Sanguinarine Induce The Apoptosis Of Lung Adenocarcinoma Cells Via The Oxidative Stress And Endoplasmic Reticulum Stress

Significant progress has been made in the lung cancer therapy currently,however, it is still one of the malignant tumors with the highest incidence,especially the middle and advanced stage is short of sensitivity to traditional chemoradiotherapy,so the development of new strategy for lung cancer treatment is still the task we are facing.Natural products are important sources of the anticancer agents.For example Taxol and Vincristine extracted from plants has been widely used in clinical cancer treatment. Sanguinarine( SAN) is a kind of alkaloids from the traditional Chinese medicine Macleaya or chelidonic. Previous studies has showed that Sanguinarine is antibacterial and anti-inflammatory which has multiple biological activities.Recent results indicate that Sanguinarine shows an anti tumor effect to prostate cancer,osteosarcoma and breast cancer.However,the mechanism is still not clear.The cancerous cells usually show a series reactions such as endoplasmic reticulum stress, mitotic stress,metabolic stress and oxidative stress to adapt the need of rapid metabolism and growth.The adaptive reaction has dual function. For one side the survival of cancerous cells is dependent on the signaling pathway related with ERS, for the other side certain drugs may induce excessive stress to promote the apoptosis by prolonged the duration or intensity of stress.Some chemical drugs including Taxol,5- fluorouracil and bortezomib can attack cancer cells to death by inducing excessive stress and apoptosis.ROS,as an signal molecule involved in regulating and carring some cell function,plays a key role in the cell fate deciding.ROS may be a potential target for tumor therapy.People try to increase the ROS level to achieve the purpose of tumor treatment. Meanwhile,many studies indicate that ROS can enhance ERS and result the cell death,which suggests the causality between ERS and ROS. It is widely concerned about the role of ROS and ERS in cell death.This paper observed the growth inhibition of lung adenocarcinoma cell line in vitro induced by sanguinarine.We analysed the interactive regulation between ERSand ROS by detecting the ROS level and the expression of the protein related with ERS signal pathway.We investigated the mechanism of sanguinarine in inhibition of the growth of lung adenocarcinoma cells, to provide theory and experiment basis for the development of anti tumor drugs.Method(1) Collect the pathological specimens of lung adenocarcinoma.Detect the expression level of GRP78、CHOP protein through immunohistochemical staining.(2) Human lung adenocarcinoma cell line SPC-A1 was obtained from the Chinese Academy of Sciences.MTT was used to detect the cell viability treated with sanguinarine.Morphology changes of nuclear chromatin were assessed with Hoechst33258 and laser scanning confocal microscope.The expression level of apoptosis and ERS related proteins(including Bax, P-PARP-1, Cleaved Caspase-3) were detected by Western blot.(3) ROS level in SPC-A1 cells treated with sanguinarine was observed by CM-H2 DCFDA staining and laser scanning confocal microscope.MTT was used to analyse the effection of NAC on the inhibition to the growth of lung adenocarcinoma cell line in vitro caused by sanguinarine. Hoechst 33258 was used to observe the effection of NAC on apoptosis induced by sanguinarine.(4) PCR array was applicated to detect the expression of 84 key genes related with UPR and the changes were showed by image form.GRP78、p-PERK、p-eIF2α、ATF-4、CHOP protein were detected by Western Blot.MTT and Hoechst 33258 were used to detecte the effection of TUDCA on the inhibition of cell proliferation and the induction of apoptosis caused by sanguinarine.(5) The level of ROS in SPC-A1 cell after treated with TUDCA and sanguinarine was analyzed by CM-H2 DCFDA.The expression level of GRP78 and CHOP protein in SPC-A1 cell after treated with NAC and sanguinarinewas was detected by Western blot.We used the immunohistochemical staining to analyze the human lung adenocarcinoma tissue achieved from lung cancer patients and diagnosied by doctor of pathology.The positive position of GRP78 were located in the cell cytoplasm,while the positive position of CHOP were located both in cytoplasm and nuclear. The brown staining also could be found in the cytoplasm of alveolar macrophage in the para carcinoma tissues, but no obvious positive staining was found in other cells.2.Sanguinarine inhibited the proliferation of human lung adenocarcinoma cell line SPC-A1 and induced apoptosis.To detect the inhibition to the growth of SPC-A1 cell induced by sanguinarine,we treated the cells with different doses of sanguinarine(0.5,1,2 and 4 μM) for 24 hours.MTT was used to analyze the cell viability.The result reflected that sanguinarine can inhibit the cell growth in a dose-dependent manner.Hoechst 33258 was used to observe the apoptosis. It was showed that there is an obvious apoptosis changes including chromatin condensation and nuclear fragmentation in the nucleus of human lung adenocarcinoma cell line SPC-A1 after treated with different doses of sanguinarine(0.5,1,2 and 4 μM) for 24 hours,which suggested that sanguinarine may play its role in anticancer activity through induction of apoptosis.3.Sanguinarine enhanced the ROS level in human lung adenocarcinoma cell line SPC-A1.We applicated CM-H2 DCFDA stain and laser scanning confocal microscope to detect the ROS level in SPC-A1 cell after treated with different doses of sanguinarine(1,2 μM) for 24 hours. The results showed that ROS increased significantly in a dose-dependent manner.Pretreatment of SPC-A1 cell with NAC(100 μM 1h) had a negative effect on the inhibition of sanguinarine to the growth of SPC-A1 cell and reduced the apoptosis induced by sanguinarine.Therefore, these results suggest that SAN-mediated growth inhibition and apoptosis partially rely on the generation of ROS.Therefore, ROS was closely related with apoptosis induced by sanguinarine.Results1.The expression of GRP78、CHOP proteins in pathological specimen of lung adenocarcinoma.4. Endoplasmic reticulum stress(ERS) induced by sanguinarine involved in the regulation of apoptosis.PCR array was applicated to detect the expression of genes related with ERS and UPR. The results showed that 32 genes were upregulated more than 2-fold after treatment with SAN(2 μM for 24 h) in the SPC-A1 cells,including GRP78, inositol requiring kinase 1α(IRE1α, ERN1), activating transcription factor 6α(ATF6α),activating transcription factor 4(ATF4), growth arrest and DNA-damage inducible gene 34(GADD34 and PPP1R15A) and CAAT/enhancer binding protein homologous protein(CHOP and DDIT3).We next confirmed ER stress by measuring protein expression by western blotting.The expression of GRP78, p-PERK, p-elF2α,ATF4 and CHOP were significantly increased in a dosedependent manner(Fig.3B). Therefore, SAN can induce ER stress in SPC-A1 cells.ER stress is thought to play an important role in the cell pathophysiology process associated with cell death.To confirm the effects of ER stress on SAN-induced growth inhibition,we blocked ER stress using TUDCA, followed by treatment with SAN, and then detected cell viability and apoptosis by the MTT assay and Hoechst 33258 staining.Results indicated that TUDCA partly reversed SAN-induced cell growth inhibition.These data suggest that ER stress is associated with SAN-induced cell growth inhibition and apoptosis in SPC-A1 cells.5.Relationship between endoplasmic reticulum stress(ERS) and ROS in lung cancer cell survival and apoptosis.High levels of ROS can induce the accumulation of misfolded and/or unfolded proteins in the ER and lead to UPR.To investigate the relationship between ER stress and ROS,we aimed to determine whether ER stress is involved in the regulation of ROS production.Notably, pretreatment with50 μM TUDCA for 1 h,followed by treatment with 2 μM SAN for 24 h, partially reversed SAN-induced ROS production. Next, the expression of t he ER stress-related proteins GRP78 and CHOP were monitored after blocking ROS with NAC.After pretreatment of SPC-A1 cells with 100 μM NAC for 1 h, followed by treatment with 2 μM SAN for 24 h,SAN-induced expression of GRP78 and CHOP was markedly attenuated. These datasuggest that blocking ER stress with TUDCA can reduce ROS production, whereas eliminating ROS by NAC can attenuate the extent of ER stress. Therefore, ER stress and ROS production promote each other and form a vicious cycle.Conclusion1.The expression of ERS-related protein GRP78 and CHOP were high in lung adenocarcinoma.Compared with the adjacent tumor tissues, the positive position of GRP78 were located in the cytoplasm of cells,while the positive position of CHOP were located both in cytoplasm and nuclear. It is suggested that ERS-related protein GRP78 and CHOP were associated with the occurrence and development of lung adenocarcinoma.2.SAN inhibits the growth and triggers apoptosis in lung cancer cells.SPC-A1 cells were treated with different doses of SAN for 24 h and the MTT assay revealed that treatment with sanguinarine inhibited cell growth in a dose-dependent manner. Hoechst 33258 staining also showed that after treatment with different doses of SAN for 24 h, the cells showed obvious features of apoptosis in a dose-dependent manner.3.SAN-induced ROS production is involved in the regulation of growth inhibition and apoptosis.Sanguinarine induced the ROS production significantly in a dose-dependent manner. NAC reverse the SAN-induced growth inhibition by means of attenuating SAN-induced apoptosis. These data suggest that sanguinarine induced an apoptosis that was partially dependent on ROS production.4.SAN-induced ER stress is associated with apoptosis in Human lung adenocarcinoma cell.PCR array assay and western blotting showed that GRP78, p-PERK, IRE1 a and ATF6 a were upregulated.TUDCA partly reversed SAN-induced cell growth inhibition and apoptosis.These data suggest that SAN-induced ER stress is associated with cell growth inhibition and apoptosis in SPC-A1 cells.5.ER stress and ROS production promote each other and form a vicious cycle.We used NAC and TUDCA to investigate the relationship between ER stress and ROS,The result suggest that ER stress could increase ROS production,whereas ROS could induce the extent of ER stress. Therefore, ER stress and ROS production promote each other and form a vicious cycle.