Expression of FXR and HRG and their clinicopathological significance in benign and malignant pancreatic lesions.

AIMS: The following study examines the FXR and HRG expression in benign and malignant lesions of the pancreas and evaluates the association between FXR and HRG expression with clinicopathological features and prognosis of pancreatic cancer. MATERIALS AND METHODS: Immunohistochemistry of FXR and HRG was performed with EnVision™ in 106 pancreatic ductal adenocarcinoma (PDAC) specimens, 35 paracancer samples (2 cm away from the tumor, when possible or available), 55 benign lesions and 13 normal tissue samples. RESULTS: The percentage of cases with positive FXR and negative HRG expression was significantly higher in PDAC compared to pericancerous tissues, benign lesions and normal tissues (P<0.05 or P<0.01). In pancreatic tissues with benign lesions, tissues with positive FXR and/or negative HRG protein expression exhibited dysplasia or intraepithelial neoplasia. The percentage of cases with positive FXR and negative HRG expressions was significantly higher in PDAC with lymph node metastasis, invasion, and TNM stage III+IV disease (P<0.05 or P<0.01). The expression of FXR was negatively correlated with HRG (P<0.05). In addition, the univariate Kaplan-Meier analysis showed that positive FXR and negative HRG expression, poor differentiation, large tumor size, high TNM stage, lymph node metastasis, and invasion were closely associated with decreased overall survival in PDAC patients (P<0.05 or P<0.01). Moreover, multivariate Cox regression analysis identified that positive FXR and negative HRG expression were independent factors for poor prognosis in PDAC. The AUC for FXR was (AUC=0.709, 95% CI: 0.632-0.787), and for HRG was (AUC=0.719, 95% CI: 0.643-0.796) in PDAC compared to benign lesions. CONCLUSIONS: Positive FXR and negative HRG expression are closely associated with the carcinogenesis, clinical, pathological and biological behaviors, and poor prognosis in PDAC.

The anti-leukemic effect of carnosic acid combined with adriamycin in a K562/A02/SCID leukemia mouse model.

The effects of carnosic acid (CA) were investigated on the acute myeloid leukemia (AML) cell growth in vivo. A NOD/SCID AML mouse model, which was set up by inoculation with K562/A02 cells, was used to study whether tumor growth in vivo can be inhibited by CA combined with adriamycin. After being inoculated with K562/A02 cells, the NOD/SCID mice were expressed positive human mdr1 and bcr/abl genes. This result indicates that the K562/A02/SCID leukemia mouse model is successfully established. The mice treated with CA combined with adriamycin exhibit a significant lower number of leukemia cells (20%) than that of untreated animals (32.5%) (P<0.05), in particular with higher percentages of apoptotic cells than the mice treated by single adriamycin (control) group. The median of 95% CI survival time is 19 (10.0-44.2) and 33 (29.4-36.6) days for the control group and the CA-treated group, respectively. The difference is statistically significant (P<0.05). It is illustrated that the natural compound CA, combined with Adriamycin, has high potential to inhibit the growth of malignant cells in vivo, and is a promising adjuvant anti-cancer drug. Prospective studies should be conducted to understand the functional mechanism of CA at the molecular level.

Synergism between carnosic acid and arsenic trioxide on induction of acute myeloid leukemia cell apoptosis is associated with modulation of PTEN/Akt signaling pathway.

OBJECTIVE: To investigate the synergistic effects of carnosic acid (CA) with arsenic trioxide (As2O3) on proliferation and apoptosis in HL-60 human myeloid leukemia cells, and the major cellular signaling pathway involved in these effects. METHODS: HL-60 cellular proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Cell cycle distribution and apoptosis were monitored by flow cytometry. The activation of casepase-9, Bcl-2-associated agonist of cell death (BAD), p-BAD, p27, phosphatase and tensin homolog deleted on chromosome ten (PTEN), Akt, p-Akt was assessed by Western blot analysis. The expression of PTEN mRNA was tested by reverse transcription polymerase chain reaction (RT-PCR) analysis. RESULTS: CA reduced HL-60 cell viability in a dose- and time-dependent manner, and induced G1 arrest and apoptosis. Moreover, CA upregulated PTEN expression, blocked the Akt signaling pathway, subsequently inhibited phosphorylation of BAD, reactivated caspase-9, and elevated levels of p27. CA also augmented these effects of As2O3. CONCLUSION: CA might be a novel candidate of the combination therapy for leukemia treatment; these effects were apparently associated with the modulation of PTEN/Akt signaling pathway.

Systematical analysis of impacts of heat stress on the proliferation, apoptosis and metabolism of mouse hepatocyte.

Heat stress will stimulate cells of living organisms to generate heat shock proteins (Hsps). In the mouse liver, impacts of heat stress on hepatocyte proliferation, apoptosis and metabolism have not been studied systematically at different temperatures. In this research, the test mice were heated to 40, 42, 44 and 46°C, respectively, for 20 min and recovered at room temperature for 8 h in normal feeding conditions; the control animals were kept at room temperature without heat stress. The expression levels of Hsp70, Pcna, Bax, Bcl2, cytochrome P450 1A2 (CYP1A2), CYP2E1 and analog of CYP3A4 (not reported in mouse before), the parameters reflecting stress strength, cell proliferation, apoptosis and metabolism, were detected by western blotting, immunohistochemistry and semi-quantitative RT-PCR in test and control mice. Haematoxylin-eosin (H&E) staining and TUNEL analysis were further used to study the impacts of heat stress at different temperatures on hepatocellular necrosis and apoptosis. Serum AST and ALT levels, the markers of liver injury, were measured after heat stress at different temperatures. The data show that Hsp70 expression was significantly increased when temperature increased (P < 0.05). At lower temperatures (40 or 42°C), expression of Pcna, CYP1A2 and analog of CYP3A4 were considerably increased (P < 0.05) while hepatocyte necrosis and apoptosis were not induced (P > 0.05). At higher temperatures (44 or 46°C), expression of Pcna was decreased while hepatocyte necrosis and apoptosis were induced (P < 0.05). Expressions of CYP1A2 and analog of CYP3A4 were decreased especially at 46°C (P < 0.05). Expression of CYP2E1 could not be detected to increase at 40°C but was at high levels at 42, 44 and 46°C (P < 0.05). Expressions of AST and ALT were not different between the test mice and control mice at 40°C while they were significantly higher in the test mice than those in the control mice at 42 (P < 0.05), 44 and 46°C (P < 0.01). In conclusion, heat stress at lower temperatures promotes hepatocyte proliferation and improves the metabolic efficiency in mouse liver while heat stress at higher temperatures inhibits hepatocyte proliferation, promotes hepatocyte apoptosis and induces hepatocyte necrosis. This may give a hint to understanding human liver injury in high temperatures. Moreover, it is the first time that the analog of CYP3A4 was detected in mouse hepatocellular cytoplasm. It is worthwhile to dissect its function in future work.

[Study on reversing mechanism of multidrug resistance of K562/A02 cell line by carnosic acid].

OBJECTIVE: To investigate the effects of carnosic acid (CA) on reversal of the multidrug resistance (MDR) of human leukemia cell line K562/A02 and its mechanism. METHODS: MTT assay was used to determine the sensitivity of K562/A02 cells to adriamycin (ADM) pre-and post-treated with CA. Flow cytometry (FCM) and laser scanning confocal microscopy (LSCM) were used to measure intracellular fluorescence intensity and concentration of ADM respectively. The expression level of mdr1 was detected by semi-quantitative RT-PCR. P-glycoprotein (P-gp) expression was detected by FCM and Western blot. RESULTS: CA decreased IC(50) of ADM in K562/A02 cells from 16.31 µg/mL to 1.35 µg/mL, being a 12.08-fold decrease. The intracellular ADM fluorescence intensity of K562/A02 was increased from 17.05 to 60.53 after treated with CA (P < 0.01). In living K562/A02 cells, after treated with CA, the diffuse distribution of intracellular ADM was recovered in both nuclear and cytoplasm, and the concentration of intracellular ADM increased from 4.93µg/mL to 15.43µg/mL. RT-PCR assay showed that CA inhibited the expressions of mdr1 mRNA in K562/A02 cells (P < 0.01). Mean fluorescence intensity of P-gp detected by FCM in CA-treated K562/A02 was decreased to 22.80 as compared with that in untreated K562/A02 cells (44.40, P < 0.05). CONCLUSION: CA can reverse the MDR of K562/A02 cells in vitro. The mechanism may be associated with down-regulation of mdr1 and inhibition of P-gp function.

Upregulation of PTEN involved in scorpion venom-induced apoptosis in a lymphoma cell line.

We investigated whether the venom of the scorpion Buthus martensii Karsch (BmK) inhibited growth of human lymphoma cells by inducing apoptosis, and studied possible signal pathways involved in this cell death. BmK venom selectively reduced the viability of Raji and Jurkat cells, and had low toxicity to human peripheral blood lymphocytes. Flow cytometry showed that BmK venom-induced apoptosis and G(0)/G(1) cell cycle arrest in Raji and Jurkat cells. In Raji cells, BmK venom upregulated the expression of PTEN accompanied by decreased levels of Akt and Bad phosphorylation. Treatment with BmK venom and LY294002 (an inhibitor of Akt) synergistically enhanced apoptosis. The expression of p27 was increased in both PTEN-positive Raji and PTEN-negative Jurkat cells exposed to BmK venom. The results indicate that key regulators in BmK venom-induced apoptosis are PTEN, acting through downregulation of the PI3K/Akt signal pathway, in Raji cells and p27 in Jurkat cells.

Reversion of P-glycoprotein-mediated multidrug resistance in human leukemic cell line by carnosic acid.

One of the common hindrances to successful chemotherapy is the development of multidrug resistance (MDR) by tumor cells to multiple chemotherapeutic agents. In this regard, P-glycoprotein (P-gp) acts as an energized drug pump that reduces the intracellular concentration of drugs, even of structurally unrelated ones. The modulators of P-gp function can restore the sensitivity of MDR cells to anticancer drugs. Therefore, to develop effective drug-resistance-reversing agents, we evaluated the P-gp modulating potential of carnosic acid (CA) in multidrug-resistant K562/AO2 cells in the present study. The reversing effect of CA was evaluated by determining the inhibition rates of cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assays. The intracellular adriamycin fluorescence intensity and the expression of P-gp were measured by flow cytometry (FCM). Meanwhile, the subcellular distribution of adriamycin was detected via Laser Scanning Confocal Microscopy (LSCM). The mRNA expression of mdrlwas then detected via semiquantitative reverse transcription polymerase chain reaction (RT-PCR). The findings showed that CA decreased apparently the Inhibition Concentration 50% (IC50) of adriamycin by increasing its intracellular concentration and thus enhancing the sensitivity of K562/AO2 cells. Adriamycin was distributed evenly in the cytoplasm when the cells were treated with CA. The expression of mdrl was decreased. Overall, the results indicated that CA can serve as a novel, non-toxic modulator of MDR, and it can reverse the MDR of K562/AO2 cells in vitro by increasing intracellular adriamycin concentration, down-regulating the expression of mdrl, and inhibiting the function of P-gp.

[Killing effect of double suicide genes mediated by retroviral vector on k562 cells].

The aim of study was to investigate the killing effect of double suicide gene system mediated by retroviral vector on K562 cells in vivo and ex vivo. CDglyTK gene was transfected into PA317 cells by using lipofectamine. K562 cells were infected with viral supernatant. K562/CDglyTK cells were treated with 5-fluorocytosine (5-FC) and/or ganciclovir (GCV). Mice were randomly divided into three groups: tumor formation, tumor inhibition and tumor therapy. Each mouse was implanted with K562/CDglyTK cells or K562 cells. The results indicated that the killing effect of 5-FC in combination with GCV on K562/CDglyTK was more significant than using 5-FC or GCV alone. In vivo study showed that after being injected subcutaneously with K562 cells and K562/CDglyTK cells, there was not obvious difference in tumor formation rate of mice, 5-FC + GCV could suppress tumor formation of the K562/CDglyTK cells. After being treated with 5-FC and GCV, the median tumor volume of mice implanted with K562/CDglyTK cells decreased obviously, compared with the control group. Their median survival was significantly prolonged. It is concluded that double suicide genes are more effective for killing effect on K562 cells in vivo and in ex vivo. It may be applicable to clinical gene therapy.

Treatment of solid tumors and metastases by fibrinogen-targeted anticancer drug therapy.

The accumulation of fibrin/fibrinogen and other coagulation factors in and around solid tumors and metastatic foci has been recognized for a century as an aspect of cancer pathology. On this basis, anticoagulants and fibrinolytic agents have been deployed as adjuvant anticancer therapies, but they have proved clinically useful for only a small proportion of tumors and they only control the functions of the coagulant components. Overuse or long-term application of anticoagulants and fibrinolytic agents often lead to undesirable side-effects. Here, we propose that anticancer drugs that act by different mechanisms can inhibit tumor-associated coagulation, and it may be possible to develop drugs that specifically targeting tumor-related coagulation, have specific cytotoxic effects on tumor and metastatic cells. We provide laboratory and clinical evidence supporting the hypothesis and offer proposals for future applications.

[Experimental study on immunotherapy with dendritic cell in leukemic mice model].

OBJECTIVE: To explore the feasibility and efficiency of immunotherapy with dendritic cell (DC) in leukemic mice model after allogeneic bone marrow transplantation (allo-BMT). METHODS: Mature DC were expanded from mice bone marrow mononuclear cells (MNC) by adding mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) and interleukin-4 (mIL-4). Three days later they were pulsed with frozen thawing L7212 leukemia-related antigen. Mice bearing leukemia received allo-BMT at d 0, and then were divided into control group (A), T cells group (B) and DC + T cells group (C) to receive respective immune therapy at d 14. The survival rate, survival time, occurrence of graft-versus-host disease (GVHD), cytotoxicity of spleen cells and serum cytokine level were observed. The survivors in each group were rechallenged with L7212 cells to observe the immune response to the leukemia. RESULTS: Mature DC were successfully induced from bone marrow MNC. In groups B and C, the relapse rates were 30% and 0%, while the long term survival rates after BMT was 30% and 70% respectively. Both of the differences were statistically significant (P < 0.05). However, the incidence of GVHD in these two groups were similar. The mean survival times were (32.95 +/- 13.29) days and (41.15 +/- 13.88) days, respectively (P < 0.01). MTT assay indicated that spleen cells from group C had specific killing activity to L7212 cells. Enzyme-labeled immunosorbent assay (ELISA) showed that the serum IL-2 level in group C was (419.75 +/- 26.66) pg/ml, being significantly higher than that in the other two groups (P < 0.01). When the survivors were rechallenged with L7212 cells, there was difference between the survival rates of groups C and B (85.7% vs 33.3%, P < 0.05). CONCLUSION: Immunotherapy with leukemia related antigen-pulsed DC in combination with donor lymphocyte infusions is an effective approach to reinforce GVL effect and decrease relapse after allo-BMT.

Individualized cancer chemotherapy integrating drug sensitivity tests, pathological profile analysis and computational coordination - an effective strategy to improve clinical treatment.

BACKGROUND: Most current cancer chemotherapy is unsatisfactory. There is a trend towards changing the norm for drug selection; one approach is to seek individualized cancer chemotherapy (ICC). METHODS AND RESULTS: ICC is an approach to maximizing the efficacy of chemotherapy and reducing its adverse effects to a minimum. It involves choosing anticancer drugs through the following critical steps: (i) performing drug sensitivity tests in vivo and/or in vitro; (ii) analyzing pathogenic information from morphology, histology and bioinformatics, so that targeted therapy can be offered to disrupt the escalating tumorigenic molecules and pathways; (iii) introducing mathematical and computational systems to assist in improving the quality of decision-making. CONCLUSION: Increasing clinical evidence indicates that drug sensitivity tests, pathological profile analyses and computational coordination are ways to improve therapeutic quality. In future, each patient should have his own unique chemotherapy protocol.

[A randomized controlled and multicenter clinical study of ferrous L-threonate in treatment of iron deficiency anemia].

OBJECTIVE: To study the clinical efficacy and side effects of ferrous L-threonate for treatment of iron deficiency anemia (IDA). METHODS: It is a multicentral, randomized, double blind, double placebo and paralled comparative study with positive control. One hundred and forty IDA patients diagnosed according to the standard criteria in three hospitals were randomly divided into a test group (ferrous L-threonate plus placebo ferrous succinate) and a positive control group (ferrous succinate plus placebo ferrous L-threonate). Some iron parameters were examined 1, 4 and 8 weeks after medication. Hemoglobin, reticulocyte and other parameters for safety observation were collected every two weeks. RESULTS: For the 2 groups, self comparison showed significant difference (P < 0.01). The total efficacy is 98.44% and 97.01% respectively with no difference. Hemoglobin rised rapidly and gradually and reached a peak in week 8, the change was statistically significant (P < 0.01). Changes of iron parameters also showed significant difference. Side-effects were similar in both groups (13.85% and 14.71%, P > 0.05). CONCLUSION: The effect of ferrous L-threonate in IDA treatment is significant and rapid. Side-effects are few and minimal.

Polymorphism of CYPIA1 and GSTM1 genes associated with susceptibility of gastric cancer in Shandong Province of China.

AIM: To explore whether polymorphisms of the CYPIA1 and GSTM1 genes are associated with susceptibility of stomach cancer. METHODS: A total of 102 stomach cancer cases and 62 healthy persons were diagnosed by pathology in 1998-2000 in the Qilu Hospital of Shandong University. Gene polymorphisms were detected by the PCR using sequence-specific primers. Data analysis of the case-control study was carried out using the unconditional logistic method. RESULTS: After adjustment for age, sex, educational levels, and occupation, the risk factors for stomach cancer were shown to be smoking, Helicobacter pylori (H pylori), and presence of the CYPIM G/G and GSTM1 O/O genotypes. Interaction was observed between the combined genotypes of either CYPIA1 G/G and GSTM1 O/O or H pylori infection, or GSTM1 O/O and H pylori infection or smoking. CONCLUSION: Polymorphisms of the CYPIA1 and GSTM1 genes, H pylori infection and smoking are related to susceptibility to stomach cancer.

[Special morphological changes of pathologic cells in two cases of Waldenström's macroglobulinemia].

Waldenström's macroglobulinemia (WM) is one of malignant hematological disease on account of abnormal proliferation of B lymphocyte clone and the pathologic cells of WM possess ability to secrete monoclonal immunoglobulin M. In this study, the diagnosis and morphological characteristics of 2 patients with WM were analyzed. The results showed that a special kind of "foam cells" were found by cytochemical staining examinations in both cases, which displayed characteristics of lymphocytes, but neither monocyte-macrophage nor fatty cells. The periodic acid-Shiff's reaction (PAS) demonstrated strong positive, especially on the inclusion bodies in pathologic cell plasma while the acid phosphatase, and alpha-butanoic acetate esterase stainings, resulted both in negative. In conclusion, the cells found in the two cases reported may be described as gemmy ring-like lymphocyte in morphology, a special subtype of ring-like lymphocyte.

[Application of pseudotyped lentivirus-mediated double suicide genes to allogeneic bone marrow transplantation in mice].

OBJECTIVE: To explore the feasibility and efficiency of cytosine deaminase (CD)/thymidine kinase (TK) gene-modified donor T cells used in allogeneic bone marrow transplantation (allo-BMT) as an approach to mitigate GVHD without compromising engraftment. METHODS: The pseudotyped lentivirus vectors containing CD and TK double suicide genes were transfected with lipofectine to donor T cells. Lethally irradiated 615 leukemia mice were transplanted with BALB/c bone marrow plus CD(+)TK(+)T cells. GVHD prophylaxis was by administration of ganciclovir (GCV) and 5-Fluoride cytosine (5-FC). RESULTS: The pseudotyped lentivirus-mediated gene transfer system could efficiently transfer CD and TK double suicide genes into donor T cells. Administration of GCV and 5-FC to the mice could markedly potentiate the CFU-S and CFU-GM yields and raise the number of peripheral white blood cells. 1 x 10(7) CD(+)TK(+) allogeneic T cells caused GVHD of a similar magnitude and time course to that of fresh, naive T cells after allo-BMT. Administration of GCV and 5-FC in mice received CD(+)TK(+)T cells reduced the severity of GVHD and resulted in significantly longer survival as compared with non-administration mice, and the effect was stronger than that of administration of GCV or 5-FC alone. CONCLUSION: Administration of CD + TK gene-modified donor T cells to recipient in allo-BMT might be an approach to mitigate GVHD without compromising alloengraftment.

[Killing effect of double suicide genes mediated by lentivirus on lymphoma cells].

BACKGROUND & OBJECTIVE: The lentiviral vectors can integrate interest genes into genome of the target cells that allow for stable transgenic expression even in non-dividing cells without evoking an immune response of the host. All the features have promised them to be used in vivo gene therapy. This study was designed to explore the killing effect of double suicide genes mediated by lentivirus on lymphoma cells (Raji). METHODS: The three plasmids expressed lentivirus, packaging plasmid pCMV 8.2, envelope plasmid pCMV.VSVG and target plasmid (pHR'CS. GFP as control group, pHR'CS.CDglytk as experiment group) were packaged into 293T cells using lipofectine method. Supernatant was harvested and concentrated. The Raji cells were infected with the concentrated virus. The gene integration and expression were confirmed by fluorescence microscopy and RT-PCR. After prodrug GCV or/and 5-FC administration, MTT method was used to detect the growth inhibition rate (GIR) of Raji cells for evaluating the killing effect of CD and HSV-tk double suicide genes on Raji cells. RESULTS: The three plasmids were effectively transferred into 293T cells. Green fluorescence on the cell was observed through fluorescence microscopy and a lot of virus particles were observed through transmission electronic microscopy. Double suicide genes mediated by lentivirus were effectively and stably expressed in Raji cells. The GIR of Raji cells using GCV or 5-FC was 51% or 50%, respectively, and it was apparently higher than that of untransfected cells(P< 0.01). When using GCV and 5-FC together, the GIR was 73%, which was apparently higher than that of group using GCV or 5-FC alone (P< 0.01). CONCLUSION: Double suicide genes mediated by lentiviral vector could transfect lymphoma cells effectively and stably. The double suicide gene system enhanced killing effect remarkably on lymphoma cells than CD/5FC or HSV-tk/GCV system alone.