Metabolite modulation of HeLa cell response to ENOX2 inhibitors EGCG and phenoxodiol.

BACKGROUND: Constituents and inhibitors of intermediary metabolism resulting in alterations in levels of cytosolic NADH, stimulation of sphingomyelinase and inhibition of sphingosine kinase were evaluated for effects on growth inhibition and induction of apoptosis by the ENOX2 inhibitors EGCG, the principal catechin of green tea, and phenoxodiol, a naturally occurring isoflavone. METHODS: Responses were evaluated from dose-response curves of the metabolites and metabolic inhibitors in which growth of HeLa cells, apoptosis based on DAPI fluorescence and cytosolic NADH levels were correlated with sphingomyelinase and spingosine kinase activities and levels of ceramide and sphingosine1-phosphate. RESULTS: Growth inhibition correlated with the modulation of localized cytosolic NADH levels by metabolites and metabolic inhibitors, the response of sphingomyelinase and sphingosine kinase located near the inner surface of the plasma membrane, and apoptosis. CONCLUSIONS: Based on findings with metabolites, we conclude that apoptosis in cancer cell lines caused by ENOX2 inhibitors such as EGCG and phenoxodiol is a direct response to elevated levels of cytosolic NADH that result from ENOX2 inhibition. GENERAL SIGNIFICANCE: The findings help to explain why increased NADH levels resulting from ENOX2 inhibition result in decreased prosurvival sphingosine-1-phosphate and increased proapoptotic ceramide, both of which may be important to initiation of the ENOX2 inhibitor-induced apoptotic cascade.

Anti-hepatitis B virus X protein in sera is one of the markers of development of liver cirrhosis and liver cancer mediated by HBV.

Hepatitis B virus X protein (HBx) plays a crucial role in the development of hepatocellular carcinoma (HCC). However, the significance of circulating antibody to hepatitis B virus X antigen (anti-HBx) in sera remains unclear. In the present study, we examined the titers of anti-HBx (IgG) in the sera from 173 patients with chronic hepatitis B (CHB), 106 liver cirrhosis (LC), and 61 HCC by enzyme-linked immunosorbent assay (ELISA), respectively. Our data showed that the positive rates of anti-HBx were higher in sera of LC (40.6%) and HCC (34.4%) than those of CHB (10.4%), P < .05. In all 40 patients with anti-HBx+ out of 340 patients, 39 (97.5%) were HBsAg/HBeAg/anti-HBc+ and 1 (2.5%) was anti-HBs+ (P < .01), suggesting that anti-HBx in sera is a marker of HBV replication rather than a protective antibody. Thus, our findings reveal that circulating anti-HBx in sera is one of the markers of development of LC and HCC mediated by HBV.

Involvement of hepatitis B X-interacting protein (HBXIP) in proliferation regulation of cells.

AIM: To investigate the effect of Hepatitis B X-interacting protein (HBXIP) on cell proliferation. METHODS: A rabbit antibody against HBXIP was generated. The RNA interference (RNAi) fragment of the HBXIP gene was constructed in the pSilencer-3.0-H1 vector termed pSilencer-hbxip. Plasmids of the pcDNA3-hbxip encoding HBXIP gene and pSilencer-hbxip were transfected into human breast carcinoma MCF-7 cells, hepatoma H7402 cells, and the normal human hepatic cell line L-O2, respectively. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and 5-bromo-2-deoxyuridine incorporation assay were applied to detect cell proliferation. MCF-7 cells and L-O2 cells in the cell cycle were examined by flow cytometry. The proteins involved in cell proliferation and cell cycle were investigated by Western blot. RESULTS: Overexpression of HBXIP resulted in the promotion of proliferation of MCF-7, H7402, and L-O2 cells. Flow cytometry showed that the overexpression of HBXIP promoted the cell proliferation of MCF-7 and L-O2 cells, and led to an increased cell proliferative index in MCF-7 cells (from 46.25% to 58.28%) and L-O2 cells (from 29.62% to 35.54%). Western blot showed that expression levels of c-Myc, Bcl-2, and proliferating cell nuclear antigen were upregulated in MCF-7, H7402, or L-O2 cells, whereas that of p27 was downregulated. However, the RNAi of HBXIP brought opposite results. CONCLUSION: One of the functions of HBXIP is its involvement in cell proliferation.

[Investigation of discrepant proteins between two breast cancer cell lines with different metastatic abilities].

BACKGROUND & OBJECTIVE: Metastasis associated proteins play important roles in metastasis. This study was to investigate proteins which may be involved in breast cancer cell metastasis and further explore the potential mechanisms. METHODS: LM-MCF-7 and MCF-7, two breast cancer cell lines with different metastatic potentials, derived from the same parent cell line, were used in our study. Proteomics and Western blot were applied to identify differentially expressed proteins. Wound healing assay was performed to observe the effect of survivin gene on breast cancer cell migration by transfecting pcDNA3-Sur plasmid into MCF-7 cells. RESULTS: Eight differently expressed proteins, which were correlated with cell structures, cellular metabolism, apoptosis, protein enfold or interaction, were identified. Protein expression of nm23 and p27 was relatively higher in MCF-7 cells; while the expression of survivin, Bcl-2 and myosin light chain kinase was relatively higher in LM-MCF-7 cells. Increased migration ability was observed in MCF-7 cells which were transfected with pcDNA3-Sur. CONCLUSION: Metastasis associated proteins exist in breast cancer cell lines with different metastatic abilities. Survivin is closely related to the metastasis in breast cancer cells.

[Screening of a sub-clone of human breast cancer cells with high metastasis potential].

OBJECTIVE: To screen a sub-clone of human breast cancer cell of the MCF-7 line with high metastasis potential. METHODS: Human breast cancer cells of the MCF-7 line were injected subcutaneously into 10 severe combined immunodeficiency (SCID) mice. Sixty-eight days after the mice were killed and their lungs were taken out. Primary cell culture was conducted. When the cells were passed on to the third generation a sub-clone was screened from the lung tissue and termed LM-MCF-7. Microscopy was performed on the lung tissues. The growth curve was drawn. Flow cytometry was used to examine the cell cycle. Chromosome analysis was done. Immunohistochemistry was used to detect the expression of breast cancer specific antigen CAI5-3. Western blotting was used to detect the protein expression of the protein associated with tumor metastasis: nm23 (a metastasis-suppressing gene), myosin light chain kinase (MLCK, a kinase related to cell movement), survivin, bcl-2 and p27 (a gene related to cell cycle). LM-MCF-7 cells were injected into other SCID mice and these mice were killed 30 days later to observe the metastasis of cancer so as to detect the tumorigenic ability of the LM-MCF-7 cells. RESULTS: When the cells from the mouse lung tissues were passed on to the third generation a sub-clone with high metastasis potential was screened and termed LM-MCF-7. The morphology of the new cell line was typically epithelioid. Flow cytometry showed that the DNA relatively contents were 53.40% of the LM-MCF-7 cells were in the G(0)/G(1) phase, a lower percentage than that of the MCF-7 cells, and 17.10% in the S phase and 23.20% in the G(2+)M phase, both percentages higher than those of the MCF-7 cells. The proliferating time of the LM-MCF-7 cell population was about 20 +/- 14 hours, much shorter than that of the parent strain cells. The chromosomes of the LM-MCF-7 cells, numbering 16-123, showed the morphology characteristic c of human chromosomes. The marker of human breast cancer CA15-3 was detected in both MCF-7 and LM-MCF-7 cells. The protein expression of nm23 and p27 was down-regulated, but the protein expression of MLCK, bcl-2 and survivin was up-regulated in LM-MCF-7 cells in comparison with those in MCF-7 cells. The tumorigenesis rate of LM-MCF-7 cells was 100% (5/5), with a latent period of 5.0 +/- 0.0 d, and the tumor metastasized to lung, kidney, spleen, bone marrow, lymph node and heart. CONCLUSION: A human breast cancer line, LM-MCF-7 cell line, with high metastasis potential has been derived from the human breast cancer cells of MCF-7 line.

tNOX is both necessary and sufficient as a cellular target for the anticancer actions of capsaicin and the green tea catechin (-)-epigallocatechin-3-gallate.

Capsaicin and the principal green tea catechin, (-)-epigallocatechin-3-gallate (EGCg), target tNOX, a tumor (cancer)-specific surface hydroquinone (NADH) oxidase with protein disulfide-thiol interchange activity (ECTO-NOX protein). Accordingly vector-forced over expression of tNOX in MCF-10A mammary epithelia or COS cells that lack tNOX or in COS cells that underexpress tNOX enhanced the susceptibility of growth and apoptosis to both EGCg and capsaicin. Additionally, the tNOX-transfected MCF-10A cells proliferated in Matrigel, a measure of invasiveness. In contrast, oligomeric antisense tNOX DNA abrogated growth inhibition by EGCg and capsaicin and reduced anchorage-dependent growth of HeLa (human cervical carcinoma) cells that naturally overexpress tNOX. The findings show cell surface expression of tNOX as both necessary and sufficient for the cellular anticancer activities attributed to both EGCg and capsaicin.

Adriamycin tolerance in human mesothelioma lines and cell surface NADH oxidase.

Adriamycin tolerant human mesothelioma cell lines derived from a single tumor prior to either chemotherapy or radiation therapy and a susceptible cell line were investigated. Not only was growth resistant to low doses of adriamycin but an unusual pattern of resistance was encountered in which cells seemed to better tolerate high adriamycin doses than intermediate doses. The differential growth susceptibility of the tolerant lines compared to A549 lung carcinoma and the bimodal dose response correlated with differences in the specific activity of a plasma membrane-associated NADH oxidase (NOX). Plasma membrane fractions of high purity were isolated by aqueous two-phase partition and assayed directly. The NADH oxidase activity of the plasma membranes for the susceptible cell line was maximally inhibited by 1 microM adriamycin whereas the NADH oxidase activity of the tolerant lines was less and was maximally inhibited by 0.1 microM adriamycin with 1 and 10 microM adriamycin being less inhibitory than 0.1 microM adriamycin. The findings suggest a relationship between the growth response to adriamycin of the adriamycin tolerant mesothelioma lines and the activity of the plasma membrane-associated NADH oxidase activity of the cell surface in these cell lines.

Biochemical basis for the biological clock.

NADH oxidases at the external surface of plant and animal cells (ECTO-NOX proteins) exhibit stable and recurring patterns of oscillations with potentially clock-related, entrainable, and temperature-compensated period lengths of 24 min. To determine if ECTO-NOX proteins might represent the ultradian time keepers (pacemakers) of the biological clock, COS cells were transfected with cDNAs encoding tNOX proteins having a period length of 22 min or with C575A or C558A cysteine to alanine replacements having period lengths of 36 or 42 min. Here we demonstrate that such transfectants exhibited 22, 36, or 40 to 42 h circadian patterns in the activity of glyceraldehyde-3-phosphate dehydrogenase, a common clock-regulated protein, in addition to the endogenous 24 h circadian period length. The fact that the expression of a single oscillatory ECTO-NOX protein determines the period length of a circadian biochemical marker (60 X the ECTO-NOX period length) provides compelling evidence that ECTO-NOX proteins are the biochemical ultradian drivers of the cellular biological clock.