Effects Of Ergosterol Peroxide On Tumor Cells In Different Proliferation Stages

Ergosterol peroxide（EP）is a kind of phytosterols with many important physiological functions,which is found from edible and medicinal fungi.At the same time,it is an oxidation product of ergosterol.The compound has a wide range of pharmacological effects such as promoting tumor cell apoptosis,anti-inflammatory,anti-oxidation,immunosuppression,and anti-atherosclerosis.In the process of tumor progression,tumor dormancy is an important stage,and the formed residual lesions will remain occult and asymptomatic for a long time,that is,in a non-proliferating state.However,tumor cells in the low-proliferation state have not lost the ability to proliferate and divide,which may be activated again,causing tumor recurrence.Therefore,research on anti-cancer drugs should not only focus on killing proliferating tumor cells,but also non-proliferating tumor cells.Based on the anti-tumor effect of ergosterol peroxide,this topic will explore the specific effects of ergosterol peroxide on tumor cells and low-proliferation tumor cells.Objective:To investigate the effects of ergosterol peroxide on proliferating tumor cells and low-proliferating tumor cells.Method:（1）Synthesis of ergosterol peroxide from ergosterol by photo-oxidation method;（2）the cytotoxicity of EP to various tumor cells was detected by MTT assay;（3）Flow cytometry test the effects of EP on human tumor cell cycle phases;（4）Glucose-free condition induced tumor cells to enter a relatively low proliferation state,and flow cytometry was performed to detect the changes in different tumor cell cycle phases after glucose-free induction;（5）Glucose-free condition induced Tumor cells enter a relatively low proliferation state,and Ed U experiment was used to detect the cell proliferation state;（6）MTT assay was used to compare the effect of EP and chemotherapeutic drug cisplatin on proliferation tumor cells and low-proliferation tumor cells;（7）Western Blot method detects the effects of EP on endoplasmic reticulum stress and UPR signal-related proteins on human tumor cells;（8）Establish a nude mice xenograft tumors model to evaluate the effect of EP on tumor cells in vivo.Result:（1）The compound was synthesized with ergosterol as raw material and eosin as catalyst.The product was analyzed by liquid chromatography and carbon spectrum analysis,which was identified as ergosterol peroxide with a purity of 96%.（2）EP has inhibitory effect on a variety of tumor cells,including breast cancer cells MCF-7,colorectal cancer cells HCT116,SW620,lung cancer cells H1299,H460,A549.The half-inhibition concentration of EP on MCF-7 for 24h and 48h were 25.37±0.83μg/m L and11.86±0.70μg/m L.The half-inhibition concentration of EP on HCT116 were17.47±0.16μg/m L and 22.8±0.12μg/m L for 24h and 48h.The half-inhibition concentration of EP on SW620 for 24h and 48h were 47.24±0.90μg/m L and16.06±1.12μg/m L.The half-inhibition concentration of EP on H1299 for 24h and 48h were 33.32±2.48μg/m L and 11.88±0.20μg/m L respectively.The 50%inhibitory concentration of EP on H460 for 24h and 48h were 17.75±0.17μg/m L and 9.199±0.48μg/m L.50%inhibitory concentration of EP on A549 for 24h and 48h were 15.32±1.09μg/m L and 21.76±3.20μg/m L.（3）EP affected the cell cycle phases of MCF-7 and A549.After administration,the proportion of G0/G1 phase cells increased significantly.So EP arrested cell cycles of MCF-7 and A549 tumor cells.（4）After glucose-free induction,the cell cycle progression of MCF-7 and A549 cells were also affected,and the proportion of cells in G2/M phase increased significantly.（5）The proliferation tumor cells of H1299,H460 and A549 cells decreased after glucose-free induction.In H1299 and H460 cells,the number of cells in S phase decreased to 57.2%and 68.9%respectively after 24 hours glucose-free culturing,moreover,after 48 hours glucose-free treatment,the proportion of S phase in A549 decreased to 70.1%.（6）The chemotherapy drug cisplatin had a weaker effect on low-proliferation tumor cells than normal tumor cells,but low-proliferation tumor cells were still sensitive to EP.（7）Under glucose-free conditions,the expression of GRP78 in tumor cells H1299,H460,and A549 were significantly increased and the Ca²⁺levels significantly changed,suggesting that unfold-protein response and endoplasmic reticulum stress may occur in cells induced by glucose-free conditions.Besides,EP can decrease the level of GRP78 in the cells induced by glucose-free culture medium.（8）A model of MCF-7 xenograft tumors was established in the experiment.Compared with the control group nude mice,the nude mice of EP-treated group had smaller tumor volumes.Conclusion:EP has inhibitory effect on a variety of tumor cells.Under the condition of glucose-free induction,the proportion of low-proliferative tumor cells has increased.The chemotherapy drug cisplatin has a weakened effect on the low-proliferating tumor cells,but the EP is different.