Effects Of Interstitial Photodynamic Therapy On Human Lung Adenocarcinoma A549 Cell Transplanted Tumour In Nude Mice

Objective:The aim of the study was to analyze the influence of interstitial photodynamic therapy(IPDT)mediated by hematoporphyrin derivatives as photosensitizers on lung adenocarcinoma A549 cells subcutaneous transplanted tumour in nude mice,and to establish a basic experimental basis for the therapeutic applications of lung cancer by IPDT.Methods:The A549 cells were cultured in vitro and injected subcutaneously into the left back of nude mice near the scapula.Twenty-four xenograft models in nude mice were constructed,which were randomly and averagely divided into four groups: the control group,the photosensitizer group,the laser group and the IPDT group.In the control group,the tumors were injected with saline for 2 m L/kg,and irradiated with natural light.In the photosensitizer group,the tumors were injected with hematoporphyrin derivative at the dose of 10 mg/kg,and irradiated with natural light after 4 hours protection from light.In the laser group,the tumors were injected with 2 m L/kg of saline and then irradiated with630 nm laser light for 15 minutes through optical fiber imported into tumor via syringe needle.In the IPDT group,the tumors were injected with hematoporphyrin derivative for10 mg/kg,and then irradiated with 630 nm laser light for 15 minutes through optical fiber imported into tumor via syringe needle after 4 hours protection from light.Each nude mouse was given a total of three treatments,with each treatment interval of one day.The tumors’ long and short diameters were measured every three days.Two weeks after completion of treatment,four groups of transplanted tumour samples were taken out for the following experiments:(1)The volume of tumours was calculated based on the long and short diameters,and the change in volume were compared between the groups.The growth curve of tumours was plotted,and the inhibition rates of tumour growth were calculated.(2)The pathological changes of transplanted tumour cells and the morphological changes of cell structures such as nuclei and chromatin in each group were observed under light microscope using HE staining.(3)Tunel staining was used to compare the apoptosis of transplanted tumour cells in different groups,the biochemical and morphological characteristics of the apoptotic cells were observed,and the apoptotic index was calculated for each group.(4)The changes in transcript levels and protein expression levels of some apoptosis-related genes,including Survivin,Bcl-2,Caspase-3and Caspase-9,in the tumour tissues were assessed by q RT-PCR assay and Western blot assay.Results:(1)After the completion of the three treatments,the tumour volume in the IPDT group was significantly smaller than that in the control group,photosensitizer group and laser group,and the difference was statistically significant(P < 0.05).The tumour inhibition rates were 0,7.51%,9.43% and 52.41% in the control group,photosensitizer group,laser group and IPDT group respectively.(2)After HE staining of tumour tissues,the tumour tissues of IPDT group showed more degenerated necrotic cells with disordered cell arrangement and partly foamy shape,while the tumour cells of control group,photosensitizer group and laser group were closely arranged with large deep stained nuclei.(3)Tunel staining showed that there were more apoptotic cells in the IPDT group,with brownish-yellow granular material visible in the nucleus.The apoptotic index was3.2%,6.5%,8.7% and 25.6% in the control group,photosensitizer group,laser group and IPDT group respectively.(4)The q RT-PCR results showed that the m RNA expression of Survivin and Bcl-2 decreased and that of Caspase-3、Caspase-9 increased in the IPDT group compared with the control,photosensitizer and laser groups,and the difference was statistically significant(P < 0.05).(5)Western blot results showed that the protein expression of Survivin and Bcl-2 decreased in the IPDT group,while the protein expression level of Caspase-3、Caspase-9 increased compared with the other three groups,and the difference was statistically significant(P<0.05).Conclusion: IPDT could significantly inhibit the growth of xenograft tumors in lung adenocarcinoma A549 cells in nude mice,which may be related to the down-regulation of apoptotic factors Survivin and Bcl-2 gene expression and up-regulation of apoptotic factor Caspase-3、Caspase-9 gene expression.