The interrelated role of TLR9 and autophagy signaling in cancer has not yet been clarified. In our previous study incubation of HT29 cancer cells with modified self-DNAs resulted in different effects on TLR9-signaling and cell differentiation. This study was designed to assess the TLR9-related activities of self-DNA sequences on cell survival and autophagy response in HT29 cells.
HT29 cells were incubated for 72 h with intact genomic (g), and arteficially hypermethylated (m), fragmented (f), and hypermethylated/fragmented (m/f) tumoral self-DNAs. Cell viability was measured by MTT assay, while induction of apoptosis by TUNEL. Cell proliferation was estimated by Ki67 immunocytochemistry. Transcriptional changes of TLR9- and autophagy pathways were assayed by qRT-PCR and immunocytochemistry. Morphologic features of apoptosis and autophagy were examined by transmission electron microscopy (TEM). The number of colonosphere-positive wells was also determined.
Following incubation with g-, m/f-, and mainly with m-DNAs viability and proliferation rate of HT29 cells decreased, while percentage of apoptotic cells increased. F-DNA resulted in an enhanced cell survival. Methylation of self-DNA decreased TLR9 expression, but it did not influence the positive effect of DNA fragmentation on MyD88 and TRAF6 overexpression, and TNFα downregulation. Fragmentation of DNA abrogated the effect of methylation on IRAK2, NFκB and IL8 mRNA upregulations. Regarding autophagy g- and f-DNAs caused significant upregulation of Beclin1, Atg16L1, and LC3 mRNAs, while m- and m/f-DNAs resulted in rather modest expressions, verified by immunocytochemistry, as well. According to TEM in each group of tumor cells varying degree of autophagy was observed. Incubation with m-DNA suppressed tumor cell survival by inducing apoptotic death, and activated mitophagy. F-DNA enhanced cell survival, and activated both macroautophagy and lipophagy. CD133+ colonospheres were detected after m-DNA incubation.
Our study provided evidence for a close interplay between TLR9-signaling and autophagy with remarkable influences on survival of HT29 cells subjected to intact or modified self-DNA treatments.
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Legal entity responsible for the study
Hungarian Scientific Research Fund (OTKA-K111743)
All authors have declared no conflicts of interest.