Docosahexaenoic Acid Reverses Epithelial-Mesenchymal Transition and Drug Resistance by Impairing the PI3K/AKT/ Nrf2/GPX4 Signalling Pathway in Docetaxel-Resistant PC3 Prostate Cancer Cells.

Drug resistance is a serious problem in cancer therapy. Growing evidence has shown that docosahexaenoic acid has anti-inflammatory and chemopreventive abilities. Studies have shown that autophagy inhibition and ferroptosis are promising therapeutic strategies for overcoming multidrug resistance. This study was aimed to examine whether docosahexaenoic acid (DHA) could reverse docetaxel resistance in prostate cancer cells. Cell survival was examined by MTT and colony formation. Protein expression was determined by Western blot. Reactive oxygen species (ROS) production was measured by flow cytometry. DHA displayed anti-cancer effects on proliferation, colony formation, migration, apoptosis, autophagy and epithelial mesenchymal transition. Glutathione-S-transferase π is an enzyme that plays an important role in drug resistance. DHA inhibited GSTπ protein expression and induced cytoprotective autophagy by regulating the PI3K/AKT signalling pathway in PC3R cells. DHA combined with PI3K inhibitor (LY294002) enhanced apoptosis by alleviating the expression of LC3B, (pro-) caspase- 3 and (uncleaved) PARP. DHA induced ferroptosis by attenuating the expression of glutathione peroxidase 4 (GPX4) and nuclear erythroid 2-related factor 2 (Nrf2). DHA-treated PC3R cells produced ROS. The ROS and cytotoxicity were reversed by treatment with ferrostatin-1. DHA combined with docetaxel inhibited EMT by regulating the expression of E-cadhein and N-cadherin. In summary, DHA reversed drug resistance and induced cytoprotective autophagy and ferroptosis by regulating the PI3K/AKT/Nrf2/GPX4 signalling pathway in PC3R cells. We propose that DHA could be developed as a chemosensitizer and that the PI3K/AKT /Nrf2/GPX4 signalling pathway might be a promising therapeutic target for overcoming cancer drug resistance.

Assembly of body-centered cubic crystals in hard spheres.

We investigate the crystallization of monodisperse hard spheres confined by two square patterned substrates (possessing the basic character of the body-centered cubic (bcc) crystal structure) at varying substrate separations via molecular dynamics simulation. Through slowly increasing the density of the system, we find that crystallization under the influence of square patterned substrates can set in at lower densities compared with the homogeneous crystallization. As the substrate separation decreases, the density, where crystallization occurs (i.e., pressure drops), becomes small. Moreover, two distinct regimes are identified in the plane of bcc particle fraction and density for the separation range investigated. For large substrate separations, the bcc particle fraction displays a local maximum as the density is increased, and the resulting formed crystals have a polycrystalline structure. However, and more importantly, another situation emerges for small substrate separations: the capillary effects (stemming from the presence of two substrates) overwhelm the bulk driving forces (stemming from the spontaneous thermal fluctuations in the bulk) during the densification, eventually resulting in the formation of a defect-free bcc crystal (unstable with respect to the bulk hard-sphere crystals) by using two square patterned substrates.

Dense packing in the monodisperse hard-sphere system: a numerical study.

We report a numerical study of the close packing of monodisperse hard spheres. The close packings of hard spheres are produced by the Lubachesky-Stillinger (LS) compression algorithm and span the range from the disordered states to the ordered states. We provide quantitative evidence for the claim that the density and structural order of the arrested close packing can be determined by the compression rate, i.e., with slower rates producing denser and more ordered structures. Through deeply analyzing the structure of the resulting arrested close packings, a transition region has been identified in the plane of density and reciprocal compression rate, in between what have been historically thought of as amorphous and crystalline packings. We also find clear system size dependences in studying the structural properties of the packings from the disordered ones to the ordered ones. These detailed investigations, on the structure of the arrested close packings, may provide a link between the glassy states and the crystalline states in the hard spheres.

Isolation of choline monooxygenase (CMO) gene from Salicornia europaea and enhanced salt tolerance of transgenic tobacco with CMO genes.

Glycinebetaine (GB) is an osmoprotectant accumulated by certain plants in response to high salinity, drought, and cold stress. Plants synthesize GB via the pathway choline --> betaine aldehyde --> glycinebetaine, and the first step is catalyzed by choline monooxygenase (CMO). In the present study, by using RT-PCR and RLM-RACE, a full-length CMO cDNA (1844 bp) was cloned from a halophyte Salicornia europaea, which showed high homology to other known sequences. In order to identify its function, the ORF of CMO cDNA was inserted into binary vector PBI121 to construct the chimeric plant expression vector PBI121-CMO. Using Agrobacterium (LBA4404) mediation, the recombinant plasmid was transferred into tobacco (Nicotiana tabacum). The PCR, Southern blot and RT-PCR analysis indicated the CMO gene was integrated into the tobacco genome, as well as expressed on the level of transcription. The transgenic tobacco plants were able to survive on MS medium containing 300 mmol/L NaCl and more vigorous than those of wild type with the same concentration salt treatment. In salt-stress conditions, transgenic plants had distinctly higher chlorophyll content and betaine accumulation than that of the control, while relative electrical conductivity of transgenic plants was generally lower. The results suggested the CMO gene transformation could effectively contribute to improving tobacco salt-resistance.

First Report of a Pestalotiopsis sp. Causing Leaf Spot of Blueberry in China.

In August 2006, leaf spots were observed on half-high blueberry (Vaccinium corymbosum) in a plant nursery in Dalian, China. The symptomatic potted 1-year-old blueberry plants were located in parts of a plant nursery with poor ventilation. The primary symptom was a leaf spot, 0.4 to 0.8 cm in diameter, with brown margins that enlarged and coalesced. Mycelium grew from symptomatic and green leaf tissue removed from the margin of a necrotic leaf spot. Plant tissues were surface disinfested with 0.1% mercuric chloride for 3 min and 70% ethyl alcohol for 30 s before plating onto potato dextrose agar. The resulting colonies were white with a regular margin and a rough surface. The cultures were covered with black and globular acervuli with a diameter of 100 to 200 µm. The base of each conidiophore was swollen and globose with phialides growing from the apical end. Mature conidia were straight to fusiform, measuring 19.0 to 27.5 × 6.3 to 9.2 µm, and five-celled with the three middle cells brown and darker than the end cells. The apical cell was triangular and hyaline with three simple setulae that were 17.2 to 29.7 µm long. The base cell terminated in a point 4.0 to 8.6 µm long. Koch's postulates were fulfilled for the fungus by spray inoculating two healthy young plants with 2 × 105 conidia per ml of sterile distilled water. As a control, two similar plants were sprayed with sterile water. Plants were placed inside plastic bags to maintain humidity and incubated in a growth chamber at 26°C under fluorescent light for 14 h and at 20°C in darkness for 10 h. After 3 days, the plastic bags were removed and plants were maintained under the same conditions. More than 20 days after inoculation, symptoms on inoculated plants were similar to those previously described in the nursery. Control plants did not show any symptoms. Cultures isolated from the lesions were similar to those isolated previously from plants in the nursery. The morphological descriptions and measurements were similar to Pestalotiopsis clavispora (1). The 5.8S subunit and flanking internal transcribed spacers (ITS1 and ITS2) of rDNA and partial ß-tubulin gene were amplified from DNA extracted from single-spore cultures using the ITS1/ITS4 and T1/Bt2b primers (2) respectively, and sequenced (GenBank Accession Nos. EF119336 and EF152585). The ITS sequences were most similar to the ITS regions of P. clavispora TA-8 (98%; GenBank Accession No. AY924264), P. clavispora TA-6 (98%; GenBank Accession No. AY924263), and P. clavispora PSHI 2002 Endo 389 (96%; GenBank Accession No. AY682929). The partial ß-tubulin gene sequence was identical to Pestalotiopsis sp. isolate PSHI 2004 Endo 86 (100%; GenBank Accession No. DQ657901). The morphology and sequence data support the identity of the causal fungus as P. clavispora. To our knowledge, this is the first report on the presence of a Pestalotiopsis sp. causing a disease of blueberry in China. References: (1) E. F. Guba. Monograph of Monochaetia and Pestalotia. Harvard University Press, Cambridge, MA, 1961. (2) W. Tao et al. Mol. Cell Biol. 27:689, 2007.

Altered expression of estrogen receptor alpha and beta in advanced gastric adenocarcinoma: correlation with prothymosin alpha and clinicopathological parameters.

AIMS: We aimed to investigate the sources of estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta) and estimate the value of both ER subtypes in gastric adenocarcinoma and analyze the possible relationship of prothymosin alpha (ProTalpha) to ERs. METHODS: ERs at the mRNA and protein levels in matched advanced gastric adenocarcinomas and surrounding non-cancerous tissues were examined by using reverse transcription-polymerase chain reaction and immunohistochemical (IHC) methods. Cell proliferation related protein ProTalpha was also detected in IHC. The immunoreactive signal, corresponding to the proteins expression level, was quantitatively analyzed. RESULTS: Both ERalpha and ERbeta mRNAs were detected in most of the cancer and matched normal tissues analyzed. At the protein level, the percentage of ERalpha and ERbeta positive cases changed. ERalpha immunoreactivity was only detected in poorly differentiated adenocarcinoma and ERalpha positive expression correlated with depth of invasion of the tumors. Compared with non-cancerous tissues, gastric tumors showed decreased ERbeta expression and lost ERbeta. Altered ERbeta in gastric adenocarcinoma correlated with decreased differentiation. And the tumors involved lymph node metastasis showed significantly lower expression level of ERbeta. ProTalpha in ERbeta-positive tumors showed higher expression than that in lost ERbeta tumors. CONCLUSIONS: Altered expression of ERalpha and ERbeta in tumors compared with corresponding normal gastric tissues was more common in poorly differentiated adenocarcinomas and related to malignant properties, such as lymph node metastasis. Decreased ERbeta and increased ProTalpha expression in advanced gastric adenocarcinoma indicated that ERbeta may play an anti-proliferation role which is opposed to the role of ProTalpha in gastric epithelium.

First Report of Alternaria tenuissima Causing Disease on Blueberry in China.

During September 2006, disease symptoms were observed on mature highbush blueberry (Vaccinium corymbosum L.) cvs. Bluecrop and Covoille in a blueberry commercial field in Dalian, China. The maximum and minimum rainfalls in June to September are 3,111.9 and 1,745.6 ml, respectively. The highest temperature during the summer is 35.3°C and relative humidity may achieve 90%. Circular to irregular, light brown-to-gray leaf spots with brownish red borders, initially 3 to 7 mm in diameter, enlarged and coalesced. Reddish, circular spots appeared on stems, developing small, insignificant cankers. A fungus was recovered on potato dextrose agar (PDA, pH nature) from the margin of necrotic leaf spots. Morphological traits of the strain that developed from a single-spore culture were as follows: colonies were regular and flat, with a rough upper surface that peripherally was olive-green with a black center and dull white spots; short conidiophores arising singly and measuring 81.6 to 163.2 × 4.1 to 8.2 µm; conidia was abundant, ovoid, and obclavate muriformly septate, which horizontal and vertical septations varied from 1 to 6 and 0 to 2, respectively, and its size varied from 26 to 48.8 × 9.7 to 16.3 µm with an average beak length of 9.6 µm, and sporulation pattern is budding. Conidia derived from conidiophores. Koch's postulates were fulfilled for the isolates by spray inoculating two healthy mature plants with 2 × 105 conidia per ml homogenized in sterile water. As a control, two plants were sprayed with sterile water. Plants were placed inside plastic bags to maintain humidity and incubated in a growth chamber at 26°C under fluorescent light for 14 h and 20°C in darkness for 10 h. After 2 days, the plastic bags were removed and plants were maintained under the same conditions for 30 days. Symptoms on inoculated plants were similar to those previously observed. Symptoms were not observed on control plants. Cultures isolated from inoculated plants had the same morphological traits as those that were isolated previously from the field plants. The morphological descriptions and measurements were similar to Alternaria tenuissima (2). The 5.8S subunit and flanking internal transcribed spacers (ITS1 and ITS2) of rDNA and partial cds histone gene were amplified from DNA extracted from single-spore cultures using the ITS1/ITS4 and H3-1a/H3-1b primers, respectively, and sequenced (GenBank Accession No. EF031053) (1,3). The ITS sequence was identical to the ITS regions of A. tenuissima strain EGS34-015 (100%; GenBank Accession No. AY751455), the partial cds histone gene sequence was similar to A. tenuissima isolate MA6 (99%; GenBank Accession No. AF404634). The morphology, secondary conidiation, and sequences of ITS and partial cds histone gene identify the causal fungus as A. tenuissima. To our knowledge, this is the first report on the presence of A. tenuissima affecting blueberry plants in China. References: (1) J. C. Kang et al. Mycol. Res. 106:1151, 2002. (2) E. G. Simmons. Mycotaxon 70:325, 1999. (3) T. J. White et al. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

Protocatechuic acid suppresses MPP+ -induced mitochondrial dysfunction and apoptotic cell death in PC12 cells.

Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia (A.) oxyphylla, showed antioxidant neuroprotective effect in our previous study. Here, we investigated the effect of PCA on the MPP(+)-induced mitochondrial dysfunction and apoptotic cell death in PC12 cells. The apoptosis in MPP(+)-induced PC12 cells was associated with loss of mitochondrial membrane potential, the formation of reactive oxygen species (ROS), GSH depletion, activation of caspase-3 and down-regulation of Bcl-2. In contrast, treatment of PC12 cells with PCA significantly prevented the above-mentioned mitochondrial dysfunction. Our data pointed to the potential clinical application/use of PCA to overcome neurodegenerative diseases such as Parkinson's disease.

Protocatechuic acid from Alpinia oxyphylla against MPP+-induced neurotoxicity in PC12 cells.

An ethyl acetate extract of Alpinia oxyphylla was found to possess neuroprotective activity against 1-methyl-4-phenylpyridinium ion (MPP(+)) induced apotosis and oxidative stress in cultured PC12 cells. From the extract, a phenolic compound was isolated through bioassay-guided fractionation and identified as protocatechuic acid (PCA) by IR, MS, and (1)H and (13)C NMR spectroscopy. It was the first time which was isolated from the kernels of A. oxyphylla. Exposure of PC12 cells to 1mM MPP(+) may cause significant viability loss and apoptotic cell death. PCA stimulated PC12 cellular proliferation and markedly attenuated MPP(+)-induced apoptotic cell death in a dose-dependent manner. By observing the nuclear morphological changes and flow cytometric analysis, PCA showed its significant effect on protecting PC12 cells against MPP(+)-induced apoptosis. Meanwhile, PCA enhanced the activities of superoxide dismutase (SOD) and catalase (CAT) in PC12 cells. In addition, PCA also dose-dependently reduced the hydrogen peroxide (H(2)O(2))- or sodium nitroprusside (SNP)-induced cell death in PC12 cells. The results suggest that PCA may be one of the primary active components in the kernels of A. oxyphylla and provide a useful therapeutic strategy for the treatment of oxidative stress-induced neurodegenerative disease such as Parkinson's disease.

First Report of Blueberry Leaf Spot Caused by Cylindrocladium colhounii in China.

During late July and early August of 2005, leaf spot symptoms were observed in a blueberry nursery at a plantation in Dalian, which to our knowledge, lies within the largest blueberry-production area in China. Symptoms were observed primarily on lowbush species, for example Blomidon, as well as half-highbush cultivars. A slow-growing, white mycelium from the margin of necrotic leaf spots was recovered on potato dextrose agar (PDA). The following morphological traits were observed: erect conidiophores that branch twice and were terminated in a stiped, clavate phialide; hyaline, cylindrical, four-celled conidia; and globose, reddish brown, aggregated chlamydospores. Conidiophores (including stipes and terminal phialides) were 305 to 420 × 5 to 9 µm; primary branches were 9 to 45 × 5 to 6.3 µm; secondary branches were 9 to 17.3 × 3.1 to 4.5 µm; phialides were 7.8 to 17.5 × 2.5 to 6 µm; stipes (from the highest branch area to vesicle) were 150 to 270 µm long; and vesicles were 13 to 30 × 2 to 4.5 µm. Conidia were 50 to 72 × 4 to 5.5 µm. Chlamydospores were 15 to 20 µm in diameter. Koch's postulates were fulfilled by spray inoculating two healthy cultivars with conidiophores homogenized in axenic water. As a control, two healthy plants were sprayed with axenic water. Plants were placed inside plastic bags to maintain humidity and incubated in a growth chamber at 26°C under fluorescent light for 14 h and 20°C in darkness for 10 h. After 2 days, the plastic bags were removed and plants were maintained under the same conditions. After 4 days, small-to-medium brown spots with purplish margins were observed on the adaxial side of leaves from inoculated plants, but not from control plants. Fungi isolated from these lesions had the same morphological traits as the ones isolated previously from field plants. The morphological descriptions and measurements were similar to Cylindorocladium colhounii (2). The 5.8S subunit and flanking internal transcribed spacers (ITS1 and ITS2) of rDNA and the ß-tubulin gene were amplified from DNA extracted from single-spore cultures using the ITS1/ITS4 primers and T1/Bt2b primers, respectively, and sequenced (1). The ITS and ß-tubulin gene sequences were similar to C. colhounii STE-U 1237 (99%; GenBank Accession No. AF231953) and C. colhounii STE-U 705 (99%; GenBank Accession No. AF231954), respectively. The morphology, secondary conidiation, and sequences of ITS and ß-tubulin gene identify the causal fungus as C. colhounii. To our knowledge, this is the first report of C. colhounii on blueberry in China or in the world. References: (1) P. W. Crous et al. Can. J. Bot. 77:1813, 1999. (2) T. Watanabe. Page 222 in: Dictorial Atlas of Soil and Seed Fungi. CRC Press, Inc., Boca Raton, Fl, 1994.

First Report of Sweet potato leaf curl virus in China.

During the 2004 growing season in the Liaoning Province in China, where there was large population of whiteflies, several sweet potato (Ipomoea batatas) breeding lines showed leaf curl symptoms. A survey was conducted to determine the incidence of Sweet potato leaf curl virus (SPLCV) in China. Sixteen plants were collected and stem scions from those plants were graft inoculated to Ipomoea nil. Three weeks later, the indicator developed symptoms of leaf curling, interveinal chlorosis, and stunting. Total nucleic acid was extracted from young leaves of sweet potato and then evaluated using polymerase chain reaction (PCR). Primers, developed by Briddon and Markham (1) and used as universal primers for amplification of the geminivirus DNA fragment, were BM-V (5'-KSG GGT CGA CGT CAT CAA TGA CGT TRT AC-3') and BM-C (5'-AAR GAA TTC ATK GGG GCC CAR ARR GAC TGG C-3'). Amplified fragments with BM primers theoretically should have sizes almost equal to the full length of the DNA A component of the bipartite genome (2). Expected DNA fragments of 2.8 kb that contained the AV1, AV2, AC1, AC2, AC3, and AC4 open reading frames were obtained from symptomatic, but not from symptomless (uninfected) plants. The 2.8-kb fragments obtained by amplification were purified and cloned into the PMD18-T vector. Recombinant plasmids were then transformed into competent cells of Escherichia coli strain DH5(. The fragment was sequenced (GenBank Accession No. DQ512731), and nucleotide sequence of corresponding regions were compared with a published sequence of SPLCV available in GenBank (Accession No. AF104036). The AC4 and AC2 genes showed the highest (92%) and the lowest (83%) identity, respectively. This virus has been reported in the United States, Taiwan, Japan, and Peru. To our knowledge, this is the first report of the natural occurrence of SPLCV in China. References: (1) R. W. Briddon and P. G. Markham. Mol. Biotechnol. 1:202, 1994. (2) M. Onuki and K. Hanada. Ann. Phytopathol. Soc. Jpn. 64:116, 1998.

Catalpol inhibits apoptosis in hydrogen peroxide-induced PC12 cells by preventing cytochrome c release and inactivating of caspase cascade.

In the present study, using a rat pheochromocytoma (PC12) cell line, the effect of catalpol on H2O2-induced apoptosis was studied. The apoptosis in H2O2-induced PC12 cells was accompanied by down-regulation of Bcl-2, up-regulation of Bax, the release of mitochondrial cytochrome c to cytosol and sequential activation of caspase-1 and caspase-3 then leading to cleavage of poly-ADP-ribose polymerase (PARP). Catalpol not only suppressed the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol, but also attenuated caspase-3 activation, PARP cleavage, and eventually protected against H2O2-induced apoptosis. Taken together, these results suggest that treatment of PC12 cells with catalpol can block H2O2-induced apoptosis by the regulation of Bcl-2 family members, as well as suppression of cytochrome c release and caspase cascade activation.

[Study on the direct interaction between aminoacylase and Cu(II) ions by spectroscopic analysis].

In this paper, the method of reconstitution was used to investigate the interaction between metalloenzymes (containing Zn (II) and metal ions. Electron paramagnetic resonance(EPR), visible spectrum (Vis) and enzyme activity assay have been employed to study the direct interactions between aminoacylase (ACY) and Cu(II) ions added in aqueous solution. The results show that a dynamic equilibrium exists between the Zn(II) in the active site of native enzymes and the added Cu(II), the added Cu(II) partly replaces the Zn(II), forming Cu(II)-enzyme derivatives. As a result, the activity of the native enzymes is influenced. In addition, the influences of pH value on this kind of interaction have also been investigated, and the results demonstrate that the decrease in the intensity of the Cu (II) EPR signal and the change of place signal in Vis were observed as increase of pH value. These results suggest that the derivative of Cu(II)-ACY exists in solution with two different conformations, and this two conformations exchanged each other depending on pH.

[Kinetics of inactivation of calf intestine alkaline phosphatase by EDTA with absorption spectrum method].

Calf intestinal alkaline phosphatase (EC.3.1.3.1) is a dimeric metalloenzyme composed of two identical subunits, the each active site of which contains a tight cluster of two zinc ions and one magnesium ion. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity previously described by Tsou has been applied for a study on the kinetics of the course of inactivation of the enzyme by EDTA. The kinetics of the substrate reaction with different concentrations of the substrate p-nitrophenylphosphate (PNPP) and inactivator EDTA suggested a competitive complexing mechanism for inactivation by EDTA, and the process of inactivation composed of the rapid initial formation of an enzyme-EDTA complex, in which the conformation of enzyme has been changed, and then zinc ions are finally removed from the enzyme.

Antioxidant enzyme levels in pathogenesis of oral squamous cell carcinoma (OSCC).

Increased oxidative stress and altered anti-oxidant defense systems have been implicated in the pathogenesis of several diseases including cancer. Therefore, the aim of our study is to evaluate the antioxidant enzyme levels such as superoxide dismutase (SOD) and catalase in blood samples and tissues collected from oral squamous cell carcinoma patients and compared with healthy controls.The collected blood samples and tumor tissues from the diseased individuals and the normal controls are analyzed for malondialdehyde (MDA) and nitric oxide (NO), which is the indicator of oxidative and nitrosative stress respectively. The anti-oxidant enzymes SOD and catalase levels are measured by UV visible spectrophotometer. Subsequently, immuno-histostaining for antioxidant enzymes were performed in oral squamous cell carcinoma biopsies and sections were analyzed.The levels of MDA and NO were significantly elevated in the blood and tissue samples of OSCC patients. The antioxidant enzymes SOD and catalase were significantly reduced in OSCC tissues; while in erythrocytes catalase level is reduced whereas the SOD level is increased. Further, the reduced immuno-histostaining was observed for catalase and SOD in OSCC tissues when compared to normal oral epithelium.The enhanced levels of MDA and NO revealed that increased oxidative stress in conjunction with the reduced antioxidant defense mechanism in OSCC patients, might be involved in cancer progression. Our results suggest that detection of reactive oxygen species (ROS) and antioxidant enzymes levels might be a valuable marker in cancer prognosis and for improving therapeutic strategies in oral cancer.

[Cloning and sequencing of the maize ribosome-inactivating protein gene].

A gene encoding maize Ribosome-inactivating protein was amplified by means of PCR using mRNA of maize leaves as a template, and cloned into pUC19 vector. The amplified DNA sequence has been determined, which consists of 828 bp and encodes 275 amino acid residues. Comparison with previously reported sequence shows 98.4% homologies in nucleotide sequence and 97.4% in amino acid sequence, respectively.

Hydrogen peroxide-induced apoptosis in pc12 cells and the protective effect of puerarin.

In this study, the effect of puerarin on hydrogen peroxide-induced apoptosis in PC12 cells was studied. Exposure of cells to 0.5mM H(2)O(2)may cause significant viability loss and apoptotic rate increase. When c-Myc, Bcl-2 and Bax expression and caspase-3 activity were measured, using Ac-DEVD-AMC as a substrate, the changes in these apoptosis regulatory and effector proteins suggested that the elevation of c-Myc, decrease in Bcl-2:Bax protein ratio, and caspase-3 activation all play a key role in apoptosis. When cells were treated with puerarin prior to 0.5 mM H(2)O(2)treatment, a reduction in viability loss and apoptotic rate was seen. In addition, c-Myc expression decreased and Bcl-2:Bax ratio increased. Puerarin also reduced the H(2)O(2)-induced elevation of caspase-3 activation. These results suggest that puerarin can protect neurons against oxidative stress. It can block apoptosis in its early stages via the regulation of anti- and pro-apoptotic proteins, as well as by the attenuation of caspase-3 activation in H(2)O(2)-induced PC12 cells.