| Objective1 To study the effects of ALA-PDT on the growth inhibition in cervical cancer cell lines, so as to search the most sensitive cervical cancer cell lines and the optimal parameters of PDT.2 To investigate the growth inhibition mechanism of ALA-PDT effecting on cervical cancer cell lines, and to build the theoretical foundation of biological mechanism for that study.3 To analyze the in vivo effects of ALA-PDT on human cervical cancer xenograft implanted in nude mice, to compare the effect of various routes of administration, so as to identify the best administration with lest phototoxic reaction.4 Combining the experiments in vivo and in vitro, to analyze the contribution of ALA-PDT on inducing apoptosis of cervical cancer cell lines.Methods1 Detecting the effects of ALA-PDT on the proliferation of cervical cancer cell lines by MTT colorometric assay, and to select the optimal parameters.2 Indentifying the growth inhibition mechanism of cervical cancer cell lines by observing cellular shape, May-Grunwald Giemsa staining, cell apoptosis and DNA content analysis using flow cytometry and Hoechst 33342, protein and mRNA detected by Western blot and relative quantification in real-time RT-PCR respectively. 3 Producing the animal model of human cervical cancer by implanting the most ALA-PDT sensitive human cervical cancer cells Me180 into the dorsum of nude mice. Comparing the effects of topical administration and caudal vein administration by tumor inhibition experiment, HE staining, immunohistochemical staining.4 Detecting the effects of ALA-PDT on the expression of xenograft mRNA by relative quantification in real-time RT-PCR, and combining the results of experiments in vitro to identify the growth inhibition mechanism and major contribution to apoptosis.Results1 The results of MTT demonstrate that, ALA-PDT does have the effects of growth inhibition on all cervical cancer lines in vitro, in which Mel 80 is the most sensitive cell line among the eight lines, the phototoxic IC50 concentration in Me180 is 6-7×10~4 M. There is no obvious phototoxic reaction under 10 mM and 30 J/cm~2. The optimal results come at 4 hours after ALA-PDT.2 The results of dual Annexin V-FITC/Propidium Iodide assay by flow cytometry demonstrate that, Me180 cell death is significantly induced post ALA-PDT, in which the pattern of early apoptosis shows in 3 hours post-PDT, and the total rate of apoptosis declines in 24 hours post-PDT, which could be considered as different phase issue. The total rate of apoptosis for 1 mM and 2 mM are 52.45% and 77.92% respectively, 3 hours post-PDT.3 Cell apoptosis is all observed by monitoring cellular shape, May-Grunwald Giemsa staining, cell apoptosis and DNA content analysis using flow cytometry and Hoechst 33342.4 The results of cell cycle analysis demonstrate that, no significant cell cycle effects were evident at 4 hours post-PDT, and the distribution of cell cycle significantly changed at 24 hours post-PDT; an appreciable accumulation of cells residing in G0/G1 phase was induced at the 1 nM～2 nM, the reverse results shows in S and G2/M phase. 5 The results of relative quantification in real-time RT-PCR demonstrate that, the experimental cell group's mRNA expression of Survivin and Bcl-2 was down-regulated versus the control cell group (P<0.001) , on the other end, the experimental cell group's mRNA expression of P53, Bax, and Bad was up-regulated (P>0.05).6 The results of Western blot demonstrate that, ALA-PDT can significantly inhibit the Survivin protein expression of Me180 cell line, the inhibition ratio of 0.1 mM, 1 mM and 2 mM are 48.42%, 71.22% and 73.84% respectively.7 The results of tumor inhibition experiment in vivo demonstrate that, the tumor volume was significantly reduced post-PDT by topical and caudal vein administration (P<0.001), in which the most significant effect shows at 7-14 days post-PDT.8 The results of Hematoxylin and Eosin staining demonstrate that, there is no appreciable change in the control group, pure vein administration group, pure topical administration group and pure laser group, however, local region necrosis show up in vein administration PDT group and topical administration PDT group.9 The results of immunohistochemical staining demonstrate that, the protein expression of Survivin and VEGF post-PDT was down-regulated significantly versus control group.10 The results of relative quantification in real-time RT-PCR demonstrate that, the experimental xenograft group's mRNA expression of Survivin and Bcl-2 was down-regulated versus the control xenograft group (P<0.001) , on the other end, the experimental xenograft group's mRNA expression of P53, Bax, and Bad was up-regulated (P>0.05). This result is coincide with that of experiment in vitro.Conclusions1 ALA-PDT can inhibit the growth of cervical cancer cell line in vitro, and the effect pattern is dose-dependent within certain concentration range. Me180 is the most sensitive cell line among the eight kind of cervical cancer cell lines.2 Early induction of apoptosis of Me180 is one of the most important mechanisms of growth inhibition post-PDT.3 The mitochondrion-dependent pathway makes important contribution in mechanism of Me180 cell line apoptosis.4 Combining experiments in vivo and in vitro, ALA-PDT is effective for treatment of cervical cancer.5 Topical administration PDT is recommended in treating cervical cancer so as to minimize th side-effects and inconvenience of phototoxic reaction brought by PDT.
|
PDF Full Text Request