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Archive > Volume 24 Issue 12 > 2017,24(12):1356-1361. DOI:10.3872/j.issn.1007-385X.2017.12.003 Prev Next
Prompt Report
Inhibitory effect of apopotin-loaded oncolytic adenovirus ATV on human cervical carcinoma HeLa cells
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Abstract:
Objective: To investigate the effect of apoptin-loaded oncolytic adenovirus ATV infection on the autophagy and apoptosis of human cervical carcinoma HeLa cells. Methods: Apoptin- loaded oncolytic adenovirus ATV and the control virus Ad-MOCK (both were constructed previously) were transfected into HeLa cells. The effect of ATV infection on proliferation of HeLa cells was measured through WST-1 assay; The effect of ATV infection on apoptosis and cell cycle of HeLa cells was detected by flow cytometry; Western blotting was used to detect the effect of ATV infection on the expression of autophagy-related proteins (LC3, P62 and mTOR) in HeLa cells;MDC staining was used to detect the effect of ATV infection on autophagy of HeLa cells. Results: ATV infection inhibited HeLa cell proliferation in a time-dependent manner; and the inhibitory rate reached 59.26% with ATV 100 MOI at 72 h, which was significantly higher than that of the control group (P<0.01); The apoptotic rate of HeLa cells in ATV group was significantly higher than that in control group[ （38.995±4.009）% vs（14.680±1.174）%,P<0.01] 48 h after infection. With the infection of ATV, flow cytometry analysis showed that the cell cycle of HeLa was blocked at S phase, which was most significant at 48 h and significantly higher than that in control group [ （58.490±2.447)% vs (43.235±4.419)%, P<0.05]. Western blotting revealed that after ATV infection, LC3 expression was gradually increase at 6, 12 h, with a highest level at 12 h（0.368±0.010，P<0.01）and a lowest level at 24 h (0.106 ± 0.023，P<0.01),P62 protein showed the highest expression level at 24 h（6.004 ± 1.423，P<0.01）, while mTOR gradually reduced（0.042±0.010，48 h，P<0.01）with the extension of infection time; under fluorescent microscope,monodansylcadaverin (MDC) positive staining of autophagosomes was observed in the peripheral region of the nucleus of HeLa cells; the number of autophagosomes of ATV group was significantly increased as the ATV infection time prolonged; compared with control group, the number of autophagosomes of ATV group significantly increased at 6 h and 12 h [ (28.000±2.828) vs (8.500±2.121), (37.000±4.243) vs (14.000±1.414), P<0.01], but relatively reduced at 24 h [ (12.000±2.828) vs (17.000±1.414), P<0.01]. Conclusion: Apoptin-loaded oncolytic adenovirus ATV can promote the autophagy and apoptosis of human cervical cancer HeLa cells and then specifically kill tumor cells.
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Project Supported:
Project supported by the National Key R&D Program (973 Program) of China（No. 2016YSC1200900），the Major New Drugs Innovation and Development （No. 2014ZX09304314）, the Key Technologies R&D Program of Jilin Province（No. 0150201002YY）, the Industrial Technology Innovation Strategic Alliance Project of Jilin Province（No. 20140309006YY），the Major Scientific and Technological Project of Changchun City（No.16ss11）, and the Technology Development Project of Jilin Province（No. 20150101124JC）
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