| BackgroundThe incidence of breast cancer ranks the second in female malignance. Though the survival rate of breast cancer has been increased through early screening and integrative therapy, breast cancer patients die of resistance to radiotherapy and chemotherapy or recurrence and metastasis. Recurrence and metastasis become the leading cause of cancer death. Therefore, the study of new therapeutic targets for breast cancer and new targeted anti-cancer drugs to prevent breast cancer recurrence and metastasis become an important subject for cancer research. The discovery of cancer stem cell provides new approach for studying new anti-cancer drugs.Cancer stem cells are sub-population of cells present in the cancer tissue or cancer cell lines, with specific surface markers. Small amount of cancer stem cells can form a tumor in immunodeficient mice, in contrast, the non-cancer stem cells have weak ability to form a tumor. Self-renewal and differentiation are two major characteristics of cancer stem cells. Self-renewal of cancer stem cell refers to the process in which cancer stem cells divide asymmetrically to form a next generation cancer stem cell identical to that of its parent generation. Differentiation of cancer stem cells refers to the process in which cancer stem cells differentiate to all the other cell types in a tumor. Recent studies have shown that cancer stem cells are resistant to chemotherapy and residual cancer stem cells survived from radiotherapy and chemotherapy in tumor tissue are responsible for tumor recurrence and involved in cancer metastasis.5-aza-2’-deoxycytidine (DAC) is a cytosine analogue, functioning in inhibition of DNA methylation. U.S. Food and Drug Administration has approved that DAC can be used for treatment of myelodysplastic syndrome and myeloid leukemia. Nevertheless, DAC also have anti-cancer effects on solid tumors as well. Preliminary clinical trial showed that the DAC had anti-tumor effect on breast cancer and non-small cell lung cancer. The anticancer mechanism of DAC remains inconclusive, though itt can be approximately based on the following two aspects:that dependent on DNA demethylation and independent of DNA demethylation. The former refers to the DAC inhibition of DNA methyltransferase in a cancer cell, demethylating the DNA and leading to expression of silenced genes in which the promoters have been methylated, especially the silenced tumor suppressors; the latter include that DAC causes DNA damage to cancer cells. Despite demethylation of the promoter region of a gene leads to gene expression, there are also reports showing that DAC could down-regulate the expression of certain genes, nevertheless, the mechanism of which is unclear. Due to its effect on the inhibition of DNA methylation, DAC is called epigenetic anti-cancer drug. Histone deacetylase inhibitor is also an epigenetic anti-cancer drug. It functions to be an anti-cancer drug mainly through inhibition of histone deacetylase. There are report that histone deacetylase inhibitor can inhibit the sternness gene expression in embryonic stem cancer cells. This research result suggests that epigenetic anti-cancer drugs could be a potential drug targeting cancer stem cells. Therefore, in the present study, we first observed the effects of DAC on the self-renewal of breast cancer stem cell, and then studied the underlying mechanism of the self-renewal inhibition.ObjectivesTo study the effect of DAC on self-renewal of cancer stem cells from different breast cancer cell lines; To study the underlying mechanisms of the self-renewal inhibition by DAC. Methods1. Adherent culture of breast cancer cell linesMDA-MB-468was cultured in DMEM medium, SK-BR-3in RIM1640medium and MDA-MB-231in L-15medium. All culture medium were supplemented with10%FBS, Penicillin and Streptomycin. When cells were grown to cover70%culture surface, cells were trypsynized using0.25%trypsin in0.02%EDTA buffer. After trypsynization termination, individualized cell suspension were made for suspension culture.2. The culture of primary mammosphere from breast cancer cell lines, DAC treatment and calculation of primary mammosphere formation efficiency.MDA-MB-468, SK-BR-3and MDA-MB-231cells cultured in adhesion condition were trypsinized to make cell suspension. After counting cell number, the cell suspension were centrifuged in15ml tube for5min at100rpm to collect cells. Cells were washed with1x PBS and centrifuged again to collect cells. The cells were finally suspended with cancer stem cell medium, followed by adjusting cell dencity to25000cells/ml. The formula of cancer stem cells culture medium is as follows:basal medium DMEM/F12(1:1for ratio), adding2%B27,20ng/ml EGF, lOng/ml bFGF. For the culture of primary mammosphere, the cell suspension in stem cell culture medium, at the cell density of25000cells/ml, were added to24well ultra-low attachment culture plate(Corning), with0.5ml cell suspension in each well for taking photograph and counting the number of primary mammosphere, and added to6well ultra-low attachment culture plate(Corning),with lml cell suspension in each well for flow cytomtric assay of CD44+/CD24" cell population, total RNA isolation and immunoblotting. Starting from the first day of the suspension culture,100nmol/L or500nmol/L DAC were added to the culture each day till the seventh day of the culture, with change of fresh stem cell medium at the third day of culture, and the control was added with0.1%DMSO. The mammosphere in the suspension culture were photographed under microscope. The mammosphere with diameter above40μm were counted. Primary mammosphere formation efficiency=number of primary mammosphere/cell number planted×100%. 3. Self-renewal ability assay of the of cancer stem cellThe self-renewal of cancer stem cell is evaluated by the secondary mammosphere formation efficiency and the percentage of CD44+/CD24"in primary mammosphere.1) The culture of secondary mammosphere and calculation of secondary mammosphere formation efficiency. The primary mammosphere were centrifuged at800g for1minute to collect cell spheroid. The cell spheroid were then trypsinized with0.25%trypsin in0.02%EDTA buffer and the trypsinization was terminated by adding10%FBS DMEM/F12medium. The cells were pipetted three times to disperse cells, followed by passing through the40μm mesh to get single cell. Cells were centrifuged for5minutes at1000rpm, and washed once with DMEM/F12. After centrifugation, cells were suspended with fresh cancer stem cell medium to the density of25000cell/ml. Cell suspension0.5ml were then cultured at24well ultra-low attachment culture plate for7days, with change of fresh medium at the third day. Cells were photographed at the7th day of culture under microscope and mammosphere size above40μm in diameter were counted. Secondary mammosphere formation efficiency=number of Secondary mammosphere/number of cells planted×100%.2) Flow cytometric assay of CD44+/CD24-cell percentage in primary mammosphere. The digestion of mammosphere and getting single cell suspension is the same above. Cells were washed once with1%FBS PBS containing Penicillin and streptomycin PBS. Cells were suspended at the density of106/100μl and FITC-CD24, PE-CD44or corresponding normal serum control were added. After incubation for40minutes in dark, cell were then washed once and fixed with1%polyformaldehyde. CD44+/CD24-cell percentage were then analyzed by flow cytometer.4. Assay of apoptosisPrimary mamsphere were digested with trypsin to get single cells. The Percentage of apoptotic cell were assayed in Flow Cytometer using Annexin-V method.5. Isolation of total RNA and quantitative PCR The total RNA of primary mammosphere were isolated using Trizol reagent: Quantitative PCR technique is used to evaluate the mRNA expression of Nanog, Oct4and Sox2gene.6. Western blotting,Primary mammosphere were lysed using RIPA buffer and the protein was isolated. The protein expression of Nanog was detected by Western blot.Results1. The effects of DAC on self-renewal of breast cancer stem cell1.1The effects of DAC on primary and secondary mammosphere formation.Three breast cancer cell lines, MDA-MB-231, MDA-MB-468and SK-BR-3, in suspension culture were treated with100nmol/L,500nmol/L DAC for seven days. The primary mammosphere formation efficiencies of the three cell lines, treated with0.1%DMSO,100nmol/L and500nmol/L DAC were as follows:MDA-MB-231,49.68±5.13%,43.68±4.63%,31.68±2.21%(F=23.940, P=0.0005); MDA-MB-468,11.43±2.37%,9.32±0.77%,8.44±0.74%(F=5.241, P=0.001); SK-BR-310.68±0.54%,8.64±0.98%,7.46±1.01%(F=17.52, P=0.0003). The primary mammosphere formation efficiencies of the three cell lines treated with500nmol/L DAC are statistically significant compared to those of the corresponding controls (P<0.05). The primary mammosphere formation efficiency of MDA-MB-231treated with100nmol/L DAC are statistically significant compared to that of the control (P<0.05). The primary mammosphere formation efficiencies of MDA-MB-468and SK-BR-3treated with100nmol/L DAC are not statistically significant compared to those of the corresponding controls (P>0.05). The secondary mammosphere formation efficiencies of the three cell lines, treated with0.1%DMSO,100nmol/L and500nmol/L DAC were as follows:MDA-MB-231,68.08±3.41%,47.28±4.01%,37.70±3.40%(F=92.15, P=0.0002); MDA-MB-468,23.72±2.08%,17.52±2.98%,8.44±2.75%(F=42.65, P=0.0001); SK-BR-3,18.84±1.67%,13.50±0.97%,6.76±0.47%(F=51.60, P=0.0001). The secondary mammosphere formation efficiencies of the three cell lines treated with two doses of DAC are statistically significant compared to corresponding controls (P<0.05).These results indicate that two doses of DAC, either at100nmol/L or500nmol/L, can inhibit the self-renewal of breast cancer stem cell.1.2The effects of DAC on the percentage of breast cancer stem cells in primary mammosphere.CD44+/CD24-is the surface marker for breast cancer stem cells. To further confirm the inhibition effect of DAC on self-renewal of breast cancer stem cell, the percentage of CD44+/CD24-cell population in primary mammosphere was assayed using Flow Cytometer. The percentages of CD44+/CD24-in the three cell lines treated with1%DMSO,100nmol/L and500nmol/L DAC were as follows: MDA-MB-231,61.46±5.60%,45.60±2.56%,38.45±2.34%(F=28.75, P=0.0008); MDA-MB-468,7.00±0.70%,2.50±0.50%,2.10±0.24%(F=83.54, P=0.0001); SK-BR-3,10.93±2.08%,3.83±0.41%,3.37±0.46%(F=34.35, P=0.0005). In three breast cancer cell lines, the percentages of CD44+/CD24-cell population in primary mammospheres treated with two doses of DAC is lower than those of their corresponding controls. The differences are statistically significant. Because primary mammosphere contains high percentage of breast cancer stem/progenitor cell, these results further indicate that DAC can inhibit the self-renewal of breast cancer stem cell.2. The effects of DAC on apoptosis of cells in primary mammosphereThe possible mechanisms by which DAC decrease the percentage of breast cancer stem cell in primary mammosphere are as follows:a. inhibition of self-renewal only; b. induction of apoptosis only; c. both inhibition of self-renewal and induction of apoptosis. To further investigate the mechanisms, we assayed the percentages of apoptotic cells in primary mammosphere with Flow Cytometer using Annexin-V method. The percentages of apoptotic cells in primary mammosphere of three breast cancer cell lines treated with0.1%DMSO,100nmol/L and500nmol/L DAC were as follows:MDA-MB-231,3.58±0.18%,3.61±0.36%,8.01±0.49%; MDA-MB-468,1.53±.15%,1.56±0.11%,3.12±0.21%; SK-BR-3,1.50±0.28%,1.66±0.20%,4.01±0.17%. The percentages of apoptotic cell in groups treated with100nmol/L DAC are not statistically significant from those of control groups (p>0.05). DAC treatment at concentration of100nmol/L does not cause apoptosis of cells in primary mammosphere. The percentages of apoptotic cell in groups treated with500nmol/L DAC are statistically significant from those of control groups (p<0.05). DAC treatment at concentration of500nmol/L causes apoptosis of cells in primary mammosphere. These results indicate that:a. The percentage decrease of breast cancer stem cell in primary mammosphere treated with100nmol/LDAC is not involved with apoptosis. but involved with inhibition of breast cancer stem cell self-renewal; b. The percentage decrease of breast cancer stem cell in primary mammosphere treated with500nmol/LDAC is involved with apoptosis and self-renewal inhibition of breast cancer stem cell.3. The effects of DAC on expression of Nanog, Oct4and sox2in primary mammosphere.3.1The effects of DAC on mRNA expression of Nanog, Oct4and sox2in primary mammosphere. The self-renewal of cancer stem cell is dependent on the expression of core transcriptional factor Nanog, Oct4and Sox2. What is the mechanisms by which DAC inhibits self-renewal of breast cancer stem cell? To answer this question, we studded the mRNA expression of Nanog, Oct4and Sox2in primary mammosphere. We found that DAC at100nmol/L concentration could down-regulate the expression of Nanog and Oct4in primary mammosphere of MDA-MB-231, MDA-MB-468and SK-BR-3, but not Sox2.3.2The effects of DAC on protein expression of Nanog in primary mammosphere. We selectively further observed the effects of DAC on protein expression of Nanog in primary mammosphere. The results showed that100nmol/L DAC could down-regulate the expression of Nanog at protein level in the primary mammosphere of MDA-MB-231, MDA-MB-468and SK-BR-3. Conclusions1. DAC inhibits the self-renewal of breast cancer stem cellBoth the low concentration of DAC(100nmol/L) and high concentration of DAC (500nmol/L) can inhibit the self-renewal of breast cancer stem cell. Low concentration of DAC inhibit the self-renewal of breast cancer stem cell only, instead, high concentration of DAC inhibit the self-renewal of breast cancer stem cell and induce apoptosis of breast cancer stem cell.2. DAC down-regulate the expression of Nanog, Oct4and sox2in primary mammosphere, suggesting that DAC could down-regulate the expression of Nanog, Oct4and sox2in breast cancer stem cell.In summary, low concentration of DAC can inhibit the self-renewal of breast cancer stem cell, suggesting that low doses of DAC could be therapeutically effective targeting cancer stem cell, and Nanog in cancer stem could be a new therapeutic target of breast cancer. |
PDF Full Text Request