Experimental Study On Irradiation Combined With Targeted Therapy

Radiation, surgery and chemotherapy are conventional therapies for cancer. Although improvements have been made to radiation, such as Three-Dimensional Conformal Radiation Therapy, Intensity-Modulated Radiation Treatment Techniques, Image-Guided Radiation Therapy and Tomotherapy, their use is often limited associated with tumor style, stage, micro-metastasis, significant toxicity, resistance to radiation and developed hypoxia areas.With the development of biochemistry and gene engineering,people realized that the effect of treatment on cancer will be improved significantly if aimed at some of its characteristic difference. So the targeted therapy comes into being. The targeted therapy takes the characteristic of tumor as its target, so it will be exclusive to cancer cells without doing any harm to normal cells. Radioimmunotherapy is one of targeted therapy. It is a new therapeutic modality including immunology, radiochemistry, radiation medicine, medical oncology and nuclear medicine. Clinical trials have showed some promising results in hematological neoplasms. But the treatment in solid tumors remains the major challenge.Oncolytic virotherapy is also one of targeted therapy. It use the genetically engineered viruses that specifically target and destroy tumor cells via their cytolytic replication cycle. viral-mediated tumor destruction is propagated through infection of nearby tumor cells by the newly released progeny. Although monotherapy using oncolytic adenoviral vectors is effective in xenograft tumor models in mice, only limited efficacy has been seen to date in clinical trials. A narrow therapeutic windows, coupled with dose-limiting toxicities and immune status of cancer patients has restricted the effectiveness of virotherapy. One approach to increase therapeutic efficacy and potentially decrease toxic side effects is combination therapy. In combined therapy, two or more types of treatment modalities are combined and take advantage of each superiority, avoiding cross-resistance. This research is emphasized on the effect of radiotherapy combined with radioimmunotherapy and oncolytic virotherapy in vitro/vivo. We hope to offer some benefit to clinical therapy.Part 1: The study of external beam radiotherapy combined with Iodine-¹³¹-labeled tumor necrosis therapy chimeric antibody (¹³¹I-chTNT) for Lewis tumor of C57BL/6 miceObjective: To investigate the biodistribution and scintigraphy in tumor-bearing mice with ¹³¹I-chTNT combined with irradiation, test the hypothesis that ¹³¹I-chTNT therapy combined with irradiation can overcome the limitations of monotherapy by studying the effects on the growth of Lewis carcinoma of mice.Methods: When the designed diameter of tumor in C57BL/6 mice bearing Lewis carcinoma in the right leg attainted, the mice were randomly assigned to different groups to receive treatment. The biodistribution of ¹³¹I-chTNT combined with or without irradiation groups was studied at various times after tail-intravenous injection in tumor-bearing mice respectively, the scintigraphy in the two groups was performed at 1,3,5,7 days respectively. The effect of treatment was assessed by tumor growth delay.Results: (1) The biodistribution data by injection of ¹³¹I-chTNT intravenously indicated that the highest uptake of tumor tissue both in ¹³¹I-chTNT group and in combined group was at 24h postinjection, and significantly higher in combined group at 24h,72h,120h (P<0.05). At the same time, There were no more side effects in the combined group(P>0.05 ) . The scintigraphy in tumor-bearing mice showed higher tumor accumulation in combined group than in ¹³¹I-chTNT group and the images of both groups were most clear at 3 day post-injection. Tumor growth delay analysis in 40 mice showed that the absolute growth delay for RT group,¹³¹I-chTNT group and the combined group was 9.1±1.86,3.1±1.75 and 13.1±2.6 days respectively. The nominal growth delay was 10 days, and the enhancement factor of ¹³¹I-chTNT for RT was 1.08.(2) The biodistribution data by injection of ¹³¹I-chTNT intratumorally indicated that the highest uptake of tumor tissue both in ¹³¹I-chTNT group and in combined group was at 24h postinjection, and significantly higher in combined group at 24h,72h(P<0.05). There were no more side effects in the combined group(P>0.05). Tumor growth delay analysis in 40 mice showed that the absolute growth delay for ¹³¹I-chTNT group, RT group and the combined group was 3.3±1.75,6.0±2.02 and 9.5±1.93 days respectively. The nominal growth delay was 6.2 days, and the enhancement factor of ¹³¹I-chTNT for RT was 1.03.Conclusion: Irradiation combined with ¹³¹I-chTNT can increase the uptake of ¹³¹I-chTNT in tumor without more side effects. ¹³¹I-chTNT can enhance radiotherapeutic effect for tumor-bearing C57 mice.Part 2: The Study of Replicating Oncolytic Adenoviruses Ad-MK Combined with Chemotherapy or Irradiation in Bladder CancerObjective: Oncolytic replication-competent adenoviral with promoter of midkine (Ad-MK) have the ability to replicate in and kill malignant cells which express midkine gene. We want to know whether the human Transitional cell carcinoma (TCC) cells BIU-87 and EJ express MK mRNA, and also to observe possible effect of using Ad-MK combined with or not radiotherapy and chemotherapeutic agent Mitomycinc (MMC) on these cells.Methods: Reverse transcription-polymerase chain reaction (RT-PCR) was employed to detect midkine mRNA expression in TCC lines BIU-87 and EJ. And also detect Adenovirus E1a mRNA expression in TCC lines BIU-87 after infected with Ad-MK. BIU-87 cells in culture medium in vitro were incubated with increasing concentration of Ad-MK,MMC. Morphological changes of cells were observed by microscopy. In addition, the survival rations of the cells were performed by tetrazolium-based colorimetric assay (MTT). Cell viability was calculated by diving the mean OD absorbance of the treated wells by the mean OD absorbance of the control wells. When the CDI<1, the interaction is considered synergy. Cell survival ratio to study the effect of Ad-MK combined with radiation. Virus yield of Ad-MK in BIU-87 in the presence and absence of radiation and MMC was determined by TCID50 (50% tissue culture infections doses).Results: The midkine mRNA express in TCC lines BIU-87 and EJ. The cells incubated with 10MOI Ad-MK have significant change at 3days. The cells were orbicular, swelling, suspending and along with cytolysis. The Morphological changes of cells are related with time. The TCC lines BIU-87 express Adenovirus E1a mRNA After infected with Ad-MK. It shows that Ad-MK can selectively replication in TCC lines BIU-87. and it is also due to the inhibition effect of Ad-MK on BIU-87 cells. Ad-MK inhibited proliferation of BIU-87 cells significantly in a dose- and time-dependent manner, and there is statistically significant difference compared with control teams(P<0.05). The potential cytostatic effects of combined treatment proved to be superior to either virus or MMC treatment alone. The data also demonstrated statistically significant difference between combined arm and either virus or MMC arm (P<0.01). The CDI=0.690 indicate the synergistic effect of Ad-MK combined with MMC. The ratio of Cell survival after 4Gy radiation team was 0.11±0.013, while Ad-MK at an MOI of 0.1 arm 0.13±0.04, and combined team was 0.003±0.002. Compared with either virus or radiation arm, The difference is statistically significant (P<0.01) . To examine the effect of radiation and MMC on virus replication, We infected BIU-87 cells with Ad-MK at an MOI of 0.1 after radiation at a dose of 4 Gy and followed by MMC at a dose of 50μg/L, then the number of infectious virus particles was determined on 293 cells by TCID50. Cells treated with Ad-MK plus radiation produced a larger burst size. Cells treated with Ad-MK plus radiation produced 4.4×103 PFU/cell 4 days after infection, cells infected with Ad-MK plus MMC produced 4.1×103 PFU/cell, whereas cells infected with Ad-MK alone generated about 3.3×103 PFU/cell at this time point. Compared Ad-MK arm with combined arm, The difference is statistically significant(P<0.05) .Conclusion: The midkine mRNA express in TCC lines BIU-87 and EJ. Ad-MK can selectively replication in TCC lines BIU-87. Ad-MK inhibited proliferation of BIU-87 cells significantly in a dose- and time-dependent manner, and there is statistically significant difference compared with control teams(P<0.05). The potential cytostatic effects of combined treatment proved to be superior to either virus,MMC treatment or radiotherapy alone and radiation and MMC can augment virus replication .Part 3: Expression of midkine and its clinical significance in transitional cell carcinoma of human urinary bladderObjective: To investigate the expression of midkine in bladder transitional cell carcinoma and analyze its relationship with clinic pathological features and prognosis.Methods: To detect the expression of midkine protein in 50 cases of bladder transitional cell carcinoma samples by SP immunohistochemical method using polyclonal antibodies against human midkine. Survival time of 40 cases were recorded. Then analyse the relationship between the expression of midkine in bladder transitional cell carcinoma and its relevant clinic pathological features and prognosis.Results: The protein expression of midkine was found in cytoplasm of tumor cells. The overall positive rate of midkine in 50 cases of bladder carcinoma was 90 % (45/50). The positive degree of midkine showed a trend of increasing with grade and stage, There was statistically significant difference among them(P<0.05), but not with sex,age,treatment,tumor number and size(P>0.05). In high expression of MK patients predicts a poor clinical outcome.Conclusion: Midkine is overexpressed in bladder transitional cell carcinoma than normal bladder. MK expression of bladder cancer is higher in less differentiated and deeper invaded cases, but it has no correlation with age,sex,treatment,tumor number and size. Patients with higher MK expression have shorter survival time than those with lower MK expression. So MK maybe becomes a useful molecular marker , prognosis factor and the target of targeted treatment to bladder neoplasm. Conclusion1. Irradiation combined with Iodine-¹³¹-labeled tumor necrosis therapy chimeric antibody (¹³¹I-chTNT) can increase the uptake of ¹³¹I-chTNT in tumor without more side effects. ¹³¹I-chTNT can enhance radiotherapeutic effect for tumor-bearing C57 mice.2. The midkine mRNA express in TCC lines BIU-87 and EJ. And Ad-MK can selectively replication in TCC lines BIU-87. Ad-MK inhibited proliferation of BIU-87 cells significantly in a dose- and time-dependent manner, and there is statistically significant difference compared with control teams(P<0.05). The potential cytostatic effects of combined treatment proved to be superior to either virus or MMC treatment alone and irradiation alone. The low dose of irradiation and MMC can augment virus replication.3. The protein expression of midkine is overexpressed in bladder transitional cell carcinoma than normal bladder. MK expression of bladder cancer is higher in less differenttiated and deeper invaded cases, but it has no correlation with age, sex, treatment, tumor number and size. Patients with higher MK expression have shorter survival time than those with lower MK expression. So it maybe a useful molecular marker in bladder cancer for prognosis and maybe a hopeful target in target therapy.