In Vivo Study Of Breast Cancer Radiosensitization By Targeting EIF4E

Objective:In the present study, we transfected pSecX-t4EBP1into mouse xenograftmodel, and then experimentally demonstrated that it could inhibit theexpression of eIF4E, leading to the down-regulation of HIF-1a expression,which resulted in inhibition of cell proliferation, promotion of apoptosis, andenhancement of radiosensitivity. The study suggests that it is feasible of usingpSecX-t4EBP1or in combination with radiotherapy for the treatment of breastcancer with tumor specifcity and high effcacy.Methods:Ninety female BALB／c mice aged6-8weeks were used for thisexperimental tumorigenicity assays. All animals were maintained in specificpathogen-free rooms and all procedures were approved by FMMU Animal Careand Use Committee. A total of2×107EMT-6cells in0.2ml were injected subcutaneously into the abdomen of each mouse. Tumor diameters wereassessed using digital Vernier callipers every day, when the tumor wereincreased up to a size of300mm3,ninety mice were assigned randomly into sixgroups, control, irradiation (IR), pSecX-t4EBP1, pSecX-t4EBP1combined withirradiation (pSecX-t4EBP1+IR), pSecX and pSecX combined with irradiation(pSecX+IR) respectively. In the groups where the mice were subjected toirradiation treatment, the mice were given three doses of local irradiation at adose of4Gy at2-day intervals. Mice which relegated to pSecX-t4EBP1andpSecX-t4EBP1+IR were performed as follows:80μg of plasmid pSecX-t4EBP1in2ml saline by intravenous, on a series of four doses at1-week intervals; Micewhich are subject to pSecX and pSecX+IR were given80μg of plasmid pSecXin2ml saline by intravenous, on a series of four doses at1-week intervals. Micewere weighed, and the tumor size was measured twice a week with anelectronic caliper. At the end of the experiments (On day29), all mice weresacrificed and the tumors were extracted, tumor tissues weighed，measured, andstored for histochemical examination. The data are reported as Means±standard deviation (SD). The statistical software SPSS13.0was performedusing in the statistical analyses. Comparisons of mean value were performed byStudent’s t-test and one-way analysis of variance (ANOVA).The statisticaldifference p<0.05was considered as significantly.Results:1．Subcutaneous transplanted tumor models on BALB／c mice could beestablished successfully after inoculation with EMT-6cells in all experimentalmice, and the rate of tumor formation is100%.2．The tumor growth in the pSecX-t4EBP1+IR, pSecX+IR and IR had a significant larger inhibitory than the pSecX-t4EBP1, pSecX and control,suggesting that Radiation therapy could significantly inhibit tumor growth. Inthe pSecX-t4EBP1, pSecX and control groups, pSecX-t4EBP1treatment had asignificant larger inhibitory effect on tumor growth than the pSecX or control(P<0.05), while the rate of tumor growth were no significant difference in thegroup of the control and pSecX (P>0.05). These data also indicate thatpSecX-t4EBP1had an effect of significant inhibition of tumor growth ascompared with control and pSecX transfected with vector only, suggesting thatconstitutively expressed pSecX-t4EBP1is sufficient to kill tumor rather thanvector only. A similar result was obtained in the three groups combined with IR,the mean relative tumor volume for the pSecX-t4EBP1+IR treatment wasinitially significantly lower (P<0.05) than pSecX+IR or IR alone and remainedlow until the trial was terminated, whereas there were no significant differencein tumor volume among the group of pSecX+IR or IR alone (P>0.05),suggesting that pSecX-t4EBP1were markedly sensitized to radiotherapy ascompared with pSecX transfected with vector only.3．The tumor weight values in pSecX-t4EBP1, pSecX and control groupswere3.02±0.849,3.48±1.220and3.51±0.963g, respectively. The tumor weightvalues in pSecX-t4EBP1+IR, pSecX+IR and IR groups were1.51±0.872,1.88±0.931and1.96±0.489g, respectively. pSecX-t4EBP1treatmentsignificantly decreased the weights of the initial tumors(P<0.05) and showedsignificantly better inhibition of tumor growth than pSecX or control.pSecX-t4EBP1+IR treatment significantly decreased the weights of the initialtumors(P<0.05) than pSecX+IR or IR alone and showed that it had significantlyenhanced the sensitivity of radiotherapy.4．Cell death was detected by FCM in the present study. The rates of cell apoptosis for the pSecX-t4EBP1treatment were initially significantly higher(P<0.05) than pSecX or control, whereas there were no obvious diferences inpSecX+IR and IR alone (P>0.05). The rates of cell apoptosis for thepSecX-t4EBP1+IR treatment were initially significantly higher (P<0.05) thanpSecX+IR or IR alone, whereas there were no obvious diferences in pSecX+IRand IR alone (P>0.05).5．In the present study, HIF-1a expression was also examined and shownto be markedly reduced in the pSecX-t4EBP1+IR in comparison with thepSecX+IR and IR group, and the pSecX-t4EBP1treatment showed to bereduced in comparison with the pSecX and control group.Conclusion:pSecX-t4EBP1can significantly inhibit tumor growth and enhance theradiosensitivity of breast carcinoma xenograft in BALB／c mice possiblyassociated with down-regulation the expression of HIF-1α,suggesting thatpSecX-t4EBP1may serve as an ideal molecular target for radiosensitization ofbreast carcinoma.