| Objective To investigated the action of zidovudine, abacavir, and lamivudine in esophageal squamous cancer cell lines, either used as a single therapeutic agent or in combination with radiation, and to elluciadate the mechanism for achieving elevated sensitivity to radiation.Methods After treated respectively with zidovudine (0.02 mM), abacavir (0.03 mM), and lamivudine (0.025 mM) for 48 h, Eca109 and Eca 9706 were used for these assays:1. Clonal efficiency assay:the survival curves were obtained with Graphpad Prism5 to establish the mean lethal dose (DO), quasi-threshold dose (Dq), and survival fractions at 2 Gy (SF2). The radiosensitivity was quantified by the sensitization enhancement ratio (SER).2.Comet assay:DNA damage was measured using alkaline electrophoresis to detect any single-stranded and double-stranded DNA breaks (SSBs and DSBs) according to the kit instructions. To quantify the DNA damage, the tail moment was calculated as the product of the migration of the DNA as well as the relative amount of DNA in the tail.3. Telomerase activity and relative telomere length were detected and evaluated by Real-Time PCR.4. Apoptosis rates were assessed by flow cytometry analysis.Results 1. Zidovudine, abacavir, and lamivudine can radio sensitize ESCC cells:to examine the effect of zidovudine, abacavir, and lamivudine on radio sensitization, clonogenic cell survival assays were performed to Eca109 and Eca9706 cancer cells. The results of the clonal efficiency assay showed that treatment with zidovudine, abacavir, and lamivudine substantially enhanced the radiation response in Eca 109 and Eca9706 cells. We obtained D0, Dq, SF2, and SERD0 by fitting the survival curves into the single-hit multi-target model Y=l-(1-EXP(-k*X))AN. The values of SF2 were 0.809 and 0.850 for Eca109 and Eca9706 cells. After the treatment with zidovudine, abacavir, and lamivudine, SF2 values were 0.682,0.678, and 0.695 in Eca109 and 0.815,0.810, and 0.794 in Eca9706 cells, respectively (P<O.05). The SERD0 data revealed that Eca 109 cells were more sensitive to the action of these three drugs than Eca9706 cancer cells. These results indicated that zidovudine, abacavir, and lamivudine may be used as radio sensitizers to improve the anticancer effect of radiotherapy.2.Increase of postirradiation DNA damage induced by zidovudine, abacavir, and lamivudine:the Comet assay was used to measure DNA damage. The values of TM were calculated by Comet Assay Software Project (CASP) Lab. Higher values of TM were obtained in the drug and radiation groups than in the radiation-only group in both Eca 109 and Eca9706 (P<0.05). However, there was no difference between any two of zidovudine, abacavir, and lamivudine (P>0.05).This experiment demonstrated that zidovudine, abacavir, and lamivudine could increase the DNA damage of irradiated Eca109 and Eca9706 cancer cells.3.Inhibition of TA by zidovudine, abacavir, and lamivudine:TA from cell extracts was evaluated by the linear equation acquired from the curve fit by using the amplification data from different concentrations of TSR8. The copy number was used to denote the TA of the experimental groups. Compared to the PBS control group, TA of Eca109 and Eca9706 was upregulated by radiation but was downregulated by zidovudine, abacavir, and lamivudine. For the drug groups, the ratios of TA inhibition of were 54.8%,54.5%, and 51.9%in Eca109 (P<O.05), and 62.5%,63.1%, and 63.3%in Eca9706 cells (P<O.05). TA of the radiation-only group was increased by 27.3%and 10.1%compared with that of the Eca109 and Eca9706cells in the PBS control group. The inhibition rates in the drug and radiation groups were 55.8%, 54.8%, and 54.3%in Eca109 and 52.0%,54.7%, and 53.3%in Eca9706 cancer cells (P<0.05). It was obvious that TA of Eca109 and Eca9706 cells was suppressed by the action of zidovudine, abacavir, and lamivudine regardless of whether or not radiation was used.Shortening of TL induced by the administration of zidovudine, abacavir, and lamivudine after radiation.The relative telomere length was measured by Real-Time PCR. There was no difference between the PBS group and drug groups, which demonstrated that zidovudine, abacavir, and lamivudine did not inhibit telomere lengthening of Eca109 (P>0.05) and Eca9706 (P>0.05) after the treatment with these drugs for 48 h. When combined with radiation, the TL in either radiation group (P=0.224 in Eca109, P=0.413 in Eca9706) or radiation and drug groups (P<0.05 for the three drugs in Eca109 and Eca9706) was shorter than that in the PBS control group. Moreover, the radiation-only group exhibited higher values of TL than did the radiation and drug groups in the two cell lines, respectively (P<0.05). The results indicated that radiation could decrease TL length, and the application of zidovudine, abacavir, and lamivudine shortened TL additionally.4.Zidovudine, abacavir, and lamivudine radiosensitize ESCC cells through induction of apoptosirthe apoptosis ratios were significantly higher in the combination treatment groups than that in the radiation alone or the separate administration of zidovudine, abacavir, and lamivudine. Radiation induced cell apoptosis in 10.8%of Eca109 and in 9.5%in of Eca9706 cells. The individual treatments with zidovudine, abacavir, and lamivudine in Eca109 cells induced apoptosis rates of approximately 3.8%, 3.4%, and 3.4%, respectively, whereas these values for Eca9706 cells were approximately 2.8%,2.7%, and 3.0%. The combination of radiation and zidovudine, abacavir, and lamivudine caused an increase in the apoptosis rates, and they reached values of 33.5%, 30.2%, and 32.2% in Eca109 cells, and 27.8%,27.2%, and 27.9% in Eca9706 cells, correspondingly, which were significantly higher than those in the radiation-only group (P<0.05). These results indicated that zidovudine, abacavir, and lamivudine sensitize Eca109 and Eca9706 to radiation by stimulating cell apoptosis.Conclusion Our study has evidenced that the combination of zidovudine, abacavir, and lamivudine exhibited pronounced effects on decreasing the colony formation ability and stimulating DNA damage of esophageal squamous cancer cells through telomere length shortening, telomerase activity reduction and promotion of apoptosis. Zidovudine, abacavir, and lamivudine may be valuable when used as radiosensitizers for cancer radiotherapy. |
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