The study on interacting factors and functions of GASA6 in Jatropha curcas L.

BACKGROUND: The gibberellic acid-stimulated Arabidopsis (GASA) gene encodes a class of cysteine-rich functional proteins and is ubiquitous in plants. Most GASA proteins are influence the signal transmission of plant hormones and regulate plant growth and development, however, their function in Jatropha curcas is still unknown. RESULTS: In this study, we cloned JcGASA6, a member of the GASA family, from J. curcas. The JcGASA6 protein has a GASA-conserved domain and is located in the tonoplast. The three-dimensional structure of the JcGASA6 protein is highly consistent with the antibacterial protein Snakin-1. Additionally, the results of the yeast one-hybrid (Y1H) assay showed that JcGASA6 was activated by JcERF1, JcPYL9, and JcFLX. The results of the Y2H assay showed that both JcCNR8 and JcSIZ1 could interact with JcGASA6 in the nucleus. The expression of JcGASA6 increased continuously during male flower development, and the overexpression of JcGASA6 was associated with filament elongation of the stamens in tobacco. CONCLUSION: JcGASA6, a member of the GASA family in J. curcas, play an important role in growth regulation and floral development (especially in male flower). It is also involved in the signal transduction of hormones, such as ABA, ET, GA, BR, and SA. Also, JcGASA6 is a potential antimicrobial protein determined by its three-dimensional structure.

Nab-paclitaxel plus S-1 versus oxaliplatin plus S-1 as first-line treatment in advanced gastric cancer: results of a multicenter, randomized, phase III trial (GAPSO study).

Background: This study aimed to investigate the superiority of nab-paclitaxel plus S-1 (AS) over oxaliplatin plus S-1 (SOX) in patients with advanced gastric cancer (AGC). Methods: In this multicenter, randomized, phase III superiority trial, eligible patients with unresectable, locally advanced gastric adenocarcinoma were recruited and randomly assigned (1:1) to receive AS (nab-paclitaxel 260 mg/m2 on day 1 or 130 mg/m2 on days 1 and 8; oral S-1 40-60 mg twice daily for 14 days) or SOX (130 mg/m2 oxaliplatin on day 1; oral S-1 40-60 mg twice daily for 14 days) every 3 weeks for up to six cycles. The primary endpoint was progression-free survival (PFS), and the secondary endpoints were overall survival, objective response rate, and safety. Results: Owing to slow enrolment, an unplanned interim analysis was performed, resulting in the early termination of the study on 31 December 2021 (data cutoff). Between March 2019 and March 2021, 97 patients (AS, n = 48; SOX, n = 49) were treated and evaluated for efficacy and safety of AS and SOX. As of the data cutoff, the median follow-up was 23.13 months [95% confidence interval (CI), 13.39-32.87]. The median PFS was 9.03 months (95% CI, 6.50-11.56) in the AS group and 5.07 months (95% CI, 4.33-5.81) in the SOX group, demonstrating a better PFS tendency following AS treatment than SOX treatment (hazard ratio = 0.59; 95% CI, 0.37-0.94; p = 0.03). The most common grade 3 or worse adverse events were anemia, neutropenia, and leukopenia in both groups, with a higher incidence of thrombocytopenia in the SOX group. Conclusion: Although this study was terminated early, the results demonstrated a better PFS tendency in patients with AGC who were treated with AS than in those treated with SOX, with controllable toxicities. Trial registration: Clinical Trials.gov identifiers: NCT03801668. Registered January 11, 2019.

Transcriptome revealing the dual regulatory mechanism of ethylene on the rhynchophylline and isorhynchophylline in Uncaria rhynchophylla.

Rhynchophylline (RIN) and isorhynchophylline (IRN) are extracted from Uncaria rhynchophylla, which are used to treat Alzheimer's disease. However, the massive accumulation of RIN and IRN in U. rhynchophylla requires exogenous stimulation. Ethylene is a potential stimulant for RIN and IRN biosynthesis, but there is no study on the role of ethylene in RIN or IRN synthesis. This study investigated the regulation of ethylene in RIN and IRN biosynthesis in U. rhynchophylla. An increase in the content of RIN and IRN was observed that could be attributed to the release of ethylene from 18 mM ethephon, while ethylene released from 36 mM ethephon reduced the content of RIN and IRN. The transcriptome and weighted gene co-expression network analysis indicated the up-regulation of seven key enzyme genes related to the RIN/IRN biosynthesis pathway and starch/sucrose metabolism pathway favored RIN/IRN synthesis. In comparison, the down-regulation of these seven key enzyme genes contributed to the reduction of RIN/IRN. Moreover, the inhibition of photosynthesis is associated with a reduction in RIN/IRN. Photosynthesis was restrained owing to the down-regulation of Lhcb1 and Lhcb6 after 36 mM ethephon treatment and further prevented supply of primary metabolites (such as α-D-glucose) for RIN/IRN synthesis. However, uninterrupted photosynthesis ensured a normal supply of primary metabolites at 18 mM ethephon treatment. AP2/ERF1, bHLH1, and bHLH2 may positively regulate the RIN/IRN accumulation, while NAC1 may play a negative regulatory role. Our results construct the potential bidirectional model for ethylene regulation on RIN/IRN synthesis and provide novel insight into the ethylene-mediated regulation of the metabolism of terpenoid indole alkaloids.

Knockdown of STIP1 inhibits the invasion of CD133­positive cancer stem­like cells of the osteosarcoma MG63 cell line via the PI3K/Akt and ERK1/2 pathways.

Osteosarcoma is the most common primary malignant tumor of the bone in adolescents and children, with high rates of metastasis and a poor prognosis. Recently, osteosarcoma cancer stem/stem­like cells (CSCs) have been identified as the main cause of recurrence and metastasis. Stress­induced phosphoprotein 1 (STIP1), a co­chaperone that binds to heat shock proteins 70 and 90, is abnormally expressed in several tumor cell lines, and may play an important role in tumor cell migration and invasion. These features indicate that STIP1 may represent a new therapeutic target for osteosarcoma CSCs. However, the role of STIP1 in osteosarcoma CSC migration and invasion remains largely unknown. In the present study, CD133­positive osteosarcoma CSCs were first isolated and cultured by magnetic cell sorting and serum­free medium suspension cell sphere culture, respectively. Knockdown of STIP1 by small interfering RNA significantly was then shown to inhibit the migration and invasion of these cells, possibly due to the regulation of the expression of matrix metalloproteinase (MMP)­2, MMP­9 and tissue inhibitor of metalloproteinase­2. Furthermore, data from the present study suggested that the knockdown of STIP1 decreased the levels of phosphorylated Akt and phosphorylated ERK1/2. In summary, these findings indicate that targeting STIP1 in osteosarcoma may constitute a viable molecular targeted therapy strategy for the inhibition of CSC invasion and migration.

Bone marrow stromal cells-derived exosomes target DAB2IP to induce microglial cell autophagy, a new strategy for neural stem cell transplantation in brain injury.

Bone marrow stromal cells (MSCs) are a useful source of stem cells for the treatment of various brain injury diseases due to their abundant supply and fewer ethical problems compared with transplant treatment. However, the clinical application of MSCs is limited due to allograft rejection and immunosuppression in the process of MSCs transplantation. According to previous studies, microglial cell autophagy occurs following co-culture with MSCs. In the present study, exosomes were obtained from MSCs and subsequently characterized using transmission electron microscopy, atomic force microscopy and dynamic light scattering particle size analysis. The type of microRNAs (miRs) found in the exosomes was then analyzed via gene chip. The results demonstrated that microglial cell autophagy could be induced by exosomes. This mechanism was therefore investigated further via reverse transcription-quantitative PCR, western blotting and luciferase assays. These results demonstrated that exosomes from MSCs could induce microglial cell autophagy through the miR-32-mediated regulation of disabled homolog 2-interacting protein, thus providing a theoretical basis for the clinical application of miRs in MSCs.

Delayed metformin treatment improves functional recovery following traumatic brain injury via central AMPK-dependent brain tissue repair.

Accumulating evidence suggests that chronic metformin posttreatment offers potent neuroreparative effects against acute brain injury. However, in previous studies, metformin was not initially administered beyond 24 h postinjury, and the effects of delayed metformin treatment in traumatic brain injury (TBI) and other types of acute brain injury and the related mechanisms are unclear. To test this, male C57BL/6 mice received once daily metformin treatment (20, 50 or 100 mg/kg/d, i.p.) at day 1-14, day 1-2, day 1-10, day 3-10, day 5-12 or day 5-28 after cryogenic TBI (cTBI). The results showed that 100 mg/kg/d metformin administered at day 1-14 postinjury significantly promoted motor functional recovery in the beam walking and gait tests and reduced the infarct volume. Metformin (100 mg/kg/d) administered at day 1-10 or day 3-10 but not day 1-2 or day 5-12 after cTBI significantly improved motor functional outcomes at day 7 and 14, and reduced the infarct volume at day 14. Interestingly, the therapeutic time window was further expanded when the duration of metformin treatment starting at day 5 postinjury was extended to 2 weeks. Furthermore, compared with cTBI, the administration of metformin at day 3-10 or day 5-28 after cTBI significantly elevated the expression of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and growth associated protein 43 (an axonal regeneration marker) and the number of vascular branch points and decreased the area of glial scar and the number of amoeboid microglia in the peri-infarct area at day 14 or 28 postinjury. The above beneficial effects of metformin were blocked by the intracerebroventricular injection of the AMPK inhibitor compound C (40 µg/mouse/d). Our data provide the first evidence that metformin has a wide therapeutic time window for at least 5 days after cTBI, during which it can improve functional recovery by promoting tissue repair and inhibiting glial scar formation and microglial activation in a central AMPK-dependent manner.

Efficacy of 50 Hz electromagnetic fields on human epidermal stem cell transplantation seeded in collagen sponge scaffolds for wound healing in a murine model.

To explore the possible efficacy of electromagnetic fields (EMF) for skin tissue engineering, effects of EMF exposure on epidermal stem cells (ESC) seeded in collagen sponge scaffolds for wound healing in a murine model were investigated. The wound models of a full-thickness defect established with 36 7 ∼ 8-week-old nude mice were randomly divided into three groups: a control group, an ESC-only group, and an ESC with EMF exposure group (frequency of 50 Hz, magnetic induction of 5 mT, 60 min per day for 20 days). ESC were separated from human foreskin and cultured in vitro, and then transplanted with collagen sponge scaffolds as a delivery vehicle to wounds of the ESC-only group, and ESC with EMF exposure group was exposed to EMF after ESC transplantation. Effects of EMF on morphological changes and expression of ß1 integrin in regenerated skins were observed. Wound healing rates and healing times were collected to evaluate the efficacy of repairment. Results showed that human ESC were successfully transplanted to nude mice, which facilitated the formation of intact skin on nude mice. In contrast to other groups, the wound healing of ESC with EMF exposure group was the fastest (P < 0.05), the structure of regenerated skins was more mature, and it contained more continuity in the number of viable cell layers and rich hair follicles' structure. These results suggest that the use of 50 Hz EMF as a non-invasive treatment can accelerate wound healing of ESC transplantation, and restore structural integrity of regenerated skin. Bioelectromagnetics. 38:204-212,2017. © 2017 Wiley Periodicals, Inc.

Predictive effects of bilirubin on response of colorectal cancer to irinotecan-based chemotherapy.

AIM: To examine the predictive effects of baseline serum bilirubin levels and UDP-glucuronosyltransferase (UGT) 1A1*28 polymorphism on response of colorectal cancer to irinotecan-based chemotherapy. METHODS: The present study was based on a prospective multicenter longitudinal trial of Chinese metastatic colorectal cancer (mCRC) patients treated with irinotecan-based chemotherapy (NCT01282658). Baseline serum bilirubin levels, including total bilirubin (TBil) and unconjugated bilirubin (UBil), were measured, and genotyping of UGT1A1*28 polymorphism was performed. Receiver operating characteristic curve (ROC) analysis was used to determine cutoff values of TBil and UBil. The TBil values were categorized into > 13.0 or ≤ 13.0 groups; the UBil values were categorized into > 4.1 or ≤ 4.1 groups. Combining the cutoff values of TBil and UBil, which was recorded as CoBil, patients were classified into three groups. The classifier's performance of UGT1A1*28 and CoBil for predicting treatment response was evaluated by ROC analysis. Associations between response and CoBil or UGT1A1*28 polymorphism were estimated using simple and multiple logistic regression models. RESULTS: Among the 120 mCRC patients, the serum bilirubin level was significantly different between the UGT1A1*28 wild-type and mutant genotypes. Patients with the mutant genotype had an increased likelihood of a higher TBil (P = 0.018) and a higher UBil (P = 0.014) level compared with the wild-type genotype. Patients were stratified into three groups based on CoBil. Group 1 was patients with TBil > 13.0 and UBil > 4.1; Group 2 was patients with TBil ≤ 13.0 and UBil > 4.1; and Group 3 was patients with TBil ≤ 13.0 and UBil ≤ 4.1. Patients in Group 3 had more than a 10-fold higher likelihood of having a response in the simple (OR = 11.250; 95%CI: 2.286-55.367; P = 0.003) and multiple (OR = 16.001; 95%CI: 2.802 -91.371; P = 0.002) analyses compared with the Group 1 individuals. Patients carrying the UGT1A1*28 (TA)7 allele were 4-fold less likely to present with a response compared with the individuals harboring a homozygous (TA)6 genotype in the simple (OR = 0.267; 95%CI: 0.100-0.709; P = 0.008) and multiple (OR = 0.244; 95%CI: 0.088-0.678; P = 0.007) analyses. Classifier's performance of CoBil and UGT1A1*28 were comparable. CONCLUSION: CoBil and UGT1A1*28 are both independent biomarkers for predicting the treatment response of mCRC patients to irinotecan-based chemotherapy. After validation, CoBil, an easily determinable index in the clinic, might be helpful in facilitating stratification of mCRC patients for individualized treatment options.

Novel Clinical Scale for Evaluating Pre-Operative Risk of Cerebral Herniation from Traumatic Epidural Hematoma.

Secondary massive cerebral infarction (MCI) is the predominant prognostic factor for cerebral herniation from epidural hematoma (EDH) and determines the need for decompressive craniectomy. In this study, we tested the clinical feasibility and reliability of a novel pre-operative risk scoring system, the EDH-MCI scale, to guide surgical decision making. It is comprised of six risk factors, including hematoma location and volume, duration and extent of cerebral herniation, Glasgow Coma Scale score, and presence of preoperative shock, with a total score ranging from 0 to 18 points. Application of the EDH-MCI scale to guide surgical modalities for initial hematoma evacuation surgery for 65 patients (prospective cohort, 2012.02-2014.01) showed a significant improvement in the accuracy of the selected modality (95.38% vs. 77.95%; p = 0.002) relative to the results for an independent set of 126 patients (retrospective cohort, 2007.01-2012.01) for whom surgical modalities were decided empirically. Results suggested that simple hematoma evacuation craniotomy was sufficient for patients with low risk scores (≤9 points), whereas decompressive craniectomy in combination with duraplasty were necessary only for those with high risk scores (≥13 points). In patients with borderline risk scores (10-12 points), those having unstable vital signs, coexistence of severe secondary brainstem injury, and unresponsive dilated pupils after emergent burr hole hematoma drainage had a significantly increased incidence of post-traumatic MCI and necessity of radical surgical treatments. In conclusion, the novel pre-operative risk EDH-MCI evaluation scale has a satisfactory predictive and discriminative performance for patients who are at risk for the development of secondary MCI and therefore require decompressive craniectomy.

Epigallocatechin-3-gallate protects HUVECs from PM2.5-induced oxidative stress injury by activating critical antioxidant pathways.

Endothelial dysfunction and oxidative stress likely play roles in PM2.5-induced harmful effects. Epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent of green tea, is a potent antioxidant that exerts protective effects on cardiovascular diseases (CVDs) in part by scavenging free radicals. The exposure to ambient fine particulate matter (PM2.5) is responsible for certain CVDs. The aim of the present study was to investigate whether EGCG could also inhibit PM2.5-induced oxidative stress by activating the nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway in human umbilical vein endothelial cells (HUVECs). PM2.5 (200 µg/mL) increased both cell death and intracellular ROS levels significantly, whereas EGCG (50-400 µM) inhibited these effects in a concentration-dependent manner. Western blotting and PCR demonstrated that EGCG increased Nrf2 and HO-1 expression in HUVECs that had been exposed to PM2.5. PD98059 (a selective inhibitor of extracellular signal regulated kinase [ERK]-1/2) and SB203580 (a selective inhibitor of p38 MAPK), but not SP600125 (a selective inhibitor of c-jun N-terminal kinase [JNK]), attenuated the EGCG-induced Nrf2 and HO-1 expression. In addition, silencing Nrf2 abolished EGCG-induced Nrf2 and HO-1 upregulation and enhancement of cell viability. The present study suggests that EGCG protects HUVECs from PM2.5-induced oxidative stress injury by upregulating Nrf2/HO-1 via activation of the p38 MAPK and the ERK1/2 signaling pathways.

Fifty-Hertz electromagnetic fields facilitate the induction of rat bone mesenchymal stromal cells to differentiate into functional neurons.

BACKGROUND AIMS: Research results have shown that bone mesenchymal stromal cells (BMSC) can different into neural cells. Electromagnetic fields (EMF) play a role in regulating cell proliferation and differentiation, but the mechanisms behind this are unknown. In the present study, we explored the efficacy of EMF on the induction of rat BMSC differentiation into neurons in vitro. METHODS: First, rat BMSC were induced in a nerve cell culture environment and divided into three groups: an EMF induction treatment group (frequency of 50 Hz, magnetic induction of 5 mT, 60 min per day for 12 days), an induction-only group and a control group. Second, we observed cell phenotypes in a confocal microscope, tested gene expression through the use of reverse transcriptase-polymerase chain reaction, and detected postsynaptic currents by means of a cell patch-clamp. We analyzed the cell cycles and the portion of cells expressing neural cell markers with the use of flow cytometry. RESULTS: The results indicated that EMF can facilitate BMSC differentiation into neural cells, which expressed neuronal-specific markers and genes; they formed synaptic junctions and pulsed excitatory postsynaptic currents. At the same time, the G0-G1 phase ratio recorded by means of flow cytometry gradually decreased under the EMF treatment, whereas there was an increase of S-phase ratio, and the portion of cells expressing neuronal-specific markers increased. CONCLUSIONS: Given that a noninvasive treatment of 50-Hz EMF could significantly facilitate BMSC to differentiate into functional neurons, EMF appears to be a promising clinical option for stem cell transplantation therapies to combat central nervous system diseases.

Transforming growth factor-1 promotes the transcriptional activation of plasminogen activator inhibitor type 1 in carcinoma-associated fibroblasts.

Carcinoma-associated fibroblasts (CAFs) play a pivotal role in promoting the growth, invasion and metastasis of tumor cells. However, to date little is known about the oncogenic mechanisms of CAFs. This study aimed to identify the microenvironmental factors involved in tumor development and progression directed by CAFs in liver metastases. Tissue samples collected from 20 patients with colorectal liver metastases were used in this study. Histological and morphological characterization of the samples was performed using hybridization and immunohistological assays. The mRNA expression of α-smooth muscle actin (α-SMA) was measured by northern blotting. The expression of plasminogen activator inhibitor type 1 (PAI-1) was measured by enzyme-linked immunosorbent assay (ELISA). As a result, co-expression of Thy-1 (CD90) and α-SMA was identified in CAFs, while normal liver samples were negative for α-SMA and Thy-1. Compared with epidermal growth factor (EGF) and tumor necrosis factor (TNF) incubation, the expression of α-SMA increased significantly following transforming growth factor-1 (TGF-1) incubation (P<0.05), while platelet-derived growth factor (PDGF) caused a significant suppression of α-SMA expression (P<0.05). PAI-1 expression was significantly lower in unstimulated fibroblasts compared to TGF-1-treated fibroblasts (P<0.01). The levels of PAI-1 transcription were significantly higher in CAFs from the patient samples compared with the healthy controls. Taken together, our findings suggest that CAFs may be important in migration, matrix degradation, invasion and angiogenesis of tumors, and TGF-1 may promote the activation of PAI-1 transcription in CAFs.

Effects of 50 Hz electromagnetic fields on human epidermal stem cells cultured on collagen sponge scaffolds.

PURPOSE: This study is to investigate the effects of electromagnetic fields (EMF) on proliferation of epidermal stem cells (ESC), which could present a viable clinical option for skin tissue engineering. MATERIALS AND METHODS: The ESC obtained from human foreskin were grafted into type-I three-dimensional collagen sponge scaffolds, and then were exposed with EMF (frequency 50 Hz, intensity 5 mT) for 14 d (30 min per d). Meanwhile, the control group was set under the same conditions without EMF. The effects of EMF on growth and proliferation of ESC were analyzed with staining of hematoxylin and eosin (H&E) and 4',6-diamidino-2-phenylindole (DAPI) under microscope or scanning electron microscope. The data of DAPI staining for 2 d, 7 d, 10 d and 14 d were collected respectively to investigate the cells proliferation. RESULTS: ESC cultured in collagen sponge scaffolds could be steady grown and EMF could promote ESC proliferation compared with control (P < 0.05). CONCLUSIONS: EMF could significantly promote proliferation of ESC, which leads to a promising clinical option for skin tissue engineering.

The correlation between IGF-II and Bcl-2 expression in colorectal adenocarcinoma.

Our aim for this study was to investigate the correlation and clinical significance between the expression of IGF-II and Bcl-2 in colorectal adenocarcinoma, especially in terms of the metastasis of colorectal adenocarcinoma. Sixty paraffin embedded samples of colorectal adenocarcinoma were selected, and fifteen normal colorectal tissues were used as controls. IGF-II mRNA was detected using in situ hybridization, and the expression of Bcl-2 along with the proliferating cell nuclear antigen (PCNA) protein was detected through immunohistochemistry. The TUNEL assay was used to detect apoptosis. Specimens with a positive cell ratio less than 30% were defined as negative. The levels of IGF-II mRNA and the Bcl-2 protein were significantly higher in colorectal adenocarcinoma (39.64 ± 7.38% and 30.74 ± 7.22%, respectively) than in normal colorectal tissues (22.56 ± 4.21% and 12.17 ± 1.94%, respectively) (P < 0.01). The levels were related to Dukes' stage and lymph node metastases, but were unrelated to patient age, gender, tumor site, tumor size, and tumor differentiation. Also, a negative correlation was observed between IGF-II mRNA and Bcl-2 protein (P < 0.05) during Dukes' stages. In addition, a positive correlation between IGF-II mRNA and PCNA or apoptosis, as well as a negative correlation between Bcl-2 and apoptosis were observed (P < 0.01). There was no correlation between Bcl-2 and PCNA (P > 0.05). The patients detected as IGF-II mRNA (+) and Bcl-2 (-) showed the worst prognosis. The expression of IGF-II and Bcl-2 was correlated with the clinical manifestation of colorectal adenocarcinoma; thus, the assessment of both IGF-II and Bcl-2's status will provide important information regarding the diagnosis and prognosis of colorectal adenocarcinoma.

Radio-activation of hTERT promoter in larynx squamous carcinoma cells: an 'indirected-activator' strategy in radio-gene-therapy.

The usefulness of human telomerase reverse transcriptase (hTERT) gene promoter has been proposed in cancer-targeted gene therapy. However, this promoter may not be strong enough to achieve therapeutic levels of transgene expression. In this study, we tested an 'indirected-activator' strategy that utilizes radiation to increase the activity of the hTERT gene promoter. We demonstrated that hTERT may participate in the process of DNA repair induced by irradiation. We found that Zidovudine (AZT, an hTERT inhibitor) can decrease the telomerase activity in human HEp-2 larynx squamous carcinoma cells and lower the survival fraction of HEp-2 cells exposed to radiation. In HEp-2 cells exposed to 6 Gy-radiation, the hTERT promoter showed 2.9-fold higher activity compared with unirradiated cells. Importantly, an increased expression of enzyme horseradish peroxidase (HRP) controlled by the hTERT promoter was found in the transfected cells after irradiation, which coincided with a higher killing rate for HEp-2 cells after prodrug indole-3-acetic acid (IAA; converted by HRP into a cytotoxin) incubation combined with irradiation or not. Our observations suggest that hTERT promoter-mediated gene therapy could be improved in combination with radiotherapy, which may be due to cellular DNA damage responses.

Efficient inhibition of human telomerase activity by antisense oligonucleotides sensitizes cancer cells to radiotherapy.

AIM: To investigate the effect of the antisense oligonucleotides (ASODN) specific for human telomerase RNA (hTR) on radio sensitization and proliferation inhibition in human neurogliocytoma cells (U251). METHODS: U251 cells were transfected with hTR ASODN or nonspecific oligonucleotides (NSODN). Before and after irradiation of (60)Co- gamma ray, telomerase activity was assayed by telomeric repeat amplification protocol ( TRAP-PCR-ELISA), and DNA damage and repair were examined by the comet assay. The classical colony assay was used to plot the cell-survival curve, to detect the D(0 )value. RESULTS: hTR antisense oligonucleotides could downregulate the telomerase activity, increase radiation induced DNA damage and reduce the subsequent repair. Furthermore, it could inhibit the proliferation and decrease the D(0 ) value which demonstrates rising radiosensitivity. However, telomere length was unchanged over a short period of time. CONCLUSION: These findings suggest that an ASODN-based strategy may be used to develop telomerase inhibitors, which can efficiently sensitize radiotherapy.

Angelica sinensis down-regulates hydroxyproline and Tgfb1 and provides protection in mice with radiation-induced pulmonary fibrosis.

Pulmonary fibrosis is a common delayed side effect of radiation therapy, and it has a poor prognosis. Tgfb1 is a potent chemoattractant for fibroblasts and stimulates the production of collagen, the protein that contains hydroxyproline. Since collagen is by far the most abundant protein in the lung, comprising 60-70% of the tissue mass, analysis of the hydroxyproline content in lung tissues provides a reliable quantitative index for pulmonary fibrosis. Thus hydroxyproline and Tgfb1 may be involved in the development of fibrosis. In this study, we investigated radiation-induced pulmonary fibrosis in a mouse model. C57BL/6 mice were assigned into four groups: no treatment, treated with Angelica sinensis treated only, X-irradiated only (a single fraction of 12 Gy to the thorax), and Angelica sinensis treatment plus radiation. We assayed expression of hydroxyproline and the mRNA and protein of Tgfb1 in the four groups. We found that Angelica sinensis down-regulated the production of Tgfb1 and hydroxyproline in mice with radiation-induced pulmonary fibrosis. This study has demonstrated for the first time that Angelica sinensis inhibits the progress of radiation-induced pulmonary fibrosis, possibly by down-regulating the expression of the proinflammatory cytokine Tgfb1. These data suggest that Angelica sinensis may be useful in preventing and/or treating radiation-induced pulmonary fibrosis in the clinic.

Chinese medicine Angelica sinensis suppresses radiation-induced expression of TNF-alpha and TGF-beta1 in mice.

Radiotherapy of thoracic cancer often causes pulmonary inflammation leading to pneumonitis and fibrosis. We favor the hypothesis that cytokine-mediated multicellular interactions may result in the overexpression of proinflammatory cytokines such as TNF-alpha and TGF-beta1, which promotes progressive radiation-induced lung injury. The root of Angelica sinensis, known as 'Danggui' in Chinese medicine, is widely used to treat radiation-induced pneumonitis in humans and shows clinical efficacy and low/no toxicity with an unclear mechanism. Using quantitative RT-PCR and immunohistochemistry (IHC) methods, we investigated radiation-induced lung injury in a mouse model. C57BL/6 mice were assigned to 4 groups: no treatment (NT), Angelica Sinensis treatment only (AS), X-ray irradiation only (XRT, single fraction of 12 Gy irradiation to the thoraces) and AS treatment plus XRT (AS/XRT). Mice in NT and AS groups exhibited low TNF-alpha and TGF-beta1 mRNA levels and few positive cell counts for TNF-alpha (8-17 cells per field, x400 magnification) and TGF-beta1 (9-31 cells per field), respectively. In XRT mice, there were increased inflammatory cells positive for TNF-alpha and TGF-beta1 in lung tissue compared with NT mice (P<0.01). However, when XRT mice received AS treatment (AS/XRT), the number of inflammatory cells in lung tissue positive for both TNF-alpha and TGF-beta1 was decreased compared with XRT-only mice (P<0.01) accompanied by moderately decreased mRNA levels of TNF-alpha and TGF-beta1. We conclude that radiation induces expression of TNF-alpha and TGF-beta1 in the inflammatory cells of irradiated lung tissue during the pneumonic phase. The predominant localization of TNF-alpha and TGF-beta1 in inflammatory cell infiltrates suggests these cytokines' involvement in the process of radiation-induced pneumonitis. Moreover, effective down-regulation of TNF-alpha and TGF-beta1 in irradiated lung tissue by Angelica Sinensis is, at least in part, indicative of its clinical efficacy in treating radiation-induced pneumonitis.

Apoptosis induced by short hairpin RNA-mediated STAT6 gene silencing in human colon cancer cells.

BACKGROUND: The relationship between signal transduction and tumors has become one of the foci in cancer research. Signal transducer and activator of the transcription 6 (STAT6) signaling pathway is found to be activated in some cancer cells. But the function of the pathway in cancer cells is unknown. This study was undertaken to investigate the effect of the Stat6 signaling pathway on apoptosis in human colon cancer cells (HT-29 cells) and the possible mechanism of Stat6 by RNA interference techniques. METHODS: Four eukaryotic expression plasmid vectors of short hairpin RNA (shRNA) specific for the STAT6 gene were designed and generated by molecular biological technology. The plasmid vectors were transfected into HT-29 cells by cation liposomes to block the Stat6 signaling pathway. The expressions of STAT6 mRNA and phosph-Stat6 protein were detected by the reverse transcriptase polymerase chain reaction (RT-PCR) method and flow cytometry respectively to screen the most effective shRNA at 72 hours after transfection. The apoptosis condition of the cells in which the expression of the STAT6 gene had been interfered was analyzed by flow cytometry and confocal microscopy. Both mRNA and protein expression of B cell lymphoma-2 (Bcl-2) and Bax were detected by RT-PCR and western blotting. RESULTS: Two effective eukaryotic expression plasmid vectors of shRNA specific for the STAT6 gene were generated successfully. One can reduce the expression of the STAT6 gene by 82.4% and the other by 56.8% (P < 0.01). The apoptotic rate of colon cancer cells in which STAT6 gene expression had been interfered was significantly higher than that in controlled colon cancer cells (P < 0.01). In the cells in which the Stat6 signaling pathway was blocked, the levels of mRNA and protein Bcl-2 were significantly decreased, whereas those of Bax were significantly increased (P < 0.01). CONCLUSIONS: The Stat6 signaling pathway can inhibit apoptosis in human colon cancer cells. The subsequent disorder of Bcl-2/Bax expression may play an important part in that process. The STAT6 gene may serve as a potential target in cancer therapy.

[Impact of STAT6 signaling pathway blockade on apoptosis of human colon cancer cells].

OBJECTIVE: To investigate the relationship between the signal transducer and activator of transcription 6 (STAT6) and human colon cancer. METHODS: Four STAT6-specific recombinant plasmid vectors, pshRNA-STAT6-1, 2, 3, and 4 were constructed and transfected into the cultured human colon cancer cells of the line HT-29. Seventy-two hours later RT-PCR was used to detect the mRNA expression of STAT6 and the apoptosis-related genes Bcl-2 and Bax, flow cytometry (FCM) was used to detect the protein expression of phopho-STAT6 (pSTAT6). HT-29 cells were inoculated into a plate and transfected with pshRNA-STAT6-1 or pshRNA-STAT6-4, and HT-29 cells without transfection were used as controls. Seventy-two hours later FCM was used to observe the cell apoptosis. Another HT-29 cells were inoculated into a plate and transfected with pshRNA-STAT6-1 or pshRNA-STAT6-4, or blank liposome as controls. Seventy-two hours later. Western blotting was used to detect the protein expression of Bcl-2 and Bax genes. RESULTS: The p-STAT6 protein expression rate was 3.2% +/- 0.6% in the pshRNA-STAT6-1 group, significantly lower than that of the blank control group (18.2% +/- 0.9%, P < 0.01) with an inhibition rate of 82.4%, and was 7.9% +/- 0.4% in the pshRNA-STAT6-4 group, significantly lower than that in the blank control group too (P < 0.01) with an inhibition rate of 56.6%. And the p-STAT6 protein expression rates of the pshRNA-STAT6-2 and pshRNA-STAT6-3 groups were 16.6% +/- 0.5% and 17.1% +/- 0.7% respectively, both not significant different from that of the blank control group (both P > 0.05). The early cell apoptosis rates of the pshRNA-STAT6-1 and pshRNA-STAT6-4 groups were 13.0% and 8.8% respectively, both significantly higher than that of the blank control group (0.4%, both P < 0.05). The mRNA expression of Bcl-2 was significantly lower and the mRNA expression of Bax was significantly higher in the pshRNA-STAT6-1 and pshRNA-STAT6-4 groups than in the blank control and blank liposome groups (all P < 0.01). The protein expression patterns of Bcl-2 and Bax was consistent with that of their protein expression. CONCLUSION: STAT6 signaling pathway inhibits the apoptosis of colon cancer cells by regulation of the Bcl-2 and Bax genes.