Cytochrome P450 SmCYP78A7a positively functions in eggplant response to salt stress via forming a positive feedback loop with SmWRKY11.

Accumulating salinity in soil critically affected growth, development, and yield in plant. However, the mechanisms of plant against salt stress largely remain unknown. Herein, we identified a gene named SmCYP78A7a, which encoded a cytochrome P450 monooxygenase and belonged to the CYP78A sub-family, and its transcript level was significantly up-regulated by salt stress and down-regulated by dehydration stress. SmCYP78A7a located in the endoplasmic reticulum. Silencing of SmCYP78A7a enhanced susceptibility of eggplant to salt stress, and significantly down-regulated the transcript levels of salt stress defense related genes SmGSTU10 and SmWRKY11 as well as increased hydrogen peroxide (H2O2) content and decreased catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) enzyme activities. In addition, SmCYP78A7a transient expression enhanced eggplant tolerance to salt stress. By chromatin immunoprecipitation PCR (ChIP-PCR), luciferase reporter assay, and electrophoretic mobility shift assay (EMSA), SmWRKY11 activated SmCYP78A7a expression by directly binding to the W-box 6-8 (W-box 6, W-box 7, and W-box 8) within SmCYP78A7a promoter to confer eggplant tolerance to salt stress. In summary, our finds reveal that SmCYP78A7a positively functions in eggplant response to salt stress via forming a positive feedback loop with SmWRKY11, and provide a new insight into regulatory mechanisms of eggplant to salt stress.

Analysis of 246 sentinel lymph node biopsies of patients with clinical primary breast cancer by application of carbon nanoparticle suspension.

AIM: This study aims to explore the accuracy, specificity and laws of axillary lymph node metastasis predicted by sentinel lymph node biopsy (SLNB) by comparing axillary lymph node status via SLNB and axillary lymph node dissection (ALND) with nanocarbon as the tracer. METHODS: Forty six patients were retrospectively analyzed. These patients underwent SLNB with nanocarbon as the tracer from March 2013 to April 2014. RESULTS: Two hundred and forty six patients of sentinel lymph node (SLN) were successfully detected. Among these patients, 8 patients had 1 SLN (3.25%), 33 patients had 2 SLN (13.41%), 46 patients had 3 SLN (18.70%), 51 patients had 4 SLN (20.73%), 40 patients had 5 SLN (16.26%), 24 patients had 6 SLN (9.76%) and 24 patients had 7 or more SLN (9.76%). The SLNB success rate of nanocarbon staining in the 246 cases was 99.59%, accuracy rate was 97.06% and sensitivity was 93.22%. Furthermore, false negatives were found in four patients, and the false-negative rate was 6.78%. The number of lymph node metastasis in the SLNB and ALND of early-stage breast cancer was analyzed. When the number of SLN dissection was 1, 2, 3, 4, 5, 6 or 7, the coincidence rate of lymph node metastasis for SLNB and ALND was 80.00, 84.36, 78.57, 88.89, 90.48, 80.00, 73.68 and 78.36, respectively. CONCLUSION: Sentinel lymph node biopsy performed using the nanocarbon staining method is simple, easy and reliable, and it can be used to predict the axillary status of breast cancer in the early stage.

AMPK activators suppress breast cancer cell growth by inhibiting DVL3-facilitated Wnt/ß-catenin signaling pathway activity.

Adenosine monophosphate-activated protein kinase (AMPK) is a principal regulator of metabolism and the conservation of energy in cells, and protects them from exposure to various stressors. AMPK activators may exhibit therapeutic potential as suppressors of cell growth; however, the molecular mechanism underlying this phenomenon in various cancer cells remains to be fully elucidated. The present study investigated the effects of AMPK activators on breast cancer cell growth and specified the underlying molecular mechanism. In the present study, the AMPK activator metformin impaired breast cancer cell growth by reducing dishevelled segment polarity protein 3 (DVL3) and ß­catenin levels. Western blotting and immunohistochemistry demonstrated that DVL3 was recurrently upregulated in breast cancer cells that were not treated with metformin, and was significantly associated with enhanced levels of ß­catenin, c­Myc and cyclin D1. Overexpression of DVL3 resulted in upregulation of ß­catenin and amplification of breast cancer cell growth, which confirmed that Wnt/ß­catenin activation via DVL3 is associated with breast cancer oncogenesis. To elucidate the underlying mechanism of these effects, the present study verified that metformin resulted in a downregulation of DVL3 and ß­catenin in a dose­dependent manner, and induced phosphorylation of AMPK. Compound C is an AMPK inhibitor, which when administered alongside metformin, significantly abolished the effects of metformin on the reduction of DVL3 and activation of the phosphorylation of AMPK. Notably, the effects of metformin on the mRNA expression levels of DVL3 remain to be fully elucidated; however, a possible interaction with DVL3 at the post­transcriptional level was observed. It has previously been suggested that the molecular mechanism underlying AMPK activator­induced suppression of breast cancer cell growth involves an interaction with, and impairment of, DVL3 proteins. The results of the present study are of future clinical importance and advocate the use of metformin as a potential therapeutic agent against breast cancer.

Interleukin-12 activated CD8+ T cells induces apoptosis in breast cancer cells and reduces tumor growth.

During the past two decades, cytokines have emerged as key molecules to modulate innate and adaptive immunity and mediate anti-tumor activity. Although multiple cytokine types are implicated for such anti-tumor activity in several cancer types, it remains largely unknown in breast cancer. In this study, cytokines that are prior known for antitumor activity in different cancer types were examined against breast cancer using a 4T1 cells based xenograft-model. Our results showed Interleukin-12 (IL-12) (500ng/mouse) significantly suppressed the growth of tumors, while other cytokines showed minimal suppression. Subsequent molecular analysis by flow cytometry and immunohistochemistry confirmed the CD8+ cells infiltration and Interferon-γ (IFN-γ) production by them in tumor environment. In addition, we observed that IFN-γ production by activated CD8+ cells directly induced apoptosis in tumor cells, which together indicate that IL-12 causes CD8+ cells to infiltrate and secrete IFN-γ in tumor environment, which induce apoptosis in them and causes tumor growth suppression. Furthermore, we showed that lower dosage of IL-12 and chemotherapy drug tamoxifen combinations enhanced the tumor suppression as opposed to single treatments, and thereby propose an alternate option for high dosage associated effects for both drug and cytokine treatments.

Production of herbicide-resistant medicinal plant Salvia miltiorrhiza transformed with the bar gene.

In this study, we successfully performed Agrobacterium-mediated genetic transformation of Salvia miltiorrhiza and produced herbicide-resistant transformants. Leaf discs of S. miltiorrhiza were infected with Agrobacterium tumefaciens EHA105 harboring pCAMBIA 3301. The pCAMBIA 3301 includes an intron-containing gus reporter and a bar selection marker. To increase stable transformation efficiency, a two-step selection was employed which consists of herbicide resistance and gus expression. Here, we put more attention to the screening step of herbicide resistance. The current study provides an efficient screening system for the transformed plant of S. miltiorrhiza harboring bar gene. To determine the most suitable phosphinothricin concentration for plant selection, non-transformed leaf discs were grown on selection media containing six different phosphinothricin concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/l). Based on the above results of non-transformed calluses, the sensitivity of phosphinothricin (0, 0.4, 0.8, 1.2, 1.6 mg/l) was tested in the screening of transgenic S. miltiorrhiza. We identified that 0.6 mg/l phosphinothricin should be suitable for selecting putatively transformed callus because non-transformed callus growth was effectively inhibited under this concentrations. When sprayed with Basta, the transgenic S. miltiorrhiza plants were tolerant to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to S. miltiorrhiza.

Decreased expression of C-erbB-2 and CXCR4 in breast cancer after primary chemotherapy.

BACKGROUND: Biological molecular markers such as proto-oncogene erbB-2 (HER-2/neu, c-erbB-2), the CXC chemokine receptor 4 (CXCR4), estrogen receptor (ER), Proliferating Cell Nuclear Antigen (PCNA), DNA topoisomerase II (topo II), P-glycoprotein (P-gp) and glutathione S-transferase (GST) were observed for changes after administration of neochemotherapy and whether these protein expression changes were correlated with response to chemotherapy. METHODS: Sixty-four patients with primary breast cancer who had undergone neo-adjuvant chemotherapy were enrolled in the present study. The expressions of C-erbB-2, CXCR4 and ER-α were measured by immunohistochemistry (IHC) on full tissue sections and on tissue microarrays (TMAs). PCNA, TopoII, P-gp and GST were measured by IHC on TMAs. On the other hand, CXCR4, C-erbB-2 and ER-α expressions were detected using western blot analysis to 16 pairs of fresh preoperative core biopsies. The final surgical specimens were obtained from patients with breast carcinoma who received neo-adjuvant chemotherapy and obtained a partial response (PR). RESULTS: Our data demonstrated that the levels of C-erbB-2, CXCR4 and ER-α in patients decreased after they received neo-adjuvant chemotherapy on full tissue sections and on TMAs. The PCNA level was down-regulated after receiving neo-adjuvant chemotherapy, and no significant change was observed for TopoII, P-gp and GST. The levels of C-erbB-2, CXCR4 and ER-α were also down-regulated after neo-adjuvant chemotherapy was administered, as detected by western blot. In addition, the change expressions of C-erbB-2 and CXCR4 in specimens tended to be correlated with pathological change to neo-adjuvant chemotherapy on full tissue sections and on TMAs in a Pearson chi-square analysis. CONCLUSIONS: As demonstrated in our study, after breast cancer patients were treated with neo-adjuvant systemic therapy, decreased expressions of C-erbB2, ER-α and CXCR4 were observed. Down-regulated expressions of c-erbB-2 and CXCR4 may be a novel mechanism of chemotherapy; the changes of these objective markers may be useful in evaluating the clinical response of neo-adjuvant chemotherapy in breast cancer.

[Effect of recombinant human granulocyte-macrophage colony stimulating factor on wound healing in patients with deep partial thickness burn].

OBJECTIVE: To evaluate the efficacy and safety of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) hydrogel in wound healing in patients with deep partial thickness burn. METHODS: The study was a multicenter, randomized, double-blind, placebo-controlled parallel clinical trial. Three hundred and twenty-one patients (302 cases finally fulfilled the protocol) with deep partial thickness burn were divided into A group (n = 200, with treatment of rhGM-CSF hydrogel, 100 microg/10 g/100 cm2/d), C group (n = 102,with treatment of placebo). Side-effect, systemic condition, wound healing time, wound healing rate, and total effective rate at different time points were observed. RESULTS: There were no obvious differences in vital signs, wound secretion, wound edge reaction, blood and urine routine, liver and kidney function between two groups (P > 0.05). No side-effect was observed. The median wound healing time was 17 days in A group, which was obviously shorter than that in C group (20 days, P < 0.01). The mean wound healing rate in A group was 24.5%, 70.5%, 95.3%, 99.6% respectively on 8th, 14th, 20th, 28th day after treatment, which were obviously higher than that in C group (15.1%, 51.4%, 84.6%, 97.1%, respectively, P < 0.01). The total effective rates in A group on 8th, 14th, 20th day after treatment were also higher than that in C group (P < 0.01). CONCLUSION: rhGM-CSF hydrogel can significantly accelerate wound healing in patients with deep partial thickness burn with certain safety.

Preferential expression of chemokine receptor CXCR4 by highly malignant human gliomas and its association with poor patient survival.

OBJECTIVE: CXCR4 is implicated in the growth, metastasis, and angiogenesis of malignant tumors. We investigated the potential role of CXCR4 in human gliomas. METHODS: The expression of CXCR4 messenger ribonucleic acid and protein by human glioma cell lines was examined by reverse-transcriptase polymerase chain reaction and immunocytochemistry analysis. Tumor cell chemotaxis and production of vascular endothelial growth factor induced by the CXCR4 ligand SDF-1beta were measured. Xenograft models were used for evaluation of glioma cell tumorigenesis. CXCR4 expression by xenografted tumors and primary human glioma specimens were evaluated for CXCR4 protein expression. The relationship between CXCR4 expression and patient survival was analyzed. A synthetic lipoxygenase inhibitor, Nordy, was tested for its effects on glioma cell expression and function of CXCR4, as well as on glioma cell tumorigenicity. RESULTS: CXCR4 expression correlated directly with the degree of malignancy of the human glioma cell lines and primary tumors. Activation of CXCR4 induced tumor cell chemotaxis and increased production of vascular endothelial growth factor. Glioma cells expressing higher levels of CXCR4 formed more rapidly growing and lethal tumors in nude mice. Primary human glioma specimens expressing CXCR4 contained high-density microvessels. Patients with CXCR4-positive gliomas had poorer prognosis after surgery. The lipoxygenase inhibitor Nordy diminished CXCR4 expression by glioma cell lines in vitro and reduced their tumorigenicity in nude mice. CONCLUSION: The level of CXCR4 expression seems to correlate with the degree of malignancy of human gliomas and may contribute to their rapid growth.

[Effect of nordy on FPR function of malignant human glioma cell line U87].

Nordy is a synthesized chrial compound. To investigate the effects of nordy (25 - 100 micromol x L(-1)) on the function of formylpeptide receptor (FPR) of malignant human glioma cells, human glioblastoma cell line U87 was used to detect its proliferation, migration, calcium mobilization, vascular endothelial growth factor (VEGF) mRNA and protein levels after activation of FPR by its agonist N-formyl-methionyl-leucyl-phenylalanine (fMLF). Cell proliferation, migration ability, VEGF mRNA, VEGF protein and calcium mobilization were evaluated by cell counting, chemotaxis assay, RT-PCR, ELISA and spectrometry. Nordy (50 - 100 micromol x L(-1)) potently inhibited the proliferation, migration and calcium mobilization of U87 cells induced by fMLF (P < 0.05). Moreover, 100 micromol x L(-1) nordy showed a significantly impaired VEGF mRNA expression and protein secretion induced by fMLF (P < 0.05). Nordy could inhibit FPR functioning in glioma cell proliferation, migration and angiogenesis, which might be a possible mechanism of its anti-cancer effects.

Activation of chemokine receptor CXCR4 in malignant glioma cells promotes the production of vascular endothelial growth factor.

Numerous studies have showed that chemokine receptors, such as CXCR4, contribute to the growth and metastasis of a variety of malignant tumors. In this study, we investigated the role of CXCR4 in the production of angiogenic factor, vascular endothelial growth factor (VEGF), in various human glioma cells from astrocytic origin. The expression of CXCR4 mRNA and protein in three glioma cell lines, U87-MG, SHG-44, and CHG-5, was determined by RT-PCR and immunocytochemistry, respectively. The malignancies of three gliomas were evaluated by expression of glial fibrillary acidic protein and vimentin, the differentiation markers of astrocytic cells. The role of functional CXCR4 in tumor cell migration was studied with chemotaxis assay. Ca2+ mobilization and VEGF production were measured in the cells after stimulation with CXCR4 ligand, SDF1beta. The results showed that the levels of functional CXCR4 expression at both mRNA and protein levels by several human glioma cell lines were correlated with the degree of differentiation of the tumor cells. Activation of CXCR4 induced glioma cell chemotaxis and could trigger the increase of intracellular [Ca2+]i. Such an activation could result in the increased production of VEGF by the stimulated tumor cells. Our results suggest that CXCR4 may contribute to the high level of VEGF produced by malignant glioma cells and thus constitute a therapeutic target for antiangiogenesis strategy.