PDK1 promotes breast cancer progression by enhancing the stability and transcriptional activity of HIF-1α.

Pyruvate dehydrogenase kinase 1 (PDK1) phosphorylates the pyruvate dehydrogenase complex, which inhibits its activity. Inhibiting pyruvate dehydrogenase complex inhibits the tricarboxylic acid cycle and the reprogramming of tumor cell metabolism to glycolysis, which plays an important role in tumor progression. This study aims to elucidate how PDK1 promotes breast cancer progression. We found that PDK1 was highly expressed in breast cancer tissues, and PDK1 knockdown reduced the proliferation, migration, and tumorigenicity of breast cancer cells and inhibited the HIF-1α (hypoxia-inducible factor 1α) pathway. Further investigation showed that PDK1 promoted the protein stability of HIF-1α by reducing the level of ubiquitination of HIF-1α. The HIF-1α protein levels were dependent on PDK1 kinase activity. Furthermore, HIF-1α phosphorylation at serine 451 was detected in wild-type breast cancer cells but not in PDK1 knockout breast cancer cells. The phosphorylation of HIF-1α at Ser 451 stabilized its protein levels by inhibiting the interaction of HIF-1α with von Hippel-Lindau and prolyl hydroxylase domain. We also found that PDK1 enhanced HIF-1α transcriptional activity. In summary, PDK1 enhances HIF-1α protein stability by phosphorylating HIF-1α at Ser451 and promotes HIF-1α transcriptional activity by enhancing the binding of HIF-1α to P300. PDK1 and HIF-1α form a positive feedback loop to promote breast cancer progression.

TROP2 is highly expressed in triple-negative breast cancer CTCs and is a potential marker for epithelial mesenchymal CTCs.

Circulating tumor cells (CTCs) are the seeds of distant metastases of malignant tumors and are associated with malignancy and risk of metastasis. However, tumor cells undergo epithelial-mesenchymal transition (EMT) during metastasis, leading to the emergence of different types of CTCs. Real-time dynamic molecular and functional typing of CTCs is necessary to precisely guide personalized treatment. Most CTC detection systems are based on epithelial markers that may fail to detect EMT CTCs. Therefore, it is clinically important to identify new markers of different CTC types. In this study, bioinformatics analysis and experimental assays showed that trophoblast cell surface antigen 2 (TROP2), a target molecule for advanced palliative treatment of triple-negative breast cancer (TNBC), was highly expressed in TNBC tissues and tumor cells. Furthermore, TROP2 can promote the migration and invasion of TNBC cells by upregulating EMT markers. The specificity and potential of TROP2 as an EMT-associated marker of TNBC CTCs were evaluated by flow cytometry, immunofluorescence, spiking experiments, and a well-established CTC assay. The results indicated that TROP2 is a potential novel CTC marker associated with EMT, providing a basis for more efficacious markers that encompass CTC heterogeneity in patients with TNBC.

PGRN inhibits CD8+T cell recruitment and promotes breast cancer progression by up-regulating ICAM-1 on TAM.

BACKGROUND: Tumor microenvironment actually reduces antitumor effect against the immune attack by exclusion of CD8+T cells. Progranulin (PGRN) is a multifunctional growth factor with significant pathological effects in multiple tumors; however, its role in immunity evasion of breast cancer (BCa) is not completely understood. METHODS: We depleted GRN (PGRN gene) genetically in mice or specifically in PY8119 murine BCa cell line, and mouse models of orthotopic or subcutaneous transplantation were used. Chimeric mice-deficient of PGRN (Grn-/-) in bone marrow (BM) compartment was also generated. Association of PGRN expression with chemokine production or BCa development was investigated by histological and immunological assays. RESULTS: We found PGRN was involved in exhaustion of cytotoxic CD8+T cell in BCa with the increasing expressions of M2 markers and intercellular cell adhesion molecule-1 (ICAM-1) on macrophages. Specifically, ablation of PGRN in PY8119 cells reduced tumor burden, accompanied by the infiltrating of cytotoxic CD8+T cells into tumor nests. Moreover, our result revealed that blockade of PD-1 in PGRN-depleted tumors exhibited better antitumor effect in vivo and significantly decreased tumor burden. CONCLUSION: These findings suggest that inhibition of PGRN may act as a potential immune-therapeutic strategy by recovering infiltration of CD8+T cell in BCa tissue and thereby enhancing the response to anti-PD-1 therapy.

Au/PANI@PtCu-based electrochemical immunosensor for ultrasensitive determination of pro-gastrin-releasing peptide.

An ultrasensitive sandwich-type electrochemical immunosensor for pro-gastrin-releasing peptide (ProGRP) detection was constructed based on PtCu nanodendrites functionalized Au/polyaniline nanospheres (Au/PANI@PtCu). The prepared Au/PANI@PtCu nanocomposites not only possessed excellent electro-catalytic activity of H2O2 reduction due to the synergistic effect between the Au/PANI and PtCu NDs but also provided large specific surface area for detection of antibodies (Ab2) immobilization. In addition, Au nanoparticles encapsulated multi-wall carbon nanotubes (AuNPs@MWCNTs) were also applied to modify the glassy carbon electrode interface for loading numerous capture antibodies (Ab1). In the presence of target ProGRP, a sandwich-type electrochemical immunosensor showed a strong current response from the electro-catalysis of Au/PANI@PtCu toward H2O2 reduction. Benefiting from the exceptional electro-catalytic performance of Au/PANI@PtCu and the high conductivity of AuNPs@MWCNTs, the sandwich-type immunoassay exhibited remarkable sensitivity in detection. The linear range extended from 100 fg/mL to 10 ng/mL, while achieving an impressively low limit of detection of 77.62 fg/mL.

The value of serum neutralizing antibody in evaluating predictability of COVID-19 after recovery and the validation of vaccine.

BACKGROUND: This work aimed to study natural humoral immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: Chemiluminescent immunoassay (CLIA) was used to detect the neutralizing antibody (Nabs) and IgG. RESULTS: Nabs peaked on days 57-96 after symptom onset and remained detected on days 97-132. The Nabs in the 32 patients who were dynamically monitored showed four changing patterns. The titers of Nabs and IgG were correlated, and three modes of relationship were found between them. CONCLUSIONS: Nabs showed a regular change in the course of coronavirus disease 2019 (COVID-19). The detection of Nabs is very important for monitoring the course of COVID-19 and predicting the strength of antibody protection.

PDK1-stabilized LncRNA SPRY4-IT1 promotes breast cancer progression via activating NF-κB signaling pathway.

Pyruvate dehydrogenase kinase 1 (PDK1) is a widely known glycolytic enzyme, and some evidence showed that PDK1 promoted breast cancer by multiple approaches. However, very few lncRNAs have been identified to be associated with PDK1 in breast cancer in previous research. In this study, we found that lncRNA sprouty4-intron transcript 1 (SPRY4-IT1) was regulated by PDK1 with correlation analysis, and PDK1 upregulated SPRY4-IT1 remarkably in breast cancer cells, as PDK1 interacted with SPRY4-IT1 in the nucleus and significantly enhanced the stability of SRPY4-IT1. Furthermore, SPRY4-IT1 was highly expressed in breast cancer, significantly promoted the proliferation and inhibited apoptosis of breast cancer cells. In terms of mechanism, SPRY4-IT1 inhibited the transcription of NFKBIA and the expression of IκBα, thus promoting the formation of p50/p65 complex and activating NF-κB signaling pathway, which facilitated survival of breast cancer cells. Therefore, our finding reveals that PDK1/SPRY4-IT1/NFKBIA axis plays a crucial role that promoting tumor progression, and SPRY4-IT1 knockdown incombined with PDK1 inhibitor is promising to be a new therapeutic strategy in breast cancer.

A simple and smart AND-gate DNA nanoprobe for correlated enzymes tracking and cell-selective imaging.

Accurate cancer diagnosis and effective drug therapy entail sensitive and dynamic monitoring of intracellular key enzymes, since their expression level is closely related to disease progression. Simultaneous monitoring of correlated enzymes is promising to help unveiling mystery of cytobiological events during tumor progression and drug response, while is challenged by lacking of a robust and simple simultaneous detection strategy. In order to construct a simple and smart strategy which is complex design-avoided and doesn't need other auxiliary enzyme, here we develop an AND-gate strategy for simultaneously monitoring correlated enzymes which both are upregulated in cancer cells (telomerase and apurinic/apyrimidinic endonuclease 1). An innovative AND-gate DNA nanoprobe has been designed to avoid mutual interference and background noise, guaranteeing an enhanced fluorescent signal output upon catalyzation of dual enzymes. This AND-gate strategy achieves sensitive detection of two enzymes in an individual manner in test tube, through which the diagnostic potential of bladder cancer has been validated by telomerase detection in clinical urine sample. The AND-gate strategy enables specific intracellular imaging of dual enzymes in different cancer cell lines. Importantly, in contrast to traditional single-targeting strategies, AND-gate imaging of dual enzymes significantly improves cancer cell selectivity. Moreover, this strategy dynamically monitors enzymatic activity changes during chemoresistance induced by chemotherapeutic treatment. This simple and smart strategy has foreseeable prospect in the fields of disease diagnosis, drug prognosis evaluation, and precise fluorescence-guided surgery.

Application of Tivantinib for Hepatocellular Carcinoma: A Meta-Analysis Study.

Objectives: The efficacy of tivantinib may have some potential in treating MET-high hepatocellular carcinoma, and we aim to compare tivantinib with placebo for the treatment of MET-high hepatocellular carcinoma. Methods: Several databases including PubMed, Cochrane Library, Web of Science, EBSCO, and EMbase have been systematically searched through March 2022, and we included studies regarding the treatment of MET-high hepatocellular carcinoma by using tivantinib versus placebo. Results: We finally include three RCTs. In comparison with placebo for MET-high hepatocellular carcinoma, tivantinib reveals no significant influence on overall survival (P=0.21), progression-free survival (P=0.13), time to progression (P=0.38), or grade ≥3 anemia (P=0.50) but increases the incidence of grade ≥3 neutropenia (P=0.04). Conclusions: Tivantinib may provide no additional benefits for MET-high hepatocellular carcinoma.

The miR-345-3p/PPP2CA signaling axis promotes proliferation and invasion of breast cancer cells.

Breast cancer is the most common malignancy among women worldwide. Functional studies have demonstrated that miRNA dysregulation in many cases of cancer, in which miRNAs act as either oncogenes or tumor suppressor. Here we report that miR-345-3p is generally upregulated in breast cancer tissues and breast cancer cell lines. Overexpression and inhibition of miR-345-3p revealed its capacity in regulating proliferation and invasion of breast cancer cells. Further research identified protein phosphatase 2 catalytic subunit alpha (PPP2CA), a suppressor of AKT phosphorylation, as a candidate target of miR-345-3p. In vitro, miR-345-3p mimics promoted AKT phosphorylation by targeting its negative regulator, PPP2CA. Blocking miR-345-3p relieved its inhibition of PPP2CA, which attenuated PI3K-AKT signaling pathway. In vivo, inhibiting miR-345-3p by miR-345-3p-inhibition lentivirus suppressed tumor growth and invasiveness in mice. Together, the miR-345-3p/PPP2CA signaling axis exhibits tumor-promoting functions by regulating proliferation and invasion of breast cancer cells. These data provide a clue to novel therapeutic approaches for breast cancer.

Enhancement of biotransformation of ginsenosides in white ginseng roots by aerobic co-cultivation of Bacillus subtilis and Trichoderma reesei.

In the present work, the biotransformation of ginsenosides in white ginseng roots was innovatively investigated using the aerobic fermentation by the co-cultivation of Bacillus subtilis and Trichoderma reesei. It is found that in the co-cultivation mode, the optimal nitrogen source was corn steep liquor, and the loading of ginseng powder and inoculation proportion of B. subtilis and T. reesei were 15 g/L and 1:4, respectively. The total ginsenoside yield and production of minor ginsenosides in the co-cultivation mode obviously enhanced in comparison to the monoculture mode. Meanwhile, the maximal total ginsenoside yield of 21.79% and high hydrolase activities were achieved using the staged inoculation at the inoculation proportion of 1:4 in the co-cultivation mode, the production of minor ginsenosides such as Rg3 and Rh1, Rh2 was significantly strengthened, and the pharmacological activities of the fermented solution obviously improved. The enhancement of ginsenoside transformation can be mainly attributed to hydrolysis of the produced hydrolases and metabolism of two probiotics. This result clearly reveals that using the staged inoculation in co-cultivation fermentation mode was favor of the ginsenoside biotransformation in ginseng due to non-synchronous cell growth and different metabolic pathways of both probiotics. This work can provide a novel method for enhancing ginsenoside transformation of ginseng.Key points• Co-cultivation fermentation significantly promoted ginsenoside biotransformation.• The staged inoculation in co-culture mode was an optimal operation method.• The pharmacological activity of the co-cultured solution was significantly enhanced.

Downregulation of PHLPP induced by endoplasmic reticulum stress promotes eIF2α phosphorylation and chemoresistance in colon cancer.

Aberrant activation of endoplasmic reticulum (ER) stress by extrinsic and intrinsic factors contributes to tumorigenesis and resistance to chemotherapies in various cancer types. Our previous studies have shown that the downregulation of PHLPP, a novel family of Ser/Thr protein phosphatases, promotes tumor initiation, and progression. Here we investigated the functional interaction between the ER stress and PHLPP expression in colon cancer. We found that induction of ER stress significantly decreased the expression of PHLPP proteins through a proteasome-dependent mechanism. Knockdown of PHLPP increased the phosphorylation of eIF2α as well as the expression of autophagy-associated genes downstream of the eIF2α/ATF4 signaling pathway. In addition, results from immunoprecipitation experiments showed that PHLPP interacted with eIF2α and this interaction was enhanced by ER stress. Functionally, knockdown of PHLPP improved cell survival under ER stress conditions, whereas overexpression of a degradation-resistant mutant PHLPP1 had the opposite effect. Taken together, our studies identified ER stress as a novel mechanism that triggers PHLPP downregulation; and PHLPP-loss promotes chemoresistance by upregulating the eIF2α/ATF4 signaling axis in colon cancer cells.

Postconditioning with Sevoflurane or Propofol Alleviates Lipopolysaccharide-Induced Neuroinflammation but Exerts Dissimilar Effects on the NR2B Subunit and Cognition.

Neuroinflammation can cause cognitive deficits, and preexisting neuroinflammation is observed frequently in the clinic after trauma, surgery, and infection. Patients with preexisting neuroinflammation often need further medical treatment under general anesthesia. However, the effects of postconditioning with general anesthetics on preexisting neuroinflammation have not been determined. In this study, adult rats were posttreated with sevoflurane or propofol after intracerebroventricular administration of lipopolysaccharide. The effects of sevoflurane or propofol postconditioning on neuroinflammation-induced recognition memory deficits were detected. Our results found that postconditioning with sevoflurane but not propofol reversed the selective spatial recognition memory impairment induced by neuroinflammation, and these differential effects did not appear to be associated with the similar anti-neuroinflammatory responses of general anesthetics. However, postconditioning with propofol induced a selective long-lasting upregulation of extrasynaptic NR2B-containing N-methyl-D-aspartate receptors in the dorsal hippocampus, which downregulated the cAMP response element-binding signaling pathway and impaired spatial recognition memory. Additionally, the NR2B antagonists memantine and Ro25-6981 reversed this neurotoxicity induced by propofol postconditioning. Taken together, these results indicate that under preexisting neuroinflammation, postconditioning with sevoflurane can provide reliable neuroprotection by attenuating lipopolysaccharide-induced neuroinflammation, apoptosis, and neuronal loss and eventually improving spatial recognition deficits. However, although posttreatment with propofol also has the same anti-neuroinflammatory effects, the neurotoxicity caused by propofol postconditioning following neuroinflammation warrants further consideration.

Clinical significance of the serum IgM and IgG to SARS-CoV-2 in coronavirus disease-2019.

OBJECTIVE: To explore the clinical value of serum IgM and IgG to SARS-CoV-2 in COVID-19. METHODS: 105 COVID-19 patients were enrolled as the disease group. 197 non-COVID-19 patients served as the control group. Magnetic chemiluminescent immunoassay (MCLIA) was used to detect the IgM and IgG. RESULTS: The peak of positive rates of SARS-CoV-2 IgM was about 1 week earlier than that of IgG. It reached to peak within 15-21 days and then began a slowly decline. The positive rates of IgG were increased with the disease course and reached the peak between 22 and 39 days. The differences in sensitivity of the three detection modes (IgM, IgG, and IgM + IgG) were statistically significant. The largest group of test cases (illness onset 15-21 days) showed that the positive rate of IgG was higher than IgM. Also, the sensitivity of IgM combined with IgG was higher than IgM or IgG. IgM and IgG were monitored dynamically for 16 patients with COVID-19, the results showed that serological transformation of IgM was carried out simultaneously with IgG in seven patients, which was earlier than IgG in four patients and later than IgG in five patients. CONCLUSION: The detection of SARS-CoV-2 IgM and IgG is very important to determine the course of COVID-19. Nucleic acid detection combined with serum antibody of SARS-CoV-2 may be the best laboratory indicator for the diagnosis of SARS-CoV-2 infection and the phrase and predication for prognosis of COVID-19.

[Value of Serum Light Chain in Diagnosis and Evaluation of Efficacy for Multiple Myeloma].

OBJECTIVE: To explore the clinical application value of serum light chain (sLC) in the diagnosis and therapeutic efficacy evaluation for multiple myeloma. METHODS: 46 patients with newly diagnosed multiple myeloma were selected as MM group and 50 healthy persons as control group. Rate scattering immunoturbidimetry was used to detect serum light chain and immunoglobulin (Ig) in two groups, serum protein electrophoresis was used to detect M protein by agarose gel. Then, the sensitivity and specificity of the two methods in MM diagnosis were analyzed and compared, and the significance of sLC detection in MM diagnosis were discussed. In addition, 15 MM patients after received conventional therapy were tracked, sLC levels in five different therapentic times were recorded, and the effect of sLC in efficacy evaluation of MM was analyzed. RESULTS: There were 11 cases of IgA type, 15 cases of IgG type, 8 cases of light chain κ type, 8 cases of light chain λ type, 2 cases of IgD type, and 2 cases of non-secretion type. The sLC-κ, sLC-λ and their ratio (including light chain type and double clone type), IgA and IgG (except IgD type), as well as albumin, beta-globulin and gamma-globulin levels showed statistically significant differences (P＜0.05) compared with the control group. The sensitivity of serum protein electrophoresis, Ig quantification, sLC and its ratio in the diagnosis of multiple myeloma were 57%, 76% and 65%, and their specificity were 83%, 61% and 90%, respectively. After the second or third chemotherapy, the sLC-κ/λ ratio gradually approached the normal range as the disease reliefes, and the sLC-κ/λ ratio continued to be on or off the line at outliers or further away from the reference value as the disease progresses in MM patients with κ type or λ type. CONCLUSION: sLC detection shows positive significance in early diagnosis of multiple myeloma, SLC monitoring can be used for the efficacy evaluation for treatment of MM patients.

Identification of a mutL­homolog 1 mutation via whole­exome sequencing in a Chinese family with Gardner syndrome.

Gardner syndrome (GS), a variant of familial adenomatous polyposis, is a rare genetic disorder with autosomal dominant inheritance, characterized by the presence of multiple intestinal polyps, multiple osteomas, dental abnormalities and soft tissue tumors. To date, only a few gene mutations have been demonstrated to be responsible for GS. To explore potential unknown mutations responsible for GS, the present study used whole­exome sequencing of two affected individuals from a family with GS to identify a candidate mutation in mutL­homolog (MLH)1. The two patients with GS were diagnosed based on a combination of clinical features, family history, physical examinations and cone­beam computed tomographic imaging. Through whole­genome sequencing, the present study subsequently identified a missense mutation in MLH1 (NM_000249.3:p.Tyr379Ser/c.1136A>C), which was further confirmed by Sanger sequencing. Furthermore, the amino acid residue p.Tyr379 was identified to be highly conserved among different species through sequence alignment with ClustalW2. In conclusion, the results identified for the first time a MLH1 missense mutation (NM_000249.3:​p.Tyr379Ser/c.1136A>C) in a Chinese family with GS, thus broadening the range of mutated genes associated with GS. This highlights the value of whole­exome sequencing in identifying disease mutations in a family.

Pyruvate kinase M2 interacts with mammalian sterile 20-like kinase 1 and inhibits tamoxifen-induced apoptosis in human breast cancer cells.

Tamoxifen has been reported to be associated with antagonism of estrogen-mediated cell growth signaling and activation of estrogen receptor-independent apoptosis events. It has been demonstrated that mammalian sterile 20-like kinase 1 is a direct target of Caspases to amplify the apoptotic signaling pathway. Here, we presented that breast cancer MCF-7 and SKBR3 cells under treatment with 4-hydroxytamoxifen displayed decreased level of pyruvate kinase M2. Western blot results also showed that 4-hydroxytamoxifen induced the activity of pro-apoptotic protein Caspase-3 in MCF-7 and SKBR3 cells, as evidenced by the cleavage of mammalian sterile 20-like kinase 1 substrate in a dose-dependent manner. Co-immunoprecipitation and immunofluorescence experiments were performed to clarify the relationship between pyruvate kinase M2 and mammalian sterile 20-like kinase 1. The results indicated that mammalian sterile 20-like kinase 1 was associated with pyruvate kinase M2 in cultured mammalian cells, and the interaction between mammalian sterile 20-like kinase 1 and pyruvate kinase M2 was decreased in response to 4-hydroxytamoxifen treatment. In addition, knockdown of pyruvate kinase M2 upregulated the level of cleaved Caspase-3 and subsequently facilitated the nuclear translocation of mammalian sterile 20-like kinase 1. Our data further supplemented the extensive functions of pyruvate kinase M2 in mediating breast cancer cell viability by substantially abating the mammalian sterile 20-like kinase 1-mediated apoptosis. In summary, our results identified that mammalian sterile 20-like kinase 1 is a novel downstream target of pyruvate kinase M2, and knockdown of pyruvate kinase M2 contributes apoptosis via promoting nuclear translocation of mammalian sterile 20-like kinase 1 by enhancing Caspase-3-dependent cleavage.

Leptin promotes epithelial-mesenchymal transition of breast cancer via the upregulation of pyruvate kinase M2.

BACKGROUND: Accumulating researches have shown that epithelial-mesenchymal transition (EMT) contributes to tumor metastasis. Leptin, a key adipokine secreted from adipocytes, shapes the tumor microenvironment, potentiates the migration of breast cancer cells and angiogenesis, and is also involved in EMT. However, the potential mechanism remains unknown. This study aims to explore the effect of leptin on EMT in breast cancer cells and the underlying mechanism. METHODS: With the assessment of EMT-associated marker expression in MCF-7, SK-BR-3, and MDA-MB-468 cells, the effect of leptin on breast cancer cells was analyzed. Besides, an array of pathway inhibitors as well as RNA interference targeting pyruvate kinase M2 (PKM2) were used to clarify the underlying mechanism of leptin-mediated EMT in vitro and in vivo. RESULTS: The results demonstrated that leptin promoted breast cancer cells EMT, visibly activated the PI3K/AKT signaling pathway, and upregulated PKM2 expression. An antibody against the leptin receptor (anti-ObR) and the PI3K/AKT signaling pathway inhibitor LY294002 significantly abolished leptin-induced PKM2 expression and EMT-associated marker expression. SiRNA targeting PKM2 partially abolished leptin-induced migration, invasion, and EMT-associated marker expression. In vivo xenograft experiments indicated that RNA interference against PKM2 suppressed breast cancer growth and metastasis. CONCLUSIONS: Our data suggest that leptin promotes EMT in breast cancer cells via the upregulation of PKM2 expression as well as activation of PI3K/AKT signaling pathway, and PKM2 might be one of the key points and potential targets for breast cancer therapy.

[Endogenous IFN-ß maintains M1 polarization status and inhibits proliferation and invasion of hepatocellular carcinoma cells].

Objective To investigate the effect of endogenous interferon ß (IFN-ß) on the polarization of M1 macrophages as well as the proliferation and invasion activities of hepatocellular carcinoma cells (HCCs) mediated by M1 macrophages. Methods U937-M1 macrophages derived from human monocytic tumor cells U937 was established and the cell phenotypes were identified by real-time quantitative PCR, ELISA and flow cytometry. After IFN-ß gene was knocked down with siRNA or IFN-ß was neutralized with IFN-ß monoantibody in U937-M1 macrophages, the change of M1/M2 phenotype was again analyzed by the above methods. The expressions of interferon regulatory factor 1 (IRF1) and IRF5 were detected by real-time quantitative PCR and Western blotting. The proliferation and invasion activities of HCCs, which were cultured with conditioned medium (CM) collected from different macrophage groups, were analyzed by CCK-8 assay and Transwell(TM) experiments, respectively. Results U937-M1 macrophages showed higher expressions of interleukin 12p35 (IL-12p35), interleukin 12p40 (IL-12p40), interleukin 12p70 (IL-12p70), interleukin 23p19 (IL-23p19), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α) and CD86 than U937-M0 did. But both U937-M0 macrophages and U937-M1 macrophages showed low expression of CD206. However, compared with the U937-M1 macrophages, the IFN-ß-blocked U937-M1 macrophages presented decreased expressions of the above M1 macrophages-associated markers, but increased expressions of M2 macrophages-associated markers IL-10 and CD206, as well as lower expressions of IRF1 and IRF5. The inhibited proliferation/invasion activities of HCCs mediated by U937-M1 macrophages were reversed by IFN-ß-blocked U937-M1 macrophages. Conclusion Blocking endogenous IFN-ß could inhibit the U937-M1 polarization status and U937-M1 macrophages-mediated anti-tumor activity of HCCs. IFN-ß might be involved in modulating the expressions of IRF1 and IRF5 as well as maintaining the M1 polarization status and its function.

[Expression Wnt2 in breast cancer tissues and sera of the patients].

OBJECTIVE: To observe the expression of Wnt2 in the breast cancer cells (MDA-MB-231, ZR-75-30 and MCF-7) and tissues, and analyze the correlation of Wnt2 expression with CA-153 in the sera of patients with breast cancer. METHODS: The expression of Wnt2 at both mRNA and protein levels was measured respectively by real-time fluorescent quantitative PCR and Western blotting in the three human breast cancer cell lines. In addition, the immunohistochemistry (IHC) was applied to detect the expression of Wnt2 in 5 corresponding tissues adjacent to cancer, 9 breast carcinoma in situ and 9 invasive breast cancer; ELISA was used to detect Wnt2 and electrochemiluminescence immunoassay was used to detect CA-153 in the sera of 15 normal women and 30 patients with breast cancer. Furthermore, we collected complete clinicopathological data from the participating subjects, and analyzed their relationships with serum Wnt2 levels. RESULTS: The mRNA and protein of Wnt2 were negative in the three human breast cancer cells, while Wnt2 in the epithelial and interstitial cells of breast cancer tissues was significantly higher than that in normal tissues (P<0.05). The expression levels of Wnt2 and CA-153 in the sera of patients with breast cancer were higher than those in normal women (P<0.05), and Wnt2 levels in the sera of breast cancer patients were positively correlated with serum CA-153 levels. CONCLUSION: Simultaneous tests of Wnt2 and CA-153 levels in the serum may help the diagnostic screening of breast cancer.

[Prokaryotic expression and purification of a tandem repeat of ovarian cancer antigen CA125 and preparation of its antiserum].

AIM: To establish a prokaryotic expression system of the tandem repeat of CA125 (CA125R), express and purify the recombinant CA125R protein, prepare its antiserum. METHODS: The full gene sequence of one tandem repeat of CA125 was synthesized and cloned into pET-32a(+) to construct a prokaryotic expression vector of the CA125R protein (pET-CA125R). The pET-CA125R was transformed into E.coli BL21 (DE3) and the soluble expression conditions were optimized; the pure recombinant CA125R protein was prepared by affinity Ni-NTA chromatography and identified by Western blotting. A rabbit was immunized with the pure recombinant CA125R protein to prepare its antiserum. RESULTS: The prokaryotic expression vector of CA125R was successfully constructed. The optimal soluble induction expression conditions were 0.5 mmol/L isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 15DegreesCelsius for 6 h. Western blotting confirmed the pure CA125R recombinant protein of high purity. The prepared antiserum specifically recognized recombinant CA125R protein and natural CA125 glycoprotein. CONCLUSION: We successfully established the efficient prokaryotic expression system of the CA125R, and prepared the recombinant CA125R protein of high purity and its antiserum.