CLIC1-mediated autophagy confers resistance to DDP in gastric cancer.

Gastric cancer has been a constant concern to researchers as one of the most common malignant tumors worldwide. The treatment options for gastric cancer include surgery, chemotherapy and traditional Chinese medicine. Chemotherapy is an effective treatment for patients with advanced gastric cancer. Cisplatin (DDP) has been approved as a critical chemotherapy drug to treat various kinds of solid tumors. Although DDP is an effective chemotherapeutic agent, many patients develop drug resistance during treatment, which has become a severe problem in clinical chemotherapy. This study aims to investigate the mechanism of DDP resistance in gastric cancer. The results show that intracellular chloride channel 1 (CLIC1) expression was increased in AGS/DDP and MKN28/DDP, and as compared to the parental cells, autophagy was activated. In addition, the sensitivity of gastric cancer cells to DDP was decreased compared to the control group, and autophagy increased after overexpression of CLIC1. On the contrary, gastric cancer cells were more sensitive to cisplatin after transfection of CLIC1siRNA or treatment with autophagy inhibitors. These experiments suggest that CLIC1 could alter the sensitivity of gastric cancer cells to DDP by activating autophagy. Overall, the results of this study recommend a novel mechanism of DDP resistance in gastric cancer.

[Research on compatibility of prescriptions including Ginseng Radix et Rhizoma and Trogopterus Dung based on complex network analysis].

The applications of prescriptions including Ginseng Radix et Rhizoma and Trogopterus Dung in contemporary literatures from 1949 to 2016 are compiled and the data mining techniques containing scale-free complex network method are utilized to explore its practical characteristics, with comparison between modern and ancient ones. The results indicate that malignant neoplasms, coronary heart disease which present Qi deficiency and blood stasis type are the main diseases treated by prescriptions including Ginseng Radix et Rhizoma and Trogopterus Dung according to the reports during 1949 to 2016. The complex network connection shows that Glycyrrhizae Radixet Rhizoma, Angelicae Sinensis Radix, Astragali Radix, Typhae Pollen, Salviae Miltiorrhizae Radix et Rhizoma are the primary drugs related to Ginseng Radix et Rhizoma and Trogopterus Dung. The next are Paeoniae Radix Alba, Atractylodis Macrocephalae Rhizoma, Persicae Semen, Foria, et al. Carthami Flos, Notoginseng Radix et Rhizoma, Cyperi Rhizoma, Bupleuri Radix are the peripheral ones. Also, Ginseng Radix et Rhizoma-Glycyrrhizae Radixet Rhizoma, Trogopterus Dung-Glycyrrhizae Radixet Rhizoma, Ginseng Radix et Rhizoma-Angelicae Sinensis Radix, Trogopterus Dung-Angelicae Sinensis Radix, Ginseng Radix et Rhizoma-Astragali Radix, Trogopterus Dung-Astragali Radix are the main paired drugs. The paired drugs including Ginseng Radix et Rhizoma-Trogopterus Dung-Glycyrrhizae Radixet Rhizoma, Ginseng Radix et Rhizoma-Trogopterus Dung-Angelicae Sinensis Radix, Ginseng Radix et Rhizoma-Trogopterus Dung-Astragali Radix, Ginseng Radix et Rhizoma-Trogopterus Dung-Typhae Pollen have a higher support degree. The main compatible drugs are different in ancient and modern prescriptions including Ginseng Radix et Rhizoma and Trogopterus Dung. Notoginseng Radix et Rhizoma, Typhae Pollen, Salviae Miltiorrhizae Radix et Rhizoma, Astragali Radix are utilized frequently in modern prescriptions while less used in ancient ones. It is also shown that more attentions are paid to the drugs contributing to invigorating Qi and promoting blood circulation in modern times with comparative results between modern and ancient prescriptions.

[Expression of myeloid-related protein complex in association with circulating endothelial cells in children with acute Kawasaki disease].

OBJECTIVE: To investigate the expression of myeloid-related protein complex (MRP-8/14) in children with acute Kawasaki Disease (KD). METHODS: A total of 41 children with acute KD and 40 age- and sex-matched control children with upper respiratory tract infection were recruited. Serum levels of MRP-8/MRP-14 complex were measured by ELISA, messenger ribonucleic acid (mRNA) abundance of MRP-8 and MRP-14 in circulating granulocytes and monocytes was determined by RT-PCR, and the number of circulating endothelial cells was determined by flow cytometry. RESULTS: When the analysis was stratified according to the presence or absence of coronary artery ectasia in the KD patient group, serum levels of MRP-8/MRP-14 complex, MRP-8 and MRP-14 mRNA abundance in granulocytes, and the number of circulating endothelial cells were all significantly higher in KD patients with coronary artery ectasia than in KD patients without coronary artery ectasia (P<0.05). Serum levels of MRP-8/MRP-14 complex were positively correlated with the number of endothelial cells in the circulation (r=0.69, P<0.05). CONCLUSIONS: Serum levels of MRP-8/MRP-14 complex are elevated in a positive association with the number of circulating endothelial cells in KD children with coronary artery ectasia, suggesting a causative role in the development of coronary artery lesions.

RNAi silencing of c-Myc inhibits cell migration, invasion, and proliferation in HepG2 human hepatocellular carcinoma cell line: c-Myc silencing in hepatocellular carcinoma cell.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Although much is known about both the cellular changes that lead to HCC and the etiological agents responsible for the majority of HCC cases, the molecule pathogenesis of HCC is still not well understood. We aimed to determine the effect of c-Myc gene expression on the proliferative, invasive, and migrative capabilities of hepatocellular carcinoma HepG2 cells. METHODS: A plasmid- based polymerase III promoter system was used to deliver and express short interfering RNA targeting c-Myc to reduce its expression in HepG2 cells. Western blot analysis was used to measure the protein level of c-Myc in HepG2 cells. The effects of c-Myc silencing on the invasion, motility, and proliferation of HepG2 cells were assessed using a Transwell chamber cell migration assay system and a growth curve assay, respectively. RESULTS: The data showed that plasmids expressing siRNA against c-Myc significantly decreased its expression in HepG2 cells by up to 85%. Importantly, pSilencer-c-Myc transfected cells showed a significantly reduced potential in migration, invasion, and proliferation. CONCLUSION: C-Myc plays an important role in the development of hepatocellular carcinoma. The data show that down-regulating the c-Myc protein level in HepG2 cells by RNAi could significantly inhibit migration, invasion and proliferation of HepG2 cells. Thus, c-Myc might be a potential therapeutic target for hepatocellular carcinoma.

Effect of combined treatment with recombinant interleukin-2 and allicin on pancreatic cancer.

This study aimed to evaluate the efficacy of combined treatment with recombinant interleukin-2 (rIL-2) and allicin on pancreatic cancer and explore the potential immunological mechanism. A total of 60 C57/BL6 nude mice pancreatic cancer xenograft models were randomized into four groups of 15 mice per group: control group, allicin treatment group, rIL-2 treatment group, combined treatment with allicin and rIL-2 group. Mice in each group were treated with saline, rIL-2, allicin, or combination of rIL-2 and allicin by weekly i.v injection for four weeks. After four weeks of treatment, eyeballs of the mice were extracted and blood was drawn, percentages of CD4+T, CD8+T and NK cell were analyzed by FACS, IFN-γ level was detected by ELISA. One mouse in each group was sacrificed to measure the weight and volume of the tumor and prepared to the paraffin section of tumor tissue. Apoptosis of the tumor cells was analyzed by TUNEL and FACS. Other mice continued to receive treatment, survival period were compared between each group. We observed a significant suppression of xenograft growth and a significant prolonged survival time in the combined treatment with allicin and rIL-2 group (P < 0.05). The most amount of apoptotic cells were observed in the combined therapy group (P < 0.05). The percentages of CD4+T, CD8+T and NK cell and serum IFN-γ level increased significantly in the combined treatment group compared with other groups (P < 0.05). Combined treatment with allicin and rIL-2 resulted in suppression of tumor growth and prolonged survival time possibly through activation of CD4+T, CD8+T and NK cell.

The expression and regulation of DFNA5 in human hepatocellular carcinoma DFNA5 in hepatocellular carcinoma.

Hepatocellular carcinoma is a primary malignancy of hepatocytes which accounts for 80 % of all primary liver cancers. DFNA5 has been identified as a tumor suppressor gene with an important role in several frequent forms of cancers, while little is known about its role in hepatocellular carcinoma. Through comparison of the DFNA5 protein expression in hepatocellular carcinoma cells (HepG2) with human fetal lung fibroblast cells (MRC5), we found that the DFNA5 protein expression in hepatocellular carcinoma cells was significantly lower than that in normal cells. The transfection of DFNA5 gene into HepG2 cells could increase DFNA5 protein expression, which subsequently led to inhibition of cell proliferation. Underlying mechanism study revealed that decreased proliferation was due to increased apoptosis and cell cycle arrest. In view of the important role of DFNA5 gene in carcinogenesis, these findings are expected to provide new understanding on development and treatment of human hepatocellular carcinoma.

Interferon-α enhances antitumor activities of oncolytic adenovirus-mediated IL-24 expression in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) has a dismal 5-year-survival rate of 10%, so novel strategies are warranted. IL-24 mediates anti-tumor activity reducing STAT3 expression, which suggests that interferon (IFN) alpha may augment tumor cell lysis and reduce angiogenesis. We investigated the antitumor activity of treatment with IFN-α, with the oncolytic adenovirus SG600-IL-24, or the combination of both in HCC in vitro and in vivo. RESULTS: RT-PCR, ELISA assay and Western-blot confirmed that the exogenous IL-24 gene was highly expressed in HCC cells infected with SG600-IL-24. Treatment with combined IFN-α and SG600-IL-24 suppressed growth and promoted apoptosis of the HepG2, MHCC97L, and HCCLM3 cell lines compared with the normal cell line L02. The combined therapy increased STAT1 and SOCS1 and apoptosis, but decreased the expression of the metastatic and angiogenic proteins MMP-2, XIAP, OPN, and VEGF, which are regulated by STAT3 in HCC cells in vitro. To assess the effects in vivo, the HCC cell line HCCLM3 was transplanted subcutaneously into the right flanks of nude mice. Mice in the IFN-α group, the SG600-IL-24 group, or the combined therapy group had significantly suppressed growth of the HCC xenografted tumors compared to the PBS control group of mice. Among the mice treated with the combination of IFN-α and SG600-IL-24, three of those eight mice had long-term survival and no evidence of a tumor. These mice also had decreased expression of the metastatic and angiogenic proteins MMP-2, XIAP, OPN, and VEGF. CONCLUSIONS: The present study demonstrated for the first time the potential antitumor activity of IFN-α combined with the oncolytic adenovirus SG600-IL-24 in HCC both in vitro and in vivo, and suggests its further development as a potential candidate for HCC cancer gene therapy.

Evaluation of in vivo antioxidant and immunity enhancing activities of sodium aescinate injection liquid.

Oxidative stress is involved in the development and progression of disease. Because sodium aescinate has been reported to have immunity enhancing and antioxidative effects, we investigated its activity by employing a hepatocellular carcinoma (HCC) mouse model. Sixty BALB/c mice were randomly divided into four groups, including a 1.4 mg/kg treated group (n = 15), a 2.8 mg/kg treated group (n = 15), an untreated hepatocellular carcinoma control group (n = 15) and a normal control group (n = 15). After H22 cells were cultured for one week, we collected 2 × 106 cells and injected them subcutaneously as 0.2 mL cell suspensions in sterile saline into the right shoulder region of every mouse. The animals were monitored for changes in activity, physical condition and body weight during the experiment. The next day after injection of H22 cells, animals in these test groups received one intraperitoneal injection of drug or physiological saline for 13 days. Results showed that in the sodium aescinate injection liquid (SAIL)-treated HCC mice, serum interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), Gamma-glutamyltransferase (γ-GT), alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) levels were significantly decreased compared with normal control mice. In addition, treatment with sodium aescinate injection liquid significantly decreased blood and liver malondialdehyde (MDA) levels, increased glutathione (GSH) levels, and antioxidant enzyme [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px)] activities in a dose-dependent manner. We conclude that sodium aescinate injection liquid can decrease oxidative injury and enhance immunity functions in HCC mice.

Ad.mda-7 (IL-24) selectively induces apoptosis in hepatocellular carcinoma cell lines, suppresses metastasis, and enhances the effect of doxorubicin on xenograft tumors.

Overexpression of the melanoma differentiation associated gene-7 (MDA-7)/IL-24 in vitro generally results in the growth suppression and induction of apoptosis of diverse human tumor cells. In this study, we investigated the effects of overexpression of the MDA-7/IL-24 gene in human hepatocellular carcinoma (HCC) cells in vitro and in vivo. Adenovirus-mediated overexpression of MDA-7 facilitated the MDA-7/IL-24-induced apoptosis and G2/M arrest in HCC cells, but not in the normal liver cell line L02, and the effect was independent of the p53 status. Inhibition of metastasis and angiogenesis was correlated with decreasing expression of STAT3, P-STAT3, MMP-2, VEGF, and TGF-beta genes, regulated by STAT3 in MHCCLM6 cells. We also showed that Ad.mda-7 combined with doxorubicin (ADM) had significantly enhanced antitumor and antimetastatic effects in vivo, accompanied by the downregulation of VEGF, MMP-2, and TGF-beta genes and the upregulation of E-cadherin genes. These data suggested that MDA-7/IL-24 induces its selective antitumor properties in HCC cells by promoting apoptosis independent of p53 status, inhibiting subcutaneous tumor growth and metastasis, and increasing the effect of chemotherapeutic agents. MDA-7/IL-24 represents a new class of cancer suppressor genes that may be useful in the targeted therapy of HCC.

Oncolytic adenovirus-mediated MDA-7/IL-24 overexpression enhances antitumor activity in hepatocellular carcinoma cell lines.

BACKGROUND: Melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24 (IL-24) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells. We investigated the effect of the replication-competent oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24, both expressing human MDA-7/IL-24 on the hepatocellular carcinoma cell lines HepG2, Hep3B, SMMC-7721, HCCLM3, and the normal liver cell line L02. METHODS: Hepatocellular carcinoma cell lines and the normal liver cell line were infected with SG600-IL24 and Ad.IL-24. The mRNA and protein expression of MDA-7/IL-24 in infected cells was confirmed by RT-PCR, ELISA, and Western blotting. MTT assay was used to investigate the proliferation effect. Hoechst staining and Annexin-V and PI staining were performed to study the MDA-7/IL-24 gene expressed in HCC cell lines and the normal liver cell line. Flow cytometry was used to analyse the cell cycle. RESULTS: RT-PCR, ELISA and Western blotting confirmed that the exogenous MDA-7/IL-24 gene was highly expressed in cells infected with SG600-IL24. MTT and apoptosis detection indicated that SG600-IL24 induced growth suppression, promoted apoptosis, and blocked cancer cell lines in the G2/M phase in hepatocellular carcinoma cell lines but not in the normal liver cell line. CONCLUSIONS: SG600-IL24 selectively induces growth suppression and apoptosis in hepatocellular carcinoma cell lines in vitro but not in the normal liver cell line L02. Compared with Ad.IL-24, SG600-IL24 dramatically enhances antitumor activity in hepatocellular carcinoma cell lines.

[Spatial distribution and interspecific associations of regenerating saplings in karst secondary forests.] / å–€æ–¯ç‰¹æ¬¡ç”Ÿæž—å¹¼æ ‘æ›´æ–°ç©ºé—´åˆ†å¸ƒæ ¼å±€åŠç§é—´å ³è”æ€§.

A 140 m×120 m plot was set in a secondary forest with more than 30 years natural reco-very after abandonment in Ziyun Miao and Buyi Autonomous County, a typical karst area in Guizhou Province. We investigated the spatial distribution and interspecific associations of regenerating sapling population using spatial point pattern analytical method. There were 1291 saplings with 39 tree species. Betula luminifera, Platycarya strobilacea, Liquidambar formosana, Pinus massoniana and Populus davidiana were the dominant populations of regenerating saplings, accounting for 83.7% of the saplings and 77.8% of the total importance value. The spatial distributions of B. luminifera, P. strobilacea and L. formosana were strongly aggregated at a spatial scale of 0-60 m, while the spatial distributions of P. massoniana and P. davidiana were aggregated at small scale and randomly distributed at large scale. The spatial associations among those dominant populations were mostly positively correlated, with positive correlations of P. massoniana with L. formosana and P. davidiana at small scale but no associations at large scale. In conclusion, the spatial distributions and interspecific associations differed among the dominant sapling populations, due to the different biological characteristics of different tree species, habitats and uses of spatial resources. Most of the stands investigated were dominated by pioneering species, with poor stand quality and unstable community structure. A mixed forest dominated by P. massoniana and B. luminifera would be the next stage of succession. We recommended that measures of forest management should be adopted to accelerate vegetation restoration.

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.

Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B.

BACKGROUND: Melanoma differentiation associated gene-7 (mda-7) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of various intracellular signaling pathways. In this study, we investigated the potential effect of mda-7 on human hepatocellular carcinoma (HCC) in vitro. METHODS: Cells from the human HCC cell line Hep3B and the human liver cell line L-02 were assigned to three groups. One was cultured in Dulbecco's modified Eagle's medium without serum (control). The others were transfected with adenovirus expressing the mda-7 gene (Ad.mda-7) or adenovirus vector serving as negative control (Ad.vec). The expression of MDA-7 and Bcl-2 proteins in Hep3B and L-02 cells was confirmed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The methyl thiazolyl tetrazolium colorimetric assay and flow cytometry were used to assess tumor cell proliferation and the cell cycle. Hoechst and Annexin-V/propidium iodide staining were used to study mda-7 gene expression in Hep3B and L-02 cells. The expression of MDA-7, Bcl-2 and Bax proteins were detected by Western blotting. RESULTS: The mda-7 gene was expressed in Hep3B and L-02 cells. The protein concentrations of MDA-7 in supernatants were 790 and 810 pg/ml, respectively. mda-7 induced Hep3B growth suppression and apoptosis, compared with Ad.mda-7 and control (P<0.01). In addition, cell block in G2/M was identified by exposure of HCC cells to secreted MDA-7 protein, but this was not found in L-02. The gene expression of Bcl-2 was markedly decreased in Hep3B but not in L-02. CONCLUSIONS: mda-7 selectively induces growth inhibition and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the anti-apoptosis protein Bcl-2. It could be an ideal gene for gene therapy in HCC.

[Mda-7-carrying replication-defective adenovirus selectively kills hepatocellular carcinoma cells by facilitating release of proapoptotic protein from mitochondria].

OBJECTIVE: To investigate the mechanism that mda-7/IL-24 selectively kills hepatocellular carcinoma (HCC) HepG2 cells in vitro. METHODS: HCC cell line HepG2 and normal liver cell line L02 were infected with Ad.mda-7. The expression of mda-7/IL-24 was detected by RT-PCR and ELISA, respectively. The apoptotic effects were confirmed by Hoechst staining and flow cytometry assay, respectively. Furthermore, Bcl-2 family proteins, cytochrome C, Smac/DIABLO and caspase-9 were determined by Western blot. RESULTS: The exogenous mda-7/IL-24 gene was expressed in HepG2 and L02 cells infected with Ad.mda-7. Ad.mda-7 induced apoptosis in HepG2 but not in L02 cells in vitro. The induction of tumor cell apoptosis is correlated with the increasing expression of Bax and decreasing expression of Bcl-2 and Bcl-xL genes, then facilitated the releasing of cytochrome C and Smac/DIABLO from mitochondria to cytoplasm and increasing the expression of caspase-9, eventually, resulted in apoptosis. CONCLUSION: Ad.mda-7 selectively induces growth inhibition and apoptosis in hepatocellular carcinoma HepG2 cells but not in normal L02 hepatocytes in vitro, and the mechanism might involve the decrease of Bcl-2 and Bcl-xL and increase of Bak expression, facilitating the release of cytochrome C and Smac/DIABLO from mitochondria in HCC cells.

[Effects of daunorubicin on the expression of brain natriuretic peptide gene in myocardial cells: an in vitro experiment with cultured myocardial cells from rats].

OBJECTIVE: To investigate the effects of daunorubicin (DNR) on the gene expression of brain natriuretic peptide (BNP) in myocardial cells. METHODS: Myocardial cells were isolated from neonatal Wistar rats, cultured, and added with DNR of the concentrations of 0, 0.02, 0.2, and 2 mg/L respectively so as to calculate the maximum non-toxic concentration of DNR. The cultured rat myocardial cells were divided into 3 groups, DNR group, added with DNR 0.2 mg/L, DNR+fructose-1, 6-diphosphate (FDP) group, added with DNR 0.2 mg/L and FDP 2 mg/ml, and control group. The viability of the myocardial cells was examined by MTT method. RT-PCR and Western blotting were used to detect the mRNA expression and protein expression of the BNP gene. RESULTS: Toxicity test showed that the maximum non-toxic concentration of DNR was 0.2 mg/L. Western blotting showed that the average grey level ratio of BNP/GAPDH of the DNR group was 0.40+/-0.09, not significantly different from that of the DNR+FDP group (0.42+/-0.11, but significantly lower than that of the control group (0.82+/-0.13, P<0.01). CONCLUSION: DNR suppresses the expression of BNP gene in myocardial cells and FDP cannot reverse the suppressing effect.

[Melanoma differentiation associated gene-7 and interleukin-24 selectively induces growth arrest and apoptosis in hepatocellular carcinoma cell lines in vitro].

OBJECTIVE: To investigate the effect of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) on the hepatocellular carcinoma cell lines and normal liver cell line in vitro. METHODS: Hepatocellular carcinoma cell lines HepG2, SMMC7721, Hep3B, MHCC97L, M6 and normal liver cell line L02 were infected with Ad.mda-7. The gene expression of mda-7/IL-24 in these cell lines was confirmed by RT-PCR and ELISA assay. MTT assay and flow cytometry were used to study tumor cell proliferation and cell cycle in vitro. Hoechst staining and cytometry assay after Annexin-V and PI staining were studied to indicate the apoptosis effect. RESULTS: It was confirmed by RT-PCR that the exogenous mda-7/IL-24 gene expressed in all of these cells. The mda-7/IL-24 protein product was confirmed by assaying the supernatant with ELISA. MTT and apoptosis test indicated mda-7/IL-24 can induce the hepatocellular carcinoma cell lines growth suppression, apoptosis in vitro but not in normal liver cell line L02, cell cycle test revealed mda-7/IL-24 can block cancer cell lines in G2/M but not in L02. CONCLUSIONS: mda-7/IL-24 selectively induces growth suppression, apoptosis in hepatocellular carcinoma lines but not in normal liver cell in vitro.

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.

[Adenovirus vector expressing MDA-7/IL-24 selectively induces growth arrests and apoptosis in human hepatocellular carcinoma cell lines independent of the state of p53 gene].

OBJECTIVE: To investigate the effect of melanoma differentiation associated gene-7/interleukin 24 (MDA/IL-24) on human hepatocellular carcinoma cell lines HepG2, MHCC97L and Hep3B and normal liver cell line L02 with a different p53 state. METHODS: The MDA-7/IL-24 gene was transfected into human hepatocellular carcinoma cell lines HepG2, MHCC97L and Hep3B and hepatocyte line L02 with a replication-incompetent adenovirus vector. The mRNA expression of MDA7/IL-24 in HepG2, MHCC97L, Hep3B and L02 cells was confirmed using RT-PCR. Protein expression was confirmed using ELISA assay. MTT assay and flow cytometry were used to study tumor cell proliferation and cell cycle in vitro. Hoechst and flow cytometry assay after annexin-V and PI staining were performed to indicate the apoptosis effect. RESULTS: Exogenous MDA-7/IL-24 gene was expressed in HepG2, MHCC97L, Hep3B and L02 cells. The protein product of MDA-7/IL-24 was confirmed in the supernatant. MTT assay and apoptosis test indicated MDA-7/IL-24 could induce growth suppression and apoptosis of HepG2, MHCC97L and Hep3B but could not in L02. Cell cycle test revealed MDA-7/IL-24 could block those cancer cells in G2/M but not in the normal cell L02. CONCLUSION: MDA-7/IL-24 selectively induces growth suppression and apoptosis in hepatocellular carcinoma lines HepG2, MHCC97L and Hep3B in vitro independent of the state of p53 gene but not in normal liver cell L02. This indicates MDA-7/IL-24 can be a perfect gene for gene therapy in hepatocellular carcinoma.

Identification of dysregulated pathways associated with pancreatic cancer by survival analysis.

In order to identify the dysregulated pathways associated with pancreatic cancer, the fourth leading cause of cancer mortality in the United States, tumor and non-tumor samples were systematically analyzed in the present study. Initially, dysregulated genes in pancreatic cancer were identified using paired t-test. Subsequently, dysregulated biological pathways involved in the development of pancreatic cancer were identified by enrichment analysis. Finally, individual survival analysis of the significantly dysregulated functions was conducted at the pathway level. Our results indicated that the pathway named ̔Pathways in cancer was significantly correlated with survival time. In addition, the mean survival time of individual and genetic variation demonstrated a significantly negative correlation, that is, the lower the genetic variation, the longer the survival time. Furthermore, detailed analysis of genes on the pathway named ̔Pathways in cancer denoted that this pathway involved multiple cancer hallmark signals and several dysregulated cancer genes, including tumor protein p53, myelocytomatosis, Kirsten rat sarcoma, phosphatidylinositol 3-kinase, v-raf murine sarcoma viral oncogene homolog B1 and cyclin-dependent kinase inhibitor 2A. According to the DrugBank database, certain oncogenes have been validated to be the targets of drugs, including Sorafenib, Trastuzumab, Imatinib and Paclitaxel or were under investigation. An improved understanding of the pathophysiology of pancreatic cancer has been achieved based on our results and the present study aimed to provide guidance for the development of drugs to treat pancreatic cancer.

Combined histological and hematological assessment of iron-induced organ damage in a gerbil model of iron overload.

BACKGROUND: Previous studies with gerbil models have suggested that excessive iron exposure causes cardiomyopathy and hepatic injury, but pathological analysis was not comprehensive, preventing a detailed understanding of how the metal induces this damage. METHODS AND RESULTS: Gerbils received single intraperitoneal injections of iron dextran (200 mg/kg) or saline and were then analyzed comprehensively for hematological and histological signs of organ damage. These tests included hematology parameters and determination of liver iron concentration, malondialdehyde levels and glutathione peroxidase activity; examination of heart and liver tissue stained with hematoxylin and eosin, Prussian-blue and Masson stain; and electron microscopy analysis of heart and liver ultrastructure. Iron-overloaded animals showed significantly different hematology parameters and significantly higher liver iron concentrations than saline-injected animals, as well as significantly higher malondialdehyde levels and significantly lower glutathione peroxidase activity. Histology analyses showed cellular damage, iron deposits, and both myocardial and liver fibrosis, while electron microscopy of heart and liver sections showed abundant iron deposition lysosomes, and disordered and swollen mitochondria. All these pathological changes increased with exposure time. CONCLUSIONS: This comprehensive assessment of iron overload in a gerbil model suggests that excessive iron deposition induces extensive cellular damage, particularly fibrosis in heart and liver. This damage may be the direct result of iron-mediated lipid peroxide damage and of iron deposition that cause compression of myocardial and liver cells, as well as vascular occlusion.