The Study Of Genistein Treatment Enhances Photodynamic Therapy Responsiveness And Its Mechanism

Objective In the last few years more and more attention has been paid to the use of photodynamic therapy (PDT) as a method with potential for the selective destruction of malignant tumors without producing damage to adjacent normal tissues. For the unique mechanism of PDT, it not only has the ability to destruct malignant tumors, but also can stimulate several signaling pathways in tumor cells, and results in various biological effects. Singlet oxygen (1O2)-induced cytotoxicity is believed to be responsible for responses to photodynamic therapy. Other cytotoxic oxidants, such as hydrogen peroxide and peroxynitrite have been shown to stimulate cell survival signaling pathways in addition to causing cell death. The activity of survival signaling pathways plays an important role in cell survival and resistance to apoptosis. On the other hand, because of the limited light penetration depth, more and more evidence have been accumulated to show that the dose of light radiation and PDT effect is changed in different depth of tumor tissues. The clinical application is limited for the same reason. It is very valuble to study the thorough mechanism and dose-effect relation of PDT for the aim at how to utilize the specificity of PDT and enhance the effect of PDT efficiently. Transcription factor hypoxia-inducible factor 1 (HIF-1) can upregulate the gene expression of enzymes or growth factors inducing angiogenesis, anaerobic glycolysis, cell survival. It has a very important role in the growth and proliferation of malignant tumors. In order to enhance the efficiency of HpD-PDT, combinations of PDT and protein tyrosine kinase (PTK) inhibitor, genistein (GEN), have been applied to improved treatment in a mouse hepatocellular carcinoma (H22) in vitro and vivo. The present study was designed to investigate the dynamic changes of hypoxia-inducible factor 1 (HIF-1) signaling pathway and its target gene vascular endothelial growth factor (VEGF) mediated by PTK, after PDT and GEN. Methods On the basis of H22 hepatocellular carcinoma cell culture, H22 cell was treated with different dose of PDT and GEN, and the proliferating status of H22 cell post HpD-PDT and GEN was determined with MTT assay. The methods as isotope assay, absorbance assay, western blot assay, electrophoretic mobility shift assay (EMSA) and reverse transcription-polymerase chain reaction(RT-PCR) were used to determine the dynamic changes of the actity of protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP), the banding activity of HIF-1, the levels of HIF-1αprotein and VEGFmRNA expression in H22 cell treated with PDT and GEN in different dose. The mice bearing H22 xenografts were treated with PDT and GEN, the tumor inhibition effect was assessed by measuring gross tumor volumes and survival time over a period of 3 weeks,the effect of PDT and GEN on HIF-1α, VEGF and intratumoral microvessel density (MVD) in transplanted tumors were examined by using immunohistochemistry technique. Results 1. When the HpD concentration was 5 μg/ml, the proliferation of H22 cell was decreased after PDT with elevation of light radiation dose from 0.25J/cm2 to 8J/cm2 in vitro, HpD alone or light radiation alone had no notable effect on H22 cell. The 50% inhibiting concentration (IC50) was 1.9957J/cm2. 2. GEN in 10-8 mol/L could not inhibit the proliferation of H22 cell alone, but enhanced the result of PDT remarkably. 3. When the HpD concentration was 5 μg/ml, the energy density of light radiation was below 0.5J/cm2 and the dose of PDT was below IC30,PDT inhibited the activity of PTP in H22 cell. It increased the ratio of PTK/PTP. When the energy density of light radiation was above 0.5J/cm2 and the dose of PDT was above IC30,PDT inhibited the activity of PTK in a dose-dependent fashion. It decreased the ratio of PTK/PTP. 4. GEN in 10-8 mol/L could increase the activity of PTP, and decrease the activity of PTK after PDT, which decreased the ratio of PTK/PTP at last. 5. When the HpD concentration was 5 μg/ml, the energy density of light radiation was below 0.5J/cm2 and the dose of PDT was below IC30,PDT increased the levels of HIF-1αprotein, the banding activity of HIF-1 and VEGFmRNA expression in H22 cell. When the energy density of light radiation was above 0.5J/cm2 and the dose of PDT was above IC30,PDT decreased the levels of HIF-1αprotein, the banding activity of HIF-1 and VEGFmRNA expression in a dose-dependent fashion on the contrary. The dose-effectcurves was like alphabet'V'which stood by head. The effects of PDT weakened in a time-dependent fashion. 6. GEN in 10-8 mol/L could decreased the levels of HIF-1αprotein, the banding activity of HIF-1 and VEGFmRNA expression after PDT in vitro. 7. When the HpD concentration was 10 mg/kg, the levels of HIF-1αand VEGF protein were incresased after PDT at 200J/cm2 in vivo. The administration of GEN decreased the levels of HIF-1α, VEGF protein and intratumoral microvessel density (MVD) remarkablly after PDT. 8. The combinations of PDT with GEN could slow down the increase of tumor volumes and prolong the survival period of mice bearing xenografts more significantly than PDT or GEN alone. Conclusions 1. The treatment of protein tyrosine kinase (PTK) inhibitor, genistein (GEN), can enhance the photodynamic therapy responsiveness in a mouse hepatocellular carcinoma (H22) in vitro and vivo. 2. In vitro, HpD-PDT can directly adjust the signaling pathway about HIF-1 which is influenced by PTK in a dose-dependent fashion: sublethal-dose PDT can induce the ratio of PTK/PTP, increase the levels of HIF-1αprotein, the banding activity of HIF-1 and VEGFmRNA expression, and lethal-dose PDT can reduce the ratio of PTK/PTP, decrease the levels of HIF-1αprotein, the banding activity of HIF-1 and VEGFmRNA expression in a dose-dependent fashion. 3. In vitro, GEN can influence the activity of PTK and PTP at the same time, decrease the ratio of PTK/PTP, enforce the degradation of HIF-1αprotein, inhibit the banding activity of HIF-1 and the expression of VEGFmRNA in lower-dose. 4. In vivo, the administration of GEN can decrease the levels of HIF-1α, VEGF protein and intratumoral microvessel density (MVD) remarkablly after PDT.