A series of chemotherapeutic drugs that induce DNA damage, such as cisplatin (DDP), are standard clinical treatments for ovarian cancer, testicular cancer, and other diseases that lack effective targeted drug therapy. Drug resistance is one of the main factors limiting their application. Sensitizers can overcome the drug resistance of tumor cells, thereby enhancing the antitumor activity of chemotherapeutic drugs. In this study, we aimed to identify marketable drugs that could be potential chemotherapy sensitizers and explore the underlying mechanisms. We found that the alcohol withdrawal drug disulfiram (DSF) could significantly enhance the antitumor activity of DDP. JC-1 staining, propidium iodide (PI) staining, and western blotting confirmed that the combination of DSF and DDP could enhance the apoptosis of tumor cells. Subsequent RNA sequencing combined with Gene Set Enrichment Analysis (GSEA) pathway enrichment analysis and cell biology studies such as immunofluorescence suggested an underlying mechanism: DSF makes cells more vulnerable to DNA damage by inhibiting the Fanconi anemia (FA) repair pathway, exerting a sensitizing effect to DNA damaging agents including platinum chemotherapy drugs. Thus, our study illustrated the potential mechanism of action of DSF in enhancing the antitumor effect of DDP. This might provide an effective and safe solution for combating DDP resistance in clinical treatment.
Female ; Male ; Humans ; Cisplatin/pharmacology* ; Disulfiram/pharmacology* ; Testicular Neoplasms/drug therapy* ; Fanconi Anemia/drug therapy* ; Alcoholism/drug therapy* ; Drug Resistance, Neoplasm ; Cell Line, Tumor ; Substance Withdrawal Syndrome/drug therapy* ; Apoptosis ; Antineoplastic Agents/therapeutic use* ; Cell Proliferation

Animals ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Disulfides ; Male ; Mice ; Mice, Inbred BALB C ; Phosphatidylinositol 3-Kinases ; Stomach Neoplasms/drug therapy* ; Sulfoxides

OBJECTIVES: To evaluate whether garlicin post-conditioning can attenuate myocardial ischemiareperfusion injury in a catheter-based porcine model of acute myocardial infarction (AMI) by affecting adhesion molecules integrin β1/CD29 and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31). METHODS: Twenty-two swine were devided into 3 groups: 6 in a sham-operation group, and 8 each in the model and garlicin groups. AMI porcine model was established in the model and garlicin groups. The distal parts of the left anterior descending coronary artery in the animals of the model and garlicin groups were occluded by dilated balloon for 2 h, followed by reperfusion for 3 h. Garlicin (1.88 mg/kg) was injected over a period of 1 h, beginning just before reperfusion, in the garlicin group. Real-time polymerase chain reaction, immunohistochemistry and Western blot were carried out to detect mRNA and protein expressions of CD29 and CD31 3 h after reperfusion. RESULTS: Hematoxylin-eosin staining showed a better myocardial structure in the garlicin group after reperfusion. Compared to the model group, garlicin inhibited both the mRNA and protein expression of CD29 and CD31 in reperfusion area and no-reflflow area (P<0.05 respectively). CONCLUSIONS Garlicin post-conditioning induced cardio-protection against myocardial ischemia-reperfusion injury in this catheter-based porcine model of AMI. The cardio-protective effect of garlicin is possibly owing to suppression of production of CD29 and CD31, by inhibition of the mRNA expression of CD29 and CD31.
Allyl Compounds ; pharmacology ; Animals ; Disease Models, Animal ; Disulfides ; pharmacology ; Integrin beta1 ; analysis ; genetics ; physiology ; Ischemic Postconditioning ; Male ; Myocardial Reperfusion Injury ; prevention & control ; Platelet Endothelial Cell Adhesion Molecule-1 ; analysis ; antagonists & inhibitors ; genetics ; RNA, Messenger ; analysis ; Swine

Objective To explore the effects of diallyl disulfide(DADS)-induced G2/M phase arrest on proliferation and apoptosis of ovarian cancer cells and its possible molecular mechanism.Methods DADS was used to incubate SK-OV-3 and OVCAR-3 cells,respectively,in different concentrations. Cell proliferation was measured by MTT assay and cell apoptosis rate was detected by flow cytometry assay. Xenograft model assay were performed to analyze the antitumor effect in vivo. Cell cycle phase distribution was detected by flow cytometry. Expressions of cell cycle G2/M phase as well as proliferation- and apoptosis-related proteins were measured by Western blotting.Results MTT assay showed that,after treatment of SK-OV-3(F=247.86,P=0.000)and OVCAR-3 cells(F=302.54,P=0.000)with different concentrations of DADS,the cell proliferation inhibition rate was significantly elevated with the increase of DADS concentrations in a concentration-dependent manner. The inhibition rate of SK-OV-3(F=335.12,P=0.000)and OVCAR-3 cells(F=347.43,P=0.000)at 24 h was significantly higher than that at 12 h and 48 h,showing a significant time-dependence manner. Flow cytometry showed that,after SK-OV-3 and OVCAR-3 cells were treated with different concentrations of DADS,the apoptosis rates increased significantly with the increase of DADS concentration in a concentration-dependent manner(P<0.05). The apoptotic rates of SK-OV-3 and OVCAR-3 cells treated with DADS at 24 h was significantly higher than that at 12 h and 48 h in a significant time-dependence manner(P<0.05). Compared with the blank treatment group,intraperitoneal injection of DADS solution significantly inhibited the xenograft volume of ovarian cancer cells in nude mice(F=548.23,P=0.000;F=311.84,P=0.000). After 30 mg/L of DADS was applied to SK-OV-3 and OVCAR-3 cells for 24 h,the percentage of cells in G2 phase of SK-OV-3 and OVCAR-3 cells increased significantly(F=375.11,P=0.000;F=256.48,P=0.000),compared with the blank cells. After 30 mg/L DADS was applied to SK-OV-3 and OVCAR-3 cells for 24 h,the expressions of p-Chk1(ser345)(F=108.89,P=0.013;F=97.58,P=0.018),p-CDC25C(ser216)(F=87.25,P=0.025;F=114.25,P=0.009),p-P53(ser15)(F=112.41,P=0.011;F=255.87,P=0.000),P21WAF1(F=246.38,P=0.001;F=141.36,P=0.005)and p-CDK1(Thr14/Tyr15)protein(F=298.12,P=0.000;F=233.15,P=0.000)were significantly increased,whereas the expressions of CDK1(F=308.24,P=0.000;F=257.55,P=0.000)and CyclinB1 protein(F=223.15,P=0.001;F=241.28,P=0.000)were significantly reduced.The expressions of proliferation and apoptosis-related proteins PCNA(F=77.36,P=0.031;F=157.28,P=0.001),Ki-67(F=205.64,P=0.007;F=315.22,P=0.000)and Survivin(F=122.13,P=0.013;F=188.24,P=0.000)were significantly decreased and Cleaved-caspase3 protein was significantly increased(F=86.46,P=0.023;F=99.11,P=0.009).Conclusion DADS can inhibit the proliferation of ovarian cancer cells and induce their apoptosis,which may be related to the activation of Chk1-CDC25C and P53-P21WAF1 signaling pathways in G2/M checkpoint,decreased kinase activity of CDK1,down-regulated expressions of CDK1 and CyclinB1 proteins,and ultimately cell cycle arrest at G2/M phase.
Allyl Compounds ; Animals ; Apoptosis ; Carcinoma, Ovarian Epithelial ; Cell Cycle Checkpoints ; Cell Line, Tumor ; Cell Proliferation ; Disulfides ; Female ; Humans ; Mice ; Mice, Nude

PURPOSE: Glioblastoma, the most common brain tumor in adults, has poor prognosis. The purpose of this study was to determine the effect of disulfiram (DSF), an aldehyde dehydrogenase inhibitor, on in vitro radiosensitivity of glioblastoma cells with different methylation status of O⁶-methylguanine-DNA methyltransferase (MGMT) promoter and the underlying mechanism of such effect. MATERIALS AND METHODS: Five human glioblastoma cells (U138MG, T98G, U251MG, U87MG, and U373MG) and one normal human astrocyte (NHA) cell were cultured and treated with DSF or 6MV X-rays (0, 2, 4, 6, and 8 Gy). For combined treatment, cells were treated with DSF before irradiation. Surviving fractions fit from cell survival based on colony forming ability. Apoptosis, DNA damage repair, and cell cycle distributionwere assayed bywestern blot for cleaved caspase-3, γH2AX staining, and flow cytometry, respectively. RESULTS: DSF induced radiosensitization in most of the glioblastoma cells, especially, in the cells with radioresistance as wildtype unmethylated promoter (MGMT-wt), but did not in normal NHA cell. DSF augmented or induced cleavage of caspase-3 in all cells after irradiation. DSF inhibited repair of radiation-induced DNA damage in MGMT-wt cells, but not in cells with methylated MGMT promoter. DSF abrogated radiation-induced G2/M arrest in T98G and U251MG cells. CONCLUSION: Radiosensitivity of glioblastoma cells were preferentially enhanced by pre-irradiation DSF treatment compared to normal cell, especially radioresistant cells such as MGMT-wt cells. Induction of apoptosis or inhibition of DNA damage repair may underlie DSF-induced radiosensitization. Clinical benefit of combining DSF with radiotherapy should be investigated in the future.
Adult ; Aldehyde Dehydrogenase ; Apoptosis ; Astrocytes ; Brain Neoplasms ; Caspase 3 ; Cell Cycle ; Cell Survival ; Disulfiram ; DNA Damage ; Flow Cytometry ; Glioblastoma ; Humans ; In Vitro Techniques ; Methylation ; Prognosis ; Radiation Tolerance ; Radiotherapy

BACKGROUND: The left internal thoracic artery (LITA) has been used as the first conduit of choice in coronary artery bypass grafting (CABG) because of excellent long-term patency and outcomes. However, no studies have examined substances other than nitric oxide that could be beneficial for the bypass conduit, native coronary artery or ischemic myocardium. This study was conducted to evaluate differences in metabolic profiles between the LITA and ascending aorta using gas chromatography-time of flight-mass spectrometry (GC-TOF-MS). METHODS: Twenty patients who underwent CABG using the LITA were prospectively enrolled. Plasma samples were collected simultaneously from the LITA and ascending aorta. GC-TOF-MS based untargeted metabolomic analyses were performed and a 2-step volcano plot analysis was used to identify distinguishable markers from two plasma metabolome profiles. Semi-quantitative and quantitative analyses were performed using GC-TOF-MS and enzyme-linked immunosorbent assay, respectively, after selecting target metabolites based on the metabolite set enrichment analysis. RESULTS: Initial volcano plot analysis demonstrated 5 possible markers among 851 peaks detected. The final analysis demonstrated that the L-cysteine peak was significantly higher in the LITA than in the ascending aorta (fold change = 1.86). The concentrations of intermediate metabolites such as L-cysteine, L-methionine and L-cystine in the ‘cysteine and methionine metabolism pathway' were significantly higher in the LITA than in the ascending aorta (2.0-, 1.4- and 1.2-fold, respectively). Quantitative analysis showed that the concentration of hydrogen sulfide (H2S) was significantly higher in the LITA. CONCLUSION: The plasma metabolome profiles of the LITA and ascending aorta were different, particularly higher plasma concentrations of L-cysteine and H2S in the LITA.
Aorta ; Chromatography, Gas ; Coronary Artery Bypass ; Coronary Vessels ; Cysteine ; Cystine ; Enzyme-Linked Immunosorbent Assay ; Humans ; Hydrogen Sulfide ; Mammary Arteries ; Mass Spectrometry ; Metabolism ; Metabolome ; Metabolomics ; Methionine ; Myocardium ; Nitric Oxide ; Plasma ; Prospective Studies ; Spectrum Analysis

Disulfiram has been used for the treatment of alcohol dependence for nearly 65 years and is approved by the Food and Drug Administration. It causes negative reinforcement by accumulating toxic acetaldehyde due to irreversible inhibition of aldehyde dehydrogenase. Disulfiram has very few side effects when taken without alcohol. Epileptic seizure induction is a rare side effect in therapeutic doses, and its mechanism is unknown. We present a patient with a single epileptic seizure which was thought to be due to disulfiram used in the treatment of alcohol dependence. We did not find it ethical to administer disulfiram again because the patient discontinued alcohol use and was afraid of epileptic seizures.
Acetaldehyde ; Alcoholism ; Aldehyde Dehydrogenase ; Disulfiram ; Epilepsy ; Humans ; Reinforcement (Psychology) ; United States Food and Drug Administration

OBJECTIVETo investigate the inducing effect of down-regulation of MCL-1 by diallyl disulfide(DADS) on the G/M arrest of human leukemia K562 cells and its mechanisms.

METHODSCCK-8 was used to detect the effect of DADS on proliferation of K562 cells, flow cytometry was employed to observe the effect of cycle arrest by DADS and RNAi silencing MCL-1 gene in K562 cells. The expressions of MCL-1, PCNA and CDK1 in K562 cells treated with DADS were detected by Western blot. The amphigamy of MCL-1 with PCNA and CDK1 was detected by Coimmunoprecipitation.

RESULTSCCK-8 detection showed that the inhibition rates of K562 cells treated with 15, 30, 60, 120, 240 µmol/L DADS were 32.48%, 59.34%, 66.42%, 77.06%, 81.05% respectively (P<0.05). Flow cytometry analysis revealed that the perecentages of G/M cells were increased to 18.6% and 34.4%, 17.5% and 28.5%, respectively at 24 and 48 h after treating K562 cells with 60 and 120 µmol/L DADS (P<0.05). And the perecentage of G/M cells of silencing MCL-1 was significantly increased (P<0.05). Silencing effects of MCL-1+DADS on the cells were enhanced more significantly as compared with DADS or MCL-1 alone (P<0.05). Western blot exhibited that DADS could markedly downregulate the expression of MCL-1, PCNA and CDK1(P<0.05). Coimmunoprecipitation revealed that MCL-1 bound with PCNA and CDK1, then forming heterodimers, which were downregulated respectively more significantly than that in the control group after treating K562 cells with DADS for 8 h (P<0.05).

CONCLUSIONDADS can inhibit the K562 cell proliferation and induce them arrest G/M through downregulation of MCL-1, then decreasing the expression of PCNA and CDK1 in hunan leukemia K562 cells. Moreover, silencing MCL-1 can enhance the effect of DADS.

Allyl Compounds ; Apoptosis ; Cell Line, Tumor ; Disulfides ; Down-Regulation ; G2 Phase Cell Cycle Checkpoints ; Humans ; K562 Cells ; Leukemia ; M Phase Cell Cycle Checkpoints ; Myeloid Cell Leukemia Sequence 1 Protein

BACKGROUND: NUP98 has numerous partner genes of which plant homeodomain (PHD) finger protein 23 (PHF23) fusion with NUP98 (NP23) can be detected by RT-PCR in patients with cytogenetically normal acute myelogenous leukemia (AML). In this fusion transcript of NP23 PHD of PHF23 is known to specifically bind H3K4me3 residues and act as a chromatic modifier. Disulfiram (DSF) which inhibits the binding of PHD to H3K4me3 residues selectively killed NP23 myeloblasts in vitro and therefore, we planned to evaluate the efficacy of DSF in vivo. METHODS: Cultured 961C cells (CD45.2), NP23 myeloblast cells were transplanted into B57BL/6 mice (CD45.1). Using limit dilution assay the number of leukemic stem cells (LSCs) could be calculated. A certain amount of 961C cells were transplanted into B57BL/6 mice and DSF was treated after 1 week. The engraftment level was monitored with CD45.2. Kaplan Meier survival curve was used to compare the survival between therapeutic and control group. RESULTS: 961C cells could be transplanted without radiation in recipient mice. Calculated LSC was estimated to be 1 out of 184 cells (95% CI range, 56–609). When treated with DSF of different doses and administration routes in 961C recipient mice no survival advantage of DSF was observed in 961C transplanted immunocompetent mouse, however it was evident that engraftment level was consistent in both groups. CONCLUSION: No survival advantage of DSF in 961C transplanted immunocompetent mouse was observed, however it was evident that 961C cells shared niche with normal hematopoietic stem cells (HSCs). We expect that 961C cells and transplanted recipient mice have the potential to be used as in vivo system for new drugs development as well as for research dealing with niche for normal HSCs and LSCs.
Animals ; Disulfiram ; Fingers ; Granulocyte Precursor Cells ; Hematopoietic Stem Cells* ; Humans ; In Vitro Techniques ; Leukemia, Myeloid, Acute ; Mice ; Plants ; Stem Cells

Algorithms ; Crystallography, X-Ray ; Disulfides ; chemistry ; Models, Molecular ; Protein Conformation ; Proteins ; chemistry