Background and objectives: The mechanisms of photodynamic therapy (PDT) have been studied on the cellular and tissue levels. However, the cellular behaviors of cancer cells survived from PDT are still not clear. Previously, we have found that PDT-derived variants A375/3A5 and A375/6A5 have reduced invasion ability. This study attempted to further elucidate the possible molecules associated with the altered invasiveness in the PDT-derived variants and cancer cells treated with PDT.
Study design/materials and methods: Scratch wound healing assay and invasion assay were performed to evaluate the migration and invasion ability of human A375 melanoma and MDA-MB-231 breast adenocarcinoma cells. Single colony selection and microarray analysis were performed to examine the differentially expressed transcripts in parental A375 and PDT-derived variants. RT-PCR and Western blots analysis were performed to examine the expression levels of matrix metalloproteinase 9 (MMP9) and chloride intracellular channel 4 (CLIC4). The MMP9 activity was examined by Zymography assay. CLIC4 expressing construct was used to examine the influence on MMP9 expression and invasion ability of cancer cells treated with PDT.
Results: Correlated with the reduced invasiveness, we found that A375/3A5 and A375/6A5 cells have decreased production of MMP9. Microarray analysis and RT-PCR showed CLIC4 was down-regulated in the PDT-derived variants. Furthermore, down-regulation of CLIC4 and MMP9 was found in cancer cells treated with PDT. Transfection of surviving cancer cells with a plasmid vector encoding CLIC4 increased MMP9 expression and cell invasion. Furthermore, overexpression of CLIC4 in A375 and MDA-MB-231 cancer cells constrains PDT-induced suppression of invasiveness.
Conclusion: Our results showed that the reduced expression of CLIC4 could further down-regulate MMP9 and result in the suppression of invasion in cancer cells treated with PDT. These results provide an insight into a new mechanism by which PDT affects the metastatic potential of cancer cells through down-regulation of MMP9 by CLIC4.
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