A correlation between radiation sensitivity and initial chromatid breaks in cancer cell lines revealed by Calyculin A-induced premature condensation

Three human malignancy cell lines were irradiated with Co-60 gamma-rays. Initial chromatid breaks were measured by using the chemically induced premature chromosome condensation technique. Survival curves of cells exposed to gamma rays was linear-quadratic while the efficiency of Calyculin A in inducing PCC of G(2) PCC was about five times more than G(1) PCC. A dose-dependent increase in radiation-induced chromatid/isochromatid breaks was observed in G(1) and G(2) phase PCC and a nearly positive linear correlation was found between cell survival and chromatin breaks. This study implies that low LET radiation-induced chromatid/isochromatid breaks can potentially be used to predict the radiosensitivity of tumor cells either in in vitro experimentation or in in vivo clinical radiotherapy.

Prostate cancer cell-specific VEGF siRNA delivery system using cell targeting peptide conjugated polyplexes

A polymeric gene carrier was developed to deliver vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) for prostate cancer cells in a target-specific manner. Prostate cancer-binding peptide (PCP) was conjugated with polyethylenimine (PEI) via a poly(ethylene glycol) (PEG) linker (PEI-PEG-PCP). The PEI-PEG-PCP conjugate could effectively condense siRNA to form stable polyelectrolyte complexes (polyplexes) with an average diameter of approximately 150 nm in an aqueous solution. VEGF siRNA/PEI-PEG-PCP polyplexes exhibited significantly higher VEGF inhibition efficiency than PCP-unmodified polycationic carriers (PEI-PEG or PEI) in human prostate carcinoma cells (PC-3 cells). The enhanced gene silencing activity of VEGF siRNA/PEI-PEG-PCP was maintained even under serum conditions, owing to the steric stabilization of the polyplexes with hydrophilic PEG grafts. Confocal microscopic studies revealed that the siRNA/PEI-PEG-PCP polyplexes were delivered into PC-3 cells in a PCP ligand-specific manner.

Co-expression of CD173 (H2) and CD174 (Lewis Y) with CD44 suggests that fucosylated histo-blood group antigens are markers of breast cancer-initiating cells

Histo-blood group antigens CD173 (H2) and CD174 (Lewis Y) are known to be developmentally regulated carbohydrate antigens which are expressed to a varying degree on many human carcinomas. We hypothesized that they might represent markers of cancer-initiating cells (or cancer stem cells, CSC). In order to test this hypothesis, we examined the co-expression of CD173 and CD174 with stem cell markers CD44 and CD133 by flow cytometry analysis, immunocytochemistry, and immunohistochemistry on cell lines and tissue sections from breast cancer. In three breast cancer cell lines, the percentage of CD173(+)/CD44(+) cells ranged from 17% to > 60% and of CD174(+)/CD44(+) from 21% to 57%. In breast cancer tissue sections from 15 patients, up to 50% of tumor cells simultaneously expressed CD173, CD174, and CD44 antigens. Co-expression of CD173 and CD174 with CD133 was also observed, but to a lesser percentage. Co-immunoprecipitation and sandwich ELISA experiments on breast cancer cell lines suggested that CD173 and CD174 are carried on the CD44 molecule. The results show that in these tissues CD173 (H2) and CD174 (LeY) are associated with CD44 expression, suggesting that these carbohydrate antigens are markers of cancer-initiating cells or of early progenitors of breast carcinomas.

The recombinant beta subunit of C-phycocyanin inhibits cell proliferation and induces apoptosis

C-Phycocyanin (C-PC) from blue-green algae has been reported to have various pharmacological characteristics, including antiinflammatory and anti-tumor activities. In this study, we expressed the beta-subunit of C-PC (ref to as C-POP) in Escherichia coli. We found that the recombinant C-PC/beta has anti-cancer properties. Under the treatment of 5 mu M of the recombinant C-PC/beta, four different cancer cell lines accrued high proliferation inhibition and apoptotic induction. Substantially, a lower response occurred in non-cancer cells. We investigated the mechanism by which C-PC/beta inhibits cancer cell proliferation and induces apoptosis. We found that the C-PC/beta interacts with membrane-associated beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Under the treatment of the C-PC/beta, depolymerization of microtubules and actin-filaments were observed. The cells underwent apoptosis with an increase in caspase-3, and caspase-8 activities. The cell cycle was arrested at the G0/G1 phase under the treatment of C-PC/beta. In addition, the nuclear level of GAPDH decreased significantly. Decrease in the nuclear level of GAPDH prevents the cell cycle from entering into the S phase. Inhibition of cancer cell proliferation and induction of apoptosis may potentate the C-POP as a promising cancer prevention or therapy agent. (c) 2006 Elsevier Ireland Ltd. All rights reserved.

Aberrant expression of X-linked genes RbAp46, Rsk4, and Cldn2 in breast cancer

The consequence of activation status or gain/loss of an X-chromosome in terms of the expression of tumor suppressor genes or oncogenes in breast cancer has not been clearly addressed. In this study, we investigated the activation status of the X-chromosomes in a panel of human breast cancer cell lines, human breast carcinoma, and adjacent mammary tissues and a panel of murine mammary epithelial sublines ranging from low to high invasive potentials. Results show that most human breast cancer cell lines were homozygous, but both benign cell lines were heterozygous for highly polymorphic X-loci (IDS and G6PD). On the other hand, 60% of human breast carcinoma cases were heterozygous for either IDS or G6PD markers. Investigation of the activation status of heterozygous cell lines revealed the presence of only one active X-chromosome, whereas most heterozygous human breast carcinoma cases had two active X-chromosomes. Furthermore, we determined whether or not an additional active X-chromosome affects expression levels of tumor suppressor genes and oncogenes. Reverse transcription-PCR data show high expression of putative tumor suppressor genes Rsk4 and RbAp46 in 47% and 79% of breast carcinoma cases, respectively, whereas Cldn2 was down-regulated in 52% of breast cancer cases compared with normal adjacent tissues. Consistent with mRNA expression, immunostaining for these proteins also showed a similar pattern. In conclusion, our data suggest that high expression of RbAp46 is likely to have a role in the development or progression of human breast cancer. The activation status of the X-chromosome may influence the expression levels of X-linked oncogenes or tumor suppressor genes.

A novel anti-tumor protein extracted from Meretrix meretrix Linnaeus induces cell death by increasing cell permeability and inhibiting tubulin polymerization

Discovery and development of new pharmaceuticals from marine organisms are attracting increasing interest. Several agents derived from marine organisms are under preclinical and clinical evaluation as potential anticancer drugs. We extracted and purified a novel anti-tumor protein from the coelomic fluid of Meretrix meretrix Linnaeus by ammonium sulphate fractionation, ion exchange and hydrophobic interaction chromatography. The molecular weight of the highly purified protein, designated MML, was 40 kDa as determined by SDS-PAGE analysis. MML exhibited significant cytotoxicity to several cancer cell types, including human hepatoma BEL-7402, human breast cancer MCF-7 and human colon cancer HCT116 cells. However, no inhibitory effect was found when treating murine normal fibroblasts NIH3T3 and benign human breast MCF-10A cells with MML. The cell death induced by MML was characterized by cell morphological changes. The induction of apoptosis of BEL-7402 cells by MML was weak by DNA ladder assay. The possible mechanisms of its anti-tumor effect might be the changes in cell membrane permeability and inhibition of tubulin polymerization. MML may be developed as a novel, highly selective and effective anti-cancer drug.