Anticancer Activity And Mechanism Of Libertellenone H,a Marine Natural Diterpenoid,Against Human Pancreatic Cancer Cells

Unique structures with extensive biological activities are important features of marine natural products,which makes them an important source for novel drug discovery and development.After nearly half a century of development,active marine natural products have provided a large number of parent structures and drug precursors for the research and development of antitumor drugs.Deeply investigation of the antitumor activity and molecular mechanisms of marine natural products is an important foundation and prerequisite for our development of innovative marine drugs.Libertellenone H(LH),a novel pimarane diterpenoid isolated from Arctic fungus Eutypella sp.D-1,has showed significant antitumor activity on several tumor cells with proliferation inhibitory effects in the preliminary activity experiments.In this study,we investigate the antitumor potentials,molecular mechanism and targets of LH on pancreatic cancer cells,provide important information for further development of LH.In the first chapter,the Arctic fungus Eutypella sp.D-1 was large-scale fermented with optimized fermentation conditions,the ferment extracts were used for LH isolation and purification,finally a total of 153 mg of LH compound monomer were obtained for the in vitro and in vivo activity experiments.Subsequently,a series of cell biological function assays were performed,which shows LH exhibits significant inhibitory activity against four human pancreatic cancer cell lines,and inhibits cancer cells clone forming and invasion,blocks the cell cycle and induces apoptosis in pancreatic cancer cell lines PANC-1 and SW1990.To elucidate the molecular mechanism of anti-tumor activity of LH,human gene expression profiling microarray was applied to screen the differential expressed genes,57significantly up-regulated genes and 90 significantly down-regulated genes were obtained.IPA analysis of the differential expression genes suggested that LH may regulate cholesterol biosynthesis and NRF2-mediated oxidative stress,plays a part in disease and function,such as morbidity or mortality,development of connective tissue and concentration of lipid.In the second chapter,we focused on the molecular mechanisms of the antitumor effects of LH.The microarray results suggesting that LH can activate oxidative stress in tumor cells,so we confirm it by LH inducing a rapid increase in ROS levels and maintaining them at high levels for a long time,accompanied by a decrease in antioxidant reducing GSH levels.Pretreatment of tumor cells with the ROS scavenger NAC and the antioxidant enzyme SOD,not only reverse the LH-induced ROS accumulation in tumor cells,but also antagonize the LH-induced tumor cell apoptosis and proliferation inhibition,suggesting that excessive reactive oxygen species accumulation underlies the anti-tumor effect of LH.To investigate how LH breaks redox imbalance in tumor cells,we examined the inhibitory effect of LH on antioxidant enzyme system,Trx system.LH showed significant inhibitory effect on Trx system in both in vitro enzymatic assay and cellular level,but have weak effect on enzyme activities of Grx and GR,which also contain sulfhydryl groups,indicating that LH can selectively inhibit Trx/Trx R.Meanwhile,the ASK1/JNK signaling cascade pathway,downstream of Trx in tumor cells and associated with apoptosis,could be activated by LH,indicating that ASK1/JNK signaling pathway was involved in LH-induced apoptosis.To investigate the role of Trx system in LH anti-tumor activity,we used si RNA to reduce the expression of Trx R in SW1990 cells,resulting in a corresponding reduction of LH-induced growth inhibition,indicating that Trx R was involved in LH-induced growth inhibition of tumor cell.To investigate the inhibitory effect of LH on the Trx system,we verified the binding modes of LH with Trx1 and Trx R proteins by molecular docking,LC-MS/MS,the results shows that the?,?unsaturated carbonyl groups in LH could interact with free sulfhydryl groups of Cys32/Cys35 residues in the active site of Trx1 and free selenol of Sec498 residues in the C-terminal redox active site of Trx R by the Michael addition reaction The BIAM experiments also confirmed the binding of LH with Sec498 in Trx R active site.In the third chapter,the potential of LH in resisting resistance tumor was mainly explored.We cultured single SW1990 tumor cell in 3D soft fibrin gels,which could grow into individual multicellular round colonies SW1990-TRCs.The expression of relevant stem marker genes are detected by q PCR experiments,the results show that compared with normal cultured SW1990 cells,SW1990-TRCs had higher expression of pancreatic cancer markers CD133,CD44 Tspan8 and the self-renewal gene Sox2,indicating that SW1990-TRCs obtained from 3D soft fibrin gel have some characteristics of embryonic stem cell,and can be used to explore the role of LH against drug-resistant tumor cells in vitro.LH showed significant growth inhibition in SW1990-TRCs clones:the IC₅₀ of day 0administration was 8.32?M,and 20?M LH treatment could stop the growth of SW1990-TRC clones.In addition,the growth inhibition of SW1990-TRCs clones by LH was also persistent,because the growth inhibition of tumor clones remained after LH eluting.The DAPI/PI staining results shows that LH could significantly promote apoptosis in SW1990-TRCs cells,but the specific molecular mechanism of inhibition in SW1990-TRCs still needs to be further deeply investigated.