1 expression using RT-PCR and visualization of acidic vesicular organelles formation.
Testing the effects on oxidative stress as reduced thiol and nitric oxide content.
The findings of the present investigation can be summarized as follows:
Expression of c-kit tyrosine kinase receptor, the target of imatinib, has been confirmed in HCT116 cells.
HCT116 cell line was found to be sensitive to imatinib and selenite. Simultaneous treatment of HCT116 cells with imatinib and selenite resulted in significant increase in cytotoxic activity relative to their individual effects. Yet, to confirm the modulatory effect of selenite on imatinib cytotoxicity, drug interaction analysis was carried out and as indicated by the calculated combination and dose reduction indices, the combination of imatinib and selenite produced synergistic cytotoxic effect in HCT116 cells.
Both imatinib and selenite induced cell cycle arrest in G0/G1 phase, in HCT116 cells. Surprisingly, their concurrent combination resulted in accumulation of the cells in S phase.
Activation of the apoptotic machinery in HCT116 treated cells has been shown as proven by increase of caspase-3 activity and DNA fragmentation in imatinib, selenite and combination treated cells.
Imatinib induced Beclin-1 dependent autophagy in HCT116–treated cells as confirmed by acidic vesicular organelles formation. Conversely, selenite decreased Beclin-1 expression and hence, reduced autophagolysosomes production. Imatinib and selenite simultaneous combination, however, resulted in inhibition of autophagy relative to the control and imatinib-treated groups.
Imatinib, selenite and their combination reduced oxidative stress as indicated by significant increase in non-protein thiol content of HCT116-treated cells relative to the control group.
Similarily, imatinib and selenite reduced nitric oxide content relative to the control group. However, their simultaneous combination failed to confer further reduction in nitric oxide content when compared to the individuals effects of imatinib and selenite when used seperately.
In conclusion, supranutritional doses of selenite, an essential micronutrient with immunostimulant effects, synergistically augment imatinib cytotoxicity in human colorectal carcinoma cell line. This is the first in-vitro study to report such a benefit associated with the combination of imatinib and selenite in treatment of colorectal cancer. The postulated mechanisms underlying this interaction include an enhancement of accumulation of cells in S phase, as well as activation of programmed cell death type I machinery. Other postulated mechanisms include switching off the cytoprotective autophagy via downregulation of Beclin-1 expression and reducing the markers of oxidative stress and angiogenesis. Because of the diverse biological effects of selenite and the complex mechanisms whereby it enhances imatinib cytotoxicity, further studies are needed to clarify other mechanisms by which selenite can enhance imatinib cytotoxicity. Moreover, pointing out to selenite immunostimulant effects which could alleviate imatinib dose-limiting safety profile, the preliminary findings of this study merit exploration of imatinib and selenite incorporation in a novel combination in prospective controlled clinical trials which is expected to be associated with improved clinical patient outcome.