The Effect Of Collagen And Its Denatured Product Gelatin On The Response To TNFα In Murine Fibrosarcoma L929 Cells

The components of the extracellular matrix(ECM)are changed during the occurrence and development of tumors and the abnormal ECM dynamies also promote tumor development.Collagen is the most abundant component of the ECM and it degrades into gelatin in physiology condition or during ECM remodeling.Although it has deeply discussed the mechanism of antitumor in vitro,the influence of ECM on tumors is ignored.Tumor necrosis factor a(TNFa)is one of the strongest cytokines for killing tumor cells and has high antitumor research value.Murine fibrosarcoma L929 cells are used to test the efficacy of TNFa and are the classical model for TNFa research.In this study,we established the collagen or gelatin culture system to investigate the effect of collagen or gelatin on the response of L929 cells to TNFα.The collagen or gelatin culture systems accurately reflect the interaction of cells and drug in vivo.Firstly,we investigated the effect of collagen molecules coated culture and collagen gel culture.L929 cells cultured on collagen molecule coated dishes showed fusiform shape and proliferated normally.The cytotoxic effect of TNFa on L929 cell cultured on collagen coated dishes was comparable with that on conventional dishes.L929 cells on collagen gel exhibited cobblestone-like morphology which was much different from the spread fusiform shape when cultured on conventional dishes as well as the cells tending to aggregate.On conventional cell culture dishes,L929 cells treated with TNFa died in a necroptotic manner and autophagy was induced,while on collagen gel culture,L929 cells were resistant to TNFα-induced death and autophagy was inhibited.Cells on collagen gel had higher content of NF-κB in nucleus and the pharmacological inhibitor of NF-κB,Pyrrolidinedithiocarbamate Ammonium(PDTC)reversed the protective effect of collagen gel on TNFα-induced death.To further evidence the effect of NF-κB,we used NF-κB siRNA to silence gene expression.On collagen gel culture,the resistance of L929 cells to TNFα was reversed by NF-κB siRNA treatment.L929 cells had high level of ROS and the ROS scavenger NAC induced cell death on collagen gel culture.These data suggested that collagen gel culture protected L929 cells from TNFa induced-death through activating NF-κB and high level of oxidation was necessary to maintain cell survival on collagen gel.Next,we investigated the effect of gelatin,the denatured form of collagen.We found that L929 cells on gelatin coated culture lost anchorage dependent growth,and cells detached from the culture dishes and form multicellular aggregates.The multicellular aggregates on gelatin coated culture were resistant to TNFα-induced death.On gelatin coated culture,L929 cells had high level of autophagy and inhibition of autophagy promoted cell death.It was different from that on collagen gel culture that NF-κB did not promote cell survival on gelatin coated culture.In conclusion,we investigated the influence of collagen molecules coated culture,collagen gel culture and gelatin coated culture which simulated the microenvironment in vivo on the response of L929 cells to TNFa.Compared to the conventional culture,L929 cells were resistant to TNFα-induced death on collagen gel culture a nd gelatin coated culture,but the protective mechanism was different between collagen gel culture and gelatin coated culture.On collagen gel culture,NF-κB promoted cell survival in the response of L929 cells to TNFa,while it had no protective effect on the cells on gelatin coated culture.On gelatin coated culture,the level of autophagy was increased and inhibition of autophagy could induce cell death.These results are conducive to clarify the influence of collagen and gelatin on tumor cell growth and shed new light of the molecular mechanism of interaction of cells and drugs,and it provides the research foundation for actually stimulate the in vivo microenvironment in future antitumor drug development.