[Morphological characteristics identification and molecular DNA barcoding analysis of Hippocampus spinosissimus].

The combination of morphological characteristics and DNA barcodes was used to a systematic study of Hippocampus spinosissimus,laying the foundation for rapid and accurate identification for the medical seahorse species. According to the reported literature and observation on seahorse samples,the typical characteristics of the H. spinosissimus include highly developed spiny,much short nose,single or double cheeks and strongly developed spines bordering pouch. Genomic DNAs of H. spinosissimus and other related seahorse species were extracted using the TIANamp Marine Animals DNA Kit. The COⅠ and ATP6 genes were amplified and sequenced in both directions. After the verification by Blast,the GC content,intraspecific and interspecific genetic distance,and the Neighbor joining( NJ) phylogenetic trees were analyzed by MEGA 7. The lengths of the COⅠ and ATP6 genes were 649 bp and 602-603 bp,respectively,with the average GC content of 39. 96% and 35. 37%. The maximum intraspecific genetic distances in H. spinosissimus based on COⅠ and ATP were both far less than the minimum interspecific genetic distance between H. spinosissimus and other seahorses,suggesting a significant barcoding gap. NJ analysis results of COⅠ and ATP6 exhibited that all H. spinosissimus species clustered together,indicating that the two DNA barcode could identify H. spinosissimus from other seahorses accurately and quickly. In addition,H. spinosissimus shared a close genetic relationship between H. kelloggi according to the NJ tree. Furthermore,there exits three stable subgroup structure of H. spinosissimus,indicating that COⅠ and ATP6 barcodes could be applied the indicator for the geographical ecology research of H. spinosissimus. The results obtained the typical morphological and molecular identification characteristics of H. spinosissimus,which played central roles for the development of species identification. This study provides an important basis data for expanding the medical seahorse resources and ensuring the safety of clinical medicine.

[Current status and future prospect of pharmacotherapy for pancreatic cancer].

Pancreatic cancer is the most common digestive tract tumor with an increasing incidence in recent years. The poor prognosis of pancreatic cancer is mainly because of the inability of detecting tumor at an early stage,its high potential for early dissemination,and its relatively poor sensitivity to chemotherapy. Most patients have lost the opportunity for surgery when they are diagnosed,which resulted in an urgent need for the development of more effective and safe therapies for pancreatic cancer. However,the current clinical cancer chemotherapy based on gemcitabine leads to poor prognosis in pancreatic patients. With the continuous research on the biological and cellular signaling pathways of pancreatic cancer,there have emerged a great many of novel agents,including new chemotherapeutic,targetable and immune-modulatory drugs,and some drugs have achieved encouraging results. Furthermore,as an alternative and supplementary method,traditional Chinese medicine has shown good application prospects in the field of pancreatic cancer treatment. This article reviews the current status of drug therapy for pancreatic cancer,summarizes the strength and weakness of existing therapeutic drugs in the application process,gives prospects of possible breakthroughs for the pharmacotherapy in the future,and provides certain new ideas and lessons for subsequent drug development.

[Inhibitory effect and mechanism of platycodin D combined with imatinib on K562/R].

Platycodin D(PD) has a significantly inhibitory effect on multiple malignant tumors, and can inhibit the proliferation of leukemia cells K562 and induce apoptosis. However, its effect in improving the sensitivity of drug-resistant cells to imatinib and their molecular mechanism remained unclear. To investigate the effect and mechanism of PD alone or combined with imatinib (IM) in inhibiting CML imatinib resistant cell line K562/R, the cell proliferation was examined by CCK8 assay to reveal the effect of PD on the inhibitory function of imatinib. Cell apoptosis was detected by Annexin V-FITC/PI double staining. Protein expressions of cleaved caspase-3, cleaved caspase-9, PARP, cleaved PARP, Bcr/abl, p-AKT and p-mTOR were detected by Western blot. The results showed that the inhibitory effect of PD combined with imatinib on the proliferation and apoptosis of K562/R cells was significantly higher than that of the control group and the single drug group. Protein expressions of cleaved caspase-3, cleaved caspase-9 and cleaved PARP were significantly up-regulated in the combination group, and protein expressions of PARP, Bcr/abl, p-AKT and p-mTOR were down-regulated. The results indicated that PD increased the sensitivity of drug-resistant cells to imatinib, and the inhibitory effect of PD combined with imatinib was significantly better than the single drug on cell proliferation, induction of apoptosis, inhibition of Bcr/abl protein and PI3K/AKT/mTOR signaling pathway.

[Progress of regulation of leukemia stem cells of chronic myeloid leukemia by autophagy].

Leukemia stem cells (LSC) that were found in chronic myeloid leukemia (CML) responsible for the abnormal proliferation with the potential of self-renewal and multi-directional differentiation are involved in the pathophysiological process for drug resistance and relapse of CML. Autophagy, a conservative lysosomal degradation process that mediates cell degradation and recycling process, plays crucial roles in maintaining the homeostasis and function of intracellular environment. Recent studies suggested that autophagy is involved in the regulation of LSC differentiation and also closely related to the chemo-sensitivity of CML. In this review, we focused on the role of autophagy on chemotherapy sensitivity of CML as well as the leukemia stem cell function for the development of new anti-leukemia drugs.

Acute upregulation of neuronal mitochondrial type-1 cannabinoid receptor and it's role in metabolic defects and neuronal apoptosis after TBI.

Metabolic defects and neuronal apoptosis initiated by traumatic brain injury (TBI) contribute to subsequent neurodegeneration. They are all regulated by mechanisms centered around mitochondrion. Type-1 cannabinoid receptor (CB1) is a G-protein coupled receptor (GPCR) enriched on neuronal plasma membrane. Recent evidences point to the substantial presence of CB1 receptors on neuronal mitochondrial outer membranes (mtCB1) and the activation of mtCB1 influences aerobic respiration via inhibiting mitochondrial cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/complex I pathway. The expression and role of neuronal mtCB1 under TBI are unknown. Using TBI models of cultured neurons, wild type and CB1 knockout mice, we found mtCB1 quickly upregulated after TBI. Activation of mtCB1 promoted metabolic defects accompanied with ATP shortage but protected neurons from apoptosis. Selective activation of plasma membrane CB1 showed no effects on neuronal metabolism and apoptosis. Activation of mtCB1 receptors inhibited mitochondrial cAMP/PKA/complex I and resulted in exacerbated metabolic defects accompanied with a higher ratio of ATP reduction to oxygen consumption decrease as well as neuronal apoptosis. Further research found the remarkable accumulation of protein kinase B (AKT) on neuronal mitochondria following TBI and the activation of mtCB1 upregulated mitochondrial AKT/complex V activity. Upregulation of mitochondrial AKT/complex V activity showed anti-apoptosis effects and alleviated ATP shortage in metabolic defects. Taken together, we have identified mtCB1 quickly upregulate after TBI and a dual role the mtCB1 might play in metabolic defects and neuronal apoptosis initiated by TBI: the inhibition of mitochondrial cAMP/PKA/complex I aggravates metabolic defects, energy insufficiency as well as neuronal apoptosis, but the coactivation of mitochondrial AKT/complex V mitigates energy insufficiency and neuronal apoptosis.

[Effect of cryptotanshinone on imatinib sensitivity and P-glycoprotein expression of chronic myeloid leukemia cells].

Cryptotanshinone (CPT), a lipid soluble active compound in Salvia miltiorrhiza, has a significant inhibitory effect on multiple malignant tumors, e. g. chronic myeloid leukemia (CML) cells and can effectively enhance imatinib's chemotherapeutic effect. However, its functional molecular mechanism remained unclear. In this experiment, the authors conducted a systematic study on the effect of CPT on the imatinib sensitivity and P-glycoprotein (P-gp) expression in CML cells by using CML cells K562 and imatinib persister K562-R. The MTT assays were performed to determine CPT's impact on the inhibitory effect of imatinib. Annexin V-FITC/PI staining analysis was used to detect the changes in the cell apoptosis rate. The active changes in apoptosis regulatory proteins Caspase-3, Caspase-9 and PARP were determined by Western blot. After the cells were pretreated with the gradient concentration of CPT, the expression of P-gp was analyzed by Western blot and flow cytometry. The changes in intracellular concentrations of imatinib were determined by HPLC analysis. The results indicated that the pretreatment with CPT significantly increased the proliferation inhibiting and apoptosis inducing effects of imatinib on K562 and K562-R cells as well as the degradation product expression of pro-apoptotic proteins Caspase-3, Caspase-9 and PARP, with a significant difference with the control group (P < 0.01). However, CPT showed no impact on the P-gp expression in CML cells and the intracellular concentrations of imatinib. In summary, the findings suggested that CPT enhanced the sensitivity of CML cells to imatinib. Its mechanism is not dependent on the inhibition in P-gp expression and the increase in intracellular drug concentration.

Saponins from Rubus parvifolius L. induce apoptosis in human chronic myeloid leukemia cells through AMPK activation and STAT3 inhibition.

BACKGROUND: Saponins are a major active component for the traditional Chinese medicine, Rubus parvifolius L., which has shown clear antitumor activities. However, the specific effects and mechanisms of saponins of Rubus parvifolius L. (SRP) remain unclear with regard to human chronic myeloid leukemia cells. The aim of this study was to investigate inhibition of proliferation and apoptosis induction effects of SRP in K562 cells and further elucidate its regulatory mechanisms. MATERIALS AND METHODS: K562 cells were treated with different concentrations of SRP and MTT assays were performed to determine cell viability. Apoptosis induction by SRP was determined with FACS and DAPI staining analysis. Western blotting was used to detect expression of apoptosis and survival related genes. Specific inhibitors were added to confirm roles of STAT3 and AMPK pathways in SRP induction of apoptosis. RESULTS: Our results indicated that SRP exhibited obvious inhibitory effects on the growth of K562 cells, and significantly induced apoptosis. Cleavage of pro-apoptotic proteins was dramatically increased after SRP exposure. SRP treatment also increased the activities of AMPK and JNK pathways, and inhibited the phosphorylation expression level of STAT3 in K562 cells. Inhibition of the AMPK pathway blocked the activation of JNK by SRP, indicating that SRP regulated the expression of JNK dependent on the AMPK pathway. Furthermore, inhibition of the latter significantly conferred resistance to SRP pro- apoptotic activity, suggesting involvement of the AMPK pathway in induction of apoptosis. Pretreatment with a STAT3 inhibitor also augmented SRP induced growth inhibition and cell apoptosis, further confirming roles of the STAT3 pathway after SRP treatment. CONCLUSIONS: Our results demonstrated that SRP induce cell apoptosis through AMPK activation and STAT3 inhibition in K562 cells. This suggests the possibility of further developing SRP as an alternative treatment option, or perhaps using it as adjuvant chemotherapeutic agent for chronic myeloid leukemia therapy.

[Mechanism of platycodin D-induced apoptosis in A549 human lung cancer cells].

OBJECTIVE: To investigate the molecular mechanism of platycodin D showing the inhibitory effect on proliferation and induced apoptosis of humane long cancer cells A549. METHOD: Humane long cancer cells A549 were cultured in vitro, with the final PD concentration of 5-20 micromol x L(-1). PD's inhibitory effect on cell proliferation was examined by MTT assay. Morphological changes in cells were observed with microscope. The cell apoptosis rate was detected by Annexin V-FITC/PI double staining. The change of mitochondrial membrane potential was detected by JC-1. The protein expressing of leaved Caspase-3, cleaved Caspase-9, cleaved PARP, Bcl-2, Bcl-xl, Bak and Bax were detected by Western blot analysis. RESULT: PD could inhibit the proliferation of A549 cells and show stronger effect with the increase of concentration and over time. Compared with the control group, PDs of different concentration showed significant increase in the cell apoptosis rate, decrease in mitochondrial membrane potential after 24 h. Protein electrophoresis inspection showed cut segments in both protein Caspase-3 and Caspase-9 and notable fractures with time. Further study found that PD decreased Bcl-2, Bcl-xl proteins and increased Bax, Bak proteins after processing A549 cells. CONCLUSION: PD shows notable effect on cytotoxicity and can induce A549 cell apoptosis. It causes decrease in mitochondrial membrane potential by regulating Bax, Bak, Bcl-2 and Bcl-xl expressions, and thus activating caspase and finally causing long cancer cell apoptosis.

[Biological activity assays and cellular imaging of anti-human sperm protein 17 immunomagnetic nanoparticles].

AIM: To prepare anti-Sperm protein 17 (Sp17) immunomagnetic nanoparticles (IMNPs), and make foundation for target diagnosis of ovarian cancer by magnetic resonance imaging. METHODS: The anti-human Sp17 IMNPs were prepared by grafting anti-Sp17 antibodies on the surface of chitosan-coated magnetic nanoparticles (MNPs) using the linker of EDC/NHS (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide/N-hydroxysuccinimide). The morphology and properties of the nanoparticles were characterized by transmission electronic microscopy (TEM), the conjugation of the antibodies was evaluated by native-polyacrylamide gel electrophoresis, the immunologic activity of IMNPs was evaluated by enzyme linked immunosorbent assay (ELISA). A set of in vitro magnetic resonance imaging (MRI) experiments were performed after incubated the IMNPs with human Sp17 gene transfected ovarian cancer HO-8910 cells. RESULTS: We had successfully grafted the MNPs with anti-Sp17 antibody and the IMNPs kept good bioactivity. The MRI showed that the IMNPs were targeted successfully to the positive cells, and no obviously non-specific adsorption was observed. CONCLUSION: The anti-Sp17 IMNPs with good specificity can used for further study of ovarian cancer target therapy.

[In vitro MR imaging of Fe2O3-arginine labeled heNOS gene modified endothelial progenitor cells].

OBJECTIVE: To explore the feasibility of in vitro magnetic resonance imaging on Fe2O3-arginine labeled heNOS gene modified endothelial progenitor cells (EPCs). METHODS: Fe2O3 was incubated with arginine to form Fe2O3-arginine complex. Rabbit peripheral blood mononuclear cells (MNCs) were isolated and EPCs were isolated by adherence method, expanded and modified with heNOS gene using Lipofectamine 2000. After 48 hours, genetically modified EPCs were incubated with Fe2O3-arginine for 24 hours. Intracellular iron was detected by Prussian blue stain. The expression of heNOS gene was detected by Western blot. MTT assay was used to evaluate cell survival and proliferation of Fe2O3-arginine labeled heNOS-EPCs. Flow cytometry was used to measure cell apoptosis. The cells underwent in vitro MR imaging with various sequences. RESULTS: Iron-containing intracytoplasmatic vesicles could be clearly observed with Prussian blue staining, and the labeling rate of labeled heNOS-EPCs were similar to that of labeled EPCs (around 100%). Survival and apoptosis rates obtained by MTT and flow cytometry analysis were similar among labeled heNOS-EPCs, labeled EPCs and unlabeled EPCs with Fe2O3-arginine. The signal intensity on MRI was equally decreased in labeled heNOS-EPCs and labeled EPCs compared with that in unlabeled cells. The percentage change in signal intensity (DeltaSI) was most significant on T2*WI and DeltaSI was significantly lower in cells labeled for 7 days than that labeled for 1 days. CONCLUSIONS: The heNOS gene can be successfully transfected into rabbit peripheral blood EPCs using Lipofectamine2000. The heNOS-EPCs can be labeled with Fe2O3-arginine without significant change in viability and proliferation capacity. The labeled heNOS-EPCs can be imaged with standard 1.5 T MR equipment. The degree of MR signal intensity may indirectly reflect the cell count, growth and division status.