Liang Yo Yang

Heme oxygenase-1 inhibits breast cancer invasion via suppressing the expression of matrix metalloproteinase-9

In the present study, we investigated the antitumor effects of the invasiveness and migration of heme oxygenase 1 (HO-1) in human breast carcinoma cells. 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced matrix metalloproteinase-9 (MMP-9) enzyme activity and gene expression at both protein and mRNA levels were examined in human breast carcinoma cells (MCF-7 and MDA-MB-231), and the addition of the MMP-9 inhibitor, SB3CT, significantly suppressed TPA-induced invasion and migration according to the in vitro Transwell assay. Elevation of HO-1 gene expression by ferric protoporphyrin IX inhibited TPA-induced invasion of MCF-7 cells, which was blocked by adding the heme oxygenase inhibitor, tin protoporphyrin IX, or transfection of cells with HO-1 short hairpin RNA. MCF-7 cells overexpressing HO-1 (MCF-7/HO-1) were established in the present study, and TPA-induced MMP-9 gene expression, tumor invasion, and colony formation were significantly reduced in MCF-7/HO-1 cells, compared with those in Neo-transfected cells. Activation of protein kinase Cα/extracellular signal-regulated kinases/AP-1 with stimulation of reactive oxygen species production was involved in TPA-induced invasion of MCF-7 cells, which was attenuated by HO-1 protein induced by ferric protoporphyrin IX or transfection of HO-1 expression vectors. Additionally, the addition of carbon monoxide, but not ferric ions, biliverdin, or bilirubin, inhibited TPA-induced invasion through suppressing MMP-9, extracellular signal-regulated kinases, and AP-1 activation stimulated by TPA. The beneficial role of HO-1 in blocking tumor invasion was first identified in this study. [Mol Cancer Ther 2008;7(5):1195–1206]

Gossypol reduction of tumor growth through ROS‐dependent mitochondria pathway in human colorectal carcinoma cells

Among 13 different cell lines, gossypol (GOS) showed the most potent cytotoxic effect against human colorectal carcinoma cells including HT29, COLO205, COLO320HSR and COLO320DM cells according to an MTT assay. The cytotoxic effect of GOS was mediated by its induction of apoptosis as characterized by the occurrence of DNA ladders, apoptotic bodies and chromosome condensation in both COLO205 and HT29 cells. Activation of caspase 3, 6, 8 and 9, but not caspase 1, accompanied by the appearance of cleaved fragments of PARP (85 kDa), and caspase 3 (p17/p15), was identified in GOS‐treated cells. Decreases in Bcl‐xL and phosphorylated Bad proteins were found in GOS‐treated cells. GOS induction of ROS production was detected by in vitro plasmid digestion, and an increase in the intracellular peroxide level was observed in GOS‐treated COLO205 cells by the DCHF‐DA assay. Antioxidants including N‐acetyl‐L‐cysteine (NAC), catalase (CAT), tempol (TEM) and melatonin (MEL), but not allopurinol (ALL), pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited GOS‐induced Reactive oxygen species (ROS) production through blocking the occurrence of apoptosis. GOS induced mitochondrial dysfunction characterized by a loss of the mitochondria membrane potential via DiOC6 staining, and the release of cytochrome c (Cyt c) and apoptosis‐inducing factor (AIF) from mitochondria to the cytoplasm was observed. Removing mitochondria by ethidium bromide (EtBr) treatment significantly reduced the apoptotic effect of GOS in COLO205 cells. Furthermore, an intraperitoneal injection of GOS or gossypol acetic acid (GAA) significantly reduced the growth of colorectal carcinoma induced by a subcutaneous injection of COLO205 cells in nude mice. Results of the present study provide the first evidences demonstrating the in vitro and in vivo antitumor effects of GOS via an ROS‐dependent mitochondrial apoptosis in colorectal carcinoma.

Resveratrol protects astrocytes against traumatic brain injury through inhibiting apoptotic and autophagic cell death.

Traumatic brain injury (TBI) is often caused by accidents that damage the brain. TBI can induce glutamate excitotoxicity and lead to neuronal and glial cell death. In this study, we investigated the mechanism of cell death during the secondary damage caused by TBI in vivo and in vitro, as well as the protective effect of resveratrol (RV). Here we report that glycogen synthase kinase-3β (GSK-3β) activation and microtubule-associated protein light chain 3 processing were induced in rat brains exposed to TBI. In the in vitro TBI model, apoptotic and autophagic cell death were induced through glutamate-mediated GSK-3β activation in normal CTX TNA2 astrocytes. The GSK-3β inhibitor SB216763 or transfection of GSK-3β small-interfering RNA increases cell survival. By contrast, overexpression of GSK-3β enhanced glutamate excitotoxicity. Administration of RV reduced cell death in CTX TNA2 astrocytes by suppressing reactive oxygen species (ROS)-mediated GSK-3β activation, the mechanism by which RV also exerted protective effects in vivo. Mitochondrial damages, including the opening of mitochondrial permeability transition pore (MPTP) and mitochondrial depolarization, were induced by glutamate through the ROS/GSK-3β pathway. Moreover, cyclosporine A, an MPTP inhibitor, suppressed mitochondrial damage and the percentages of cells undergoing autophagy and apoptosis and thereby increased cell survival. Taken together, our results demonstrated that cell death occurring after TBI is induced through the ROS/GSK-3β/mitochondria signaling pathway and that administration of RV can increase cell survival by suppressing GSK-3β-mediated autophagy and apoptosis. Therefore, the results indicated that resveratrol may serve as a potential therapeutic agent in the treatment of TBI.

Hyaluronic acid inhibits the glial scar formation after brain damage with tissue loss in rats.

BACKGROUND Brain tissue scarring (gliosis) was believed to be the major cause of epileptic focus after brain injury, and prevention of scarring could reduce the incidence of seizure. We tried the HA coating onto the cortical brain defect of Spraque-Dawley rats to reduce the marginal glial scarring. METHODS A 4 x 2 x 2 mm(3) cortical defect was created in the brain of Spraque-Dawley rats. Three percent HA gel was coated onto the lesion for the experimental groups and normal saline solutions for the control groups. The brain was retrieved 4, 8, and 12 weeks after treatment. The brains were then sectioned and processed for H&E and GFAP staining, and the thickness of the scarring and the number of GFAP+ cells were analyzed. RESULTS The thickness of cutting marginal gliosis was significantly decreased in the HA groups. The 12-week HA group showed the smallest thickness of gliosis, whereas the 12-week control group exhibited the largest thickness of gliosis. The significant difference in the thickness of gliosis was also noted between the HA and the control groups 8 weeks after treatment. The number of GFAP+ cells was also significantly decreased in the HA groups when compared to the respective control group 4, 8, and 12 weeks after the surgery. CONCLUSION The results support the hypothesis that HA inhibits glial scarring not only by decreasing the thickness of gliosis but also by reducing the number of the glial cells. Furthermore, our results suggest that HA might be used to reduce glial scar formation in central nervous system surgery, which subsequently prevents the post-operation or posttraumatic seizure incidence.

IGF-I plus E2 induces proliferation via activation of ROS-dependent ERKs and JNKs in human breast carcinoma cells

Induction of 17β‐estradiol (E2) and insulin‐like growth factor‐I (IGF‐I) has been detected in breast carcinoma, however the interaction between E2 and IGF‐I in the proliferation of breast carcinoma cells is still unclear. In the present study, we found that IGF‐I enhances the E2‐induced proliferation in MCF‐7 human breast carcinoma cells in accordance with stimulation of colony formation via a soft agar assay. Activation of insulin receptor substrate‐1 (IRS‐1) protein and extracellular signal‐related kinases (ERKs) and c‐Jun N‐terminal kinases (JNKs), but not p38 mitogen‐activated protein kinase (MAPK), via phosphorylation induction was detected in MCF‐7 cells treated with IGF‐I plus E2 (E2/IGF‐I). E2/IGF‐I‐induced proliferation was blocked by chemical inhibitors of ERKs (PD98059) and JNKs (SP600125). An increase in the expression of c‐Jun protein was detected in E2/IGF‐I‐treated MCF‐7 cells, and this was inhibited by PD98059 and SP600125. Transfection of the dominant negative MEKK and JNK plasmids significantly reduced E2/IGF‐I‐induced proliferation with suppression of c‐Jun protein expression. An increase in peroxide production was detected in E2/IGF‐I‐treated cells, and N‐acetyl‐L‐cysteine (NAC) and Tiron (TIR) addition significantly inhibited E2/IGF‐I‐induced cell proliferation with blocking of the phosphorylation of ERKs and JNKs, and the expression of c‐Jun protein. Additionally, 3‐OH flavone, baicalein, and quercetin showed effective inhibitory activities against E2/IGF‐I‐induced proliferation through suppressing proliferative events such as phosphorylation of IRS‐1, ERKs, and JNKs proteins, and induction of c‐Jun protein and colony formation. These results indicate that IGF‐I interacts with E2 to promote the proliferation of breast carcinoma cells via ROS‐dependent MAPK activation and c‐Jun protein expression. The structure‐related inhibition of E2/IGF‐I‐induced proliferative events by flavonoids is elucidated. J. Cell. Physiol. 212:666–674, 2007.

Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As +3 - and MMA +3 -induced apoptosis through inhibition of telomerase activity via JNK activation

The effects of six arsenic compounds including As(+3), MMA(+3), DMA(+3), As(+5), MMA(+5), and DMA(+5) on the viability of NIH3T3 cells were examined. As(+3) and MMA(+3), but not the others, exhibited significant cytotoxic effects in NIH3T3 cells through apoptosis induction. The apoptotic events such as DNA fragmentation and chromosome condensation induced by As(+3) and MMA(+3) were prevented by the addition of NAC and CAT, and induction of HO-1 gene expression in accordance with cleavage of the HSP90 protein, and suppression of telomerase activity were observed in NIH3T3 cells under As(+3) and MMA(+3) treatments. An increase in the intracellular peroxide level was examined in As(+3)- and MMA(+3)-treated NIH3T3 cells, and As(+3)- and MMA(+3)-induced apoptotic events were blocked by NAC, CAT, and DPI addition. HSP90 inhibitors, GA and RD, significantly attenuated the telomerase activity in NIH3T3 cells with an enhancement of As(+3)- and MMA(+3)-induced cytotoxicity. Suppression of JNKs significantly inhibited As(+3)- and MMA(+3)-induced apoptosis by blocking HSP90 protein cleavage and telomerase reduction in NIH3T3 cells. Furthermore, Hb, SnPP, and dexferosamine showed no effect against As(+3)- and MMA(+3)-induced apoptosis, and overexpression of HO-1 protein or inhibition of HO-1 protein expression did not affect the apoptosis induced by As(+3) or MMA(+3). These data provide the first evidence to indicate that apoptosis induced by As(+3) and MMA(+3) is mediated by an ROS-dependent degradation of HSP90 protein and reduction of telomerase via JNK activation, and HO-1 induction might not be involved.

The influence of masticatory hypofunction on developing rat craniofacial structure.

The purpose of this study was to use botulinum neurotoxin type A (BoNT/A) selectively to evaluate the influence of localized masticatory atrophy and paresis on craniofacial growth and development. 60 growing rats, 4 weeks old, weighing approximately 120g, were randomly divided according as follows (Long-Evans, N=15 per group): I (Mb+Tns); II (Mns+Tb); III (Mb+Tb); IV (Mns+Tns), where Mb or Tb is the BoNT/A-injected masseter or temporalis muscles (1.0U/muscle, 2.5ml) and Mns or Tns is the saline-injected muscles (2.5ml). After 7 weeks, the mature rats were killed, the muscles dissected and mean muscle mass recorded. Anthropometric cranial, maxillary and mandibular measurements were taken from the dried skulls. Changes in animal weight during the growth period were not statistically significant. The mean masticatory muscle mass was smaller for the BoNT/A-injected muscles of Mb and Tb. Anthropometric measurements of bony structures inserted by masseter and temporalis muscles revealed a significant treatment effect. The measurements showed a facial morphology typical of a dolichofacial profile: short upper face accompanied by a long lower face with an extended mandibular length and ramus height and constricted bicoronoidal and bigonial widths. The results suggest that induction of localized masticatory muscle atrophy with BoNT/A alters craniofacial growth and development.

Reactive Oxygen Species-Dependent Nitric Oxide Production in Reciprocal Interactions of Glioma and Microglial Cells

Conditioned mediums (CMs) from glioma cells U87, GBM‐8401, and C6 significantly induced iNOS protein and NO production by microglial cells BV‐2 but without altering the cell viability or cell‐cycle progression of BV2 microglia. Significant increases in intracellular peroxide by U87‐CM and C6‐CM were detected by a DCHF‐DA assay, and vitamin (Vit) C and N‐acetyl cysteine (NAC)‐reduced intracellular peroxide levels elicited by CMs lead to inhibition of iNOS/NO production The extracellular signal‐regulated kinase (ERK) inhibitor, U0126, and c‐Jun N‐terminal kinase (JNK) inhibitor, SP600125, suppressed U87‐CM‐ and C6‐CM‐induced iNOS/NO production by respectively blocking phosphorylated ERK (pERK) and JNK (pJNK) protein expressions stimulated by U87‐CM and C6‐CM. Increased migration of U87 and C6 glioma cells by a co‐culture with BV‐2 microglial cells or adding the nitric oxide (NO) donor, sodium nitroprusside (SNP) was observed, and that was blocked by adding an NO synthase (NOS) inhibitor, N‐nitro L‐arginine methyl ester (NAME). Contributions of ROS, pERK, and pJNK to the migration of glioma cells was further demonstrated in a transwell coculture system of U87 and C6 gliomas with BV‐2 microglial cells. Furthermore, expressions of tumor necrosis factor (TNF)‐α and monocyte chemoattractant protein (MCP)‐1 messenger (m)RNA in U87 and C6 cells were detected by an RT‐PCR, and TNF‐α and MCP‐1 induced iNOS protein expression in time‐ and concentration‐dependent manners. Neutralization of TNF‐α or MCP‐1 in U87‐CM and C6‐CM using a TNF‐α or MCP‐1 antibody inhibited iNOS protein expression, and increased intracellular peroxide by TNF‐α or MCP‐1 was identified in BV‐2 cells. The reciprocal activation of glioma cells and microglia via ROS‐dependent iNOS/NO elevation at least partially mediated by TNF‐α and MCP‐1 is elucidated. J. Cell. Physiol. 229: 2015–2026, 2014.

Morphologic and Bony Structural Changes in the Mandible After a Unilateral Injection of Botulinum Neurotoxin in Adult Rats

PURPOSE Previous animal experiments showed how reduced masticatory function plays an important role in the craniofacial and mandibular regions in the growing period. However, articles focusing on the effects of reduced masticatory function in adult animals are quite scarce. The purpose of this study was to evaluate the effects of reduced masticatory muscle activity on the jaw bones of adult rats. MATERIALS AND METHODS Botulinum neurotoxin type A (BTX-A) was injected into the left masseter muscle to reduce masticatory muscle function, and sequential changes in the jaw bones were investigated. Ten male 60-day-old Sprague-Dawley rats with a mean body weight of about 300 g were used. The following procedures were performed on each sample: 25 U/mL (0.3 mL) of BTX-A was injected into the left masseter muscle, whereas the right masseter muscle received an equal amount of sterilized saline solution. Ninety days after the procedure, the rats were sacrificed, and the weights of the masseter muscles were recorded. Meanwhile, direct anthropometric measurements were taken, and the cortical thickness, trabecular thickness, and bone mineral content of the skull and mandible were measured. All measurements were evaluated with statistical software. RESULTS There were significant differences in all measurements on the BTX-A side compared with those on the control side. The differences included a decreased ramus height, increased gonial angle, and increased crown height of the posterior teeth on the injection side. Significant decreases in the muscle weight, bone mineral content, cortical thickness, and trabecular thickness were also seen on the paralyzed side compared with the non-injection side. CONCLUSIONS We found that reduced masticatory function in adult rats affected the weight of the masseter muscle and the bony structure and dentition.

Quercetin enhancement of arsenic-induced apoptosis via stimulating ROS-dependent p53 protein ubiquitination in human HaCaT keratinocytes.

Abstract:  In this study, QUE, but not the structurally related chemical, rutin, enhanced the cytotoxicity of arsenic trioxide (As+3) against the viability of normal human HaCaT keratinocytes via induction of apoptosis. QUE enhancement of As+3‐mediated apoptosis was accompanied by increased intracellular peroxide production according to a DCFH‐DA analysis, and DNA ladders induced by QUE/As+3 were inhibited by adding the antioxidative compound, N‐acetyl cysteine (NAC). A loss of the mitochondrial membrane potential by QUE/As+3 was observed according to DiOC6 staining in concert with increased Bax protein and cytosolic cytochrome (Cyt) c protein expression in HaCaT cells, which was prevented by the addition of NAC. A decrease in the p53 protein with increased protein ubiquitination was detected in QUE/As+3‐treated HaCaT cells, and this was prevented by the addition of NAC. The decrease in the p53 protein by QUE/As+3 was reversed by adding the proteasome inhibitor, MG132. L‐Buthionine sulphoximine (BSO) enhanced the cytotoxicity of As+3 against the viability of HaCaT cells with reduced p53 protein through inducing protein ubiquitination and reactive oxygen species (ROS) production, and disrupting the mitochondrial membrane potential in HaCaT cells. Additionally, QUE and BSO enhanced the cytotoxic effects of monomethylarsonous acid (MMA+3) but not other arsenic compounds in accordance with increased p53 protein ubiquitination in HaCaT cells. QUE plus As+3 stimulation of apoptosis in human HaCaT keratinocytes via activating ROS‐dependent p53 protein ubiquitination may offer a rationale for the use of QUE to improve the clinical efficacy of arsenics in treating psoriasis.