Tumor repressor gene chondroadherin oppose migration and proliferation in hepatocellular carcinoma and predicts a good survival.

The molecular that used as prognosis and potential therapy target is urgently needed in hepatocellular carcinoma (HCC). In current work, we found the expression of CHAD (chondroadherin) was significantly reduced in hepatocellular carcinoma compared to the normal tissue, on both mRNA and protein levels, in three independent datasets. Survival analysis was implemented on these datasets, and low expression of CHAD was found to be significantly associated with poor survival. Furthermore, metastasis-averse HCC and metastasis-incline HCC group comparison, and protein abundance evaluation of normal-tumor-portal vein tumor thrombus pairs indicate that metastatic tendentiousness is reduced along with CHAD abundance. Correlation analysis was also carried out and CHAD was shown to be significantly associated with differentiation and metastasis. Multivariable cox regression analysis showed that CHAD expression is more important for prognosis, compared to the other clinical indicators. To facilitate the utilization of CHAD clinically, a nomogram was plotted to estimate the three-year survival rate. Functional assays testing the migration and proliferation ability following knock down of CHAD in two cell lines, SMMC7721 and HCCLM3, were performed and discovered that reduction of CHAD level significantly enhance both proliferation and migration in both cell lines. Gene Set Enrichment Analysis (GSEA) comparing the CHAD-low and CHAD-high group showed that KEGG signaling pathways including "focal adhesion", "ECM receptor interaction", and "regulation of actin cytoskeleton" were significantly enriched. In conclusion, as a potential prognostic biomarker, tumor suppressor gene CHAD represses migration and proliferation of hepatocellular carcinoma cells, probability via mediating cell-cell adhesion.

Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation, Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells.

Treatments for triple-negative breast cancer (TNBC) are limited; intermediate-conductance calcium-activated potassium (SK4) channels are closely involved in tumor progression, but little is known about these channels in TNBC. We aimed to investigate whether SK4 channels affect TNBC. First, by immunohistochemistry (IHC) and western blotting (WB), increased SK4 protein expression in breast tumor tissues was detected relative to that in non-tumor breast tissues, but there was no apparent expression difference between various subtypes of breast cancer (p>0.05). Next, functional SK4 channels were detected in the TNBC cell line MDA-MB-231 using WB, real-time PCR, immunofluorescence and patch-clamp recording. By employing SK4 specific siRNAs and blockers, including TRAM-34 and clotrimazole, in combination with an MTT assay, a colony-formation assay, flow cytometry and a cell motility assay, we found that the suppression of SK4 channels significantly inhibited cell proliferation and migration and promoted apoptosis in MDA-MB-231 cells (p<0.05). Further investigation revealed that treatment with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) caused MDA-MB-231 cells to undergo the epithelial-mesenchymal transition (EMT) and to show increased SK4 mRNA expression. In addition, the down-regulation of SK4 expression inhibited the EMT markers Vimentin and Snail1. Collectively, our findings suggest that SK4 channels are expressed in TNBC and are involved in the proliferation, apoptosis, migration and EMT processes of TNBC cells.

Effects of BRAF(V600E) mutation on Na(+)/I(-) symporter expression in papillary thyroid carcinoma.

Radioiodine ablation (RIA) therapy is one of the most important treatments for papillary thyroid carcinoma (PTC), but some patients who received (131)I have radioiodine-refractory disease caused by the decreased expression of the Na(+)/I(-) symporter (NIS). BRAF(V600E) mutation is one possible risk factor that can disturb the NIS expression, but the roles are unclear in clinical practice. This research discussed the association of BRAF(V600E) mutation and NIS expression in PTC tissue and the clinical implications in RIA therapy. 134 PTC samples were collected between June 2013 and June 2014 from Tongji Hospital affiliated to Tongji Medical College, and their clinical characteristics were analyzed. RT-PCR was used to detect the BRAF(V600E) mutation from formalin-fixed paraffin-embedded samples, and immunohistochemistry was applied to detect the NIS expression. IPP software was used to calculate the relative expression quantity of NIS. We found that there was no significant correlation between the absorbance (A) values of NIS and clinicopathologic features in these cases, even thyroid stimulating hormone. BRAF(V600E) mutation showed inhibitory effect on the NIS expression without statistically significant difference in all PTC cases (ß=-0.0195, P=0.085), but in the subgroup without hashimoto's thyroiditis (HT), BRAF(V600E) mutation could significantly inhibit the NIS expression (ß=-0.0257, P=0.046). The results indicate that BRAF(V600E) mutation is correlated with a lower expression of NIS in PTCs without HT, suggesting the radioiodine-refractory effects during RIA therapy in these patients.

miR-153 inhibits epithelial-to-mesenchymal transition in hepatocellular carcinoma by targeting Snail.

Epithelial-to-mesenchymal transition (EMT) has been implicated as a dynamic cellular process in embryonic development and invasion of human cancers. Snail1 is a critical convergence hub in EMT regulation which transcriptionally represses E-cadherin expression. Currently, published data indicate that upregulation of Snail is mainly due to transcriptional activation and regulation of protein stability and cellular location. However, whether there is an alternative regulatory mechanism remains unclear. Our study showed that the expression of miR-153 was noticeably downregulated in hepatocellular carcinoma (HCC) cell lines and tissues, compared with normal liver epithelial cells (NLCs) and matched adjacent normal HCC tissues. Ectopic expression of miR-153 inhibited the migration and invasion ability of HCC cells, while suppression of miR-153 rescued this inhibitory effect. In addition, upregulation of miR-153 in HCC cells resulted in a decrease in epithelial markers, E-cadherin and α-catenin, and an increase in mesenchymal markers, N-cadherin and vimentin, and vice versa. Moreover, we demonstrated that miR-153 downregulated Snail expression by directly targeting the 3'-untranslated region (3'UTR) of Snail. Taken together, our results suggest that miR-153 plays a critical role in suppressing EMT and HCC progression by direct suppression of Snail expression.

Inhibitory effects of blockage of intermediate conductance Ca(2+)-activated K (+) channels on proliferation of hepatocellular carcinoma cells.

The roles of intermediate conductance Ca(2+)-activated K(+) channel (IKCa1) in the pathogenesis of hepatocellular carcinoma (HCC) were investigated. Immunohistochemistry and Western blotting were used to detect the expression of IKCa1 protein in 50 HCC and 20 para-carcinoma tissue samples. Real-time PCR was used to detect the transcription level of IKCa1 mRNA in 13 HCC and 11 para-carcinoma tissue samples. The MTT assay was used to measure the function of IKCa1 in human HCC cell line HepG2 in vitro. TRAM-34, a specific blocker of IKCa1, was used to intervene with the function of IKCa1. As compared with para-carcinoma tissue, an over-expression of IKCa1 protein was detected in HCC tissue samples (P<0.05). The mRNA expression level of IKCa1 in HCC tissues was 2.17 times higher than that in para-carcinoma tissues. The proliferation of HepG2 cells was suppressed by TRAM-34 (0.5, 1.0, 2.0 and 4.0 µmol/L) in vitro (P<0.05). Our results suggested that IKCa1 may play a role in the proliferation of human HCC, and IKCa1 blockers may represent a potential therapeutic strategy for HCC.

Endothelial progenitor cells homing to the orthotopic implanted liver tumor of nude mice.

This study investigated the "homing" phenomenon in hepatocellular carcinoma (HCC). The "homing" specificity of endothelial progenitor cells (EPC) by establishing an orthotopic implantation model in nude mice. EPCs harvested from the marrow cells were separated by density gradient centrifugation. Fluorescence microscope, flow cytometry (FCM) and double fluorescence staining with FITC-UEA-I and DiI-ac-LDL, were employed to identify the cells. 4',6-diamidino-2-phenylindole (DAPI) labelling and real-time PCR were used for detecting the expression of CD133 and chemokines to trace and observe the distribution of EPCs. Our results showed that the distribution rate of EPCs was obviously higher than that in other important organs and the negative control group. Detection of CD133 and chemokines yielded similar results in difference tissues. Our experiment confirmed that the chemotaxis of EPCs does exist in HCC. Moreover, HIF-1α, SDF-1 and VEGF might play important roles in the "homing" of EPCs in HCC. EPCs might be a potential candidate for targeting vector of HCC for gene therapy.

[Effect of protecting parathyroid in situ in the operation of total thyroidectomy].

OBJECTIVE: To evaluate the effect of protecting parathyroid glands in situ in the operation of total thyroidectomy by detecting parathyroid hormone after the operation. METHODS: In the surgical team, 1019 consecutive patients with thyroid diseases were treated with total thyroidectomy. During the operation, parathyroid glands were protected in situ with correctly identifying the parathyroid glands, precisely dissecting its envelope and protecting its blood supply. Serum calcium level and parathyroid hormone were measured before and 24 hours after operation. The patients who had symptomatic hypocalcemia or hypoparathyroidism were given supportive treatment and followed-up. RESULTS: At least one of the parathyroid glands was preserved and remained in situ in all cases. Eighty-nine cases (8.7%) had decreased parathyroid hormone levels and 42 cases (4.1%) had complicated symptomatic hypocalcemia. The symptoms of hypocalcemia in all these cases could be controlled by supportive treatment, and serum calcium level and parathyroid hormone had all recovered 1 - 6 months later. If 3 and 4 parathyroid were conserved in situ, the postoperative complication rate was significantly lower than those with 1 and 2 parathyroid conserved (decreased PTH 69/999 vs 20/20, symptoms of hypocalcemia 25/999 vs 17/20, all P < 0.01). CONCLUSION: The techniques to protect parathyroid glands in situ are effective measure to prevent the postoperative hypoparathyroidism in total thyroidectomy.

Isolation of cultured endothelial progenitor cells in vitro from PBMCs and CD133(+) enriched cells.

Two isolation methods for sorting of endothelial progenitor cells (EPCs): from peripheral blood mononuclear cells (PBMCs) and CD133(+) enriched cells were compared, by defining the cell morphology, phenotype, reproductive activities and function in vitro, to provide a reference for clinical application of EPCs. PBMCs from healthy subjects were used either directly for cell culture or for CD133(+) sorting. The two groups of cells were cultured in complete medium 199 (M199) for 7 to 14 days and the phenotypes of EPCs were analyzed by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and the VEGF concentration was measured using an ELISA kit. ECM gel experiment and migration assay were performed in vivo. The results showed that PBMCs produced more colony-forming units (CFU) than CD133(+) enriched cells from the same volume of blood (P<0.01). From day 7 to 14, the two groups showed decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144(+) cells in CD133(+) group were less than in PBMCs group (P<0.01). PBMCs group secreted more VEGF than CD133(+) group on the day 7 (P<0.01). As compared with CD133(+) group, PBMCs group had more potent potential of proliferation and vascularization in vitro. It was concluded that CD133(+) sorted cells showed a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, suggesting that CD133-negative cells may be a preferential way to get EPCs for clinical therapy.

Increased DJ-1 and its prognostic significance in hepatocellular carcinoma.

BACKGROUND/AIMS: To investigate the expression profile of DJ-1 gene and its clinical relevance and prognostic value in hepatocellular carcinoma (HCC). METHODOLOGY: Specimens from 149 HCC patients were applied for DJ-1 expression through immunohistochemistry. The correlation of DJ-1 levels with clinicopathologic variables and prognosis was analyzed. 32 paired HCC and para-carcinomatous liver tissue (PCLT) specimens from 149 HCC patients plus 10 hepatic cirrhosis specimens and 10 normal liver specimens were detected by semi-quantitative polymerase chain reaction (PCR) and Western blot. RESULTS: DJ-1 was up-regulated significantly in HCC by semi-quantitative PCR and Western blot. DJ-1 expression closely correlated with preoperative AFP, liver cirrhosis, vein invasion, differentiation and Edmondson grade in HCCs by Pearson Chi-square test. Both of tumor-free survival time and overall survival time in the DJ-1 high expression group were shorter than those in the low expression group. DJ-1 was adopted as an independent prognostic factor for overall survival of HCC patients through multivariate Cox proportional hazard model analysis (HR, 2.568; p = 0.003). Additionally, immunohistochemistry analysis revealed that expression of DJ-1 negatively correlated with expression of tumor suppressor gene phosphatase and tensin homolog deleted on chromosome ten (PTEN) in HCC (r = -0.836; p < 0.001). CONCLUSIONS: DJ-1 expression is significantly upregulated in HCC, and its expression level correlates with clinicopathological variables and prognosis of HCC patients, which suggests that DJ-1 maybe a candidate prognostic biomarker of HCC.

A novel immunotherapy to hepatocellular carcinoma: CD40-activated B lymphocytes transfected with AFPmRNA.

Alpha-fetoprotein (AFP) is overexpressed in the majority of hepatocellular carcinomas (HCCs), and thus may offer attractive target for immunotherapy against this neoplasm. CD40 ligand (CD40L) is the major signal that induces B cells to efficiently present antigen to T cells, and CD40-activated B (CD40-B) lymphocyte cells may boost cytotoxic T lymphocytes (CTLs) when they are pulsed with tumour antigens. CTL is considered to be a promising therapeutic means for the treatment of cancers. Here, we intend to build a plasmid pGEM4Z/AFP/A64 and to prepare AFPmRNA, then separate B lymphocyte cells. These CD40-B cells are pulsed with AFPmRNA, and they may boost robust T-cell responses, but more importantly, they also may prime naive T-cell responses against hepatocarcinoma. These CD40-B cells will be a powerful source of APCs generated by simple and reliable technology that may be applied to antigen responses, immune treatment for cancer, vaccination approaches, and ex vivo T-cell expansion for adoptive therapy. AFPmRNA-transfected B cells may represent a broadly applicable vaccine strategy to induce potentially therapeutic CTL responses against AFP-positive target cells in HCC. Vaccine strategies such as these may contribute to the effective future treatment of HCC.

[Conservative therapy in the treatment of cervical chylous leakage].

OBJECTIVE: To explore and evaluate the combined conservative managements in the treatment of cervical chylous leakage. METHODS: Thirty nine cases of cervical chylous leakage from June 1992 to June 2008 were retrospectively analyzed in this hospital. All of the 39 cases were cured by treating with conservative individualized therapy, including the applying of diet with high calorie, high protein and low fat and fatty food should only contains medium-chain triglycerides, total parenteral nutrition, keep the balance of hydrogen and electrolyte and correct hypoproteinemia, local pressure dressing, high persistent vacuum drainage (-50 approximately -80 kPa) and/or somatostatin analogue. RESULTS: All the cases of chylous leakage happened 2nd to 5th days after the operation. Among the 39 cases, 7 were high flow (drainage>or=500 ml/d) chylous leakage, the amount of drainage reached as high as 1440 ml per day. The time of chylous leakage closure was 3 approximately 12 days, and the mean time was 7 days. No one experienced re-operation, wound hydrops or wound infection. CONCLUSIONS: The conservative individualized therapy may play a key role in the treatment of cervical chylous leakage.

Ultrasound may be exploited for the treatment of microbial diseases.

In view of the global problems caused by drug-resistant bacteria and pathogenic viruses, current methods are not satisfying. It is urgent need for novel antimicrobial strategies. It has now been confirmed that illumination of the photosensitizers (PS) by light at an appropriate wavelength can lead to inactivation of the pathogen through the production of highly reactive free radical species, which induce oxidative damage to lipid, proteins and DNA/RNA; But for the limited tissue penetration of visible light and the problems of systemic light delivery, the use of light in treatment of microbial diseases is still in its infancy. Recently, it has been found that some PS also produces free radicals on irradiation with ultrasound whose mechanism is similar to Laser. Ultrasound can penetrate into tissue more deeply than light and can be focused into a small region to activate some PS. Due to the unique advantage of ultrasound and mechanism of producing free radicals similar to light, we hypothesized that ultrasound may be exploited for the treatment of bacterial and virus infectious diseases. We proposed a new concept of "sonodynamic antimicrobial chemotherapy (SACT)", and hypothesized that it may be a promising novel antimicrobial strategy.

Combination sonodynamic therapy with immunoadjuvant may be a promising new modality for cancer treatment.

Sonodynamic therapy (SDT) is a new cancer therapy basing on photodynamic therapy (PDT). Some chemicals produce free radicals on irradiation with laser (photosensitizers) or ultrasound (sonosensitizers). These active molecules destroy biological tissues, thus producing therapeutic effects. Although PDT has been adopted in clinical cancer therapy especially for superficial cancers, this modality is under continued investigation for improved efficacy and expanded use. For example, PDT-generated tumor cell lysates are effective cancer vaccines; treatment of PDT in conjunction with immunoadjuvant, called "PDT-immunoadjuvant therapy" (PIT), "photoimmunotherapy" or "laser immunotherapy", is considered to be a promising therapeutic interventions for the treatment of cancers. Ultrasound, especially focused ultrasound, can penetrate deeply into tissues and can be focused into a small region of a tumor to activate the cytotoxicity of sonosensitizers. This is a unique advantage in the non-invasive treatment of nonsuperficial tumors when compared to laser light used for PDT. For the similar mechanism of PDT and SDT, we hypothesize that SDT may be exploited for the generation of effective therapeutic cancer vaccines like PDT; and combination SDT with Immunoadjuvant may be a promising systemic treatment modality, not only for superficial cancers but also for deep-seated tumors, which would surpass PIT.

Effect of fragile histidine triad gene transduction on proliferation and apoptosis of human hepatocellular carcinoma cells.

AIM: To evaluate the inhibitory effects of human fragile histidine triad (FHIT) gene on cell proliferation and apoptosis in human hepatocellular carcinoma line Hep3B in vitro. METHODS: A recombinant pcDNA3.1 (+)/FHIT including the functional region of FHIT gene was constructed and transferred into human hepatocellular carcinoma cells in vitro. mRNA and protein expression of the FHIT gene in the transfected cells was detected by RT-PCR and Western blot, respectively. The effect of FHIT on proliferation was detected by MTT assay. Changes in cell cycle and apoptosis were assayed by flow cytometry. Five mice received subcutaneous transplantation of Hep3B-FHIT; 5 mice received subcutaneous transplantation of normal Hep3B and Hep3B-C as controls. The body weight of nude mice and tumor growth were measured. RESULTS: RT-PCR and Western blot analysis showed that the expression level of FHIT-mRNA and FHIT protein was higher in Hep3B cells after infection with pcDNA3.1 (+)/FHIT. The growth of Hep3B cells treated with pcDNA3.1 (+)/FHIT was significantly inhibited. The pcDNA3.1 (+)/FHIT-transfected Hep3B cells showed a significantly higher cell rate at G(0)-G(1) phase and increased apoptosis in comparison with controls (P < 0.05). The growth of transplanted tumor was inhibited markedly by FHIT. Tumors arising from the Hep3B-FHIT cells occurred much later than those arising from the Hep3B and Hep3B-C cells. The growth of Hep3B-FHIT cells was slow and the tumor volume was low. CONCLUSION: Transduction of FHIT gene inhibits the growth of human hepatocellular carcinoma cells and induces cell apoptosis in vivo and in vitro.

Replication-incompetent adenovirus vector-mediated MDA-7/IL-24 selectively induces growth suppression and apoptosis of hepatoma cell Line SMMC-7721.

In order to investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis of human hepatocellular carcinoma (HCC) cell line SMMC-7721 and normal liver cell line L02, the recombinant replication-incompetent Ad.mda-7 virus vector was constructed and infected into the HCC cell line SMMC-7721 and normal liver cell line L02. RT-PCR was performed to examine the expression of MDA-7 mRNA. The concentrations of MDA-7/IL-4 in culture supernatants were determined by using ELISA. MTT and Hoechst staining assay were applied to observe the inhibitory and killing effects of MDA-7 on the HCC cells. By using flow cytometry, the apoptosis, cell cycle and proliferation of SMMC-7721 and L02 cells were measured. The results showed recombinant replication-incompetent virus expressing MDA-7/IL-24 was constructed successfully, and RT-PCR revealed that it could mediate the high expression of the exogenous gene MDA-7/IL-24 in SMMC-7721 and L02 cells. The expression of MDA-7/IL-24 proteins in the culture supernatant was detectable by ELISA. Ad.mda-7 infection induced apoptosis and growth suppression in SMMC-7721 cells and an increased percentage of HCC cells in the G2/M phase of the cell cycle, but not in L02 cells. It was concluded that mda-7/IL-24 gene, mediated with replication-incompetent adenovirus vector, could selectively induce growth suppression and apoptosis in HCC cell line SMMC-7721 but without any toxic side-effect on normal liver line L02.

Clinicopathologic significance of BAG1 and TIMP3 expression in colon carcinoma.

AIM: To explore the expression of BAG1 and tissue inhibitor of metalloproteinase 3 (TIMP3) in colon carcinoma and their correlation and clinicopathologic significance. METHODS: SABC immunohistochemistry was used to detect the expression of BAG1 and TIMP3 in 80 colon carcinoma tissues and 20 normal colonic mucosa. RESULTS: Positive rate of BAG1 in colon carcinoma tissue (80%) was notably higher compared to normal colonic mucosa (10%) (P < 0.05). However, no significant difference was observed in positive rate of TIMP3 in colon carcinoma tissue (43.75%) as compared with normal colonic mucosa (60%) (P > 0.05). Expression of BAG1 and TIMP3 was strongly associated with colon carcinoma differentiation, Duke's staging, lymph node metastasis and survival rate (P < 0.05), but not associated with gender and age. Moreover, BAG1 expression was not correlated with TIMP3. CONCLUSION: Our results suggest that over-expression of BAG1 or attenuated expression of TIMP3 may play an important role in genesis and development of colon carcinoma. The protein expression levels of BAG1 and TIMP3 are related to the malignant degree, infiltration and metastasis of colon carcinoma. BAG1 and TIMP3 might be new biological parameters in predicting invasion and metastasis of colon carcinoma.

Relationship between the changes of VEGF level and dendritic cells in peripheral blood of patients with hepatocellular carcinoma after transcatheter arterial chemoembolization.

In order to investigate the relationship between the VEGF level and the counts of dendritic cells (DCs) in peripheral blood of patients with hepatocellular carcinoma (HCC) before and after transcatheter arterial chemoembolization (TACE), the peripheral blood was obtained from 37 patients with HCC who treated by TACE. The blood was obtained on the day before TACE, the first day, the 7th day and the 15th day after TACE respectively. The counts of DCs were quantified by flow cytometry. The plasma VEGF level was measured by ELESA kit. It was shown after TACE, the counts of DCs in peripheral blood were decreased significantly (P<0.05), and the VEGF level in peripheral blood was increased significantly (P<0.05). The counts of DCs in peripheral blood had an inverse correlation with the plasma VEGF level (r=-0.57, P<0.05) after TACE. It was concluded that in patients with HCC after TACE, the increased plasma VEGF level appeared to have the effect to suppress the maturation of DCs, which may contribute to reduction of the body's anti-tumor immunity effect, with a consequence of recur and metastasis of tumor.

[Optimized expression of a single-chain Fv antibody against human asialoglycoprotein receptor and determination of its affinity constant].

AIM: To express and purify a single chain Fv antibody (scFv) C1 against human hepatic asialoglycoprotein receptor (ASGPR) and to determine affinity constant of the purified scFv C1. METHODS: The specific anti-ASGPR phage clone C1 was transfected into E. coli HB2151. The single colony was chosen to be inoculated into 2 x TY medium and shaken (250 r/min) overnight at 37 degrees C. After 1 in 100 dilution in 2 x TY medium and induced for secreted expression, scFv C1 was induced at different concentrations (0.25, 0.5 and 1.0 mmol/L IPTG) overnight at 37 degrees C, 25 degrees C and 20 degrees C, respectively. The supernatant was precipitated with saturated ammonium sulfate and its sediment was analyzed by SDS-PAGE. In addition, the sediment was resuspended in 30 mL PBS and dialyzed against PBS overnight at 4 degrees C. The expressed scFv C1 was purified by Ni(2+) chelating HiTrap HP column and the purity of the purified scFv C1 was identified by SDS-PAGE. Then affinity constant of scFv C1 was determined by noncompetitive enzyme immunoassay. RESULTS: After induced with 0.5 mmol/L IPTG overnight at 25 degrees C, the amount of expressed scFv C1 increased greatly and its relative molecular mass was about 28,000, and it existed in culture supernant in soluble form. The purity of scFv C1 by nickel-agarose column was above 95% and its yield was about 0.8 mg/L. The affinity constant of the purified scFv C1 was confirmed to be (2.31+/-0.36)x10(-7) mol/L. CONCLUSION: The E. coli HB2151 infected with phage C1 clones may express soluble scFv C1 with low affinity, which has potential applications to gene therapy of hepatocellular carcinoma.

Melanoma differentiation-associated gene-7, MDA-7/IL-24, selectively induces growth suppression, apoptosis in human hepatocellular carcinoma cell line HepG2 by replication-incompetent adenovirus vector.

AIM: To investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis in human hepatocellular carcinoma (HCC) cell line HepG2 and normal liver cell line L02. METHODS: We constructed the recombinant replication-incompetent Ad.mda-7 virus vector and infected it into the human HCC cell line HepG2 and normal liver cell line L02. RT-PCR was performed to detect the mRNA expressing in cells. by ELISA was used to detect MDA-7/IL-24 protein expression in the culture supernatant. The effect of apoptosis induced by Ad.mda-7 was confirmed by Hoechst staining and flow cytometry assay with Annexin-V and PI staining. MTT assay was used to determine growth inhibition of HepG2 cells, and cell-cycle and hypodiploidy analyses were performed by flow cytometry. RESULTS: Recombinant replication-defective virus expressing MDA-7/IL-24 was constructed successfully. RT-PCR showed that the Ad.mda-7 could mediate the expression of the exogenous gene MDA-7/IL-24 into HepG2 and L02. The concentration of MDA-7/IL-24 protein in supernatant was 130 pg/mL and 110 pg/mL in Ad.mda-7-infected L02 and HepG2 cells, respectively. Ad.mda-7 infection obviously induced apoptosis (from 2.60%+/-0.72% to 33.6%+/-13.2%, P=0.00012) and growth suppression in HepG2 (inhibition ratio IR=68%) and an increase in the percentage of specific cancer cell types at the G2/M phase of the cell cycle (from 6.44% to 32.29%, P<0.01), but not in L02 cells. CONCLUSION: These results confirm selectively induction of apoptosis and growth suppression by the mda-7/IL-24 gene with replication-incompetent adenovirus vector in human hepatocellular carcinoma cell line HepG2.

Relationship between PTEN and VEGF expression and clinicopathological characteristics in HCC.

To investigate the expressions and significance of the tumor suppressor gene phosphatase and tensin homolog deleted on chromosome ten protein (PTEN) and vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC), and to analyze the relationship between their expressions and the tumor's invasion and their peri-carcinomatous tissues, the correlation of their expressions with the tumor's clinicopathological characteristics and invasion potential were studied. Our study showed that the expression level of PTEN in HCC was remarkably lower than that in peri-carcinomatous liver tissues, while the expressions of both VEGF and MVD were higher than that in peri-carcinomatous liver tissues. Correlation analysis revealed that the expression of PTEN was negatively related to the progression of the pathological differentiation and invasion of tumor, whereas the expressions of VEGF and MVD were positively related. Moreover, there was a negative relationship between the expression of PTEN and the expressions of VEGF and MVD, and a positive one between VEGF and MVD. The expressions of PTEN and VEGF may reveal the degree of differentiation and the invasive potential of HCC tissues. The mechanism by which the lack of PTEN expression probably induces abnormal hyperexpression of VEGF may play an important role in the invasion and metastasis of HCC.