The molecular mechanism of GADD153 in apoptosis of keloid fibroblasts exposed to botulinum toxin type A.

Apoptosis plays a key role in keloids. Growth arrest and DNA damage-inducible gene 153 (GADD153) is regulated by apoptosis. Botulinum toxin type A (BTXA) can induce apoptosis in keloid fibroblasts. This research aimed to explore the hypothesis that GADD153 mediates apoptosis in keloid fibroblasts exposed to BTXA. BTXA significantly induced GADD153 protein and mRNA expression in keloid fibroblasts. Treatment with c-Jun N-terminal kinase (JNK) inhibitor SP600125, JNK small interfering RNA (siRNA) and tumour necrosis factor-alpha (TNF-α) antibodies reversed the BTXA-induced GADD153 expression. BTXA enhanced the transcriptional activity of GADD153, whereas the GADD153 mutant plasmid, JNK siRNA and anti-TNF-α antibody treatment abolished the BTXA-induced transcriptional activity of GADD153. The addition of TNF-α to keloid fibroblasts markedly increased GADD153 protein expression. The addition of GADD153 siRNA, SP600125 and anti-TNF-α antibodies reversed cell death and caspase 3 and 9 activity induced by BTXA.

Hinokitiol Exhibits Antitumor Properties through Induction of ROS-Mediated Apoptosis and p53-Driven Cell-Cycle Arrest in Endometrial Cancer Cell Lines (Ishikawa, HEC-1A, KLE).

Hinokitiol is a natural tropolone derivative that is present in the heartwood of cupressaceous plants, and has been extensively investigated for its anti-inflammatory, antioxidant, and antitumor properties in the context of various diseases. To date, the effects of hinokitiol on endometrial cancer (EC) has not been explored. The purpose of our study was to investigate the anti-proliferative effects of hinokitiol on EC cells. Cell viability was determined with an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the quantification of apoptosis and reactive oxygen species (ROSs) was performed by using flow cytometry, while protein expression was measured with the Western blotting technique. Hinokitiol significantly suppressed cell proliferation through the inhibition of the expression of cell-cycle mediators, such as cyclin D1 and cyclin-dependent kinase 4 (CDK4), as well as the induction of the tumor suppressor protein p53. In addition, hinokitiol increased the number of apoptotic cells and increased the protein expression of cleaved-poly-ADP-ribose polymerase (PARP) and active cleaved-caspase-3, as well as the ratio of Bcl-2-associated X protein (Bax) to B-cell lymphoma 2 (Bcl-2). Interestingly, except for KLE cells, hinokitiol induced autophagy by promoting the accumulation of the microtubule-associated protein light chain 3B (LC3B) and reducing the sequestosome-1 (p62/SQSTM1) protein level. Furthermore, hinokitiol triggered ROS production and upregulated the phosphorylation of extracellular-signal-regulated kinase (p-ERK1/2) in EC cells. These results demonstrate that hinokitiol has potential anti-proliferative and pro-apoptotic benefits in the treatment of endometrial cancer cell lines (Ishikawa, HEC-1A, and KLE).

Chrysin boosts KLF2 expression through suppression of endothelial cell-derived exosomal microRNA-92a in the model of atheroprotection.

PURPOSE: Atherosclerosis and its related clinical complications are the leading cause of death. MicroRNA (miR)-92a in the inflammatory endothelial dysfunction leads to atherosclerosis. Krüppel-like factor 2 (KLF2) is required for vascular integrity and endothelial function maintenance. Flavonoids possess many biological properties. This study investigated the vascular protective effects of chrysin in balloon-injured carotid arteries. MATERIALS AND METHODS: Exosomes were extracted from human coronary artery endothelial cell (HCAEC) culture media. Herb flavonoids and chrysin were the treatments in these atheroprotective models. Western blotting and real-time PCRs were performed. In situ hybridization, immunohistochemistry, and immunofluorescence analyses were employed. RESULTS: MiR-92a increased after balloon injury and was present in HCAEC culture media. Chrysin was treated, and significantly attenuated the miR-92a levels after balloon injury, and similar results were obtained in HCAEC cultures in vitro. Balloon injury-induced miR-92a expression, and attenuated KLF2 expression. Chrysin increased the KLF2 but reduced exosomal miR-92a secretion. The addition of chrysin and antagomir-92a, neointimal formation was reduced by 44.8 and 49.0% compared with balloon injury after 14 days, respectively. CONCLUSION: Chrysin upregulated KLF2 expression in atheroprotection and attenuated endothelial cell-derived miR-92a-containing exosomes. The suppressive effect of miR-92a suggests that chrysin plays an atheroprotective role. Proposed pathway for human coronary artery endothelial cell (HCAEC)-derived exosomes induced by chrysin to suppress microRNA (miR)-92a expression and counteract the inhibitory effect of miR-92a on KLF2 expression in HCAECs. This provides an outline of the critical role of the herbal flavonoid chrysin, which may serve as a valuable therapeutic supplement for atheroprotection.

Effect of atorvastatin on cardiomyocyte hypertrophy through suppressing MURC induced by volume overload and cyclic stretch.

MURC (muscle-restricted coiled-coil protein) is a hypertrophy-related gene. Hypertrophy can be induced by mechanical stress. The purpose of this research was to investigate the hypothesis that MURC mediates hypertrophy in cardiomyocytes under mechanical stress. We used the in vivo model of an aortocaval shunt (AV shunt) in adult Wistar rats to induce myocardial hypertrophy. We also used the in vitro model of cyclic stretch in rat neonatal cardiomyocytes to clarify MURC expression and the molecular regulation mechanism. The flexible membrane culture plate seeding with cardiomyocytes Cardiomyocytes seeded on a flexible membrane culture plate were stretched by vacuum pressure to 20% of maximum elongation at 60 cycles/min. AV shunt induction enhanced MURC protein expression in the left ventricular myocardium. Treatment with atorvastatin inhibited the hypertrophy induced by the AV shunt. Cyclic stretch markedly enhanced MURC protein and mRNA expression in cardiomyocytes. Addition of extracellular-signal-regulated kinase (ERK) inhibitor PD98059, ERK small interfering RNA (siRNA), angiotensin II (Ang II) antibody and atorvastatin before stretch, abolished the induction of MURC protein. An electrophoretic mobility shift assay showed that stretch enhanced the DNA binding activity of serum response factor. Stretch increased but MURC mutant plasmid, ERK siRNA, Ang II antibody and atorvastatin reversed the transcriptional activity of MURC induced by stretch. Adding Ang II to the cardiomyocytes also induced MURC protein expression. MURC siRNA and atorvastatin inhibited the hypertrophic marker and protein synthesis induced by stretch. Treatment with atorvastatin reversed MURC expression and hypertrophy under volume overload and cyclic stretch.

Atorvastatin alleviates cardiomyocyte apoptosis by suppressing TRB3 induced by acute myocardial infarction and hypoxia.

BACKGROUND/PURPOSE: TRB3 (tribbles 3), an apoptosis-regulated gene, increases during endoplasmic reticulum stress. Hypoxia can induce inflammatory mediators and apoptosis in cardiomyocytes. However, the expression of TRB3 in cardiomyocyte apoptosis under hypoxia is not thoroughly known. We investigated the regulation mechanism of TRB3 expression and apoptosis induced by hypoxia in cardiomyocytes. METHODS: An in vivo model of acute myocardial infarction (AMI) was applied in adult Wistar rats to induce myocardial hypoxia. Rat neonatal cardiomyocytes were subjected to 2.5% O2 to induce hypoxia. RESULTS: The expression of TRB3 was evaluated in cultured rat neonatal cardiomyocytes subjected to hypoxia. Hypoxia significantly enhanced TRB3 protein and mRNA expression. Adding c-jun N-terminal kinase (JNK) inhibitor SP600125, JNK small interfering RNA (siRNA), tumor necrosis factor-α (TNF-α) antibody, and atorvastatin 30 minutes before hypoxia reversed the induction of TRB3 protein. A gel-shift assay showed the DNA-binding activity of growth arrest and DNA damage-inducible gene 153 (GADD153), which increased after hypoxia. Hypoxia increased, whereas the TRB3-mut plasmid, SP600125, and TNF-α antibody abolished the hypoxia-induced TRB3 promoter activity. Hypoxia increased the secretion of TNF-α from cardiomyocytes. Exogenous administration of TNF-α recombinant protein to the cardiomyocytes without hypoxia increased TRB3 protein expression, similar to that observed after hypoxia. Hypoxia-induced cardiomyocyte apoptosis is inhibited by TRB3 siRNA, the TNF-α antibody, and atorvastatin. Atorvastatin reduced the TRB3 expression and cardiomyocyte apoptosis induced by AMI. Hypoxia induces TRB3 through TNF-α, JNK, and the GADD153 pathway. CONCLUSION: Treatment of atorvastatin inhibits the expression of TRB3 and cardiomyocyte apoptosis induced by AMI and hypoxia.

MicroRNA-208a Increases Myocardial Endoglin Expression and Myocardial Fibrosis in Acute Myocardial Infarction.

BACKGROUND: MicroRNAs (miRs) play a role in cardiac remodelling, and acute myocardial infarction (AMI) can regulate miR expression. MiR-208a is essential for the expression of the genes involved in cardiac hypertrophy and fibrosis. MiR-208a activates endoglin expression and may result in cardiac fibrosis. The role of miR-208a and endoglin in AMI is not known. We sought to investigate the regulation of miR-208a and endoglin in AMI. METHODS: Ligation of the proximal left anterior descending artery was performed in adult Sprague-Dawley rats to induce AMI. Echocardiography was used to measure heart size and left ventricular function. The TaqMan miR real-time quantitative assay was used to quantitate miR-208a. Myocardial fibrosis was detected by Masson trichrome staining. RESULTS: AMI and overexpression of miR-208a in the sham group without infarction significantly increased myocardial miR-208a, endoglin, and ß-myosin heavy chain (ß-MHC) expression. Overexpression of antagomir-208a significantly inhibited the increase of myocardial endoglin and ß-MHC protein expression induced by infarction. Overexpression of mutant miR-208a in the sham group did not induce myocardial endoglin and ß-MHC expression. Pretreatment with atorvastatin and the angiotensin-receptor antagonist valsartan significantly attenuated the increase of endoglin and ß-MHC induced by infarction. AMI and overexpression of miR-208a in the sham group significantly increased the area of myocardial fibrosis compared with the sham group. Overexpression of antagomir-208a and pretreatment with atorvastatin and valsartan in the AMI group significantly decreased the area of myocardial fibrosis induced by infarction. CONCLUSIONS: MiR-208a increases endoglin expression to induce myocardial fibrosis in rats with AMI. Treatment with atorvastatin and valsartan can decrease myocardial fibrosis induced by AMI through attenuating miR-208a and endoglin expression.

Mechanical Stretch Induces Apoptosis Regulator TRB3 in Cultured Cardiomyocytes and Volume-Overloaded Heart.

The expression of TRB3 (tribbles 3), an apoptosis regulated gene, increases during endoplasmic reticulum (ER) stress. How mechanical stress affects the regulation of TRB3 in cardiomyocytes during apoptosis is not fully understood. An in vivo model of aorta-caval shunt in adult rats demonstrated the increased TRB3 protein expression in the myocardium. The tumor necrosis factor-alpha (TNF-α) antagonist etanercept reversed the TRB3 protein expression and cardiomyocyte apoptosis induced by AV shunt. An in vitro model of cyclic stretch in neonatal rats was also used to investigate TRB3 expression. We hypothesized that cardiomyocyte apoptosis induced by cyclic stretch is TRB3 dependent. Neonatal rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. Cyclic stretch significantly increased TRB3 protein and mRNA expression. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125, TNF-α antibody and etanercept 30 min before stretch reversed the induction of TRB3 protein induced by stretch. Cyclic stretch induced the DNA-binding activity of growth arrest and DNA damaged inducible gene-153 (GADD153) by electrophoretic mobility shift assay. SP600125, JNK siRNA, TNF-α antibody and etanercept abolished the binding activity induced by stretch. TRB3 promoter activity was enhanced by stretch and TRB3-mut plasmid, SP600125, TNF-α antibody and etanercept attenuated TRB3 promoter activity induced by stretch. Exogenous administration of TNF-α recombinant protein to the non-stretched cardiomyocytes increased TRB3 protein expression similar to that seen after stretch. Cyclic stretch induced cardiomyocyte apoptosis is inhibited by TRB3 siRNA and etanercept. The stretch-induced TRB3 is mediated by TNF-α、JNK and GADD153 pathway. These results indicate that TRB3 plays an important role in stretch-induced cardiomyocyte apoptosis.

Hypoxia activates muscle-restricted coiled-coil protein (MURC) expression via transforming growth factor-ß in cardiac myocytes.

The expression of MURC (muscle-restricted coiled-coil protein), a hypertrophy-regulated gene, increases during pressure overload. Hypoxia can cause myocardial hypertrophy; however, how hypoxia affects the regulation of MURC in cardiomyocytes undergoing hypertrophy is still unknown. The aim of the present study was to test the hypothesis that hypoxia induces MURC expression in cardiomyocytes during hypertrophy. The expression of MURC was evaluated in cultured rat neonatal cardiomyocytes subjected to hypoxia and in an in vivo model of AMI (acute myocardial infarction) to induce myocardial hypoxia in adult rats. MURC protein and mRNA expression were significantly enhanced by hypoxia. MURC proteins induced by hypoxia were significantly blocked after the addition of PD98059 or ERK (extracellular-signal-regulated kinase) siRNA 30 min before hypoxia. Gel-shift assay showed increased DNA-binding activity of SRF (serum response factor) after hypoxia. PD98059, ERK siRNA and an anti-TGF-ß (transforming growth factor-ß) antibody abolished the SRF-binding activity enhanced by hypoxia or exogenous administration of TGF-ß. A luciferase promoter assay demonstrated increased transcriptional activity of SRF in cardiomyocytes by hypoxia. Increased ßMHC (ß-myosin heavy chain) and BNP (B-type natriuretic peptide) protein expression and increased protein synthesis was identified after hypoxia with the presence of MURC in hypertrophic cardiomyocytes. MURC siRNA inhibited the hypertrophic marker protein expression and protein synthesis induced by hypoxia. AMI in adult rats also demonstrated increased MURC protein expression in the left ventricular myocardium. In conclusion, hypoxia in cultured rat neonatal cardiomyocytes increased MURC expression via the induction of TGF-ß, SRF and the ERK pathway. These findings suggest that MURC plays a role in hypoxia-induced hypertrophy in cardiomyocytes.

Mechanical stretch via transforming growth factor-ß1 activates microRNA-208a to regulate hypertrophy in cultured rat cardiac myocytes.

BACKGROUND/PURPOSE: MicroRNA-208a (miR208a) and mechanical stress play a key role in cardiac hypertrophy. The relationship between miR208a and mechanical stress in cultured cardiomyocytes has not been investigated. The molecular mechanisms underlying miR208a-induced hypertrophy of cardiomyocytes by mechanical stress is poorly understood. This study investigated whether miR208a is a critical regulator in cardiomyocyte hypertrophy under mechanical stretch. METHODS: Neonatal rat cardiomyocytes grown on a flexible membrane base were stretched at 60 cycles/minute. MiR real-time quantitative assays were used to quantify miRs. A quantitative sandwich enzyme immunoassay technique was used to measure transforming growth factor-ß1 (TGF-ß1). A (3)H-proline incorporation assay was used to measure protein synthesis. RESULTS: Mechanical stretch significantly enhanced miR208a expression. Stretch significantly induced cardiomyocyte hypertrophic protein expression such as ß-myosin heavy chain (MHCß), thyroid hormone receptor-associated protein 100, myostatin, connexin 40, GATA4, and brain natriuretic peptide. MHCα was not induced by stretch. Overexpression of miR208a significantly increased MHCß protein expression while pretreatment with antagomir208a significantly attenuated MHCß protein expression induced by stretch and overexpression of miR208a. Mechanical stretch significantly increased the secretion of TGF-ß1 from cultured cardiomyocytes. Exogenous addition of TGF-ß1 recombinant protein significantly increased miR208a expression and pretreatment with TGF-ß1 antibody attenuated miR208a expression induced by stretch. Mechanical stretch and overexpression of miR208a increased protein synthesis while antagomir208a attenuated protein synthesis induced by stretch and overexpression of miR208a. CONCLUSION: Cyclic stretch enhances miR208a expression in cultured rat cardiomyocytes. MiR208a plays a role in stretch-induced cardiac hypertrophy. The stretch-induced miR208a is mediated by TGF-ß1.

Regulation of PUMA induced by mechanical stress in rat cardiomyocytes.

BACKGROUND: PUMA (p53-up-regulated modulator of apoptosis), an apoptosis regulated gene, increased during endoplasmic reticulum stress. However, the expression of PUMA in cardiomyocytes under mechanical stress is little known. We aimed to investigate the regulation mechanism of PUMA expression and apoptosis induced by mechanical stress in cardiomyocytes. METHODS: Aorta-caval (AV) shunt was performed in adult Wistar rats to induce volume overload. Rat neonatal cardiomyocytes were stretched by vacuum to 20% of maximum elongation at 60 cycles/min. RESULTS: PUMA protein and mRNA were up-regulated in the shunt group as compared with sham group. The increased PUMA protein expression and apoptosis induced by shunt was reversed by treatment with atorvastatin at 30 mg/kg/ day orally for 7 days. TUNEL assay showed that treatment with atorvastatin inhibited the apoptosis induced by volume overload. Cyclic stretch significantly enhanced PUMA protein and gene expression. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125, JNK small interfering RNA (siRNA) and interferon-γ (INF-γ) antibody 30 min before stretch reduced the induction of PUMA protein. Gel shift assay demonstrated that stretch increased the DNA binding activity of interferon regulatory factor-1. Stretch increased, while PUMA-Mut plasmid, SP600125 and INF-γ antibody abolished the PUMA promoter activity induced by stretch. PUMA mediated apoptosis induced by stretch was reversed by PUMA siRNA and atorvastatin. CONCLUSIONS: Mechanical stress enhanced apoptosis and PUMA expression in cardiomyocytes. Treatment with atorvastatin reversed both PUMA expression and apoptosis induced by mechanical stress in cardiomyocytes.

Mechanism of the inhibitory effect of atorvastatin on leptin expression induced by angiotensin II in cultured human coronary artery smooth muscle cells.

Leptin contributes to the pathogenesis of atherosclerosis. Ang II (angiotensin II), a proatherogenic cytokine, increases leptin synthesis in cultured adipocytes. Statin suppresses leptin expression in adipocytes and human coronary artery endothelial cells. However, the effect of Ang II and statin on leptin expression in VSMCs (vascular smooth muscle cells), the major cell types in atheroma, is poorly understood. Thus the aim of the present study was to investigate the molecular mechanism of atorvastatin for reducing leptin expression after Ang II stimulation in VSMCs. VSMCs from human coronary artery were cultured. Ang II stimulation increased leptin protein and mRNA and phospho-JNK (c-Jun N-terminal kinase) expression. Exogenous addition of Dp44mT (2,2'-dipyridyl-N,N-dimethylsemicarbazone) and mevalonate increased leptin protein expression similarly to Ang II. Atorvastatin, SP600125, JNK siRNA (small interfering RNA) and NAC (N-acetylcysteine) completely attenuated the leptin and phospho-JNK protein expression induced by Ang II. Ang II significantly increased ROS (reactive oxygen species) formation in human VSMCs. Addition of atorvastatin and NAC significantly attenuated the formation of ROS induced by Ang II. Addition of atorvastatin and SP600125 inhibited the phosphorylation of Rac1 induced by Ang II. The gel shift and promoter activity assay showed that Ang II increased AP-1 (activator protein-1)-binding activity and leptin promoter activity, while SP600125, NAC and atorvastatin inhibited the AP-1-binding activity and leptin promoter activity induced by Ang II. Ang II significantly increased the migration and proliferation of cultured VSMCs, while addition of atorvastatin, SP600125, NAC and leptin siRNA before Ang II stimulation significantly inhibited the migration and proliferation of VSMCs induced by Ang II. Ang II significantly increased secretion of leptin from human VSMCs, and addition of SP600125, atorvastatin and NAC before Ang II stimulation almost completely inhibited the leptin secretion induced by Ang II. In conclusion, Ang II induces leptin expression in human VSMCs, and atorvastatin could inhibit the leptin expression induced by Ang II. The inhibitory effect of atorvastatin on Ang II-induced leptin expression was mediated by Rac, ROS and JNK pathways.

Mechanical stretch induces the apoptosis regulator PUMA in vascular smooth muscle cells.

AIMS: The expression of PUMA (p53-up-regulated modulator of apoptosis), an apoptosis-regulating gene, increases during endoplasmic reticulum stress. The mechanisms by which cyclic stretch influences the regulation of PUMA in vascular smooth muscle cells (VSMCs) during apoptosis remain unclear. We hypothesized that cyclic stretch enhances PUMA expression in VSMCs undergoing apoptosis. METHODS AND RESULTS: Human VSMCs grown on a Flexcell I flexible membrane base were stretched via vacuum to 20% of elongation at a frequency of 1 Hz. An in vivo model of volume overload with aorta-caval shunt and pressure overload with aortic banding in adult rats was used to study PUMA expression. Cyclic stretch markedly enhanced PUMA protein and gene expression after stretch. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125 and interferon-γ (IFN-γ) antibody 30 min before stretch inhibited PUMA expression. Gel shift assay demonstrated that stretch increased the DNA binding activity of interferon regulatory factor-1 (IRF-1). SP600125, JNK small interfering RNA, and IFN-γ antibody attenuated the DNA binding activity induced by stretch. PUMA-Mut plasmid, SP600125, and IRF-1 antibody attenuated the promoter activity. Stretch increased secretion of IFN-γ from VSMCs, and conditioned media from stretched VSMCs increased PUMA protein expression. The in vivo model of aorta-caval shunt and aortic banding also showed increased PUMA protein expression in the aorta. CONCLUSION: Cyclic mechanical stretch increases PUMA expression in cultured human VSMCs. The PUMA expression induced by stretch is mediated by IFN-γ, JNK, and IRF-1 pathways. These findings suggest that PUMA is an important mediator in VSMC apoptosis induced by stretch.

The molecular regulation of GADD153 in apoptosis of cultured vascular smooth muscle cells by cyclic mechanical stretch.

AIMS: The expression of GADD153 (growth arrest and DNA damage-inducible gene 153), an apoptosis-regulated gene, increases during endoplasmic reticulum (ER) stress. How mechanical stretch affects the regulation of GADD153 in vascular smooth muscle cells (VSMCs) during apoptosis is not fully understood. We aimed to test the hypothesis that mechanical stretch induces GADD153 expression in VSMCs undergoing apoptosis. METHODS AND RESULTS: Rat VSMCs grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. An in vivo model of aorta-caval shunt in adult rats was used to investigate GADD153 expression. Cyclic stretch significantly increased GADD153 protein and mRNA expression after 18 h of stretch. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125, JNK siRNA, tumour necrosis factor-alpha (TNF-alpha) and TNF-alpha receptor antibody 30 min before stretch inhibited the induction of GADD153 protein. Gel shift assay showed that DNA-binding activity of activating factor 1 (AP-1) increased after stretch. SP600125, JNK siRNA and TNF-alpha antibody abolished the binding activity induced by stretch. Stretch increased while GADD153-Mut plasmid, SP600125, and c-jun antibody abolished the promoter activity. Both conditioned media from stretched VSMCs and exogenous administration of TNF-alpha recombinant protein to the non-stretched VSMCs increased GADD153 protein expression similar to that seen after stretch. An in vivo model of aorta-caval shunt in adult rats also demonstrated the increased GADD153 protein expression in the aorta. CONCLUSION: Cyclic stretch enhanced GADD153 expression in cultured rat VSMCs. The stretch-induced GADD153 is mediated by TNF-alpha, at least in part, through the JNK and AP-1 pathway. These findings suggest that GADD153 plays a role in stretch-induced VSMC apoptosis.

Antitumor activity of a novel bis-aziridinylnaphthoquinone (AZ4) mediating cell cycle arrest and apoptosis in non-small cell lung cancer cell line NCI-H460.

AIM: The cytotoxic activities of a series of bis-aziridinylnaphthoquinone, AZ1 to AZ4, on human lung carcinoma cell lines, H460, and normal lung cells fibroblast cell line, MRC-5, and the mechanisms of H460 cells induced by AZ4 were investigated. METHODS: The MTT assay was used to determine the cell proliferation. Cell cycle was analysed by FACS. The activity of caspase 3, 8 and 9 was determined by cell-permeable fluorogenic detection system. Western blot assay was used to evaluate the regulation of cyclin B, Cdc-2, p53, p21, and the Bcl-2 protein. RESULTS: AZ1 to AZ4 displayed various cytotoxicity activities against H460 and MRC-5 cells. Compared to those compounds, AZ4 was with the most effective agent among the 5 tested analogues at reducing H460 cell viability with an IC(50) value of 1.23 micromol/L; it also exhibited weak cytotoxicity against MRC-5 cells with an IC(50) value of 12.7 micromol/L. The results show that growth arrest on the G2-M phase of H460 cells induced by AZ4 for 24 h was discovered, and this might be altered with the reduced Cdc-2 protein expression of 47% at 2.0 micromol/L AZ4, but not with cyclin B protein expression. The AZ4 treated cells were then led to apoptosis after 48 h. This was associated with the activation of apoptotic enzyme caspase 3 and mediated by caspase 8, but not caspase 9 at various concentrations of AZ4 after being cultured for 48 h and 30 h, respectively. The anti-apoptotic protein (Bcl-2) expression in H460 cells altered by 39% with downregulation, and the p53 protein by 25% with upregulation after being cultured with 2.0 micromol/L AZ4 for 48 h. In a time-dependent manner, the expression of the p53 and p21 proteins were increased to the maximum at 24 h, and then decreased at 48. CONCLUSION: AZ4 represents a novel antitumor aziridinylnaphthoquinone with therapeutic potential against the non-small cell lung cancer cells.