Study On The Combination Of DHA Based On The Mechanism Of Synergistic Induction Of Ferroptosis In Tumor Cells

Tumor is a major disease that endangers people’s health.Reducing the side effects of chemotherapeutic drugs,enhancing the curative effect,overcoming the problems of drug resistance,as well as the combination of multi-target drugs and the application of new anti-tumor mechanisms are still the key issues of tumor therapy.Ferroptosis is a newly discovered regulatory mode of cell death.The core pathological mechanism of ferroptosis is the imbalance between iron-dependent lipid peroxidation pathway and lipid membrane antioxidant system,resulting in excessive accumulation of lipid peroxides,structural damage and loss of lipid membrane function,and then death.The discovery of this death pattern has aroused great concern in the biological community,and opened up a new field for the research and development and treatment of tumor chemotherapeutic drugs.In order to maintain rapid material and energy metabolism,cancer cells have an increased demand for iron compared with normal non-cancer cells.This iron dependence makes cancer cells more vulnerable to iron-catalyzed death,that is,ferroptosis.The content of iron in different tissues of human body plays different roles in the metabolic processes of iron absorption,transport,storage,release and excretion,in which the intestinal tract,liver,blood cells and other tissues have abundant cells and exuberant iron metabolism.and their corresponding tumors are in the forefront of the tumor spectrum harmful to human health,is this kind of tumor more sensitive to ferroptosis inducers?Is the combination of drugs based on ferroptosis mechanism more effective?These are all very important questions that need to be answered urgently.In recent years,a large number of studies have confirmed that artemisinin and its derivatives(ARTs)also has a definite broad-spectrum anti-tumor effect.The anti-tumor effect of ARTs has relatively less toxic and side effects;its mechanism is different from that of traditional chemotherapeutic drugs,and it can achieve good synergism in combination with commonly used chemotherapeutic drugs;it can reverse the multidrug resistance of tumor cells and improve the sensitivity of tumor cells to radiotherapy and chemotherapy;it is effective for some tumors(such as pancreatic cancer)which are lack of effective therapeutic drugs at present.Although artemisinin and its commonly used derivatives have the above anticancer advantages,ARTs have difficulties in preparation because of their special molecular structure and chemical properties.In addition,ARTs has some defects such as low anticancer potency and relatively low selectivity,but these characteristics undoubtedly provide a broad research space for synergism and toxicity reduction of combined drugs.Therefore,ARTs should be a routine drug for clinical tumors in the future,but it may be used as part of a combination therapy.Therefore,on the basis of inducing ferroptosis of tumor cells,DHA was combined with first-line anti-tumor drugs or some precursor compounds to screen the combination of drugs with synergism,and the synergistic mechanism was discussed at the same time.In order to achieve the effect of synergism and toxicity reduction,and provide basic data for clinical application.1.Screening of DHA combined with anticancer drugs in vitroAfter incubation HepG2 cells with DHA,All-trans Retinoic Acid(ATRA),13-cis Retinoic acid(13-CRA),Fenretinide(Fen),Arotinoid Acid(TTNPB),Sorafenib(Sora),Dabrafenib(Dab),Vemurafenib(Vem),PLX-4720(PLX),Gemcitabine HCl(Gem)respectively for 24 h,48 h and 72 h,MTT method was used to analysis the inhibition rate of tumor cells.Found that the IC50 of DHA,ATRA,13-CRA,Fen,Sora,PLX,TTNPB,Dab,Vem and Gem in HepG2 cells for 48 h was 19.46 μM,107.33 μM,104.58μM,6.21 μM,6.21 μM,55.54 μM,44.49 μM,121.43 μM,18.79 μM and 0.82 μM respectively.According to the IC50 value of each drug,the concentration gradient was set at a fixed concentration ratio,and each drug was combined with DHA respectively.After co-incubating with cells for 48 h,the inhibition rate of tumor cells was detected by MTT method.The synergistic effect was analyzed by Calcusyn software,and the CI index was calculated.We found that in HepG2 cells,DHA combined with Sora,ATRA,Fen,PLX showed a synergistic inhibitory effect on the proliferation of HepG2 cells at 24 h,48 h and 72 h,respectively(CI<0.9).2.Different tumor cell lines have different sensitivity to DHA and SoraDHA and Sora can be used as ferroptosis inducers.In order to explore the sensitivity of different tumor cell lines to ferroptosis inducers.,we used the MTT method to detect the inhibitory effects of DHA and Sora in human myeloid leukemia K562 cells,human liver cancer HepG2 cells,human colon cancer SW480 cells,human lung cancer A548 cells,and human glioma U251 cells.Found that K562 cells were most sensitive to DHA and the IC50 of DHA at 24 h was 2.76 μM;the IC50 of DHA at 24 h were 16.16 μM and 24.29 μM in HepG2 cells and SW480 cells respectively;and A549 cells and U251 cells are less sensitive to DHA,the IC50 of DHA at 24 h were 96.7μM and 154.2 μM respectively.The IC50 of Sora at 24 h in K562,HepG2,SW480,A548 and U251 cells was 5.38 μM,12.55 μM,11.79 μM,20.61 μM and 23.1 μM respectively.In the correlation analysis of the death percentage of a group of human cancer cells induced by Sora and DHA,we found that the cell inhibition induced by Sora 10 μM was positively correlated with the cell inhibition induced by DHA 20 μM.Therefore,compared with other parts of the human body,DHA and Sora may have a more obvious inhibitory effect on tumor cells in the small intestine,liver and blood.3.Antitumor effect of DHA combined with Sora on xenograft in vivo1.5 ×106 HepG2 cells were inoculated into the right flanks of the nude BALB/c mice.When the tumor size reached 300 mm3.The mice were randomly divided into 4 groups with 7 mice in each group and administered DHA 40 mg/kg alone,Sora 10 mg/kg alone,the combinations of DHA and Sora and the same volume of PBS every day.The size of tumor nodules was measured with digital caliper once a week,and the weight of nude mice was weighed.After three weeks of administration,the mice were killed,the tumors were dissected and weighed,and the tumors were stained with H&E.After 21 days,no significant change in body weight was found in mice that received treatment compared with control mice at the end of the experiment.The combination treatment resulted in significantly reduced tumor growth compared with either treatment alone in mice injected with HepG2 cells(COM 420.60±175.65 mm3 vs.Sora 707.95±202.17 mm3,P<0.05;COM vs.DHA 902.59±226.84 mm3,P<0.01).The weight of tumors from the mice treated with both drugs(0.57 ±0.19 g)was significantly reduced compared with that noted for untreated mice(1.2±0.20 g,P<0.01),Sora-treated mice(0.81 ± 0.14 g,P<0.05),and DHA-treated mice(1.04 ± 0.22 g,P<0.01).The results of H&E staining showed that about 30%,57%and 47%of the cell death areas were found in the control group,Sora alone group and DHA alone group,respectively,DHA combined with Sora showed a large area of blurred cell morphology and disappearance of nuclear staining,and about 80%of the cells died in the section.4.Effects of DHA alone and combined with Sora on L-ROS,MDA,LIP and GSH in HepG2 cellsAfter incubation drugs with cells for 24 h,using different fluorescent probes Calcein AM and BODIPY 581/591 C11 detect LIP and ROS in HepG2 cells respectively;and intracellular MDA level was detected by lipid peroxidation(MDA)detection kit and intracellular GSH level was detected by GSH/GSSG ratio detection assay kit.Compared with the control group we found DHA 20 μM and Sora 12 μM alone could significantly increase the level of intracellular ROS(P<0.05),increase level of intracellular MDA(P<0.05)and LIP(P<0.01),and significantly reduce the level of reduced GSH(P<0.05).Compared with DHA 10 μM alone,DHA 10 μM combined with Sora 12 μM significantly increased the levels of intracellular ROS,MDA and LIP(P<0.01),and significantly decreased the level of reduced GSH(P<0.01).The results showed that both DHA and Sora could increase the levels of ROS,MDA and LIP,decrease the level of reduced GSH,destroy the iron homeostasis and redox balance,and accelerate the production of lipid peroxidation in HepG2 cells.Therefore,DHA and Sora may have a synergistic effect through the same mechanism of inducing ferroptosis in cells.5.Effects of DHA alone and combined with Sora on ferroptosis related proteins in HepG2 cellsAfter the drug and HepG2 cells were incubated for 24 hours,the cells were lysed.After the total protein was extracted,the BCA protein Assay Reagent kit was used for quantification.Western Blot was used to detect the relative level of GPX4,HO-1/HMOX1,IREB2,GCLC and SLC7A11/xCT.We found that compared with the control group,DHA could significantly reduce the levels of intracellular xCT,GPX4,GCLC and HO-1 under the condition of 10 μM(P<0.01);Sora could significantly reduce the levels of intracellular xCT,GPX4,GCLC,IREB2 and HO-1 under the condition of 6 μM(P<0.01).Compared with the group treated with DHA 10 μM alone,the levels of intracellular xCT,GPX4,GCLC,IREB2 and HO-1 decreased significantly after the combination of DHA 10 μM and Sora 6 μM.The results showed that DHA and Sora could induce ferroptosis by interfering with the balance of iron-dependent lipid peroxidation and cysteine-GPX4 anti-lipid peroxidation in HepG2 cells.6.Effects of DHA alone and combined with Sora on aerobic glycolysis and oxidative phosphorylation in HepG2 cellsAfter the drugs were incubated with the cells for 8 h,the glycolysis rate of HepG2 cells was detected by Seahorse XF Glycolytic Rate Assay Kit,and the oxidative phosphorylation ability of Seahorse XF cells was detected by Seahorse XF Cell Mito Stress Test Kit.We found that compared with the control group,DHA at the dose of 10μM,20 μM and 30 μM significantly decreased the glycolysis rate of HepG2 cells in a dose-dependent manner(P<0.05),and the glycolysis rate of DHA 10 μM combined with Sora 12 μM group was significantly lower than that of DHA 10 μM group(P<0.01).Compared with the control group,DHA at the doses of 8 μM,12 μM and 15 μM significantly decreased the maximum and spare respiratory capacity of cells(P<0.01,P<0.01),decreased the ATP production ability of aerobic respiration(P<0.01),increased proton leak(P<0.01);Compared with the group treated with DHA 10 μM alone,the combination of DHA 10 μM and Sora 5 μM significantly decreased the maximum respiratory capacity(P<0.01),spare respiratory capacity(P<0.01)and ATP production ability of HepG2 cells(P<0.01),and accelerated the damage of mitochondria causing proton leakage(P<0.01)and further decreased the ability of oxidative phosphorylation of mitochondria.The results showed that DHA could inhibit the aerobic glycolysis and oxidative phosphorylation of HepG2 cells,and the combination of DHA and Sora could synergistically inhibit energy metabolism.