CD206 modulates the role of M2 macrophages in the origin of metastatic tumors.

Tumor metastasis is a key factor affecting the life of patients with malignant tumors. For the past hundred years, scientists have focused on how to kill cancer cells and inhibit their metastasis in vivo, but few breakthroughs have been made. Here we hypothesized a novel mode for cancer metastasis. We show that the phagocytosis of apoptotic tumor cells by macrophages leads to their polarization into the M2 phenotype, and that the expression of stem cell related as well as drug resistance related genes was induced. Therefore, it appears that M2 macrophages have "defected" and have been transformed into the initial "metastatic cancer cells", and thus are the source, at least in part, of the distal tissue tumor metastasis. This assumption is supported by the presence of fused cells with characteristics of both macrophage and tumor cell observed in the peripheral blood and ascites of patients with ovarian cancer. By eliminating the expression of CD206 in M2 macrophages using siRNA, we show that the growth and metastasis of tumors was suppressed using both in vitro cell line and with experimental in vivo mouse models. In summary, we show that M2 macrophages in the blood circulation underwent a "change of loyalty" to become "cancer cells" that transformed into distal tissue metastasis, which could be suppressed by the knockdown of CD206 expression.

Establishment of an optimized CTC detection model consisting of EpCAM, MUC1 and WT1 in epithelial ovarian cancer and its correlation with clinical characteristics.

Objective: Emerging studies have demonstrated the promising clinical value of circulating tumor cells (CTCs) for diagnosis, disease assessment, treatment monitoring and prognosis in epithelial ovarian cancer. However, the clinical application of CTC remains restricted due to diverse detection techniques with variable sensitivity and specificity and a lack of common standards. Methods: We enrolled 160 patients with epithelial ovarian cancer as the experimental group, and 90 patients including 50 patients with benign ovarian tumor and 40 healthy females as the control group. We enriched CTCs with immunomagnetic beads targeting two epithelial cell surface antigens (EpCAM and MUC1), and used multiple reverse transcription-polymerase chain reaction (RT-PCR) detecting three markers (EpCAM, MUC1 and WT1) for quantification. And then we used a binary logistic regression analysis and focused on EpCAM, MUC1 and WT1 to establish an optimized CTC detection model. Results: The sensitivity and specificity of the optimized model is 79.4% and 92.2%, respectively. The specificity of the CTC detection model is significantly higher than CA125 (92.2% vs. 82.2%, P=0.044), and the detection rate of CTCs was higher than the positive rate of CA125 (74.5% vs. 58.2%, P=0.069) in early-stage patients (stage I and II). The detection rate of CTCs was significantly higher in patients with ascitic volume ≥500 mL, suboptimal cytoreductive surgery and elevated serum CA125 level after 2 courses of chemotherapy (P<0.05). The detection rate of CTCEpCAM + and CTCMUC1+ was significantly higher in chemo-resistant patients (26.3% vs. 11.9%; 26.4% vs. 13.4%, P<0.05). The median progression-free survival time for CTCMUC1+ patients trended to be longer than CTCMUC1- patients, and overall survival was shorter in CTCMUC1+ patients (P=0.043). Conclusions: Our study presents an optimized detection model for CTCs, which consists of the expression levels of three markers (EpCAM, MUC1 and WT1). In comparison with CA125, our model has high specificity and demonstrates better diagnostic values, especially for early-stage ovarian cancer. Detection of CTCEpCAM+ and CTCMUC1+ had predictive value for chemotherapy resistance, and the detection of CTCMUC1+ suggested poor prognosis.

Putative Prevention of XML Injection Against Myocardial Ischemia Is Mediated by PKC and PLA2 Proteins.

Background: Xinmailong (XML) injection is a CFDA-approved traditional Chinese medicine with clinical value for heart failure treatment. The present investigation was aimed to evaluate the potential protective roles of this injection on myocardial ischemia and the underlying molecular mechanism. Methods: In our study, we selected two models of myocardial ischemia rats. Rats were randomly divided into six groups, with saline or XML administrated 4 days before ischemia model establishment. ECG of different time intervals and biochemical parameters of end point were measured. The potential mechanisms of the protective role of XML were explored using system pharmacology and molecular biology approaches. Results: Myocardial ischemia rats demonstrated abnormal ECG and serum levels of cTnT. Pretreatment with XML significantly attenuated these damages, especially the medium doses. GO and KEGG analysis revealed that the 90 putative target genes were associated with pathways of fatty acid absorption/metabolism, inflammation, RAAS, and vascular smooth muscle. Further network pharmacology method identified five main chemical ingredients and potential targets of XML injection for myocardial ischemia. Mechanically, the beneficial effect of XML injection was mediated by the reactive oxygen species (ROS) inhibition and inflammation attenuation via regulating the expression levels of targets of PKC and PLA2. Conclusion: These findings indicate that XML exerts protective effects against myocardial injury, with attenuated ROS production, apoptosis, and inflammation. Therefore, we speculate that XML may be an alternative supplementary therapeutic agent for myocardial ischemia prevention.

Overexpression of DUSP6 enhances chemotherapy-resistance of ovarian epithelial cancer by regulating the ERK signaling pathway.

Objective: DUSP6 is a negative regulator of the ERK signaling pathway and plays an important role in chemotherapy-resistance. Previously we showed that DUSP6 is overexpressed in ovarian cancer side population (SP) cells that possess cancer stem cell-like properties and are quiescent and chemotherapy-resistant. Here, we explore the effects of DUSP6 on chemotherapy-resistance by examining its regulation of the ERK signaling pathway and G0/G1 cell cycle arrest. Methods: mRNA and protein expression of DUSP6 and G0/G1 cell cycle checkpoint regulating proteins (CyclinD1, CyclinD3 and CyclinE2) was evaluated among ovarian cancer cell lines and tissue samples. Ovarian cancer cells were transiently transfected to overexpress DUSP6. After treatment with cisplatin, cell viability was measured by the MTS assay at 48 hours and the half maximal inhibitory concentration (IC50) for each cell line was calculated. Subcellular localization and cell cycle analysis were determined by using immunofluorescence and FACS, respectively. Results: SKOV3 and OVCAR8 SP cells were shown to express higher levels of DUSP6 and lower levels of CyclinD3 compared with non-SP (NSP) cells (P<0.001). Among 39 ovarian cancer tissue samples, expression of DUSP6 in the chemotherapy-resistant group (12 samples) was higher than in the chemotherapy-sensitive group (27 samples) (P<0.05). While a lower level of expression of CyclinD3 was seen in the chemotherapy-resistant group, it was not statistically different from the chemotherapy-sensitive group. HO8910 cells where shown to have higher IC50 to cisplatin than SKOV3 or OVCAR8 cells, and this correlated with higher levels of DUSP6 expression. Overexpression of DUSP6 in SKOV3 cells led to an increase in cisplatin IC50 values (P<0.05), and also markedly reduced the expression levels of phospho-ERK1/2 and CyclinD3 and to the predominance of cells in the G0/G1 phase. Conclusion: Our findings reveal an enhancement of chemotherapy-resistance and a predominance of cells in G1 cell cycle arrest in DUSP6-overexpressing ovarian cancer cells. This suggests that overexpression of DUSP6 promotes chemotherapy-resistance through the negative regulation of the ERK signaling pathway, increasing the G0/G1 phase ratio among ovarian cancer cells, and leading to cellular quiescence.

Apoptotic SKOV3 cells stimulate M0 macrophages to differentiate into M2 macrophages and promote the proliferation and migration of ovarian cancer cells by activating the ERK signaling pathway.

Ovarian cancer has a high rate of recurrence, with M2 macrophages having been found to be involved in its progression and metastasis. To examine the relationship between macrophages and ovarian cancer in the present study, M0 macrophages were stimulated with apoptotic SKOV3 cells and it was found that these macrophages promoted tumor proliferation and migration. Subsequently, the mRNAs and proteins expressed at high levels in these M2 macrophages were examined by RNA­Seq and quantitative proteomics, respectively, which revealed that M0 macrophages stimulated by apoptotic SKOV3 cells also expressed M2 markers, including CD206, interleukin­10, C­C motif chemokine ligand 22, aminopeptidase­N, disabled homolog 2, matrix metalloproteinase 1 and 5'­nucleotidase. The abundance of phosphorylated Erk1/2 in these macrophages was increased. The results indicate that apoptotic SKOV3 cells stimulate M0 macrophages to differentiate into M2 macrophages by activating the ERK pathway. These results suggest possible treatments for patients with ovarian cancer who undergo chemotherapy; inhibiting M2 macrophage differentiation during chemotherapy may reduce the rate of tumor recurrence.

Stromal vascular fraction promotes migration of fibroblasts and angiogenesis through regulation of extracellular matrix in the skin wound healing process.

BACKGROUND: A refractory wound is a typical complication of diabetes and is a common outcome after surgery. Current approaches have difficulty in improving wound healing. Recently, non-expanded stromal vascular fraction (SVF), which is derived from mature fat, has opened up new directions for the treatment of refractory wound healing. The aim of the current study is to systematically investigate the impact of SVF on wound healing, including the rate and characteristics of wound healing, ability of fibroblasts to migrate, and blood transport reconstruction, with a special emphasis on their precise molecular mechanisms. METHODS: SVF was isolated by digestion, followed by filtration and centrifugation, and then validated by immunocytochemistry, a MTS proliferation assay and multilineage potential analysis. A wound model was generated by creating 6-mm-diameter wounds, which include a full skin defect, on the backs of streptozocin-induced hyperglycemic mice. SVF or human adipose-derived stem cell (hADSC) suspensions were subcutaneously injected, and the wounds were characterized over a 9-day period by photography and measurements. A scratch test was used to determine whether changes in the migratory ability of fibroblasts occurred after co-culture with hADSCs. Angiogenesis was observed with human umbilical vein endothelial cells. mRNA from fibroblasts, endotheliocyte, and skin tissue were sequenced by high-throughput RNAseq, and differentially expressed genes, and pathways, potentially regulated by SVF or hADSCs were bioinformatically analyzed. RESULTS: Our data show that hADSCs have multiple characteristics of MSC. SVF and hADSCs significantly improved wound healing in hyperglycemic mice. hADSCs improve the migratory ability of fibroblasts and capillary structure formation in HUVECs. SVF promotes wound healing by focusing on angiogenesis and matrix remodeling. CONCLUSIONS: Both SVF and hADSCs improve the function of fibroblast and endothelial cells, regulate gene expression, and promote skin healing. Various mechanisms likely are involved, including migration of fibroblasts, tubulogenesis of endothelial cells through regulation of cell adhesion, and cytokine pathways.

Exosomes from Macrophages Exposed to Apoptotic Breast Cancer Cells Promote Breast Cancer Proliferation and Metastasis.

Exosomes have recently become the subject of increasing research interest. Interactions between tumor and host cells via exosomes play crucial roles in the initiation, progression and invasiveness of breast cancer. In our study, we used exosomes isolated from a co-culture model of THP-1-derived macrophages exposed to apoptotic MCF-7 or MDA-MB-231 breast cancer cell line cells to investigate their effects on naïve MCF-7 or MDA-MB-231 cells in vitro and in vivo. This post-chemotherapy tumor microenvironment model allowed us to explore possible mechanisms that explain increased proliferation and metastasis of breast cancer seen in some patients. Our results suggest that while exosomes derived from macrophages normally inhibit proliferation and metastasis of MCF-7 or MDA-MB-231 cells, exposure of macrophages to breast cancer cells that have experienced chemotherapy are modified them to promote these processes. Exosomes from macrophages exposed to apoptotic cancer cells have increased amounts of IL-6 that increases the phosphorylation of STAT3, which likely explains the increased transcription of STAT3 target genes such as CyclinD1, MMP2 and MMP9. These observations suggest that the inhibition of exosome secretion and STAT3 signaling pathway activation might suppress the growth and metastasis of malignant tumors, and provide new targets for therapeutic treatment of malignant tumors after chemotherapy.

Construction of a sensitive pyrogen-testing cell model by site-specific knock-in of multiple genes.

A pyrogen test is crucial for evaluating the safety of drugs and medical equipment, especially those involved in injections. As existing pyrogen tests, including the rabbit pyrogen test, the limulus amoebocyte lysate (LAL) test and the monocyte activation test have limitations, development of new models for pyrogen testing is necessary. Here we develop a sensitive cell model for pyrogen test based on the lipopolysaccharides (LPS) signal pathway. TLR4, MD2, and CD14 play key roles in the LPS-mediated pyrogen reaction. We established a new TLR4/MD2/CD14-specific overexpressing knock-in cell model using the CRISPR/CAS9 technology and homologous recombination to detect LPS. Stimulation of our TLR4/CD14/MD2 knock-in cell line model with LPS leads to the release of the cytokines IL-6 and TNF-alpha, with a detection limit of 0.005 EU/ml, which is greatly lower than the lower limit of 0.015 EU/ml detected by the Tachypleus amebocyte lysate (TAL) assay.

Application of a TLR overexpression cell model in pyrogen detection.

Pyrogens are components derived from microorganisms that induce complex inflammatory responses. Current approaches to detect pyrogens are complex and difficult to replicate, thus there is a need for new methods to detect pyrogens. We successfully constructed a pyrogen-sensitive cell model by overexpressing Toll-like receptor (TLR)2, TLR4, MD2, and CD14 in HEK293 cells. Since the cytokine IL-6 is specifically released upon stimulation of the TLR2 and TLR4 signaling pathways in response to pyrogen stimulation, we used it as a read out for our assay. Our results show that IL-6 is released in response to trace amounts of pyrogens in our cell model. Pyrogen incubation times and concentrations were explored to determine the sensitivity of our cell model, and was found to be sensitive to 0.05 EU/ml of LPS and 0.05 ug/ml of LTA after stimulation for 5 hr. Our TLR overexpressing cell model, with IL-6 as readout, could be a new method for in vitro testing of pyrogens and applicable for evaluating the safety of drugs.

Neuroprotective Effect of Anethole Against Neuropathic Pain Induced by Chronic Constriction Injury of the Sciatic Nerve in Mice.

Neuropathic pain is an intractable disease with few definitive therapeutic options. Anethole (AN) has been confirmed to possess potent anti-inflammatory and neuroprotective properties, but its effect on neuropathic pain has not been reported. The present study was designed to investigate the antinociceptive effect of AN on chronic constriction injury (CCI)-induced neuropathic pain in mice. AN (125, 250, and 500 mg/kg) and pregabalin (40 mg/kg) were intragastric administered for 8 consecutive days from the 7th day post-surgery. Behavioral parameters were measured on different days, namely, 0, 7, 8, 10, 12, and 14, from CCI operation. Additionally, electrophysiological and histopathological changes were analyzed on the 14th day. Afterward, immunofluorescence and Western blot were utilized to examine the activation of glial cells and the expression of inflammatory cytokines, respectively. AN treatment of CCI mice considerably alleviated hyperalgesia and allodynia, ameliorated abnormal sciatic nerve conduction, and restored injured sciatic nerves in a dose-dependent manner. Furthermore, AN suppressed the activation of glial cells, down-regulated pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin (IL-6, and IL-1ß), and up-regulated the anti-inflammatory cytokine (IL-10). These assays first indicated that AN exerted an antinociceptive effect on CCI-induced neuropathic pain, and might be attributed to the anti-inflammatory and neuroprotective activities of AN.

Analysis of Circulating Tumor Cells in Ovarian Cancer and Their Clinical Value as a Biomarker.

BACKGROUND/AIMS: Monitoring the appearance and progression of tumors are important for improving the survival rate of patients with ovarian cancer. This study aims to examine circulating tumor cells (CTCs) in epithelial ovarian cancer (EOC) patients to evaluate their clinical significance in comparison to the existing biomarker CA125. METHODS: Immuomagnetic bead screening, targeting epithelial antigens on ovarian cancer cells, combined with multiplex reverse transcriptase-polymerase chain reaction (Multiplex RT-PCR) was used to detect CTCs in 211 samples of peripheral blood (5 ml) from 109 EOC patients. CTCs and CA125 were measured in serial from 153 blood and 153 serum samples from 51 patients and correlations with treatment were analyzed. Immunohistochemistry was used to detect the expression of tumor-associated proteins in tumor tissues and compared with gene expression in CTCs from patients. RESULTS: CTCs were detected in 90% (98/109) of newly diagnosed patients. In newly diagnosed patients, the number of CTCs was correlated with stage (p=0.034). Patients with stage IA-IB disease had a CTC positive rate of 93% (13/14), much higher than the CA125 positive rate of only 64% (9/14) for the same patients. The numbers of CTCs changed with treatment, and the expression of EpCAM (p=0.003) and HER2 (p=0.035) in CTCs was correlated with resistance to chemotherapy. Expression of EpCAM in CTCs before treatment was also correlated with overall survival (OS) (p=0.041). CONCLUSION: Detection of CTCs allows early diagnose and expression of EpCAM in CTC positive patients predicts prognosis and should be helpful for monitoring treatment.

Betaine Attenuates Monocrotaline-Induced Pulmonary Arterial Hypertension in Rats via Inhibiting Inflammatory Response.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by increased pulmonary vascular resistance, leading to right ventricular failure and death. Recent studies have suggested that chronic inflammatory processes are involved in the pathogenesis of PAH. Several studies have demonstrated that betaine possesses outstanding anti-inflammatory effects. However, whether betaine exerts protective effects on PAH by inhibiting inflammatory responses in the lungs needs to be explored. To test our hypothesis, we aimed to investigate the effects of betaine on monocrotaline-induced PAH in rats and attempted to further clarify the possible mechanisms. METHODS: PAH was induced by monocrotaline (50 mg/kg) and oral administration of betaine (100, 200, and 400 mg/kg/day). The mean pulmonary arterial pressure, right ventricular systolic pressure, and right ventricle hypertrophy index were used to evaluate the development of PAH. Hematoxylin and eosin staining and Masson staining were performed to measure the extents of vascular remodeling and proliferation in fibrous tissue. Monocyte chemoattractant protein-1 (MCP-1) and endothelin-1 (ET-1) were also detected by immunohistochemical staining. Nuclear factor-κB (NF-κB), tumor necrosis factor alpha (TNF-α), and interleukin-1ß (IL-1ß) were assessed by Western blot. RESULTS: This study showed that betaine improved the abnormalities in right ventricular systolic pressure, mean pulmonary arterial pressure, right ventricle hypertrophy index, and pulmonary arterial remodeling induced by monocrotaline compared with the PAH group. The levels of MCP-1 and ET-1 also decreased. Western blot indicated that the protein expression levels of NF-κB, TNF-α, and IL-1ß significantly decreased (p < 0.01). CONCLUSION: Our study demonstrated that betaine attenuated PAH through its anti-inflammatory effects. Hence, the present data may offer novel targets and promising pharmacological perspectives for treating monocrotaline-induced PAH.

Tumacrophage: macrophages transformed into tumor stem-like cells by virulent genetic material from tumor cells.

Tumor-associated macrophages are regarded as tumor-enhancers as they have key roles in the subversion of adaptive immunity and in inflammatory circuits that promote tumor progression. Here, we show that cancer cells can subvert macrophages yielding cells that have gained pro-tumor functions. When macrophages isolated from mice or humans are co-cultured with dead cancer cell line cells, induced to undergo apoptosis to mimic chemotherapy, up-regulation of pro-tumor gene expression was identified. Phagocytosis of apoptotic cancer cells by macrophages resulted in their transformation into tumor stem (initiating)-like cells, as indicated by the expression of epithelial markers (e.g., cytokeratin) and stem cell markers (e.g., Oct4) and their capability to differentiate in vitro and self-renew in serum-free media. Moreover, we identified a subset of monocytes/macrophages cells in the blood of cancer (breast, ovarian and colorectal) patients undergoing chemotherapy that harbor tumor transcripts. Our findings uncover a new role for macrophages in tumor development, where they can be transformed into tumor-like cells, potentially by horizontal gene transfer of tumor-derived genes, thus, by taking advantage of chemotherapy, these transformed macrophages promote tumor metastasis by escaping immune surveillance.

Differences in anti-inflammatory effects between two specifications of Scutellariae Radix in LPS-induced macrophages in vitro.

Scutellariae Radix (SR), the root of Scutellaria baicalensis Georgi, is used as an antipyretic drug and has been demonstrated to have anti-inflammatory activity. SR is divided into two specifications, "Ku Qin" (KQ) and "Zi Qin" (ZQ), for use against different symptoms (upper energizer heat or lower portion of the triple energizer), according to the theory of traditional Chinese medicine (TCM). However, differences in the efficacies of these two specifications have not been determined. In the present study, we aimed to characterize the differences in the anti-inflammatory activities between KQ and ZQ and to explore how their differences are manifested in lipopolysaccharide (LPS)-induced macrophages. Our results showed that, in RAW264.7 cells (a mouse macrophage cell line derived from ascites), KQ and ZQ displayed anti-inflammatory effects by inhibiting the release of nitric oxide (NO), inducible NOS (iNOS), and nuclear factor-κB (NF-κB) in a dose-dependent manner without distinction. In NR8383 cells (a rat alveolar macrophage cell line), KQ and ZQ displayed similar effects on NO, iNOS, and NF-κB as seen in RAW264.7 cells, but KQ showed a higher inhibition rate for NO and iNOS than that shown by ZQ at the same concentration. These results indicated that there were differences in efficacy between KQ and ZQ in treating lung inflammation. Our findings provided an experimental evidence supporting the different uses of KQ and ZQ in clinic, as noted in ancient herbal records.

Excessive Autophagy Activation and Increased Apoptosis Are Associated with Palmitic Acid-Induced Cardiomyocyte Insulin Resistance.

Diabetic cardiomyopathy (DCM) remains the major cause of death associated with diabetes. Researchers have demonstrated the importance of impaired cardiac insulin signaling in this process. Insulin resistance (IR) is an important predictor of DCM. Previous studies examining the dynamic changes in autophagy during IR have yielded inconsistent results. This study aimed to investigate the dynamic changes in autophagy and apoptosis in the rat H9c2 cardiomyocyte IR model. H9c2 cells were treated with 500 µM palmitic acid (PA) for 24 hours, resulting in the induction of IR. To examine autophagy, monodansylcadaverine staining, GFP-LC3 puncta confocal observation, and Western blot analysis of LC3I-to-LC3II conversion were used. Results of these studies showed that autophagic acid vesicles increased in numbers during the first 24 hours and then decreased by 36 hours after PA treatment. Western blot analysis showed that treatment of H9c2 cells with 500 µM PA for 24 hours decreased the expression of Atg12-Atg5, Atg16L1, Atg3, and PI3Kp85. Annexin V/PI flow cytometry revealed that PA exposure for 24 hours increased the rate of apoptosis. Together, this study demonstrates that PA induces IR in H9c2 cells and that this process is accompanied by excessive activation of autophagy and increases in apoptosis.

Xinmailong mitigated epirubicin-induced cardiotoxicity via inhibiting autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Using insects, such as the cockroach, for the treatment of disease has a long history in traditional Chinese medicine. Xinmailong (XML) Injection, a bioactive composite extracted from Periplaneta americana (a species of cockroach), shows reasonable protective effects against cardiovascular injury and was approved for the use in the treatment of cardiac dysfunction in 2006, yet its cardio protective mechanisms remain unclear. AIM: The present study aims to examine the protective effects of XML against epirubicin-induced cardiotoxicity in vivo and determine its underlying mechanisms. MATERIALS AND METHODS: The chemical characteristics of XML were identified using high performance liquid chromatography (HPLC). Rats were intraperitoneally injected with epirubicin and then treated with XML for 14 days. Survival rate, echocardiography, electrocardiographic recordings and Masson staining were used to evaluate the cardioprotective activity of XML. Western blot and quantitative real time reverse transcriptase polymerase chain reaction (RT-PCR) analyses were used to investigate the molecular mechanisms underlying the actions of XML. RESULTS: XML treatment significantly enhanced the survival rate of rats from epirubicin-induced heart failure. XML prevented left ventricle dilatation, improved cardiac function. Furthermore, treatment with XML also significantly inhibited the accumulation of collagen, reduced the levels of mRNA for matrix metalloproteinases-9 (Mmp9) and transforming growth factor-ß 1(Tgfb1). This action of XML therefore might be responsible, at least in part, for the attenuation of cardiac fibrotic remodeling. XML inhibited autophagy as evidenced by the decreased accumulation of Beclin1 and autophagy related 7 (Atg7), which are necessary to form autophagosome structures. Protein kinase B (PKB/Akt), phosphatidylinositol 3 kinase (PI3K) and B cell lymphoma2 (Bcl2) levels were up-regulated, while significantly decreased protein levels for phosphorylated P38 and extracellular regulated protein kinases 1/2 (Erk1/2) were observed in the XML treated rats. The autophagy related results suggested that the increase in PI3K/Akt levels and inhibition of the phosphorylation of P38 MAPK and Erk1/2 contributed to the anti-autophagic activity of XML. CONCLUSIONS: Our data suggest that XML may be effective for mitigating epirubicin-induced cardiomyopathy and inhibits autophagy via activating the PI3K/Akt signaling pathway and inhibiting the Erk1/2 and P38 MAPK signaling pathways.

Trans-cinnamaldehyde protected PC12 cells against oxygen and glucose deprivation/reperfusion (OGD/R)-induced injury via anti-apoptosis and anti-oxidative stress.

Ischemia stroke is the major cause of mortality and permanent neurological disability with little definitive therapeutic options. This cerebral ischemic injury leads to the oxidative stress and eventually cell death. We hypothesized that treatment of this condition with the trans-cinnamaldehyde(TC) could protect cells from ischemic and reperfusion injury. Oxygen and glucose deprivation/reperfusion (OGD/R) was used as an in vitro model of hypoxic ischemic injury in present study. MTT was used to evaluate the protective effects of TC. Next, we tested whether TC reduced the production of reactive oxygen species (ROS). Besides, experiments were performed to determine whether or not the mitochondrial membrane potential was affected. Furthermore, the inhibiters of NO and PI3 K were used to determine the initial mechanisms. TC treatment improved cell viability, reduced intracellular ROS, and increased MMP. Further, the inhibition of NO or PI3 K significantly reduced TC's protective effects. These findings suggest that TC might be a promising agent for ischemic stroke.

Alpha-Actinin-4 is a Possible Target Protein for Aristolochic Acid I in Human Kidney Cells In Vitro.

Aristolochic acid I (AA-I) is a strong nephrotoxin, carcinogen, and mutagen found in plants such as the Aristolochia species. The mechanisms underlying AA-I toxicity in the kidneys are poorly understood. In this study, we aimed to gain insight into the mechanism of AA-I nephrotoxicity by analyzing the uptake, subcellular distribution, and intracellular targets of AA-I in the human kidney cell line HK-2 using immunocytochemistry, immunoprecipitation, and LC-MS/MS. In HK-2 cells incubated with 20[Formula: see text][Formula: see text]g/mL AA-I for different periods of time (up to 12[Formula: see text]h), AA-I was detected by a specific monoclonal antibody (MAb) against AA-I, both in the cytoplasm and nuclei. Nuclear localization depended on the exposure time. A protein with the molecular weight of 100 kDa was immunoprecipitated with the anti-AA-I MAb from the AA-I-treated cell lysates and was identified by LC-MS/MS as [Formula: see text]-actinin-4 after digestion of the protein, and was confirmed by immunoblotting with a specific anti-[Formula: see text]-actinin-4 MAb. This evidence shows, for the first time, that [Formula: see text]-actinin-4 is a protein targeted by AA-I in kidney cells. Our findings strongly suggest an association between [Formula: see text]-actinin-4 and AA-I nephrotoxic activity.

Staurosporine Induced Apoptosis May Activate Cancer Stem-Like Cells (CD44(+)/CD24(-)) in MCF-7 by Upregulating Mucin1 and EpCAM.

Malignant tumors recur after chemotherapy. A small population of cancer stem-like cells within tumors is now generally considered the prime source of the recurrence. To better understand how cancer stem-like cells induce relapse after fractionated chemotherapy, we examined changes in the CD44(+)/CD24(-) cancer stem-like cells population and behavior using the breast cancer cell line MCF-7. Our results show that apart from an increase in the CD44(+)/CD24(-) population, proliferation and clone formation, but not migration, were enhanced after recovery from apoptosis induced by two pulses of staurosporine (STS). The distribution of cells in the cell cycle differed between acutely induced apoptosis and fractionated chemotherapy. Sorted CD44(+)/CD24(-) stem-like cells from MCF-7 cells recovered from STS treatment possessed greater proliferation abilities. We also observed that mucin1 (MUC1) and Epithelial cell adhesion molecule (EpCAM) were up-regulated in abundance coincidently with proliferation and clone formation enhancement. Our findings suggest that fractionated chemotherapy induced apoptosis could stimulate cancer stem-like cell to behave with a stronger malignant property than cancer cells themselves and MUC1 and EpCAM are important factors involving in this process. By demonstrating changes in cancer stem cell during chemotherapy and identifying the crucial factors, we potentially can target them, to eradicate tumors and overcome cancer relapse.

Development of a mitochondria-based centrifugal ultrafiltration/liquid chromatography/mass spectrometry method for screening mitochondria-targeted bioactive constituents from complex matrixes: Herbal medicines as a case study.

Mitochondria are an important intracellular pharmacological target because damage to this organelle results in a variety of human disorders and because mitochondria are involved in complex processes such as energy generation, apoptosis and lipid metabolism. To expedite the search for natural bioactive compounds targeting mitochondria, we initially developed an efficient mitochondria-based screening method by combining centrifugal ultrafiltration (CU) with liquid chromatography/mass spectrometry (LC/MS), which is called screening method for mitochondria-targeted bioactive constituents (SM-MBC) and is compatible with the search of mitochondria-targeted compounds from complex matrixes such as herbal medicines extracts. Functionally active, structurally intact and pure mitochondria were obtained from rat myocardium using an optimized protocol for mitochondrial isolation comprising organelle release followed by differential and Nycodenz density gradient centrifugation. After evaluating the reliability of the method using thiabendazole (TZ), rotenone (RN), amiodarone (AR) and trimetazidine (TD) as positive controls, this method was successfully applied to screen bioactive constituents from extracts of Polygoni Cuspidati Rhizoma et Radix (PCRR) and Scutellariae Radix (SR). Nineteen active compounds were detected and identified by LC/MS, of which 17 were new mitochondria-targeted compounds. The activity of 9 of the 19 hit compounds was confirmed by in vitro pharmacological trials. These results demonstrate that SM-MBC can be used for the efficient screening of mitochondria-targeted constituents in complex preparations used to treat mitochondrial disorders, such as PCRR and SR. The results may be meaningful for an in-depth understanding of drug mechanism of action and drug discovery from medicinal herbs.