Mechanisms and in vitro effects of cepharanthine hydrochloride: Classification analysis of the drug-induced differentially-expressed genes of human nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is the most commonly diagnosed head and neck malignancy and is prevalent worldwide. Previous studies have demonstrated the antitumor properties of cepharanthine hydrochloride (CH) in several human cancer cells. However, the action of CH in NPC cells has yet to be determined. In the present study, we investigated the effects of CH in human NPC cell lines including CNE-1 and CNE-2 on cell growth and apoptosis in vitro. Using MTT and ATP-tumor chemosensitivity assays it was found that CH inhibited cell viability. Additionally, flow cytometric and analysis electron microscopy revealed the inhibition of cell cycle progression and reduction of apoptosis, respectively, in human NPC cell lines including CNE-1 and CNE-2 in vitro. To identify the potential action mechanisms of CH, the cDNA microarray analysis results were confirmed by quantitative PCR analysis using a number of genes, including CDKN1A/P21, NR4A1/TR3 and DAXX. In total, 138 upregulated and 63 downregulated genes in CNE-2 cells were treated with CH. According to their biological function, the genes were classified as: i) cell cycle-related genes; ii) DNA repair­related genes; iii) apoptosis-related genes and iv) nuclear factor-κB (NF-κB) transcription factors signal pathways. The results of the present study showed that CH is a potential therapeutic agent against human NPC, and provide rational explanations and a scientific basis for the study of the development of CH in the treatment of NPC.

[The effect of smoking and smoking cessation on the phosphorylation of IKK-ß in type 2 diabetic rats].

OBJECTIVE: To investigate the effect of smoking and smoking cessation on the phosphorylation of IKK-ß in type 2 diabetic rats. METHODS: Forty-two six-week-old Wistar rats were randomly divided into 4 groups: normal control (NC, n = 7), diabetes control (DC, n = 7), diabetes with smoking (DS, n = 14) and diabetes with smoking cessation (SC, n = 14). Rats in DS and SC groups were further assigned randomly into 8w and 12w subgroups. DS group was given passive smoking twice a day for 8 or 12 weeks, while SC group ceased passive smoking for 4 weeks after 8 or 12 weeks of smoking. Western blot method was used to detect the level of IKK-ß phosphorylation in skeletal muscle. RESULTS: Compared with the NC group, the phosphorylation of IKK-ß protein in DC group was increased (0.16 ± 0.05 vs 0.30 ± 0.08, P < 0.01). There was an increasing trend with the phosphorylation level of IKK-ß in the DS (8w) subgroup, but there was no statistical difference between the DC group and SC (8w) subgroup (0.40 ± 0.09 vs 0.30 ± 0.08, 0.36 ± 0.10, P > 0.05). The phosphorylation level of IKK-ß in DS (12w) group increased obviously, being significantly higher than that in the DC group and SC (12w) subgroup (0.74 ± 0.11 vs 0.30 ± 0.08, 0.35 ± 0.07, P < 0.01). CONCLUSION: With the prolongation of smoking duration, the phosphorylation of IKK-ß in type 2 diabetic rats increased. After smoking cessation, the phosphorylation of IKK-ß decreased. The phosphorylation of IKK-ß may be involved in the mechanism by which smoking causes type 2 diabetes.

[The biological function of peroxiredoxin II on Hep3B cells and its underlying mechanism].

OBJECTIVES: To study the biological function and its possible underlying mechanism of peroxiredoxin II (PrxII) in liver cancer cell line Hep3B. METHODS: Two pairs of double-stranded small interfering RNA (siRNA) targeted on PrxII gene were transfected into Hep3B cells using LipofectamineTM 2000. After confirming the inhibited effects of these siRNAs through Quant SYBR Green polymerase chain reaction and Western blot, the biological characters of Hep3B cell were analyzed by flow cytometry analysis, MTT and colony formation assays. Furthermore, dichlorodihydrofluorescein diacetate (DCFH-DA) and thiobarbituric acid (TBA) assays, for measuring the products of oxidative reaction, such as the reactive oxygen species (ROS) and malondialdehyde (MDA), were applied to explore whether the antioxidant mechanism was involved in the effects of PrxII functioning on Hep3B cell. RESULTS: The two pairs of siRNA significantly inhibited PrxII mRNA and protein expression. Compared to the mock and blank control groups, the two PrxII-silent groups showed decreased rates of cell growth and clone formation and increased rates of cell apoptosis. The numbers of the formed colonies were 42.0+/-2.8 and 40.5+/-0.7 respectively in the two PrxII-silent groups, while they were 121.5+/-2.1 and 130.0+/-1.4 in the mock and blank control groups (P less than 0.05). The levels of endogenous ROS and MDA were significantly higher in the two PrxII-silent groups than those in the mock and blank control groups (P less than 0.05). CONCLUSION: PrxII might play an important role in the hepatocarcinogenesis, possibly through an antioxidant function which may provide a favorable microenvironment for cancer cell survival and progression.

Proteome analysis of aflatoxin B1-induced hepatocarcinogenesis in tree shrew (Tupaia belangeri chinensis) and functional identification of candidate protein peroxiredoxin II.

In order to explore the proteins responsible for hepatocellular carcinoma (HCC), aflatoxin B(1)-induced hepatocarcinogenesis in tree shrew (Tupaia belangeri chinensis) was analyzed with 2-DE and MS. By comparing HCC samples with their own precancerous biopsies and HCC-surrounding tissues, a group of candidate proteins that differentially expressed in HCC were obtained. Peroxiredoxin (Prx) II, one of the candidates with distinct alteration, was further investigated and validated. Western blot and RT-PCR assays confirmed the overexpression of Prx II in both tree shrew and human HCC tissues. RNA interference for silencing Prx II was employed subsequently to explore the function and underlying mechanism of Prx II on liver cancer cell line Hep3B. Results showed the cell proliferation and clone formation decreased obviously when Prx II expression was inhibited, while the flow cytometer analysis showed the percentage of cell apoptosis enhanced. Inhibition of Prx II expression also obviously increased the generation of ROS and malondialdehyde, both are the products from peroxidation. These results imply the important role of Prx II in hepatocarcinogenesis, possibly through its function in regulating peroxidation and hereby to provide a favorable microenvironment for cancer cell surviving and progressing.

Candidate genes responsible for human hepatocellular carcinoma identified from differentially expressed genes in hepatocarcinogenesis of the tree shrew (Tupaia belangeri chinesis).

AIM: To explore gene expression profiles during hepatocarcinogenesis of the tree shrew, and to find the genes responsible for human hepatocellular carcinoma (HCC). METHODS: Tree shrews were used as an animal model for HCC induction employing aflatoxin B(1) (AFB(1)) alone or AFB(1) plus hepatitis B virus (HBV) as etiological factors. Gene expression profiles from the tissues of HCC, HCC-surrounding liver tissues (para-HCC) and the corresponding biopsies taken from the same animals before HCC had developed (pre-HCC) were analyzed by cDNA microarray assay to identify differentially expressed genes. Two genes, CuZn-superoxide dismutase (SOD1) and glutathione S-transferase A1 (GSTA1), were further investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical (IHC) assays were done on tree shrew and human HCC samples. RESULTS: RESULTS from the cDNA microarray analysis indicated that the gene expression profiles of HCC between AFB(1)and AFB(1) + HBV treatment groups were markedly different. A total of 11 genes, including SOD1 and GSTA1, were found changing in expression levels in all detected samples from both groups. RESULTS from RT-PCR and IHC assays indicated that mRNA and protein levels of SOD1 and GSTA1 were markedly downregulated in both tree shrew and human HCC, and downregulation of SOD1 and GSTA1 proteins in human HCC samples was closely correlated with the histopathological grading (P < 0.05). CONCLUSION: The differentially expressed genes found in all HCC cases induced by different etiological factors among different species should be considered as good candidate genes responsible for HCC. Downregulation of SOD1 and GSTA1 might play an important role in hepatocarcinogenesis.

[The expression of peroxiredoxin II in hepatocellular carcinoma and its significance].

OBJECTIVE: To evaluate the mRNA and protein expressions of peroxiredoxin II (PrxII) in hepatocellular carcinoma (HCC) and their significance. METHODS: HCC was induced by aflatoxin B1 (AFB1) in 6 tree shrews (Tupaia belangeri chinensis). The expression levels of PrxII mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot on HCC tissues and on their surrounding liver tissues (para-HCC). Biopsied liver tissues were taken before the HCC induction (pre-HCC) from the same animals and from a group of blank controlled animals that served as controls. Liver biopsy specimens from 18 cases of human HCC and from 17 healthy human volunteers were studied using the same methods. RESULTS: The mRNA and protein expressions of PrxII in tree shrew HCC tissues were significantly higher than those in para-HCC and pre-HCC tissues, and also higher than those in the liver tissues from the control animals (all P < 0.05). The expression levels of PrxII mRNA and protein in human HCC tissues were also significantly higher than those in their para-HCC tissues and in the human normal liver tissues (P < 0.05). CONCLUSION: PrxII might play an important role in hepatocarcinogenesis and might be used as a molecular target for HCC prevention and treatment.

Dynamic expression of apoptosis-related genes during development of laboratory hepatocellular carcinoma and its relation to apoptosis.

AIM: To explore the expression of p53, bcl-2, bax, survivin and the cell apoptosis during the development of tree shrew hepatocellular carcinoma (HCC), the relationship between expression of these genes, its impact on HCC development, and its relation to cell apoptosis. METHODS: Tree shrew HCC was induced with aflatoxin B1 (AFB1), and regular biopsy of liver tissues was carried out and the biopsy tissues were collected during cancer inducement. Liver biopsy tissue and HCC tissue were collected from 35 pre-cancerous experimental animals at wk 30 and 60 and at the 30th-, 60th-, and 90th-wk. Liver biopsy tissues were collected from 13 blank control animals at wk 30, 60, and 90. Expression of p53, bcl-2, bax, and survivin at each stage was examined by immunohistochemistry method. Apoptotic cells were detected in situ by the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) technique. RESULTS: The apoptosis rate of normal hepatic cells was extremely low, whereas it increased during the formation of HCC. Expression of the apoptosis-related genes p53, bcl-2, bax, and survivin during the formation of HCC presented an increasing tendency. Expression of p53 did not noticeably relate to that of bcl-2, bax, and survivin, whereas expression of bcl-2 and bax was closely related. In HCC, p53 did not present a distinct relation to cell apoptosis, whereas its high level expression was probably related to liver cell proliferation. Survivin negatively correlated apoptosis index, and its overexpression could inhibit cell apoptosis. CONCLUSION: Apoptosis-related genes p53, bcl-2, bax, and survivin are all related to the occurrence of HCC. The anti-apoptosis effect of bcl-2 is influenced by bax, and ratio bcl/bax reflects more correctly the extent of cell apoptosis.

Differential expression of genes during aflatoxin B(1)-induced hepatocarcinogenesis in tree shrews.

AIM: Through exploring the regulation of gene expression during hepatocarcinogenesis induced by aflatoxin B(1) (AFB(1)), to find out the responsible genes for hepatocellular carcinoma (HCC) and to further understand the underlying molecular mechanism. METHODS: Tree shrews (Tupaia belangeri chinensis) were treated with or without AFB(1) for about 90 weeks. Liver biopsies were performed regularly during the animal experiment. Eight shares of total RNA were respectively isolated from 2 HCC tissues, 2 HCC-surrounding non-cancerous liver tissues, 2 biopsied tissues at the early stage (30th week) of the experiment from the same animals as above, 1 mixed sample of three liver tissues biopsied at the beginning (0th week) of the experiment, and another 1 mixed sample of two liver tissues from the untreated control animals biopsied at the 90th week of the experiment. The samples were then tested with the method of Atlas(TM) cDNA microarray assay. The levels of gene expression in these tissues taken at different time points during hepatocarcinogenesis were compared. RESULTS: The profiles of differently expressed genes were quite different in different ways of comparison. At the same period of hepatocarcinogenesis, the genes in the same function group usually had the same tendency for up- or down-regulation. Among the checked 588 genes that were known to be related to human cancer, 89 genes (15.1%) were recognized as "important genes" because they showed frequent changes in different ways of comparison. The differentially expressed genes during hepatocarcinogenesis could be classified into four categories: genes up-regulated in HCC tissue, genes with similar expressing levels in both HCC and HCC-surrounding liver tissues which were higher than that in the tissues prior to the development of HCC, genes down-regulated in HCC tissue, and genes up-regulated prior to the development of HCC but down-regulated after the development of HCC. CONCLUSION: A considerable number of genes could change their expressing levels both in HCC and in HCC-surrounding non-cancerous liver tissues. A few modular genes were up-regulated only in HCC but not in surrounding liver tissues, while some apoptosis-related genes were down-regulated in HCC and up-regulated in surrounding liver tissues. To compare gene-expressing levels among the liver tissues taken at different time points during hepatocarcinogenesis may be helpful to locate the responsible gene (s) and understand the mechanism for AFB(1) induced liver cancer.

[Differentially expressed genes in hepatocellular carcinoma of tree shrew induced by different factors].

BACKGROUND & OBJECTIVE: Previous studies on differentially expressed genes in hepatocellular carcinoma (HCC) used to perform with para-cancerous tissues as normal control. However, the para-cancerous tissue of HCC is actually abnormal because they frequently contain hepatitis, cirrhosis, hyperplastic nodules or foci, etc. In order to explore the molecular mechanism and the responsible genes for hepatocarcinogenesis, through applying the HCC model of tree shrew (Tupaia belangeri chinensis), this study was designed to compare gene expression levels between HCC induced by different factors and their corresponding biopsies taken before HCC formation. METHODS: Tree shrews were divided into two groups. Group AFB(1) was fed with aflatoxin B1 (AFB(1)). Group AFB(1)+HBV was infected firstly with human hepatitis B virus (HBV) and then fed with AFB(1) as group AFB(1). Serum tests for HBV markers and liver biopsies were performed periodically during the experiment. After appearance of HCC, 2 HCC samples from each group and their corresponding 30th-week biopsies were tested respectively by cDNA microarray assay. The gene expression levels were compared between each HCC and the corresponding biopsies, and the differentially expressed genes from the two groups of HCC induced by different factors were analyzed. RESULTS: The incidence rates of HCC in group AFB(1) and group AFB(1)+HBV were 73.3% and 77.8%, respectively. A considerable number of genes in both groups showed changes in their expression levels, which were mainly up-regulated in group AFB(1) but down-regulated in group AFB(1)+HBV. On the other hand, among the 588 checked genes (16 functional classifications) that were known related to human cancer, 11 genes were similarly expressed in all of the 4 HCC from the two animal groups. Most of these 11 genes belonged functionally to 3 types, namely "apoptosis-associated protein","DNA synthesis,repair and recombination proteins", and "growth factors, cytokines and chemokines". CONCLUSION: (1)HBV can affect AFB(1)-induced gene expression in certain extent. (2)The gene expression profiles of HCC induced by different factors are different. (3)The common differentially expressed genes in these two HCC groups are worthwhile for further study as the possibly responsible genes for hepatocarcinogenesis.