Anti-tumor effect of suicide gene therapy using chimeric promoter plus radiotherapy on cancer cell lines / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the synergistic anti-tumor effect of radiotherapy and horseradish peroxidase/prodrug indole-3-acetic acid (HRP/IAA) gene therapy system using chimeric hTERT promoter responsive to ionizing radiation.</p><p><b>METHODS</b>The synthetic hTERT promoters containing four tandem-repeat copies of radio-inducible CArG elements, and the chimeric promoter containing cytomegalovirus (CMV) early promoter were both constructed. The activities of the chimeric promoters in cancer cell lines (HeLa, A549, and MHCC97) and normal cell line (MRC-5) were detected using luciferase reporter gene expression analysis after a (60)Co γ-irradiation treatment at a series of doses(a single dose of 0 to 10 Gy). The anti-tumor effect of combining irradiation with HRP/IAA gene-directed enzyme prodrug therapy system controlled by the chimeric promoter was tested by colony formation assay, cell counting and apoptosis analysis.</p><p><b>RESULTS</b>The chimeric promoters were ineffective in normal human cells, even after irradiation, but the expression of luciferase gene in tumor cells was significantly higher. The activity of the chimeric promoter in MRC-5 cells was 22.3%, 12.9% and 13.6% of that in HeLa, A549 and MHCC97 cells, respectively. After irradiation, the ratios were 11.7%, 8.7% and 8.8%, respectively. Furthermore, the chimeric promoters could successfully induce the expression of luciferase gene following different doses of radiation, with maximal inducible activity seen after 6 Gy irradiation. The chimeric promoter containing four tandem-repeat copies of radio-inducible CArG elements and CMV early promoter showed the highest activity with 6 Gy irradiation. The relative luciferase activities in HeLa, A549 and MHCC97 cells were 1.7 ± 0.2, 2.3 ± 0.2 and 2.3 ± 0.1, respectively. The chimeric promoter mediated suicide gene therapy system could increase radio-sensitivity in different cancer cells. Compared with the control system, it plus irradiation showed stronger cell proliferation inhibition, 67.3% vs. 26.1% in HeLa, 69.0% vs. 28.3% in A549, 64.6% vs. 20.8% in MHCC97 cells, and also higher apoptosis-inducing effect, 39.6% vs. 14.2% in HeLa, 33.0% vs. 12.4% in A549, and 33.2% vs. 14.2% in MHCC97 cells.</p><p><b>CONCLUSIONS</b>Chimeric promoter containing hTERT promoter, CArG elements and CMV promoter preserve the tumor-specificity in telomerase-positive tumor cells, and irradiation-responsive to low dose of radiation. The suicide gene therapy using this promoter plus radiotherapy show a strong anti-tumor effect in vitro. It is expected to have a good potential for future application in gene radiotherapy.</p>

hTERT promoter enhances the radiosensitivity to gene-radiation therapy of human laryngeal carcinoma transplanted in nude mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To explore the therapeutic efficiency of human telomerase reverse transcriptase promoter (hTERTp) mediated horseradish peroxidase (HRP) catalyzed effects of indole-3-acetic (IAA) on laryngeal squamous cell carcinoma with different radiosensitivity in vivo.</p><p><b>METHODS</b>Human laryngeal squamous cell carcinoma Hep-2 and Hep-2R cells were transplanted into nude mice. After growing to about 30 approximately 50 mm3, the tumor-bearing mice were randomly divided into eight groups: Hep-2 line: combined group (A), gene group (B), radiation group (C) and blank group (D); Hep-2R line: combined group (AR), gene group (BR), radiation group (CR) and blank group (DR). The phTERTp-HRP was delivered by intratumoral injection and the IAA by intraperitoneal injection, combined with 2 Gy daily radiation to a total dose of 30 Gy. The tumor volume was recorded. The cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The expression of HRP protein was detected by AP immunohistochemisty.</p><p><b>RESULTS</b>The tumor growth of combined groups was attenuated significantly and the tumor volume of Hep-2R blank group was the largest. The inhibition rate of each group was: A: 54.8%, B: 10.0%, C: 31.9%; AR: 52.7%, BR: 24.8%, CR: 17.0%. In the combined groups, necrosis and apoptosis of tumor cells were observed under the light microscope and the apoptotic index [A (16.6 +/- 1.3)% vs. AR (17.6 +/- 1.3)%] of tumor cells was highest (P < 0.05). The HRP protein expression of BR (33.3 +/- 8.9)% was higher than that of B (21.9 +/- 5.7)%, which was directly up-regulated in the tumors (45.0% vs. 54.8%, P < 0.05) after radiation.</p><p><b>CONCLUSION</b>In the Hep-2- and Hep-2R-transplantation tumors in nude mice, hTERTp can be induced by radiation and enhance the expression of horseradish peroxidase (HRP) gene according to telomerase activity. hTERTp-HRP/IAA system, which has synergistic effects with radiation and inhibits the tumor growth by induction of apoptosis and necrosis, may be a new gene-radiation strategy for the treatment of laryngeal carcinoma.</p>

Effective inhibition of hTERT expression by RNA interference on the radiosensitivity of human laryngeal cancer Hep-2 cell line / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To construct an eukaryotic expression vector of human telomerase reverse transcriptase (hTERT) gene specific shRNA, and investigate the effect of pshRNA-hTERT combined with gamma-irradiation on cell survival and telomerase activity.</p><p><b>METHODS</b>According to the coding sequence of hTERT mRNA, the target of RNAi was designed, and recombinant expression plasmid pshRNA-hTERT was constructed. The vector was transfected into Hep-2 cells. The radiosensitivity of Hep-2 cells was determined by clonogenic assay. Telomeric repeat amplification protocol (TRAP-PCR-ELISA) was used to observe the telomerase activity in each group. Results Recombinant expression vector pshRNA-hTERT was successfully transfected into Hep-2 cells. The hTERT expression inhibition rate reached 60. 8%. pshRNA-hTERT not only inhibited telomerase activity of Hep-2, but also inhibited the raise of telomerase activity induced by gamma-irradiation. Exposure of Hep-2 cells to pshRNA-hTERT for 24 hrs before irradiation resulted in a decrease in mean surviving fraction of Hep-2 cells compared with cells of group with irradiation alone (67. 7% vs 85. 7%, P <0. 05) .</p><p><b>CONCLUSION</b>RNAi showed a significant inhibitory effect to the expression of hTERT. The results indicate that pshRNA-hTERT can effectively inhibit telomerase activity of Hep-2 cells treated or untreated with 2 Gy gamma-irradiation and significantly enhance the radiosensitivity of Hep-2 cells in vitro. The role of radiosensitization of pshRNA-hTERT may be related with the inhibition of telomerase activity.</p>

Protection of Angelica sinensis against radiation-induced pulmonary fibrosis in mice / 中华预防医学杂志

<p><b>OBJECTIVE</b>To evaluate the Angelica Sinensis as a protecting agent affecting the radiation-induced pulmonary fibrosis in an animal model,</p><p><b>METHODS</b>The thoraces of C57BL/6 mice were exposed to either sham irradiation or single fraction of 12 Gy. Four groups were defined: that received neither irradiation nor Angelica Sinensis (N group), that received Angelica Sinensis but no irradiation (A group), that underwent irradiation without Angelica Sinensis (NX group) and that received both Angelica Sinensis and irradiation (AX group). Mice were sacrificed at 1, 24, 72 hours and 1, 2, 4, 8, 16, 24 weeks post-irradiation. The lungs tissue were removed and processed for definitive analysis, including hydroxyproline content, HE and Masson staining, and the TGF-beta1, (Transforming Growth Factor beta1, TGF-beta1) mRNA expressions.</p><p><b>RESULTS</b>Compared with N and A group, there was some differences in the AX group, but a significant histological and pathologic changes in NX group. Non-irradiated groups (N and A group) exhibited low levels of hydroxyproline (0.775 +/- 0.024) microg/mg and (0.751 +/- 0.034) microg/mg, and there was a significantly elevated level of hydroxyproline in NX group (0.875 +/- 0.009) microg/mg (P < 0.05). AX group (0.782 +/- 0.010) microg/mg was in between the non-irradiated groups (N and A group) and the radiation-only group (NX group), and the difference between AX group and NX group was significant (P < 0.01). The results of real-time quantitative RT-PCR showed that the relative mRNA expressions of cytokine TGF-beta1 in NX group(249.655 +/- 16.320) was significantly higher than that in group A (1.254 +/- 0.061) and N (1.324 +/- 0.057) (P < 0.01), and that in AX group (108.076 +/- 9.870) decreased than that of NX group (P < 0.01).</p><p><b>CONCLUSION</b>An animal model of mice with radiation-induced lung injure was established successfully. This study has demonstrated that Angelica sinensis in Hibits the progress of radiation-induced pulmonary fibrosis, Possibly by down-regulating the expression of the proinflammatory cytokine Tgfb1. These data suggest that Angelica sinensis maybe useful in preventing and/or treating radiation-induced pulmonary fibrosis in the clinic.</p>