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Background: Oral squamous cell carcinoma (OSCC) has a poor prognosis when treated with surgery and chemotherapy. Therefore, a new therapy and preventative strategy for OSCC and its underlying mechanisms are desperately needed. The purpose of this study was to examine the chemopreventive effects of sanggenol L on oral squamous cell carcinoma (OSCC). The research focused on molecular signalling pathways in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis., Aim: The purpose of this study was to look at the biochemical and chemopreventive effects of sanggenol L on 7,12-dimethylbenz(a)anthracene (DMBA)-induced HBP (hamster buccal pouch) carcinogenesis via cell proliferation and the apoptotic pathway., Methods: After developing squamous cell carcinoma, oral tumours continued to progress leftward into the pouch 3 times per week for 10 weeks while being exposed to 0.5 % reactive DMBA three times per week. Tumour growth was caused by biochemical abnormalities that induced inflammation, increased cell proliferation, and decreased apoptosis., Results: Oral sanggenol L (10 mg/kg bw) supplementation with cancer-induced model DMBApainted hamsters prevented tumour occurrences, improved biochemistry, inhibited inflammatory markers, decreased cell proliferation marker expression of tumour necrosis factor-alpha (TNF- α), nuclear factor (NF-κB), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and induced apoptosis., Conclusion: Sanggenol L could be developed into a new medicine for the treatment of oral carcinogenesis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Emerging evidence has indicated that circular RNAs (circRNAs) play pivotal roles in the regulation of cellular processes and are found to be aberrantly expressed in a variety of tumors. However, the clinical role of circRNAs in bladder cancer (BC) and the molecular mechanisms have yet to be fully understood. In this study, the clinical specimens were obtained and the expression level of a circRNA BCRC4 was detected by real-time PCR in both BC tissues and cell line. The circular RNA over-expression plasmid was constructed and transfected into BC cells and related cell line. The cell cycles and apoptosis were observed using inverted microscope and flow cytometry. Western blotting was used to compare the relative protein expression of groups with different treatments. It was found that circRNA BCRC4 expression was lower in BC tissues than in adjacent normal tissues. Furthermore, consequences of forced-expression of BCRC4 promoted apoptosis and inhibited viability of T24T and UMUC3 cells, and up-regulated BCRC4-increased miR-101 level, which suppressed EZH2 expression in both RNA and protein levels. In addition, gambogic acid (GA) is a promising natural anticancer compound for BC therapy, and GA treatment increased the BCRC4 expression in T24T and UMUC3 cells in a dose-dependent manner. Altogether, our findings suggest that BCRC4 functions as a tumor suppressor in BC, and mediates anticancer function, at least in part, by up-regulating the expression of miR-101. Targeting this newly identified circRNA may help us develop a novel strategy for treating human BC.

Olfactory ensheathing cells (OECs) are a type of glia from the mammalian olfactory system, with neuroprotective and regenerative properties. β-Amyloid peptides are a major component of the senile plaques characteristic of the Alzheimer brain. The amyloid beta (Aβ) precursor protein is cleaved to amyloid peptides, and Aβ 25-35 is regarded to be the functional domain of Aβ, responsible for its neurotoxic properties. It has been reported that Aβ 25-35 triggers reactive oxygen species (ROS)-mediated oxidative damage, altering the structure and function of mitochondria, leading to the activation of the mitochondrial intrinsic apoptotic pathway. Our goal is to investigate the effects of OECs on the toxicity of aggregated Aβ 25-35 , in human neuroblastoma SH-SY5Y cells. For such purpose, SH-SY5Y cells were incubated with Aβ 25-35 and OEC-conditioned medium (OECCM). OECCM promoted the cell viability and reduced the apoptosis, and decreased the intracellular ROS and the lipid peroxidation. In the presence of OECCM, mRNA and protein levels of antioxidant enzymes (SOD1 and SOD2) were upregulated. Concomitantly, OECCM decreased mRNA and the protein expression levels of cytochrome c, caspase-9, caspase-3, and Bax in SH-SY5Y cells, and increased mRNA and the protein expression level of Bcl-2. However, OECCM did not alter intracellular Ca 2+ concentration in SH-SY5Y cells. Taken together, our data suggest that OECCM ameliorates Aβ 25-35 -induced oxidative damage in neuroblastoma SH-SY5Y cells by inhibiting the mitochondrial intrinsic pathway. These data provide new insights into the functional actions of OECCM on oxidative stress-induced cell damage.

Neuronal cell death following spinal cord injury (SCI) is an important contributor to neurological deficits. The purpose of our work was to delineate the function of mammalian sterile 20-like kinase 1 (Mst1), a pro-apoptotic kinase and key mediator of apoptotic signaling, in the pathogenesis of an experimental mouse model of SCI. Male mice received a mid-thoracic spinal contusion injury, and it was found that phosphorylation of Mst1 at the injured site was enhanced significantly following SCI. Furthermore, when compared to the wild-type controls, Mst1-deficient mice displayed improved locomotor function by increased Basso mouse scale score. Deletion of Mst1 in mice attenuated loss of motor neurons and suppressed microglial and glial activation following SCI. Deletion of Mst1 in mice reduced apoptosis via suppressing cytochrome c release and caspase-3 activation following SCI. Deletion of Mst1 attenuated mitochondrial dysfunction and increased ATP formation following SCI. Deletion of Mst1 in mice inhibited local inflammation following SCI, evidenced by reduced activities of myeloperoxidase and protein levels of TNF-α, IL-1β, and IL-6. In conclusion, the present study demonstrated that deletion of Mst1 attenuated neuronal loss and improved locomotor function in a mouse model of SCI, via preserving mitochondrial function, attenuating mitochondria-mediated apoptotic pathway, and suppressing inflammation, at least in part.

BACKGROUND Endothelial outgrowth cells (EOCs) are terminal endothelial progenitor cells (EPCs). Asymmetric dimethylarginine (ADMA) has been identified as a novel risk factor for cardiovascular diseases. Our aim in the present study was to investigate the effect of regulation of asymmetric dimethylarginine (ADMA) on EOCs apoptosis and to explore the underlining mechanisms of c-Jun N-terminal protein kinase (JNK) pathway in the process. MATERIAL AND METHODS EOCs were harvested from umbilical cord blood and obtained by using density gradient centrifugation and adhesive culture methods. Endothelial characteristics were identified by immunohistochemistry and fluorescence staining. EOCs were treated with different concentrations of ADMA and detected by flow cytometry. After JNK specific inhibitor (SP600125) was added, EOCs apoptosis protein expressions were measured by Western blot analysis. Proliferation, migration, and vascularization were detected by CCK-8 assay, wound healing assay, and tube-like formation assay, respectively. RESULTS EOCs were successfully extracted from umbilical cord blood and different concentrations of ADMA aggravated EOCs apoptosis. ADMA distinctly activates the phosphorylation activity of JNK. Supplementation of JNK-specific inhibitor (SP600125) decreased expression of Bax and cleaved caspase 3/9, and alleviated ADMA-induced apoptosis. SP600125 also promoted angiogenesis viability. CONCLUSIONS The JNK pathway participates in the apoptosis-promoting process of EOCs, and targeted inhibition of the JNK pathway can alleviate ADMA-induced injury, which I s the potential underlying mechanism of vascular endothelium injury in ischemic stroke.

Objective: To study the effect of silencing pyruvate kinase M2 (PKM2) on gambogic acid (GA)-induced apoptosis of human prostate cancer PC3 cells., Methods: Three specific PKM2 siRNAs and one negative control siRNA (si-NC) were transfected into PC3 cells. The silencing effect of PKM2 siRNAs was determined by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, and the effects of PKM2 siRNA on the vitality and apoptosis of GA-stimulated PC3 cells detected by MTT and AO/EB double staining, respectively. The mRNA and protein levels of c-myc and cyclin D1 were analyzed by qRT-PCR and Western blot, respectively., Results: All the 3 PKM2 siRNAs effectively reduced the mRNA and protein expressions of PKM2, and PKM2 siRNA-1 exhibited the strongest silencing effect. At 24 h after transfection, the expression levels of PKM2 mRNA and protein were reduced by 70% and 85%, respectively (P < 0.05). Twenty-four hours of treatment with GA (0.5 micromol/L) following transfection with PKM2 siRNA-1 inhibited the vitality of the PC3 cells by 68%, increased their apoptosis, and significantly down-regulated the mRNA and protein levels of c-myc (50% and 35%) and cyclin D1 (60% and 20%) (P < 0.05)., Conclusion: Inhibition of PKM2 sensitized PC3 cells to GA-induced apoptosis, suggesting that PKM2 may be a potential therapeutic target for sensitizing human prostate cancer to GA.

Background: Spontaneous and stress-induced germ cell apoptosis during spermatogenesis of multicellular organisms have been investigated broadly in mammals. Spermatogenetic process in urodele amphibians was essentially like that in mammals in spite of morphological differences; however, the mechanism of germ cell apoptosis in urodele amphibians remains unknown. The Chinese fire-belly newt, Cynops orientalis, was an excellent organism for studying germ cell apoptosis due to its sensitiveness to temperature, strong endurance of starvation, and sensitive skin to heavy metal exposure., Methodology/principal Findings: TUNEL result showed that spontaneous germ cell apoptosis took place in normal newt, and severe stress-induced apoptosis occurred to spermatids and sperm in response to heat shock (40°C 2 h), cold exposure (4°C 12 h), cadmium exposure (Cd 36 h), and starvation stress. Quantitative reverse transcription polymerase chain reactions (qRT-PCR) showed that gene expression of Caspase3 or Caspase7 was obviously elevated after stress treatment. Apaf1 was not altered at its gene expression level, and p53 was significantly decreased after various stress treatment. Caspase assay demonstrated that Caspase-3, -8, -9 enzyme activities in newt testis were significantly elevated after heat shock (40°C 2 h), cold exposure (4°C 12 h), and cadmium exposure (Cd 36 h), while Caspase3 and Caspase8 activities were increased with Caspase9 significantly decreased after starvation treatment., Conclusions/significance: Severe germ cell apoptosis triggered by heat shock, cold exposure, and cadmium exposure was Caspase3 dependent, which probably involved both extrinsic and intrinsic pathways. Apaf1 may be involved in this process without elevating its gene expression. But starvation-induced germ cell apoptosis was likely mainly through extrinsic pathway. p53 was probably not responsible for stress-induced germ cell apoptosis in newt testis. The intriguing high occurrence of spermatid and sperm apoptosis probably resulted from the sperm morphology and unique reproduction policy of Chinese fire-belly newt, Cynops orientalis.

Methyl jasmonate (MJ) has recently attracted attention as a promising antitumoral compound because of its highly specific proapoptotic properties in a wide range of malignancies. However, the high doses required to achieve a therapeutic benefit have limited its clinical development. Here, we hypothesize that the family of inhibitor of apoptosis proteins (IAPs) may inhibit MJ-mediated apoptosis in cancer cells. We combined MJ with the IAPs inhibitor, the second mitochondria-derived activator of caspases (Smac) peptide to treat bladder cancer cells. The results showed that the combination of MJ and Smac peptide enhanced the apoptosis-inducing effect in a synergistic manner by releasing and activating IAPs-bounding caspase-3. These findings suggest that the inhibition of IAPs could overcome the resistance of cancer cells to MJ.

Recent evidence indicates that methyl jasmonate, a plant stress hormone, exhibits anticancer activity on human cancer cells. Whether methyl jasmonate could inhibit the growth of human neuroblastoma cells still, however, remains largely unknown. In this study, administration of methyl jasmonate to cultured neuroblastoma cell lines, SK-N-SH and BE(2)-C, resulted in a decrease of cell viability in a dose-dependent and time-dependent manner as demonstrated by MTT colorimetry and colony formation assay. The results from RT-PCR indicated that the expression of proliferating cell nuclear antigen, but not of cyclin D1, was downregulated by methyl jasmonate. Accordingly, the cell cycle of methyl jasmonate-treated neuroblastoma cells was arrested at the G0/G1 phase. Moreover, incubation of SK-N-SH and BE(2)-C cells with methyl jasmonate resulted in characteristic changes of apoptosis, as demonstrated by acridine orange-ethidium bromide (AO/EB) staining, Hoechst 33258 staining and flow cytometry. Moreover, methyl jasmonate decreased the expression of the X-linked inhibitor of apoptosis protein and survivin, critical members of the inhibitors of apoptosis protein family, in neuroblastoma cells. These findings indicate that methyl jasmonate suppresses the growth of cultured human neuroblastoma cells associated with downregulation of proliferating cell nuclear antigen, and induces apoptosis accompanied by downregulation of the X-linked inhibitor of apoptosis protein and survivin, which lays the groundwork for further investigation into the mechanisms of methyl jasmonate-mediated anticancer activities.

This study is to explore the inhibitory effect of methyl jasmonate on cell proliferation and expression of XIAP and survivin of human neuroblastoma cell line BE(2)-C. After cultivation of 1 - 2 mmol x L(-1) jasmonates with BE (2) -C cells for 6 - 24 h, the growth inhibiting rates of BE (2) -C cells were studied by MTT colorimetry. Cell proliferation was detected by colony formation assay. Cell cycle phases were assayed by propidium iodide staining flow cytometery. Cell apoptosis was inspected by acridine orange-ethidium bromide fluorescent staining, Hoechst 33258 fluorescent staining, and Annexin V-FITC and propidium iodide staining flow cytometry. Expressions of cyclin D1, XIAP and survivin were determined by RT-PCR and real-time RT-PCR. Methyl jasmonate inhibited the growth of BE(2)-C cells in a dose- and time-dependent manner. After addition of 1, 1.5 and 2 mmol x L(-1) of methyl jasmonate for 24 h, the inhibiting rates of cell growth reached 20.6% - 85.5% (P < 0.01), and the IC50 was 1.35 mmol x L(-1). The cell cycles were arrested at S phase. A part of cells presented the characteristic morphological changes of apoptosis. The early apoptotic rates were 13.51%, 17.32%, 24.59% (P < 0.01) and the cell death rates were 29.36% , 54.73% , 75.52% (P < 0.01), respectively. The expression of XIAP and survivin mRNA were downregulated by 18.5% - 68.9% , 22.4% - 48.7% (P < 0.05), respectively, without change in that of cyclin D1. The results indicated that methyl jasmonate could significantly inhibit the growth of BE(2) -C cells through inducing cell cycle arrest and apoptosis, downregulating the expression of XIAP and survivin might be one of its molecular mechanisms of action.

Objective: To observe the effects of hydroxycamptothecin (HCPT) on the apoptosis of prostate cancer cell line PC-3 and to explore the possible mechanism., Methods: The influence of different concentrations (1 x 10(-1), 1 x 10(-2), 1 x 10(-3), 1 x 10(-4) mg/ml) of HCPT on PC-3 cell proliferation at different time (12, 24, 48 h) was determined by tetrazolium (MTT) assay. The morphologic changes of the apoptotic cells were observed by acridine orange/ethidium bromide dyeing. The DNA of the apoptotic cells was analyzed with agarose gel electrophoresis. The apoptosis rate of HCPT on prostate cancer cells was analyzed by flow cytometry (FCM)., Results: The growth of PC-3 was inhibited by HCPT in a time- and dose- dependent manner. The values of IC50 were 6.50 x 10(-2) mg/ ml (12 h), 2.35 x 10(-2) mg/ml (24 h) and 5.31 x 10(-3) mg/ml (48 h) respectively. The typical apoptotic cells under the fluorescence microscope showed budding phenomena and apoptotic bodies. And the DNA ladder was observed in ultraviolet light. FCM analysis showed that the apoptosis rate of PC-3 cells increased with the increasing dose of HCPT, which reached the peak (35.76%) at 1 x 10(-3) mg/ml., Conclusion: HCPT could suppress PC-3 cell proliferation significantly by inducing the apoptosis of PC-3 cells. However, the mechanism is yet to be further studied.

Aim: To characterize the molecular mechanisms of nitrofen-induced pulmonary hypoplasia., Methods: After administration of nitrofen to cultured type II A549 pneumocytes, cell proliferation and DNA synthesis were investigated by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetry, colony formation assay, flow cytometry and [3H]-thymidine incorporation assay. Apoptosis was measured by terminal transferase-mediated dUTP nick-end-labeling, acridine orange-ethidium bromide staining and flow cytometry. Expression of proliferating cell nuclear antigen (PCNA) and apoptosis-related genes was assayed by immunofluorescence, RT-PCR and Western blot., Results: Nitrofen inhibited the cell proliferation of A549 cells in a dose- and time-dependent manner, accompanied by downregulation of PCNA. As a result, the DNA synthesis of nitrofentreated A549 cells decreased, while cell cycle was arrested at G0/G1 phase. Moreover, nitrofen induced apoptosis of A549 cells, which was not abolished by Z-Val-Ala- Asp(OCH3)- fluoromethylketone. In addition, nitrofen decreased the expression of Bcl-x( L), but not of Bcl-2, Bax, and Bak, resulting in a loss of mitochondrial membrane potential and the nuclear translocation of apoptosis-inducing factor (AIF). Meanwhile, nitrofen strongly activated the p38 mitogen-activated protein kinase (p38-MAPK). Pretreatment of cells with SB203580 (5 micromol/L) blocked nitrofen-induced phosphorylation of p38-MAPK and abolished nitrofen-induced AIF translocation and apoptosis in A549 cells., Conclusion: Nitrofen suppresses the proliferation of cultured type II pneumocytes accompanied by the downregulation of PCNA, and induces mitochondria-mediated apoptosis involving the activation of p38-MAPK.

Background & Objective: The interaction between pro-apoptotic factors and anti-apoptotic factors is closely related to the genesis and development of tumors. Omi/HtrA2 is a novel gene involved in the regulation of apoptosis. PED/PEA-15 is a widely expressed anti-apoptotic protein. This study was to explore the effects of Omi/HtrA2 on PED/PEA-15 expression and apoptosis of prostate cancer cell line PC-3., Methods: Omi/HtrA2 expression and specific siRNA vectors were constructed and transiently transfected into PC-3 cells. The effect of Omi/HtrA2 on PED/PEA-15 expression was assayed by Western blot, and its effect on apoptosis of PC-3 cells was analyzed by ELISA. Caspase-8 activity was assayed using Caspase-8 colorimetric assay kit. The effects of Omi/HtrA2-specific siRNA sequence on its transcription and translation were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The sensitivity of PC-3 cells to cisplatin (DDP) after Omi/HtrA2 gene silencing was determined by flow cytometry., Results: Enzyme digestion analysis and DNA sequencing confirmed Omi/HtrA2 expression, and specific siRNA vectors were successfully constructed. After transfection of Omi/HtrA2 expression vector, PED/PEA-15 expression was inhibited, Caspase-8 activity was promoted, and the apoptosis of PC-3 cells was enhanced. The sensitivity of PC-3 cells to DDP was suppressed after Omi/HtrA2 gene silencing., Conclusion: Omi/HtrA2 can promote the apoptosis of PC-3 cells through inhibiting PED/PEA-15 expression.

Our aim was to prepare curcumin derivatives and study their apoptosis-inducing effects on bladder cancer cells in order to establish a basis for targeted chemotherapy of cancer. n-Maleoyl-L-valine-curcumin (NVC) and n-maleoyl-glycine-curcumin (NGC) were chemically synthesized. Intracellular esterase activity of the human bladder cancer EJ cell line and renal tubular epithelial (HKC) cells was examined by 6-carboxyfluorescein diacetate fluorometry. After incubation with NVC or NGC for 6-24 h, cell viability was detected by MTT colorimetry. Cell apoptosis and apoptotic rates were measured by acridine orange/ethidium bromide staining, TUNEL labeling and flow cytometry. Intracellular caspase-3 activities were determined by spectrophotometry. The esterase activity within EJ cells was 10.2-fold higher than that of HKC cells, which was abolished by bis-p-nitrophenylphosphate, an esterase inhibitor, resulting in decreases in NVC- and NGC-mediated cell viability arrest. For EJ cells, the IC50 values of NVC (20.1 micromol/l) and NGC (18.7 micromol/l) were close to curcumin (16.5 micromol/l). Meanwhile, their IC50 values on HKC cells were, respectively, 4.06- and 3.23-fold higher than curcumin. Moreover, NVC and NGC induced apoptosis of EJ cells by 10.13-23.36 and 12.42-28.56%, respectively. Administration of these two derivatives resulted in decreased apoptosis of HKC cells compared with curcumin. The caspase-3 activities of EJ cells, but not of HKC cells, were 5.21- and 5.63-fold enhanced by NVC and NGC, respectively. Thus, novel esterase-sensitive curcumin derivatives were synthesized, which induced extensive apoptosis of bladder cancer EJ cells, but not normal cells.

X-linked inhibitor of apoptosis (XIAP) is the most potent member of the inhibitor of apoptosis protein (IAP) gene family in terms of its ability to inhibit caspases and suppress apoptosis. Recent evidence has suggested that XIAP is a key determinant in chemoresistance of cancer cells. To explore a novel approach for ameliorating chemotherapy of gastric cancer, the antisense expression vector for the XIAP gene was constructed and transferred into gastric cancer cell lines, MKN-45 (wild-type p53) and MKN-28 (mutant-type p53). This transfer resulted in significant downregulation of XIAP expression, decreased in vitro cell viabilities, and induced apoptosis. In transferred cells, inactive caspase-3 precursors were cleaved into the active subunits (p20 and p17) during apoptosis induced by downregulation of XIAP. The inhibitory effects of cisplatin and mitomycin C on the growth of XIAP downregulated cancer cells were significantly enhanced. In addition, this process occurred only in wild-type p53 (MKN-45), but not in mutant-type p53 (MKN-28) gastric cancer cells. The data presented suggest that downregulation of XIAP via antisense RNA can lead to apoptosis of gastric cancer cells in vitro, correlating with cellular p53 status and activation of caspase-3. This finding could lead to a potential strategy for improving the efficiency of therapies for gastric cancer.

Stem cell factor (SCF) is essential for the development of primordial follicles. One of its functions is to prevent oocytes from apoptosis. However, the underlying mechanism remains largely unknown. By using cultured ovaries that are rich in primordial follicles, the anti-apoptotic action of SCF and the potential signal transduction pathways were investigated. The apoptosis was evaluated by means of in situ 3'-end labeling. The expressions of proteins were analyzed with immunohistochemistry and Western blot. The data showed that SCF significantly prevented oocytes from apoptosis in the cultured organs. Addition of a specific pharmacological inhibitor of PI3K abolished the anti-apoptotic action of SCF while that of a MEK inhibitor did not. The phosphorylation of two mitogen activated protein kinases (MAPKs) (p42 and p44) and AKT, the respective substrates of MEK and PI3K, were enhanced by SCF treatment. Not surprisingly, the MAPK activation occurred only in theca cells. The expressions of apoptosis-related gene products, the Bcl-2 family proteins, in response to SCF treatment were also investigated. While SCF up-regulated the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, it did the opposite to the pro-apoptotic factor Bax. The PI3K inhibitor reversed the regulation of SCF on Bcl-xL and Bax but not on Bcl-2. Therefore, it seemed that SCF initiated an anti-apoptotic signal starting from its membrane receptor c-kit to Bcl-2 family members through PI3K/AKT and other signaling cascades in the oocytes of primordial follicles., ((c) 2004 Wiley-Liss, Inc.)

E. sinensis is an animal model for studying the reproduction and development of crustaceans. In this study, we knocked down the Es-Kif2a gene by injecting dsRNA into E. sinensis and inhibited Es-Plk1 gene expression by injecting PLK1 inhibitor BI6727 into E. sinensis. Then, the cell proliferation level, apoptosis level, and PI3K/AKT signaling expression level were detected. Our results showed that the proliferation level of spermatogenic cells decreased, while the apoptosis level increased after Es-Kif2a knockdown or Es-Plk1 inhibition. In order to verify whether these changes are caused by regulating the PI3K/AKT pathway, we detected the expression of PI3K and AKT proteins after Es-Kif2a knockdown or Es-Plk1 inhibition. Western Blot showed that in both the Es-Kif2a knockdown group and the Es-Plk1 inhibition group, the expression of PI3K and AKT proteins decreased. In addition, immunofluorescence showed that Es-KIF2A and Es-PLK1 proteins were co-localized during E. sinensis spermatogenesis. To further explore the upstream and downstream relationship between Es-KIF2A and Es-PLK1, we detected the expression level of Es-PLK1 after Es-Kif2a knockdown as well as the expression level of Es-KIF2A after Es-Plk1 inhibition. Western Blot showed that the expression of Es-PLK1 decreased after Es-Kif2a knockdown, while there was no significant change of Es-KIF2A after Es-Plk1 inhibition, indicating that Es-PLK1 may be a downstream factor of Es-KIF2A. Taken together, these results suggest that Es-KIF2A upregulates the PI3K/AKT signaling pathway through Es-PLK1 during the spermatogenesis of E. sinensis, thereby affecting the proliferation and apoptosis levels of spermatogenic cells.

Simple Summary: The kinesin superfamily is an important member of motor proteins, playing important roles in cell growth and development. Studies on KIF2A of the kinesin-13 family have mainly focused on cell division, while the relationship between KIF2A and signaling pathways remains unclear. The purpose of the present study was to determine whether KIF2A has a regulatory effect on the PI3K/AKT pathway, thus affecting spermatogenic cell proliferation and apoptosis during spermatogenesis, and whether its interacting protein PLK1 is involved in this process. RNAi experiments were performed in our study. Our results showed that KIF2A upregulates the PI3K/AKT signaling pathway through its interacting protein PLK1 during the spermatogenesis of Eriocheir sinensis. E. sinensis is an animal model for studying the reproduction and development of crustaceans. In this study, we knocked down the Es-Kif2a gene by injecting dsRNA into E. sinensis and inhibited Es-Plk1 gene expression by injecting PLK1 inhibitor BI6727 into E. sinensis. Then, the cell proliferation level, apoptosis level, and PI3K/AKT signaling expression level were detected. Our results showed that the proliferation level of spermatogenic cells decreased, while the apoptosis level increased after Es-Kif2a knockdown or Es-Plk1 inhibition. In order to verify whether these changes are caused by regulating the PI3K/AKT pathway, we detected the expression of PI3K and AKT proteins after Es-Kif2a knockdown or Es-Plk1 inhibition. Western Blot showed that in both the Es-Kif2a knockdown group and the Es-Plk1 inhibition group, the expression of PI3K and AKT proteins decreased. In addition, immunofluorescence showed that Es-KIF2A and Es-PLK1 proteins were co-localized during E. sinensis spermatogenesis. To further explore the upstream and downstream relationship between Es-KIF2A and Es-PLK1, we detected the expression level of Es-PLK1 after Es-Kif2a knockdown as well as the expression level of Es-KIF2A after Es-Plk1 inhibition. Western Blot showed that the expression of Es-PLK1 decreased after Es-Kif2a knockdown, while there was no significant change of Es-KIF2A after Es-Plk1 inhibition, indicating that Es-PLK1 may be a downstream factor of Es-KIF2A. Taken together, these results suggest that Es-KIF2A upregulates the PI3K/AKT signaling pathway through Es-PLK1 during the spermatogenesis of E. sinensis, thereby affecting the proliferation and apoptosis levels of spermatogenic cells. [ABSTRACT FROM AUTHOR]

Olfactory ensheathing cells (OECs) are a type of glia from the mammalian olfactory system, with neuroprotective and regenerative properties. β-Amyloid peptides are a major component of the senile plaques characteristic of the Alzheimer brain. The amyloid beta (Aβ) precursor protein is cleaved to amyloid peptides, and Aβ is regarded to be the functional domain of Aβ, responsible for its neurotoxic properties. It has been reported that Aβ triggers reactive oxygen species (ROS)-mediated oxidative damage, altering the structure and function of mitochondria, leading to the activation of the mitochondrial intrinsic apoptotic pathway. Our goal is to investigate the effects of OECs on the toxicity of aggregated Aβ, in human neuroblastoma SH-SY5Y cells. For such purpose, SH-SY5Y cells were incubated with Aβ and OEC-conditioned medium (OECCM). OECCM promoted the cell viability and reduced the apoptosis, and decreased the intracellular ROS and the lipid peroxidation. In the presence of OECCM, mRNA and protein levels of antioxidant enzymes (SOD1 and SOD2) were upregulated. Concomitantly, OECCM decreased mRNA and the protein expression levels of cytochrome c, caspase-9, caspase-3, and Bax in SH-SY5Y cells, and increased mRNA and the protein expression level of Bcl-2. However, OECCM did not alter intracellular Ca concentration in SH-SY5Y cells. Taken together, our data suggest that OECCM ameliorates Aβ-induced oxidative damage in neuroblastoma SH-SY5Y cells by inhibiting the mitochondrial intrinsic pathway. These data provide new insights into the functional actions of OECCM on oxidative stress-induced cell damage. [ABSTRACT FROM AUTHOR]

Both tyrosine kinase and topoisomerase II (TopII) are important anticancer targets, and their respective inhibitors are widely used in cancer therapy. However, some combinations of anticancer drugs could exhibit mutually antagonistic actions and drug resistance, which further limit their therapeutic efficacy. Here, we report that HMNE3, a novel bis-fluoroquinolone chalcone-like derivative that targets both tyrosine kinase and TopII, induces tumor cell proliferation and growth inhibition. The viabilities of 6 different cancer cell lines treated with a range of HMNE3 doses were detected using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular apoptosis was determined using Hoechst 33258 fluorescence staining and the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. The expression of activated Caspase-3 was examined by immunocytochemistry. The tyrosine kinase activity was measured with a human receptor tyrosine kinase (RTK) detection kit using a horseradish peroxidase (HRP)-conjugated phosphotyrosine (pY20) antibody as the substrate. The topoisomerase II activity was measured using agarose gel electrophoresis with the DNA plasmid pBR322 as the substrate. The expression levels of the P53, Bax, Bcl-2, Caspase-3, -8, -9, p-cSrc, c-Src and topoisomerase II proteins were detected by western blot analysis. The proliferation of five of the six cancer cell lines was significantly inhibited by HMNE3 at 0.312 to 10 μmol/L in a time- and dose-dependent manner. Treatment of the Capan-1 and Panc-1 cells with 1.6 to 3.2 μM HMNE3 for 48 h significantly increased the percentage of apoptotic cells (P<0.05), and this effect was accompanied by a decrease in tyrosine kinase activity. HMNE3 potentially inhibited tyrosine kinase activity in vitro with an IC50 value of 0.64±0.34 μmol/L in Capan-1 cells and 3.1±0.86 μmol/L in Panc-1 cells. The activity of c-Src was significantly inhibited by HMNE3 in a dose- and time-dependent manner in different cellular contexts. Compared with the control group, HMNE3 induced increased expression of cellular apoptosis-related proteins. Consistent with cellular apoptosis data, a significant decrease in topoisomerase IIβ activity was noted following treatment with HMNE3 for 24 h. Our data suggest that HMNE3 induced apoptosis in Capan-1 and Panc-1 cells by inhibiting the activity of both tyrosine kinases and topoisomerase II. [ABSTRACT FROM AUTHOR]

Background: Spontaneous and stress-induced germ cell apoptosis during spermatogenesis of multicellular organisms have been investigated broadly in mammals. Spermatogenetic process in urodele amphibians was essentially like that in mammals in spite of morphological differences; however, the mechanism of germ cell apoptosis in urodele amphibians remains unknown. The Chinese fire-belly newt, Cynops orientalis, was an excellent organism for studying germ cell apoptosis due to its sensitiveness to temperature, strong endurance of starvation, and sensitive skin to heavy metal exposure. Methodology/Principal Findings: TUNEL result showed that spontaneous germ cell apoptosis took place in normal newt, and severe stress-induced apoptosis occurred to spermatids and sperm in response to heat shock (40°C 2 h), cold exposure(4°C 12 h), cadmium exposure(Cd 36 h), and starvation stress. Quantitative reverse transcription polymerase chain reactions (qRT-PCR) showed that gene expression of Caspase3 or Caspase7 was obviously elevated after stress treatment. Apaf1 was not altered at its gene expression level, and p53 was significantly decreased after various stress treatment. Caspase assay demonstrated that Caspase-3, -8,-9 enzyme activities in newt testis were significantly elevated after heat shock (40°C 2 h), cold exposure(4°C 12 h), and cadmium exposure(Cd 36 h), while Caspase3 and Caspase8 activities were increased with Caspase9 significantly decreased after starvation treatment. Conclusions/Significance: Severe germ cell apoptosis triggered by heat shock, cold exposure, and cadmium exposure was Caspase3 dependent, which probably involved both extrinsic and intrinsic pathways. Apaf1 may be involved in this process without elevating its gene expression. But starvation-induced germ cell apoptosis was likely mainly through extrinsic pathway. p53 was probably not responsible for stress-induced germ cell apoptosis in newt testis. The intriguing high occurrence of spermatid and sperm apoptosis probably resulted from the sperm morphology and unique reproduction policy of Chinese fire-belly newt, Cynops orientalis. [ABSTRACT FROM AUTHOR]

Aim: To characterize the molecular mechanisms of nitrofen-induced pulmonary hypoplasia. Methods: After administration of nitrofen to cultured type IIA549 pneumocytes, cell proliferation and DNA synthesis were investigated by 3- (4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide colorimetry, colony formation assay, flow cytometry and [3H]-thymidine incorporation assay. Apoptosis was measured by terminal transferase-mediated dUTP nick-end-labeling, acridine orange-ethidium bromide staining and flow cytometry. Expression of proliferating cell nuclear antigen (PCNA) and apoptosis-related genes was assayed by immunofluorescence, RT-PCR and Western blot. Results: Nitrofen inhibited the cell proliferation of A549 cells in a dose- and time-dependent manner, accompanied by downregulation of PCNA. As a result, the DNA synthesis of nitrofentreated A549 cells decreased, while cell cycle was arrested at G0/G1 phase. Moreover, nitrofen induced apoptosis of A549 cells, which was not abolished by Z-Val-Ala-Asp(OCH3)- fluoromethylketone. In addition, nitrofen decreased the expression of Bcl-xL, but not of Bcl-2, Bax, and Bak, resulting in a loss of mitochondrial membrane potential and the nuclear translocation of apoptosis-inducing factor (AIF). Meanwhile, nitrofen strongly activated the p38 mitogen-activated protein kinase (p38-MAPK). Pretreatment of cells with SB203580 (5 μmol/L) blocked nitrofen-induced phosphorylation of p38-MAPK and abolished nitrofen-induced AIF translocation and apoptosis in A549 cells. Conclusion: Nitrofen suppresses the proliferation of cultured type II pneumocytes accompanied by the downregulation of PCNA, and induces mitochondria-mediated apoptosis involving the activation of p38-MAPK. [ABSTRACT FROM AUTHOR]

The serine protease Omi/HtrA2 is released from mitochondria into the cytosol after apoptotic stimuli, inducing apoptosis in a caspase-independent manner through its protease activity and in a caspase-dependent manner by neutralizing the inhibition of inhibitor of apoptosis proteins (IAPs) on caspases. Alteration of apoptosis is essential for cancer development, and cancer cell death by radiation and chemotherapy is largely dependent upon apoptosis. Thus, analysis of the expression status of Omi/HtrA2, a regulator of apoptosis, in cancer tissues is needed for an understanding of cancer development. In the current study we analyzed the expression of Omi/HtrA2 in 65 prostate cancer, 40 benign prostatic hyperplasia and 10 normal prostate specimens by immunohistochemistry. Omi/HtrA2 mRNA levels of in vivo prostate cancer and benign prostatic hyperplasia samples were also assayed by semiquantitative reverse transcription-polymerase chain reaction. Immunopositivity (defined as ≥30%) was observed for Omi/HtrA2 in most of the prostate cancers, and the positive rate of Omi/HtrA2 was lower in the well-differentiated group than in the poorly and moderately differentiated groups (p<0.005). By contrast, the cells in the normal prostate and benign prostatic hyperplasia groups showed no or only weak expression of Omi/HtrA2. Meanwhile, the Omi/HtrA2 mRNA level of prostate cancer is much higher than that of benign prostatic hyperplasia (p<0.001). Taken together, these results suggest that prostate cancer cells in vivo may need Omi/HtrA2 expression for apoptosis, and that Omi/HtrA2 expression might be involved in prostate cancer development. [ABSTRACT FROM AUTHOR]

X-linked inhibitor of apoptosis (XIAP) is the most potent member of the inhibitor of apoptosis protein (IAP) gene family in terms of its ability to inhibit caspases and suppress apoptosis. Recent evidence has suggested that XIAP is a key determinant in chemoresistance of cancer cells. To explore a novel approach for ameliorating chemotherapy of gastric cancer, the antisense expression vector for the XIAP gene was constructed and transferred into gastric cancer cell lines, MKN-45 (wild-type p53) and MKN-28 (mutant-type p53). This transfer resulted in significant downregulation of XIAP expression, decreased in vitro cell viabilities, and induced apoptosis. In transferred cells, inactive caspase-3 precursors were cleaved into the active subunits (p20 and p17) during apoptosis induced by downregulation of XIAP. The inhibitory effects of cisplatin and mitomycin C on the growth of XIAP downregulated cancer cells were significantly enhanced. In addition, this process occurred only in wild-type p53 (MKN-45), but not in mutant-type p53 (MKN-28) gastric cancer cells. The data presented suggest that downregulation of XIAP via antisense RNA can lead to apoptosis of gastric cancer cells in vitro, correlating with cellular p53 status and activation of caspase-3. This finding could lead to a potential strategy for improving the efficiency of therapies for gastric cancer.Cancer Gene Therapy (2005) 12, 509-514. doi:10.1038/sj.cgt.7700813 Published online 11 February 2005 [ABSTRACT FROM AUTHOR]