Study Of Molecular Biology Changing Induced By Low Dose Of¹²⁵I Seeds Brachytherapy On Lung Adenocarcinoma A549Cells And Detecting Apoptosis Using⁹⁹Tc^m-AnnexinV Imaging Combined With MR-DWI

Objective:To investigate the tumor inhibitory effect, possible mechanism and the influence ofregulatory proteins’ expression related to molecular biological characteristics andradiosensitivity of continuous low-dose¹²⁵I irradiation seeds interstitial brachytherapy onimplanted A549lung adenocarcinoma cells in nude mice, and explore the feasibility ofnoninvasive way detecting apoptosis induced by¹²⁵I ionization radiation using⁹⁹Tc^m-AnnexinV combined with diffusion weighted magnetic resonance imaging（MR-DWI）.Methods:36BALB/c-nu nude mice bearing A549cells were randomly stratified into three groups:group A, B and C（control group）. One conventional dose¹²⁵I seed with the apparent activityof （25.5²7.8, mean26.9）MBq was implanted into each tumor in group A，extremelylow-dose¹²⁵I seed with the apparent activity was （4.4⁶.3, mean5.1）MBq was implantedinto group B, while the control group received "cold seeds" treatment. After treatment,the volume of the tumors was measured every two days. Both⁹⁹Tc^m-Annexin V imaging andMR-DWI were performed before and after14days of the brachytherapy, then all mice weresacrificed for pathological examination, routine pathological slides of tumor tissue wereobserved under light microscope to evaluate the range of tumor tissues damaged induced by¹²⁵I seeds, the weight of tumors was measured, tumor control rate was calculated, apoptosiswas detected in tumor tissue by TUNEL immunofluorescence, the expression of molecularbiomarkers such as NF-κB, HIF-1α, survivin, caspase-3, cyclinD1, p27and HSP90was alsoassayed by immunohistochemical（S-P）determination.Results:1The inhibition rate of tumor volume were51.2%and20.9%in group A and group B,respectively, not any inhibitory effect could be found in group C which handled with "coldseeds".2Pathological examination showed diffuse necrosis dramatically presented aroundingactive¹²⁵I seeds both in groups A and B, but damage induced by¹²⁵I ionization radiation wasmore serious in groups A than groups B with a much larger area of necrosis, no pathologicalchanges but slight fibroplasia were observed at the periphery of dummy seeds（"cold seeds"）,more over, A549lung cell could be seen infiltrating into the nearing muscle tissue directly.Tumor cells which arounding blood vessels avoided from¹²⁵I ionization radiation both in the A group and group B.3The results of immunohistochemistry showed, the expression rate of NF-κB, HIF-1α,survivin, caspase-3, cyclinD1, p27and HSP90in A549lung cells were not the same amongthe three groups after treatment.The expression of HIF-1α and HSP90in group A weredifferent from that of group A（P<0.05）, the expression of survivin, caspase-3and p27weresignificantly different （P<0.05） between group A and group C, the expression of cyclinD,p27and HSP90were also significantly different（P<0.05）between group B and group C.Positive or nagtive correlation to some degree among these molecular biomarkers were found,cell apoptosis index（AI）detected by TUNEL in group A was significantly higher than that ingroup B and group C （0.39±0.20,0.26±0.15and0.17±0.11repectively, P<0.05）.4Positive rate of⁹⁹Tc^m-Annexin V imaging after14days of¹²⁵I brachytherapy was58.3%（7/12）, which was significantly higher than that of before brachytherapy [8.3%（1/12）, P<0.05)]. The uptake ratio of⁹⁹Tc^m-Annexin V（RI）before the treatment in groupsA, B and C was1.30±0.39,1.72±0.71and1.39±0.42, respectively（P>0.05）. However, RIafter14days’ treatment（3.03±1.69）was increased significantly compared with that of thebefore brachytherapy in groups A（t=3.346, P=0.007）.5Apparent diffusion coefficient（ADC） value of the tumor in groups A, B and C was（1.35±0.38）×10^-3mm²/s,（1.24±0.28）×10^-3mm²/s and（1.51±0.46）×10^-3mm²/s respectivelybefore the treatment, no significant difference were found among them（P>0.05）, ADC ingroup A after the brachytherapy was （2.50±1.08）×10^-3mm²/s, which significantlyincreased compared with that of before the treatment（t=3.924, P=0.007）. In all, the value ofADC was correlated with RI（r=0.310, P<0.05）. Positive relationship between RI and AI afterthe brachytherapy was found（r=0.566, P<0.05）, and ADC was positively correlated with RIand AI in some degree（r=0.311and0.329, repectively, P<0.05）.Conclusion:1The interstitial brachytherapy with¹²⁵I seeds of the dosage was25.5²7.8MBq in eachseed could significantly inhibit the growth of A549solid tumor in mice, with the inhibitionrate of tumor volume was51.2%after14days’ treatment, one of the mechanisms of¹²⁵Iinterstitial brachytherapy may be the apoptosis induced by¹²⁵I ionization radiation.2⁹⁹Tc^m-Annexin V imaging combined with magnetic resonance diffusion weightedimaging （MR-DWI）could effectively evaluating the apoptosis of lung adenocarcinoma cellsinduced by¹²⁵I interstitial brachytherapy in a noninvasive way, hence,⁹⁹Tc^m-Annexin V combined with MR-DWI is conductive to determining the early efficacy of25I seedsbrachytherapy.3Some key molecular proteins such as NF-κ B, HIF-1α, survivin, caspase-3, cyclinD1,p27and HSP90which playing an important role in tumor molecular biological behavior andthe radiosensitivity were expressed diversely under the low dose of¹²⁵I irradiation on A549lung cancer.