[T lymphocytes with chimeric receptor induce carcinoembryonic antigen-positive specific gastric carcinoma cells apoptosis].

OBJECTIVE: To develop a method to generate T lymphocytes that identify tumor specific carcinoembryonic antigen (CEA), and induce anti-tumor response such as apoptosis. METHODS: Peripheral blood samples were collected from 10 healthy persons and peripheral blood mononuclear cells (PBMCs) were isolated. 1, then CD8(+) T cells were isolated from the PBMCs by magnetic activated cell sorting (MACS) using magnetic beads-conjugated anti-CD8 monoclonal antibodies. Then the recombinant vector anti-CEA-scFv-CD3zeta-pcDNA3.0 was transfected into the CD8(+) T cells by lipofectamine 2000 and T lymphocytes with chimeric receptor were generated and cultivated. The T lymphocytes' activation was assessed by detecting the expression of CD69, an early signal of T cell activation. Human gastric carcinoma cells of the lines HGC-27 (CEA(+)) and SGC-7901 (CEA(-)) were cocultivated with the T lymphocytes with chimeric receptor. Flow cytometry (FCM) with annexin V-FITC/PI double staining was used to detect the expressions of annexin V, a signal of cell apoptosis, so as to observe the apoptosis of the gastric carcinoma cells. T cells activated by phytohemagglutinin (PHA) were used as positive controls, and T cells transfected with blank vector pcDNA3.0 were used as negative controls. RESULTS: T lymphocytes with chimeric receptor directed towards CEA(+) gastric carcinoma cells were generated. 12 and 24 hours after the co-culture of these T cells and HCG-27 cells, the CD69 expression rates were 40.5% +/- 3.4% and 48.3% +/- 2.8% respectively. However, the CD69 expression rates of the HGC-27 and SGC-7901 cells transfected with the T lymphocytes transfected with blank vector were both 0%. FCM showed that the apoptotic rates of the CEA(+) gastric tumor HGC-27 cells was 47.8% +/- 4.2%, significantly higher than that of the CEA(-) gastric tumor SGC-7901 cells (18.7% +/- 2.8%, P < 0.05). CONCLUSION: T lymphocytes with chimeric receptor targeting CEA specifically identify CEA positive gastric carcinoma cells and promote the apoptosis thereof, thus providing a promising method of cellular immunotherapy for gastric carcinomas.

Utility of adenovirus-mediated Fas ligand and bcl-2 gene transfer to modulate rat liver allograft survival.

BACKGROUND: Expression of Fas ligand (FasL) on the graft by gene transduction is expected to introduce apoptosis to lymphocytes to protect rejection, but the FasL-expressing graft cells may also induce apoptosis as the graft usually expresses Fas antigens. In this study, a strong antiapoptotic gene, bcl-2, was cotransfected with the FasL gene in rat liver graft to protect against Fas-mediated cell death and to prolong recipient survival. METHODS: Orthotopic liver transplantation was done in a strain combination of DA to LEW rats. After donor vascular isolation, adenovirus-mediated FasL and bcl-2 genes were cotransfected in the liver graft. RESULTS: Intragraft expression of FasL mRNA was constitutively expressed after adenovirus-mediated transduction, although expression of FasL increased mildly in control grafts. Bcl-2 mRNA was highly expressed at 2 days after reperfusion. In contrast, lower expression of bcl-2 was observed in the control group. The average survival of the gene transferred allografts increased from (9.8+1.3) days to (18.5+8.7) days compared with the control group. CONCLUSION: Our results indicate that rat liver allografts can be protected against host immune responses by adenovirus-mediated FasL and bcl-2 transfection, and that bcl-2 expression prevents the graft from Fas-mediated apoptosis.

[Enhancement of invasion of hepatocellular carcinoma cells by lysophosphatidic acid through Rho signal transduction pathway].

OBJECTIVE: To investigate the mechanism of enhancement of invasion of hepatocellular carcinoma (HCC) cells by lysophosphatidic acid (LPA). METHODS: Human HCC cells of the line SMMC7721 were cultured. LPA at different concentrations (2, 5, and 25 micromol/L) was added into the culture fluid. The Rho activity was detected with Rho activity detection kit. Western blotting was used to detect the protein expression of Rho. Adhesion test was conducted to calculate the adhesion percentage of the SMMC7721 cells. The invasion potential of the SMCC7721 cells was observed using transwell cell test. RESULTS: LPA at the concentrations of 5, and 25 micromol/L increases the activity of Rho protein. When the concentration of LPA was 25 micromol/L the activity of Rho protein was 400 times that of the control group (P < 0.01). The Rho protein expression in the SMCC7721 cells increased when stimulated by LPA, peaked 20 approximately 25 hours after stimulation, and then gradually decreased. When the concentration of LPA was 25 micromol/L the Rho protein expression level was 242% higher than that of the control group. LPA at the concentration of 5 micromol/L and over increased the migratory and invading potential of the SMCC7721 cells and increased the adhesiveness of the SMCC7721 cells time-dependently. CONCLUSION: LPA increases the migratory and invading potential of HCC cells through Rho signal transduction pathway.

Overexpression of ribosomal protein L15 is associated with cell proliferation in gastric cancer.

BACKGROUND: Ribosomal proteins are the components of ribosome, which also exhibit various secondary functions in DNA repair, apoptosis, drug resistance and proliferation. In our previous study of microarray, ribosomal protein L15 (RPL15) was identified as an upregulated gene in gastric cancer. METHODS: We investigated the expression of ribosomal protein L15 in gastric cancer and the effect of RPL15 on proliferation of gastric cancer. RESULTS: It was found that the expression of RPL15 was markedly up-regulated in gastric cancer tissues. RPL15 was also highly expressed in gastric cancer cell lines AGS, MKN45, MKN28, SGC7901 and KATOIII. Inhibition of RPL15 expression by siRNA vector transfection suppressed the growth of SGC7901 cells significantly, which was independent of the expression of Cyclin D1 and B1. Down-regulation of RPL15 expression inhibited SGC7901 cell growth in soft agar and its tumorigenicity in nude mice. CONCLUSION: RPL15 promotes cell proliferation and may be a potential target for anticancer therapy of gastric cancer.

[Treatment of rabbits bearing advanced VX2 tumors in the mammary gland with nano-sized liposomal adriamycin administered by various routes].

OBJECTIVE: To investigate the therapeutic effect of nano-sized liposomal adriamycin (NLADR) administered by various routes on rabbits bearing advanced breast tumors. METHODS: NLADR with a mean diameter of 120 nm was prepared by pH gradient-driven drug encapsulation method. VX(2) tumor mass suspension was injected into the breast tissues of 50 female New Zealand White rabbits. Six weeks after the inoculation 38 surviving tumor-bearing rabbits were randomly divided into 5 groups: group A (control group), receiving a sham treatment; group B, receiving subcutaneous injection of NLADR with a dose of 1 mg/kg into the areas adjacent to the implanted tumor; group C, receiving intravenous injection of NLADR with a dose of 1 mg/kg; group D, receiving NLADR with a dose of 1 mg/kg administered by subcutaneous injection combined with intravenous injection with half of the whole dose for both routes; and group E, receiving intravenous injection of free adriamycin (FADR). The treatment was repeated every 48 hours. The rabbits were killed 48 hours after the third treatment. The breast tumors, axillary lymph nodes, and all of the metastatic tumors anatomically detected in distant organs were collected. The sizes of tumors and axillary nodes before and after treatment were measured. Necrosis of tumor tissue was assessed pathologically. The mRNA expression of proliferating cell nuclear antigen (PCNA) was determined using RT-PCR. Apoptosis was identified and quantified as apoptosis index (AI) using TUNEL method. RESULTS: The average growth rate of tumor was the highest in group A (1.58) and the lowest in group C (1.33). The average growth rate of axillary lymph nodes was the highest in group A (3.70), significantly higher than in any other groups (all P = 0.00); and was the lowest in group B, significantly lower than groups A, C, and E (all P < 0.01), however without a significant difference between groups B and D (P = 0.148). The PCNA mRNA expression level of the implanted tumor in groups C was the lowest, significant lowest then those in group A and B (both P < 0.01). The sequence of PCNA mRNA in the axillary lymph nodes was group B < group D < group C < group E < group A with significant differences between group B and groups A, C, and E. The sequence of PCNA mRNA in the mediastinal lymph nodes was group B < Group E < group D < group C < group A. The PCNA mRNA expression level was the lowest in group D, significantly lower than that in group A (P = 0.011). Necrosis of implanted tumor, metastatic foci in lung and liver, and lymph nodes was obvious in groups C, D, and E. Necrosis of implanted tumor and metastatic foci in lung and liver was significantly obvious in group C than in group D (P = 0.000 and P = 0.022). Necrosis of the implanted tumor was significantly more obvious in group C than in group F (P = 0.033). Necrosis of axillary lymph nodes was significantly more obvious in group than in groups C and E (P = 0.000 and P = 0.000). The values of AI of the implanted tumor in groups C and D were 16.74% and 18.04%, both significantly higher than those in group E (P = 0.02 and P = 0.04), and those of group E were significantly higher than those in groups A and B (both P < 0.01). The AI value of the axillary lymph nodes was 21.73%, significantly than those in groups A, C, and E (all P < 0.01). The average AI values of the metastatic foci were 16.52%, 15.77%, and 14.50%, all significantly higher than that in group B (all P < 0.01), and that of group B was significantly higher than that in group A (P = 0.039). CONCLUSION: NLADR, especially intravenous administration combined with subcutaneous administration, is effective in treatment of advanced breast carcinoma with lymphatic and distant metastases.

Expression of MUC1 and its significance in hepatocellular and cholangiocarcinoma tissue.

AIM: To investigate the relation between MUC1 expression, distribution, and prognosis in hepatocellular and cholangiocarcinoma (HCC and CC) and cirrhotic liver tissues, and their significance in HCC and CC diagnosis. METHODS: Expression and distribution of MUC1 were examined by immunohistochemical assay with anti-MUC1 mAb in 59 samples of HCC and 37 samples of CC, 20 samples of cirrhotic liver tissues, and 10 samples of normal liver tissues, seeking possible associations between MUC1 positive expression, distribution in HCC and CC (primary liver cancer, PLC) cases and the studied clinical data. RESULTS: Immunohistochemical analysis of MUC1 expression showed that in the 96 PLC samples, 68 (70.8%) were strong positive, and 6 (6.2%) were weak positive. Only 4 in the 20 cirrhotic liver tissues were found to be weak positive, while no expression of MUC1 was detected in normal liver tissues. Apparently, the high expression rate of MUC1 in PLC tissues was statistically significant in comparison to that in cirrhotic and normal liver tissues. The expressed MUC1 protein, stained in dark brownish or brownish-yellow particles, chiefly localized on the cancer cell membranes or in cytoplasm. In the 68 strong positive samples, 40 were detected on cell membrane and the other 28 were in cytoplasm. In addition, follow-up studies of those PLC cases demonstrated that MUC1 expression on cell membrane or in cytoplasm was closely associated with PLC prognosis. The expression of MUC1 in PLC had little statistical significance in respect of the pathological types and sizes of the tumors, but a strong relationship regarding histological differentiation, metastasis of lymph nodes, portal canal emboli, and post-operational recurrence of the carcinomas. After 3 years of tumor excision, the metastasis rate in MUC1 positive expression group (67.6%) was much higher than that in MUC1 weak expression group (33.3%) and negative expression group (31.8%), and thus the survival rate in MUC1-positive expression group was significantly different from that in weak and negative expression groups. CONCLUSION: Expression and localization of MUC1 proteins in primary liver carcinomas (PLCs) may act as prognostic markers, and MUC1 molecules might be helpful in differential diagnosis.

Overexpression of Bax induces apoptosis and enhances drug sensitivity of hepatocellular cancer-9204 cells.

AIM: To investigate the role of overexpression of Bax in apoptotic pathways and the response of human hepatocellular cancer (HCC)-9204 cells to cell death induced by adriamycin. METHODS: The whole length of Bax cDNA was transfected into human HCC-9204 cells by the method of lipofectamine transfection. An inducible MT-II regulatory system was constructed, which allowed controlled expression of protein upon addition of ZnSO4 (100 micromol/L) as an external inducer. Stable transfecting inducible expression vector containing Bax gene was performed. Expression of Bax in protein was analyzed by immunohistochemistry and Western blotting. TUNEL and flow cytometry were used to assess the effect of Bax on apoptosis. Colony assay and tetrazolium blue (MTT) assay were used to evaluate the difference in drug sensitivity of HCC-9204 cells after Bax-transfection. RESULTS: Immunohistochemistry and Western blotting demonstrated that the expression of Bax protein markedly increased in Bax-transfected cells 4 h after the addition of ZnSO4. Bax positive signal was frequently found on the cytoplasm and perinuclear region of HCC-9404 cells, and there was ectopic expression in cells with marked condensation of chromatin and cytoplasm (apoptotic cells). Apoptotic index significantly increased in Bax-transfected HCC-9204/Bax cells (3.6 vs 27.2, 4.2 vs 32.3, P<0.05). Flow cytometry analysis showed a significant sub-G1 peak and apoptosis in 15.4% HCC-9204/Bax cells 24 h after treatment. Furthermore, colony survival rate decreased from 66% (HCC-9204/pMD) to 45% (HCC-9204/Bax) 2 d after ADR withdrawal. MTT assay result showed that the effects of Bax on cell viability following ADR exposure were significant as compared to the vehicle-transfected HCC-9204/pMD cells (21% vs 44%, P<0.01). CONCLUSION: Overexpression of Bax not only induces apoptosis, but also sensitizes HCC-9204 cells to cell death induced by adriamycin.

[Expression and purification of rat betacellulin with biological activity].

AIM: To obtain recombinant rat betacellulin with biological activity. METHODS: A 534 bp of rat betacellulin gene fragment was amplified by RT-PCR from rat kidney and cloned into pET28a(+) vector to construct recombinant plasmid pET28a-rBTC. The recombinant plasmid was transformed into E. coli BL-21(DE3) and the betacellulin was expressed under IPTG induction. The expressed betacellulin was detected by SDS-PAGE and Western blot. The expressed protein was purified by Ni(2+) affinity chromatography and then renatured by dialysis. The effect of the renatured protein on proliferation of NIH3T3 cells was detected by MTT colorimetry. RESULTS: Rat betacellulin protein with M(r) being 20 000 was expressed under IPTG induction. The purity of purified protein reached over 96%. After renaturation, the expressed protein could significantly stimulate the proliferation of NIH3T3 cells. CONCLUSION: Rat betacellulin gene is successfully cloned into the expression vector pET28a(+) and highly expressed in E.coli. Purified and refolded betacellulin protein can obviously stimulate the proliferation of NIH3T3 cells.

[Drug concentration in axillary lymph nodes of patients with breast cancer after lymphatic chemotherapy].

BACKGROUND & OBJECTIVE: Lymph node status is one of the decisive prognostic factors of breast cancer. Chemotherapy targeting regional lymphatic tissues has emerged as a promising therapy for malignancies with high tendency to disseminate lymphatically. This study was to detect drug concentration in axillary lymph nodes of patients with breast cancer after lymphatic chemotherapy (LC), and to investigate effect of LC on accumulation of antitumor drugs in regional lymph nodes through comparing with the effect of intravenous chemotherapy (VC). METHODS: Sixty patients with breast cancer, confirmed by preoperative puncture biopsy, were randomized into 2 groups, 30 (LC group) were subcutaneously injected with 4 ml of carboplatin-activated carbon suspension (containing 20 mg of carboplatin) around the primary tumor, the other 30 (VC group) were intravenously injected with an equal dose of aqueous carboplatin. Every 6 patients from each group received modified radical mastectomy 1, 12, 24, 36, or 48 h after injection. Axillary lymph nodes were removed for pathologic examination. The concentration of carboplatin in nodes was detected by Zeeman atomic absorption spectrometry. RESULTS: A total of 275 axillary lymph nodes were resected, with 154 in LC group and 121 in VC group. Of the 275 lymph nodes, 136(49.5%) were from 23 patients (38.3%) had pathologically detected metastases. The concentrations of carboplatin were significantly higher in LC group than in VC group 1, 12, 24, 36, and 48 h after injection [(11.82+/-3.50) microg/g vs. (0.06+/-0.02) microg/g, (23.58+/-7.34) microg/g vs. (0.11+/-0.05) microg/g, (18.22+/-4.93) microg/g vs. (0.10+/-0.02) microg/g, (16.70+/-5.15) microg/g vs. (0.05+/-0.02) microg/g, and (14.62+/-4.29) microg/g vs. 0, respectively, P < 0.001]. Lymph node metastasis had no correlation with drug concentration (P > 0.05). CONCLUSION: Compared with VC, LC can effectively and continuously improve drug concentration in axillary lymph nodes of patients with breast cancer.

Effect of p27(KIP1) on cell cycle and apoptosis in gastric cancer cells.

AIM: To elucidate the effect of p27(KIP1) on cell cycle and apoptosis regulation in gastric carcinoma cells. METHODS: The whole length of p27(KIP1) cDNA was transfected into human gastric cancer cell line SCG7901 by lipofectamine. Expression of p27(KIP1) protein or mRNA was analyzed by Western blot and RNA dot blotting, respectively. Effect of p27(KIP1) on cell growth was observed by MTT assay and anchorage-independent growth in soft agar. Tumorigenicity in nude mice was used to assess the in vivo biological effect of p27(KIP1). Flow cytometry, TUNEL, and electron microscopy were used to assess the effect of p27(KIP1) on cell cycle and apoptosis. RESULTS: Expression of p27(KIP1) protein or mRNA increased evidently in SCG7901 cells transfected with p27(KIP1). The cell growth was reduced by 31% at 48 h after induction with zinc determined by cell viability assay. The alteration of cell malignant phenotype was evidently indicated by the loss of anchorage-independent growth ability in soft agar. The tumorigenicity in nude mice was reduced evidently (0.55+/-0.14 cm vs 1.36+/-0.13 cm, P<0.01). p27(KIP1) overexpression caused cell arrest with 36% increase (from 33.7% to 69.3%, P<0.01) in G1 population. Prolonged p27(KIP1) expression induced apoptotic cell death reflected by pre-G1 peak in the histogram of FACS, which was also confirmed by TUNEL assay and electron microscopy. CONCLUSION: p27(KIP1) can prolong cell cycle in G1 phase and lead to apoptosis. p27(KIP1) may be a good candidate for cancer gene therapy.

[Expression of human telomerase reverse transcriptase gene in HCC does not depend on proliferating cell nuclear antigen and P53].

AIM: To investigate the expression of human telomerase reverse transcriptase(hTERT) gene and its relationship with proliferating cell nuclear antigen and P53. METHODS: Immunohistochemical staining was used to detect the expressions of hTERT, PCNA and P53 in 42 hepatocellular carcinoma(HCC) tissues. The relationship between hTERT expression, pathological characters of HCC and PCNA and P53 expression was analyzed. RESULTS: The expression rates of hTERT, PCNA and P53 in HCC tissues were 71.4%(30/42),76.2%(32/42) and 73.8%(31/42), respectively. hTERT was not expressed in normal liver tissues. The expression rates of hTERT gene in HCC tissues of different pathological grades (I, II and III)were 40.0%(4/10), 70.0%(14/20) and 100%(12/12), respectively. The expression of hTERT was correlated with HCC recurrence. CONCLUSION: The expression of hTERT gene may relate to the genesis and progression of HCC. There is no significant correlation between the expression of hTERT and PCNA and P53. The detection of hTERT gene expression may be regarded as a marker for recurrence of HCC.

[Enhanced inhibitory effect of MUC1 gene vaccine on breast cancer growth by GM-CSF].

AIM: To investigate if GM-CSF adjuvant could enhance the inhibitory effect of the MUC1 gene vaccine on EMT6 breast cancer growth. METHODS: Female BALB/c mice were immunized intramuscularly with 100 microgram MUC1 gene vaccine 3 times at 3 week intervals. On 1, 3 and 5 days after intramuscular immunization, GM-CSF 100 microL(1 microg/100 microL) was given s.c. respectively. Three weeks after the last immunization, tumor challenge experiments were performed by using MUC1 expressing tumor cell line EMT6. Tumor growth inhibition was observed two weeks later. After 43 d of challenge experiments, all mice were killed and tumors were weighted. CTL-specific cytotoxicity was detected by (51)Cr release assay. RESULTS: After 43 d of challenge experiments, the size of EMT6 tumor in MUC1 DNA+GM-CSF, MUC1 DNA, pcDNA3.1(+)+GM-CSF and pcDNA3.1(+) group were (135+/-33.8) mm(3), (250+/-34.3) mm(3), (568+/-43.6) mm(3) and (596+/-48.2) mm(3), respectively. The average weight (g) of EMT6 tumors in those four groups were 0.81+/-0.42, 1.23+/-0.41, 2.30+/-0.48 and 2.28+/-0.58, respectively. EMT6 breast cancer growth in mice of MUC1 gene vaccine group was suppressed significantly(P<0.05), compared with that in pcDNA3.1(+) control group. Co-delivery of GM-CSF adjuvant and MUC1 DNA immunization enhanced the antitumour effects(P<0.05). The cytotoxicity of MUC1-specific CTLs to EMT6 target cells was different at various ratios of effector cells to target cells. At ratios of 100:1, 50:1, 25:1 and 12.5:1, the specific lysis for MUC1 DNA+GM-CSF group reached 68.5%, 53.4%, 35.9% and 28.5%, MUC1 cDNA group 54.1%, 39.8%, 26.4% and 20.1%, while for control groups 13.2%, 10%, 8.2%, 7.2% and 11.7%, 9.8%, 7.7%, 7.0%, respectively. GM-CSF enhanced the cytotoxicity of CTL induced by MUC1 DNA immunization(P<0.05). CONCLUSION: GM-CSF adjuvant significantly enhanced the inhibitory effect of the MUC1 gene vaccine on EMT6 breast cancer growth.

Protective effects of tumor necrosis factor alpha antibody and ulinastatin on liver ischemic reperfusion in rats.

AIM: To study the protective effects of tumor necrosis factor alpha (TNF alpha) antibody and ulinastatin on liver ischemic reperfusion in rats. METHODS: One hundred and twenty male SD rats were randomly divided into four groups: normal control group, ischemic group, TNF alpha antibody group and TNF alpha antibody + ulinastatin group. The animals were killed at 0, 3, 6, 9, 12 h after ischemia for 60 min and followed by reperfusion. Serum alanine aminotransferase (ALT), malondialdehyde (MDA) and liver histopathology were observed. RESULTS: After ischemic reperfusion, the serum ALT and MDA were remarkably increased, and the hepatic congestion was obvious. Treatment of TNF alpha antibody and ulinastatin could significantly decrease serum ALT and MDA levels, and relieve hepatic congestion. CONCLUSION: Ulinastatin and TNF alpha antibody can suppress the inflammatory reaction induced by hepatic ischemic reperfusion, and have protective effects on rat hepatic ischemic reperfusion injury.

Tumor necrosis factor alpha antibody prevents brain damage of rats with acute necrotizing pancreatitis.

AIM: To study the protective effects of tumor necrosis factor alpha (TNFalpha) antibody on pancreatic encephalopathy in rats. METHODS: One hundred and twenty SD rats were randomly divided into normal control group, acute necrotizing pancreatitis group and TNFalpha antibody treated group. Acute hemorrhage necrotizing pancreatitis model in rats was induced by retrograde injection of 50 g/L sodium taurocholate into the pancreatobiliary duct. Serum TNFalpha was detected and animals were killed 12 h after drug administration. Changes in content of brain water, MDA and SOD as well as leucocyte adhesion of brain microvessels were measured. RESULTS: In TNFalpha antibody treated group, serum TNFalpha level was decreased. Content of brain water, MDA and SOD as well as leucocyte adhesion were decreased significantly in comparison with those of acute necrotizing pancreatitis group (P<0.05). CONCLUSION: TNFalpha antibody can alleviate the brain damage of rats with acute hemorrhage necrotizing pancreatitis.

Relationship between urokinase-type plasminogen activator receptor and vascular endothelial growth factor expression and metastasis of gallbladder cancer.

AIM: To investigate the relationship of urokinase type plasminogen activator receptor (uPAR) and vascular endothelial growth factor (VEGF) expression with clinical and pathological characteristics of human gallbladder cancer. METHODS: uPAR and VEGF expressions in 68 gallbladder cancer tissues were detected with anti-receptor immunohistochemical stain. RESULTS: Expression rate of uPAR was 57.4% (39/68), and VEGF 51.5% (35/68) in gallbladder cancer tissues. Expression of both uPAR and VEGF was significantly related to metastasis, but not significantly correlated with differentiation stage and size of gallbladder cancer. CONCLUSION: Expression of uPAR and VEGF may be an invasive phenotype of gallbladder cancer and indicator for predicting prognoses, and uPAR expression is significantly correlated with the expression of VEGF.

Enhancement of migration and invasion of hepatoma cells via a Rho GTPase signaling pathway.

AIM: Intrahepatic extension is the main cause of liver failure and death in hepatocellular carcinoma patients. The small GTPase Rho and one of its effector molecules ROCK regulate cytoskeleton and actomyosin contractility, and play a crucial role in cell adhesion and motility. We investigated the role of small GTPase Rho in biological behaviors of hepatocellular carcinoma to demonstrate the importance of Rho in cancer invasion and metastasis. METHODS: Using Western blotting, we quantitated Rho protein expression in SMMC-7721 cells induced by Lysophosphatidic acid (LPA). Furthermore, we examined the role of Rho signaling in regulating the motile and invasive properties of tumor cells. RESULTS: Rho protein expression was stimulated by LPA. Using the Rhotekin binding assay to assess Rho activation, we observed that the level of GTP-bound Rho was elevated transiently after the addition of LPA, and Y-27632 decreased the level of active Rho. LPA enhanced the motility of tumor cells and facilitated their invasion. Rho played an essential role in the migratory process, as evidenced by the inhibition of migration and motility of cancer cells by a specific inhibitor of ROCK, Y-27632. CONCLUSION: The finding that invasiveness of hepatocellular carcinoma is facilitated by the Rho/Rho-kinase pathway is likely to be relevant to tumor progression and Y-27632 may be a new potential effective agent for the prevention of intrahepatic extension of human liver cancer.

[Apoptosis of human hepatoma HepG2 cells induced by emodin in vitro].

BACKGROUND & OBJECTIVE: Emodin (3-methyl-1,6,8-trihydro- xyanthrax-quinone) is the main effective composition of some Chinese herbs. Previous studies showed that emodin could inhibit the proliferation of some kind of tumor cells, such as breast cancer and lung cancer, while the mechanism(s) by which emodin suppresses tumor growth remains unknown. The study was designed to investigate the inhibitory effects and mechanisms of emodin-induced cell death in human hepatoma cell HepG2. METHODS: MTT assay was used to evaluate the IC(50) of emodin on HepG2 cells. Through soft agar assay, the ability of cell proliferation when exposed to emodin at various dosages was detected. DNA fragmentation (ladder) and flow cytometry analysis were applied to investigate the effects and mechanisms of emodin on HepG2 cells. RESULTS: Emodin could inhibit the growth of HepG2 cells significantly with IC(50) of 36+/-2.6 microg/ml; and could inhibit the colony formation of the cells in soft agar. After treatment of emodin,extraction of cancer cells exhibited typical DNA fragmentation, and flow cytometry analysis showed apoptosis in a dosage- dependent manner. As the concentration of emodin raised from 10 microg/ml to 20 microg/ml,the ratio of apoptotic cells increased from 27.3% to 59.6%. Under the concentration of 40 microg/ml, there were almost no living cells detected. CONCLUSION: Emodin may inhibit the growth and proliferation of HepG2 cells through the way of apoptosis introduction.

Hepatocellular apoptosis after hepatectomy in obstructive jaundice in rats.

AIM: To investigate the hepatocellular apoptosis after hepatectomy in obstructive jaundice and biliary decompression rats. METHODS: After bile duct ligation for 7 days, rats were randomly divided into OB group in which the rats underwent 70% hepatectomy, OB-CD group in which the rats underwent hepatectomy accompanied by choledochoduodenostomy, CD-Hx group in which the rats underwent choledochoduodenostomy and then received 70% hepatectomy on the fifth day after biliary decompression. The control group (Hx group) only underwent hepatectomy. RESULTS: The level of total serum bilirubin and serum enzymes was significantly lower in CD-Hx group than in OB-CD and OB groups on day 1, 3 and 5 after hepatectomy. The apoptotic index was significantly lower in CD-Hx group than in OB-CD and OB groups on day 3 and 5. The oligonucleosomal DNA fragments and Caspase-3 activity were also lower in CD-Hx group than in OB-CD and OB groups 3 days after hepatectomy, without differences between CD-Hx and Hx groups. CONCLUSION: Hepatocellular apoptosis plays vital roles in jaundice rats, and biliary decompression is more effective in treatment of patients with severe jaundice before operation.

Influence of liver nonparenchymal cell infusion combined with cyclosporin A on rejection of rat small bowel transplantation.

AIM: To investigate the effect of liver nonparenchymal cell infusion combined with cyclosporin A (CsA) on rejection of heterostrain rat small bowel transplantation. METHODS: The liver nonparenchymal cell suspension was prepared by density gradient centrifugation method with Percoll centrifugal solution. Heterotopic small bowel transplantation was performed. Then the rats were divided into four groups. Group one: homogenic transplantation (F344/N-F344/N), group two: allotransplantation (F344/N-Wistar), group three: allotransplantation (F344/N-Wistar) + CsA, with CsA 10 mg/kg(-1)/d(-1) after transplantation, group four: allotransplantation + CsA (F344/N-Wistar) + liver nonparenchymal cell infusion + CsA (F344/N-Wistar), in which recipient Wistar rats had been injected with 2x10(8) F344/N liver nonparenchymal cells 20 days before transplantation, and treated with CsA after transplantation. Finally, the survival time after small bowel transplantation, gross and histopathological examination, and IL-2 levels in serum were observed. RESULTS: The survival time after small bowel transplantation was 7.14 +/- 0.33 d, 16.32 +/- 0.41 d and 31.41 +/- 0.74 d in group 2, 3, and 4, respectively. The survival time was significant longer (P<0.01) in group 4. The gross and histopathological examination showed that the rejection degree in group 4 was lower than those in groups 2 and 3. Serum IL-2 level in group 4 was also lower than those in groups 2 and 3 (P<0.01). CONCLUSION: Liver nonparenchymal cell infusion combined with CsA can prolong the survival time of rat small bowel transplantation, and the anti-rejection effect is good.

Expression of PCNA and CD44mRNA in colorectal cancer with venous invasion and its relationship to liver metastasis.

AIM: To investigate the expression of proliferating cell nuclear antigen (PCNA) and CD44mRNA in colorectal cancer with venous invasion and its relationship with liver metastasis. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of PCNA and CD44mRNA in 31 cases of colorectal cancer with venous invasion. RESULTS: Positive expression rates of PCNA and CD44mRNA in colorectal cancer were higher than those without liver metastasis (P<0.05 and P<0.01). In case of colorectal cancer with liver metastasis, strongly positive rates of PCNA and CD44mRNA were 94.1% and 70.6%, respectively, significantly higher than those without liver metastasis. There was a positive relationship between the expressions of PCNA and CD44mRNA (r=0.67, P<0.05). CONCLUSION: Detection of PCNA and CD44mRNA expression in colorectal cancer may be useful for evaluating liver metastasis of cancer cells.