Azurin And Its Combination With Chemotherapy Drugs Induced Apoptosis Of Human Osteosarcoma Cell By P53Mediated ROS And Mitochondrial Pathway

Backgroud:Osteosarcoma because of its high degree of malignancy, early metastasis, clinical treatment with surgery combined with chemotherapy and radiotherapy treatments, but limited to the surgical treatment of high morbidity and poor function of the prosthesis, put the side effects of chemotherapy, advanced tumors easy to form a tolerance to chemotherapeutic drugs, vulnerable to relapse, re-treatment is also extremely difficult, and therefore lower long-term survival of osteosarcoma difficult to satisfactory efficacy, the limitations of traditional therapy makes osteosarcoma new therapies such as biological therapy, immunotherapy therapy, gene therapy, and microbial treatment of the purified product are increasingly becoming the focus of research and clinical and hotspots.Yamada reported in recent years by Pseudomonas bacteria secrete copper-containing bacterial redox protein Azurin, can combine to form complexes with the P53protein, inhibition of P53degradation, thereby stabilizing the P53protein.It can be induced in vitro cell assays and in vivo tumor-bearing nude mice P53wild type mononuclear macrophages, melanoma cells, breast cancer cells, while P53mutation or deletion of melanoma cells in the rendered breast cancer cells, the cytotoxicity is greatly reduced, its cytotoxic obvious p53selectivity.Particularly critical in malignant melanoma cells, breast cancer cells in nude mice transplanted tumor, tumor size, and the wild-type Azurin can promote the degeneration of the transplanted tumors, inhibiting the growth of transplanted tumors tumor block induction of tumor cell apoptosis, while nude mice The body was found no significant side effects.This finding viable cells product purified protein the Azurin used in clinical tumor therapy to bring hope and prospects.Zhejiang Second Orthopaedic Institute Professor Yang Disheng and Miao Xudong et al Azurin protein in human osteosarcoma cells to carry out a series of in vitro experimental study.The results are as follows:Azurin inhibited the growth of osteosarcoma induced P53wild type U2OS cell apoptosis, while normal liver L-02the P53mutant human osteosarcoma MG-63cells less toxic.Further study its molecular mechanisms:P53protein is play the key factors of the apoptotic effects the Bax mitochondrial shift of Bcl-2/Bax ratio downward, caspase-3,8increased activity, mitochondrial, extracellular Fas by pathway Azurin-induced the U2OS cell apoptosis molecular mechanisms are involved.Tumor cells escape apoptosis caused apoptosis resistance (resistance to apoptosis) is one of the main reasons of tumor drug resistance, restore the system state level of intracellular reactive oxygen species (ROS) and glutathione (GSH) change is closely related to the apoptosis.The wild-type p53gene is a tumor suppressor gene, the main function of p53is to arrest the cell cycle or induce apoptosis.There are three key steps in the p53-induced apoptosis signaling pathways:(i) oxidation reduction related gene transcription induced;(ii) the formation of reactive oxygen species (ROS) is formed; component (iii) mitochondrial oxidative degradation, and ultimately activation caspase leading to apoptosis.In osteosarcoma chemotherapy drug toxicity reaction, while tumor recurrence rate was high due to repeated administration can easily acquire resistance, and therefore the long-term survival rate is lower.Studies have shown that the the ROS level differences related to the resistance of tumor cells to their cells.Through the regulation of intracellular GSH levels or elevated ROS levels can enhance the apoptosis-inducing effects of chemotherapy drugs.Accordingly, we speculate Azurin raised p53transcriptional activity, triggered intracellular oxidation reduction state of imbalance through the mitochondrial pathway induction of apoptosis, and at the same time, Azurin of osteosarcoma tumor chemotherapy drug cis-platinum (Cisplatin, of CDDP), methotrexate chatter methotrexate (MTX). adriamycin (ADM) and ifosfamide (IFO) with chemotherapy sensitizing effect?And to explore the the p53, GSH, ROS in Azurin chemosensitizing effect of the specific mechanism of action.Methods:In our study, three different genotypes of P53osteosarcoma cell line (p53wild-type U2OS, p53mutant MG-63, p53-deficient SAOS-2) and a p53wild-type normal Human peripheral blood mononuclear cells (PMBCs), by MTT colorimetric assay of cell viability, application of Annexin V and PI double staining flow cytometry apoptosis rate Annexin V positive cell (%) by flow cytometry, fluorescence microscopy detection of mitochondrial membrane potential (ΔΨm) and mitochondrial inner membrane heart phospholipids oxidation by immunofluorescence techniques, laser confocal subcellular protein separation Cytochrome C (cytochrome C) Western blot analysis of mitochondrial outer membrane protein, AIF (apoptosis-inducing factor) release into the cytoplasm, cytoplasmic Bax shift mitochondria, Western blot analysis of mitochondrial core apoptotic protein Bcl-xL of MCL-1, XIAP, survivin, Bak, Bid, tBid of expression by flow cytometry reactive oxygen species (H202), superoxide anion (O2-) and nitric oxide (NO), through the the microplate colorimetric detection intracellular GSH, extracellular fluid GSH, glutathione peroxidase (GPX), GSSG (oxidized form of GSH), glutathione reductase (GR) level, pp53-TA-luc reporter plasmid and the internal reference Renilla-luc transiently co-transfected osteosarcoma cells SAOS-2(null), MG-63(mut), U2OS (wt), the fluorescence detector detects the above cell luciferase activity, namely the transcriptional activity of the p53gene.And mitochondrial membrane PT pore inhibitor cyclosporine A (CsA); reductant Catalase, NAC, MnTBAP; GSH depleting agent BSO; P53signal blocking agent PFT-a verified from two positive and negative direction to clear mitochondria ROS, P53specific mechanism of action.Result:First, we observed Azurin P53pathway induced tumor cell apoptosis.Further clear of Azurin the P53selectively kill tumor mechanism, we have selected different P53genotype background of tumor cells in other tissue sources:53people carrying wild-type P hepatoma cell line HepG2cell lines, lung cancer cell line A549, flesh and blood tumor cell line U2OS; P53mutant lung cancer cell line H322, hepatoma cell line Huh-7cells, osteosarcoma cell line MG-63; render P53deletion lung cancer cell lines HI299hepatoma cell line Hep3B cells, osteosarcoma SAOS-2cell lines; carrying the wild-type P53normal human peripheral blood the monocytes (PMBCs).Azurin proliferation of these cell lines by MTT assay.By MTT assay, we found that the tumor cells in the background of P53gene in lung cancer, liver cancer, osteosarcoma cell lines, Azurin also significantly inhibited with wild-type P53the HepG2, A549, U2OS cells proliferation activity, whom In contrast, in the cells of P53functional inactivation AZURIN cell toxicity is significantly weakened.In these cells, Azurin perform equally obvious P53selective cytotoxic effect, simultaneously less toxic of normal PMBCs cells.Azurin treatment in p53wild-type U2OS cells, caspase-3activity was significantly increased, start caspase cascade, PARP occurred shear induced apoptosis.Conversely, p53mutated MG-63, p53-deficient SAOS-2cell lines Azurin upregulation of caspase-3kinase activity, Annexin V positive cell (%) were significantly weakened; trials further a P53signal blocker PFT-a preincubation U2OS cells, were observed on the the Azurin role Cleaved-PARP protein kinase caspase-3activity, Annexin V positive cell (%), MTT indicators, we find that PFT-a almost completely inhibited Azurin induced Caspase-3activation, PARP a shear, Annexin V positive (%) of the raised; thus confirming the molecular mechanism of Azurin apoptosis presented P53-dependent manner.Secondly, we detect the the Azurin induced mitochondrial damage.P53wild-type cells U2OS after Azurin role, JC-1red and green fluorescence ratio decreased mitochondrial membrane potential (ΔΨm) happened; mitochondrial NAO fluorescence intensity was significantly reduced, and reduce mitochondrial endometrial cardiolipin content endometrial produce a shift gathered mitochondrial outer membrane lipid peroxidation, causing increased permeability of the outer mitochondrial membrane; cytoplasmic protein Bax monomer targeted protein conformational change occurred oligomerization to form across the mitochondrial outer membrane pore, lead to mitochondrial further increase in membrane permeability.Bcl-2family inhibiting apoptotic protein Bcl-xL and MCL-1, XIAP protein expression levels of self-8h time point reduced to promote the expression of apoptotic protein Bak presents a time-dependent increase, while the truncated Bid (tBid) from the4h time to point began to appear, the24h time point tBid the protein expression levels peak of Bid Protein shear.Cytochrome C combined with cardiolipin be uncoupling between the inner and outer mitochondrial membrane protein Cytochrome C (Cytochrome C), AIF (apoptosis inducing factor) protein pore massive release into the cytoplasm, start caspase cascade activation of downstream effector caspase-3, AIF thus translocation nucleus and DNA binding in the nuclear cause staining quality gathering and fracture.It is noteworthy that in p53mutant MG-63, p53-deficient SAOS-2cells and normal human peripheral blood mononuclear cells (PMBCs) Azurin treatment, mitochondrial membrane potential ΔΨm, mitochondrial inner membrane center phospholipid content did not change significantly, suggesting that p53signaling pathways Azurin-induced mitochondrial damage.To clear the mitochondrial pathway Azurin induced apoptosis, mitochondrial membrane PT pore inhibitor cyclosporine A (CsA) pre-incubated U2OS cells, CsA significantly inhibited the the Azurin enhanced caspase-3activity, Cleaved-PARP The upregulation of the protein, and apoptosis.But raised on Azurin induced intracellular ROS levels, GSH levels decreased almost no effect, suggesting that mitochondrial channel opening events later, in the redox system imbalance occurs downstream events.Again, we observed Azurin-induced cell redox state of imbalance.Azurin in a P53wild-type U2OS cells, quickly lead to the DCF (H2O2), ethidium (O2-) significant upregulation of the fluorescence intensity, but then the fluorescence intensity of the DAF-FM (NO) does not affect, i.e. The Azurin mainly caused too hydrogen peroxide (H2O2) and superoxide anion (O2-) of these two kinds of radicals rise, but then the nitric oxide (NO) no effect.Meanwhile, we observed the Azurin U20S cells GSH reduction system, we found Azurin suppression Synthesis and enzyme activity of glutathione peroxidase (GPX), oxidized GSSG content increased greatly reduces the antioxidant capacity of the GSH reduction system, to induce GSH release into the extracellular fluid, further depletion of intracellular GSH, promote hair redox system imbalance.Application of GSH precursor antioxidants NAC pretreatment p53wild-type U20S cells, we found that NAC significantly inhibited the Azurin induced intracellular GSH level reduced and ROS upward, also prevent a decline in mitochondrial membrane potential and intimal Azurin priming heart phospholipid peroxidation, and eventually suppressed caspase activation mediated apoptosis.Which prompted Azurin triggered intracellular redox state imbalance between mitochondrial damage occurs before the upstream events. Test further reducing agent, hydrogen peroxide (H2O2) the enzyme Catalase pretreatment P53wild-type cells U20S Catalase suppression Azurin induced intracellular ROS levels increase, and apoptosis, but then the Azurin due to the reduced GSH levels did not impact, which indicates that the decline in the level of GSH Azurin first initiator in U2OS cells, leading to oxygen radicals of hydrogen peroxide (H2O2) accumulation.GSH decreased upstream events prior to ROS accumulation occurred.We also used the superoxide anion scavenger MnTBAP pretreatment P53wild type cells U2OS the the Azurin induced intracellular oxidative stress, caspase activity had no effect, suggesting that superoxide anion levels presented raised, but did not participate in The Azurin induced apoptosis.Compared with U2OS cells, P53mutant cells of MG63of p53-deficient SAOS-2after Azurin processing, hydrogen peroxide (H2O2), superoxide anion (O2-) cell extracellular fluid GSH intracellular GSH content, glutathione Gan peptide peroxidase (GPX) activity showed no significant change.The BSO inhibition of intracellular GSH synthesis P53mutant of MG63and p53-deficient SAOS-2cells after BSO role of intracellular GSH was significantly decreased, and a sharp increase in the level of oxygen free radicals, significantly decreased the ΔΨm, mitochondrial inner membrane lipid peroxidation occurred, Caspase activation and induce apoptosis.The BSO analog Azurin in U2OS cytotoxic effect.The above results indicate that GSH depletion, accumulation of ROS-induced apoptosis in Azurin plays an important role, and oxidative stress may be affected by the p53pathway regulation.Finally, we observed p53signaling pathway, the relationship between oxidative stress, mitochondrial damage, apoptosis.We will pp53-TA-luc reporter plasmid and the internal reference Renilla-luc instantaneous co-transfected lung cancer cell lines H1299(null), H322(mut), A549(wt); hepatoma cell line Hep3B (null), Huh-7(mut was), HepG2(wt); osteosarcoma cell line, SAOS-2(null), MG-63(MUT), U2OS,(by weight), the fluorescent detector detecting the luciferase activity, we found to The Azurin enhance the transcriptional activity of the p53gene.Further test p53inhibitor PFT-α (pifithrinalpha, PFT-α) interference test, blocked U2OS P53signaling pathways, and results showed that PFT-a pretreatment inhibits Azurin triggered GPX activity decreased, GSH decreased, ROS raised; mitochondrial membrane potential ΔΨm lower endometrial cardiolipin oxidation; the Bax shift mitochondria; cytochrome C, AIF release into the cytoplasm.This indicates that the redox state of imbalance Azurin mediated mitochondrial damage by the p53signaling pathway regulation.In addition, the results also show that:U2OS cells, NAC pretreatment inhibited Azurin upregulation of p53transcriptional activity, GSH depletion agent BSO in U2OS cells to simulate the the Azurin raised p53transcriptional activity, suggesting the redox state of imbalance Positive feedback activation of the p53signaling pathway.The results of the study show that the P53signaling pathway and its regulation of GSH metabolism, ROS levels, mitochondrial pathway in the Azurin induced target cell apoptosis process plays an important role.We selected the commonly used drugs such as osteosarcoma chemotherapy Cisplatin (Cisplatin, CDDP), methotrexate (MTX), adriamycin (ADM) and Ifosfamide (IFO), U2OS MG-63, SAOS-2cells, Azurin drug-sensitizing activity screening to verify that small doses Azurin combined with low concentrations of chemotherapy drugs osteosarcoma cells destruction sensitizing effect?And P53, ROS, GSH in which to further explore the molecular mechanisms.Susceptibility testing of cisplatin, methotrexate, U2OS cells, MTT showed that low-dose the Azurin significant increase cisplatin, methotrexate cytotoxicity, administered in combination have a synergistic effect.At the same time, it also raised GSH decline, ROS increase.Reducing agent NAC, p53inhibitor PFT-a (pifithrinalpha, PFT-a) after pretreatment U2OS significantly inhibited due to the co-administered group of GSH decreased ROS raised as well as the reduction in cellular viability.In P53loss of function of the remaining two MG-63, SAOS-2cells, combination group lack of synergy effects, the MG-63, SAOS-2intracellular GSH of ROS levels are almost no change.These data suggest that the P53and its regulation of the redox state of imbalance is particularly critical in Azurin combination with cisplatin, methotrexate group sensitizing effect.Doxorubicin, ifosfamide susceptibility testing, it is interesting that three kinds of MG-63, SAOS-2, U2OS cells, small doses of Azurin significantly enhanced doxorubicin, ifosfamide cytotoxicity, co-administration, we have not observed GSH decline, ROS increases, reducing agent NAC p53inhibitor PFT-a (pifithrinalpha, PFT-a) pretreatment cell viability due to the co-administered group decline in almost no effect, which prompted the MG-63, SAOS-2, U2OS, Azurin doxorubicin, different sensitizing effect of cyclophosphamide cytotoxicity P53, ROS pathway unrelated.This shows that in the p53functional deletion of tumor cells, Azurin also be synergistic sensitizing effect of cytotoxic chemotherapy drugs, thus avoiding its limited to P53wild type tumor cells.In addition, the tests also found that the cells of the human peripheral blood mononuclear cells (PBMC), AZURIN cisplatin, methotrexate, adriamycin, ifosfamide administration group cytotoxic relatively small.This means that the co-administration group only avoids acquired drug resistance of tumor cells when administered alone, but also makes its toxic side effects on normal cells greatly reduced.Conclusion:In In summary, on the basis of the work of Professor Yang Disheng, Miao Xudong et al, we further investigate the P53, ROS, mitochondrial pathway of the molecular mechanisms of cytotoxicity in Azurin:Azurin enhanced p53transcriptional activity↑â†'p53intracellular levelsâ†'GSH↓of ROS, the↑â†'glutathione peroxidase (GPX)↓↑â†'mitochondrial oxidation of endometrial cardiolipinâ†'mitochondrial membrane potential (ΔΨ m)↓â†'Bax shift mitochondrialâ†'cytochrome C, AIF release into cell the qualityâ†'activation of the mitochondrial pathwayâ†'caspase activationâ†'apoptosis of osteosarcoma.Redox state of the P53gene regulatory imbalance (GSH depletion and increased ROS) in which is particularly critical.In addition, the P53-dependent or non-dependent pathway of P53Azurin with low doses of chemotherapy drugs such as cisplatin (Cisplatin, CDDP), methotrexate methotrexate (MTX), adriamycin (ADM) and ifosfamide (IFO) to the United The drugs have a synergistic effect, Azurin sensitizing effect of the chemotherapy drugs osteosarcoma cells in vitro cytotoxic, oxidative stress is closely related to its P53-dependent the sensitizing mechanism with P53control, which does not depend on the P53sensitizing mechanism has nothing to do with the level of oxygen free radicals.The findings of this paper will not only help to identify the Azurin the possible targets of signaling pathways also provide sufficient experimental evidence of Azurin the preclinical development and application, and also for the future Azurin used in clinical osteosarcoma chemotherapy combination provides a safe and effective molecular strategies.