| Objective Colorectal cancer is one of the commonest malignant tumors in gastrointestinal tract and the third leading cause of cancer death in Western communities. But only a small proportion of these patients may be treated by surgical resection or radiochemical therapy. PDT is a promising novel treatment modality for cancer. The second-generation photosensitizer 5-aminolevulinate acid could possibly establish a more selective accumulation in tumor tissue and, when absorbing at longer wavelength, could result in larger volumes of necrosis with less phototoxicity and side effects. We studied ALA-PDT in an human colon carcinoma SW480 in vitro and vivo, assessed short-term and long-term effects of ALA-PDT treatment in a nude mice tumor xenograft. Although the primary mechanism of tumor regression by PDT is still not completely understood, it is known to induce tumor cell cycle arrest and apoptosis. G1/S checkpoint is the key restriction point of cell cycle which is disrupting in tumor cell. In this study, we provided evidence for the involvement of G1/S cell cycle regulation factors during PDT-mediated cell cycle arrest.Methods 1. SW480 cell was treated with 1mM ALA and 6 h later was irradiated with 9 J/cm2 631-nm light from a laser. After ALA-PDT, the inhibition effects were observed by typan blue exclusion test; the inhibiting effect was detected by observing morphologic changes and drawing growth curve; 2. Nude mice bearing xenografts were treated with 250mg/kg ALA and 3 h later were irradiated with 9 J/cm2 631-nm light from a laser. After ALA-PDT, the tumor inhibition effect was assessed by measuring tumor volumes over a 3-4 weeks period, the morphologic changes were observed by macroscopy and microscopy by histological examination; 3. The analysis of cell cycle arrest by ALA-PDT was performed by flow cytometer; 4. The effect of ALA-PDT on G1/S cell cycle regulators Chk2,cyclinD1,p21WAF1/Cip1/Sdi1 was examined by using immunocytochemistry technique and in situ hybridization technique.Results 1. Cell viability dropped down in post-PDT(ALA 1mM,9J/cm2) earlierperiod,but in late time after ALA-PDT,SW480 cell growth resumed slowly,treatment with ALA alone or light alone did not exert any detectable effect on tumor cell. 2. Compared with control groups, ALA-PDT-treated tumors had regressed significantly in earlier period with the inhibiting rate 64.1% ; in the later period post-PDT, tumors growth resumed with a slower rate. Treatment with ALA alone or light alone did not exert any significant effect on tumor cell. 3. Compared with control groups, ALA-PDT prolonged the survival time of the treated goup with 34.0±6.0 days. 4. ALA-PDT resulted in G0-G1 phase arrest of the tumor cell cycle in post-PDT and a time-dependent fashion. 5. Immunocytochemistry and in situ hybridization analysis revealed that PDT resulted in an induction of G1/S checkpoint regulation factors such as Chk2 and p21WAF1/Cip1/Sdi1,and a down-regulation of cyclinD1 in a time-dependent fashion.Conclusions 1. ALA-PDT results in inhibition of SW480 colon carcinoma cell growth in post-PDT earlier period in vitro and in vivo, and can prolong the survival period of nude mice bearing xenografts significantly, in the later period, tumor growth resumed with a slower rate, compared with control groups. 2. ALA-PDT causes G0-G1 phase arrest of SW480 cell cycle in post-PDT earlier period; PDT-mediated induction of G1/S checkpiont regulators Chk2 and p21WAF1/Cip1/Sdi1 results in the imposition of artificial checkpoint at G1-S transition thereby leading to an arrest of cells in G0-G1 phase of the cell cycle through inhibition in cyclinD1 in a time-dependent fashion; 3. With the presence of mutant p53 in SW480 cells, PDT results in the induction of p21WAF1/Cip1/Sdi1 and arrest of cell cycle in G0-G1 phase. The arrest of SW480 cell in G0-G1 phase by ALA-PDT may be mediated by activated G1-S checkpoint pathway independent of mutated p53...
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