| Objective: Breast cancer is currently the most common cancer that threatens women's health.In the United States,one in eight women will have breast cancer in their lifetime.At world level,annual new cases can reach 1.4 million,and Chinese patients accounts for 280,000.Fortunately,clinical treatment level have improved in recent years,breast cancer survival rates can reach 73%.Breast cancer is still the leading cause of death in women,with about 520,000 deaths each year,accounting for 20-30% of patients who died from cancer.Triple-negative breast cancers usually have high histological grade and with transfer rates.TNBC is more aggressive than hormone receptor-positive tumors,and has high recurrent and metastatic rate.Because of the lack of hormone receptor expression and its aggressive phenotype,chemotherapy has become the primary treatment for TNBC.TNBC is highly sensitivity to chemotherapy,but have short remission stage.It is not ideal with chemotherapy.Due to the unsatisfactory effect of chemotherapy,there is an urgent need to develop new therapies to control triple negative breast cancer cell proliferation.FEN1(Flap endonuclease-1),a member of the RAD2 structure-specific nuclease family,has multiple enzymatic activities that play an important role in DNA synthesis,apoptosis and DNA fragmentation effect.Several studies have reported that the DNA repair system have been strengthened after receiving chemotherapy or targeted therapy,which cause cancer cells to be resistant to drugs.FEN1 can be raised afer the use of DNA damage agents,which plays a key role in DNA repair system.Our previous findings showed that inhibition of FEN1 expression increases the sensitivity of breast cancer cells to Herceptin.Other studies have shown that decreasing the expression of FEN1 making lung cancer and gastric cancer cells more sensitive to cisplatin.In addition,we found that FEN1 protein was highly expressed in the tumor tissue of patients with TNBC,and that the high expression of FEN1 protein was associated with shorter DMSF and OS.Therefore,it is of great significance to study whether FEN1 can be a new target for TNBC and to explore new therapeutic methods for the treatment of TNBC.Oxidative stress is the imbalance between the production of reactive oxygen radicals(ROS)and the antioxidant defense systems.The imbalance of redox state is the common characteristic of tumor cells including breast cancer,however,high level of ROS can lead to cell death.The existing research shows that a variety of chemotherapeutic drugs and targeted treatment agents can increase the ROS in tumor cells.ROS overload cause oxidative stress,which results in oxidative damage of proteins,lipids and DNA.Repair of oxidative DNA damage caused by ROS needs to recruite base excision repair pathway related proteins to DNA damage site.As a key protein of BER passway,FEN1 has been proved to be necessary for repairing oxidative damage of DNA.And previous studies have reported that inhibition of FEN1 phosphorylation may reduce the adaptability of myocardial cells to oxygen in nude mice,but it is not clear whether FEN1 is involved in the oxidative stress of TNBC.In order to clear whether FEN1 is involved in regulation of ROS in TNBC cells,we choose ATO as positive control,which has been proved can induce ROS in other cancer cells and has therapeutic potentialities for TNBC.The first application of ATO in cancer treatment was in the 1970 s,for acute promyelocytic leukemia(APL).ATO can induce APL cell differentiation and apoptosis.In recent years,a number of studies have shown that ATO has potential antitumor activity in a variety of solid tumors,including hepatocellular carcinoma,lung cancer and prostate kidney neoplasm.ATO can induce the increase of intracellular ROS,apoptosis and DNA damage.ATO have already apply to the treatment for liver cancer.The combined alllication of ATO and hepatic arterial embolization can inhibit the tumor more effectively.But its clinical application has been limited due to its side effects including hepatotoxicity,neurotoxicity,nephrotoxicity,cardiotoxicity and dermatosis.The key to solving the problem is to reduce the dosage of ATO while maintaining anti-tumor efficacy.?-elemene,can inhibit the proliferation of malignant tumors,including lung cancer,liver cancer,esophageal cancer,nasopharyngeal cancer and so on,is Curcuma extract.?-elemene can make cancer cells more sensitive to a variety of chemotherapy drugs and reduce side effects of radiotherapy and chemotherapy,which probably has therapeutic potentialites.Recent studies on ?-elemene show that it can induce more ROS in osteosarcoma and cervical cancer cells.We studied the role of FEN1 in oxidative injury of TNBC induced by ?-elemene.In order to study whether FEN1 was involved in the regulation of ROS in TNBC cells,we choose ATO and ?-elemene as positive inducer,which could induce the increase of ROS in tumor cells.To explore new targets and new methods for the treatment of TNBC,in this paper,the effects of FEN1 on oxidative stress induced by ATO and ?-elemene,and the inhibitory effect of ATO and ?-elemene on the proliferation inhibition of TNBC were studied in combination with the inhibition of FEN1 expression.The experimental dose is much lower than the IC50 of ATO and ?-elemene on the cells,in order to reduce drug-related side effects of ATO and ?-elemene while ensuring the anti-tumor efficacy of the two drugs.To provide theoretical and experimental evidence for the application of FEN1 inhibitor combined with ATO or ?-elemene in TNBC.Methods: 1.The cell viability was measured by MTT assay,and the drug IC50 was calculated.2.Using Invitrogen ? H2 DCFDA molecular probes to detect intracellular ROS.3.Apoptosis cells were detected by e BioscienceTM Annexin V-FITC apoptosis kit.4.Utilize the ACTIVE MOTIFTM kit to extract cellular nucleoprotein.5.Western Blotting was used to detect the expression of BAX,BCL-2,PARP,?H2AX,FEN1,p-P38,P38,p-JNK,JNK,NRF2,LAMIN A/p-ERK,ERK,ACTIN.6.Immunofluorescence was used to detect the expression of ?H2AX protein.7.Lipofectamine 2000 reagent used for si RNA transiently transfection of FEN1.8.Animal experiments to study the inhibition of decreasing FEN1 expression combined with ATO and ?-elemene to the mouse tumor growing.9.Use Invitrogen ? CMFDA reagents to detect intracellular GSH levels.10.The related action target of ?-elemene was extracted by BATMAN-TCM database.Protein-protein interaction(PPI)analysis was performed using String 10.5 database.Use the web Gestalt database for passway analysis.11.Each experiment result repeats 3 times.All experimental data were shown as mean±standard deviation.SPSS 17.0 statistical analysis software was used for statistical analysis.The statistically significant was considered when P value<0.05.Results: 1.ATO inhibits triple negative breast cancer cell growth in a time and dose dependent manner.The cell viability was detected by MTT assay in two TNBC cell lines,MDA-MB-231 and MDA-MB-468,following ATO treatment different concentrations for 24 and 48 h.The results showed that the cell survival decreased with the increase of dosage and duration of drug action,from that we can know ATO suppressed TNBC cells growth in a time-and dose-dependent manner.2.The increasing ROS induced by low concentration ATO can lead to cell apoptosis and DNA damage.The ROS generation was detected by flow cytometry after given increasing concentration of ATO after following treatment for 48 h.ROS production was significantly inhibited after pretreatment with ROS inhibitor NAC.Under the same treatment conditions,the cell apoptosis also increased with the increased of ATO,which could also be inhibited by NAC.The results show that ATO induced TNBC apoptosis caused by increased levels of intracellular ROS.The expression of BAX and PARP-cleaved increased and Bcl-2 decreased with the increasing concentration of ATO in MDA-MB-231 cell.There is evidence that ROS can induce oxidative DNA damage.The expression of ?H2AX increased with the extended exposure time.3.Expression of FEN1 is upregulated in ATO treated TNBC cells,inhibiting FEN1 can make the low concentration of arsenic trioxide have stronger antitumor effect in cell and tumor xenograft models.Giving arsenic trioxide to MDA-MB-231 And MDA-MB-468 cell line,FEN1 expression gradually increased with the increase of drug action time.The percentage of cell survival was detected by MTT assay after MDA-MB-231 and MDA-MB-468 cells were silent FEN1 and then with different concentrations of arsenic trioxide.The results showed that silent FEN1 combined with ATO could effectively inhibit the proliferation of TNBC cells.In vivo experiments also proved that inhibition of FEN1 expression can make ATO have better inhibit tumor effect.After injection MDA-MB-231 cells into the mammary fat pads of mice,the mice were randomly divided into 4 groups,and each group of mice was intraperitoneally injected with different drugs.The results showed that FEN1 inhibitor C20 and ATO both had effect on inhibiting tumor growth.And the effect of arsenic trioxide on tumor growth was much better than C20.The combination of C20 and ATO had the highest tumor inhibition rate.In addition,the tumor weight was measured,the control group highest,followed by the C20 group,the ATO group and the C20 + ATO group,respectively.In short,inhibition of FEN1 expression combined with ATO may result in the effective inhibition of tumor-bearing balb/c mice.4.Inhibition of FEN1 expression with low concentration of ATO can induce more ROS and apoptosis in TNBC cells.After transfected with FEN1 si RNA and NC si RNA,MDA-MB-231 and MDA-MB-468 cells were treated with increasing concentrations of arsenic trioxide.After treatment with the same concentration of ATO,the FEN1-si RNA group produced more ROS than the NC-si RNA group,and that could be reversed by NAC.And under the same processing conditions and groupings,the rate of apoptosis was significantly higher in FEN1-si RNA group,and the change of apoptosis was in accordance with ROS change,which could be reversed by NAC.5.FEN1 silencing induces apoptosis by increasing ROS generation to activate P38/JNK following arsenic trioxide treatment.The cells were divided into 6 groups.Western Blot was performed to detect BAX,BCL-2,PARP-Cleaved and ?H2AX protein.In FEN1-si RNA + ATO group,the ratio of BAX / BCL-2,PARP-Cleaved and the expression of ?H2AX were further increased than those in ATO group,NAC reversible.Previous studies have shown that ATO induces apoptosis through activation of P38 and JNK.ATO can make P38 and JNK phosphorylation increased,but silent FEN1 can't.Giving arsenic trioxide to the knocking-down FEN1 cells,P38 and JNK phosphorylation levels further increased,and can also be reversed by NAC.Treatment cells with 5?M of SB203580 and 10?M SP600125 respectively,the ratio of BAX / BCL-2 and PARP-Cleaved decreased and the cell apoptosis was inhibited.The above results indicate that inhibition of FEN1 expression can further increase the production of ROS following arsenic trioxide treatment,which could elevate phosphorylation levels of P38 and JNK lead to increased apoptosis.6.Reducing the expression of FEN1 in TNBC can enhance the inhibition of GSH and Nrf2 nuclear translocation by arsenic trioxide.The MDA-MB-231 and MDA-MB-468 cells were transfected with FEN1 si RNA or control si RNA respectively.The cells were treated with increasing concentrations of arsenic trioxide with or without NAC.Flow cytometry was used to detect the intracellular GSH level.The experimental results showed that the intracellular GSH gradually decreased with the increase of the concentration of ATO in NC-si RNA and FEN1-si RNA group.With the same ATO concentration,FEN1-si RNA group had lower GSH,which could be reversed by NAC.The expression of Nrf2 protein in MDA-MB-231 cells was increased after silencing FEN1 or giving ATO.And the expression of Nrf2 was further increased by silencing FEN1 banded with ATO.The cell core protein was extracted after different treatments.Nrf2 nuclear translocation in FEN1-si RNA + ATO group was significantly reduced compared with ATO group and FEN1-si RNA group.Nrf2 nuclear translocation did not reverse by NAC.7.?-elemene suppressed triple-negative breast cancer cells growth in a time-and dose-dependent manner.MTT method was used to test the cell survival rate after exposing to ?-elemene for 24 or 48 hours.The results showed that the cell survival rate decreased with the increase of the dosage or the duration of the action time.Thus,we can know that ?-elemene inhibits the growth of TNBC in a time-and dose-dependent manner.8.In TNBC,?-elemene induces cell apoptosis,accompanied by MET and MAPK / ERK signaling inhibition and cell DNA damage.The apoptotic cells were measured by flow cytometry after giving ?-elemene with different concentrations.The degree of apoptosis increased along with rising concentrations of ?-elemene,the same result has been achieved by Western Blot.The phosphorylation of MET and ERK was gradually inhibited,by giving increasing concentration of ?-elemene or extending the exposure time of ?-elemene in MDA-MB-231 cells.The Western Blot results showed that the ?-H2 AX level increased with the increase of the dosage or the duration of the action time.The result indicated that ?-elemene could induce DNA damage in TNBC cells.9.?-elemene can increase the expression of FEN1 in TNBC,inhibit FEN1 can make ?-elemene have stronger antitumor effect in cell and tumor xenograft models.FEN1 expression increased by giving increasing concentrations of ?-elemene in MDA-MB-231 cells.We used FEN1-knockdown MDA-MB-231 and MDA-MB-468 cells following ?-elemene treatment.MTT assay was used to detect the percentage of cell survival,and the results showed that silence FEN1 increased the sensitivity of TNBC to ?-elemene.The results indicated that inhibition of FEN1 expression combined with ?-elemene could effectively inhibit the proliferation of TNBC cells.In vivo experiments also proved that inhibition of FEN1 expression can make ?-elemene have better inhibit tumor effect.After injection MDA-MB-231 cells into the mammary fat pads of mice,the mice were randomly divided into 4 groups,and each group of mice was intraperitoneally injected with different drugs.The results showed that FEN1 inhibitor C20 and ?-elemene both had effect on inhibiting tumor growth.And the effect of ?-elemene on tumor growth was much better than C20.The combination of C20 and ?-elemene had the highest tumor inhibition rate.In addition,the tumor weight was measured,the control group highest, followed by the C20 group,the ?-elemene group and the C20 + ?-elemene group,respectively.In short,inhibition of FEN1 expression combined with ?-elemene may result in the effective inhibition of tumor-bearing balb/c mice.10.Silencing FEN1 can further increase ?-elemene-induced ROS and lead cell death.By giving the increasing concentration of ?-elemene in MDA-MB-231 and MDA-MB-468 cells,the generation of ROS gradually increased.ROS production was significantly inhibited by NAC.ROS production is further increased in si-FEN1 group and the ROS could be reversed by NAC.Under the same treatment conditions,flow cytometry was used to detected cell apoptosis.With the same concentration of ?-elemene,the apoptosis cells of FEN1-si RNA group was significantly higher than that of NC-si RNA group,and that could be reversed by NAC.The results was consistent with the change of ROS.11.Silent FEN1 further increased inhibition of survival pathways induced and DNA damage by ?-elemene.The cells were divided into 6 groups.Western Blot was performed to detect BAX,BCL-2,Caspase 3-Cleaved and ?H2AX protein.In FEN1-si RNA + ?-elemene group,the ratio of BAX / BCL-2,Caspase 3-Cleaved and the expression of ?H2AX were further increased than those in ?-elemene group,NAC reversible.The expression of p-ERK and p-MET was reduced to a certain extent in both ?-elemene and FEN1-si RNA group,and further decreased in FEN1-si RNA+ ?-elemene group.But it couldn't reverse by NAC.12.Bioinformatics analysis showed that ?-elemene could affect the antioxidant ability of tumor cells through GSH metabolic pathway.30 target sites associated with ?-elemene were obtained using the BATMAN-TCM database,and the 30 target genes were analyzed using String 10.5 database for protein-protein interaction(PPI),and 406 proteins interacting with target genes were obtained.Then we use the web Gestalt database to make pathway analysis for the intersection target of ?-elemene interpenetrating protein network.The results show that ?-elemene can affect some antioxidant molecules in cells and can target GSH metabolic pathway.Conclusion: 1.ATO inhibits triple negative breast cancer cell growth in a time and dose dependent manner.2.In TNBC cells,arsenic trioxide can induce cell apoptosis and DNA damage.3.In TNBC cells,ATO mediated apoptosis by inducing oxidative stress.4.In TNBC cells,arsenic trioxide can induce FEN1 expression up-regulation,and silencing FEN1 can enhance the anti-tumor effect of low concentration of arsenic trioxide in vivo and in vitro assays.5.Silencing FEN1 can further increase the ROS induced by arsenic trioxide,which could induce increasing the P38 and JNK phosphorylative level and DNA damage.6.Silencing FEN1 can cooperate with arsenic trioxide to reduce intracellular GSH and Nrf2 nuclear translocation.7.?-elemene inhibits triple negative breast cancer cell growth in a time and dose dependent manner.8.In TNBC,?-elemene induces cell apoptosis,accompanied by MET and MAPK / ERK signaling inhibition and cell DNA damage.9.In TNBC,?-elemene mediated apoptosis by inducing oxidative stress.10.Silencing FEN1 can further increase ROS induced by ?-elemene,resulting in the increase of apoptosis and necrosis of TNBC cells.11.Silent FEN1 further increased inhibition of survival pathways induced and DNA damage by ?-elemene.12.Bioinformatics analysis showed that ?-elemene could affect the antioxidant ability of tumor cells through GSH metabolic pathway. |
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