Suspension culture combined with chemotherapeutic agents for sorting of breast cancer stem cells.

BACKGROUND: Cancer stem cell (CSC) hypothesis has not been well demonstrated by the lack of the most convincing evidence concerning a single cell capable of giving rise to a tumor. The scarcity in quantity and improper approaches for isolation and purification of CSCs have become the major obstacles for great development in CSCs. Here we adopted suspension culture combined with anticancer regimens as a strategy for screening breast cancer stem cells (BrCSCs). BrCSCs could survive and be highly enriched in non-adherent suspension culture while chemotherapeutic agents could destroy most rapidly dividing cancer cells and spare relatively quiescent BrCSCs. METHODS: TM40D murine breast cancer cells were cultured in serum-free medium. The expression of CD44+CD24- was measured by flow cytometry. Cells of passage 10 were treated in combination with anticancer agents pacilitaxel and epirubicin at different peak plasma concentrations for 24 hours, and then maintained under suspension culture. The rate of apoptosis was examined by flow cytometry with Annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining method. Selected cells in different amounts were injected subcutaneously into BALB/C mice to observe tumor formation. RESULTS: Cells of passage 10 in suspension culture had the highest percentage of CD44+CD24- (about 77 percent). A single tumor cell in 0.35 PPC could generate tumors in 3 of 20 BALB/C mice. CONCLUSION: Suspension culture combined with anticancer regimens provides an effective means of isolating, culturing and purifying BrCSCs.

Suspension culture combined with anticancer regimens for screening breast cancer stem cells.

Breast cancer stem cells (BrCSCs)have been first identified in solid tumors. To be more exact, they are defined as tumorigenic cancer cells which exhibit the capacity to form tumors distinct from the majority non-tumorigenic cancer cells. However it is only a minor subset that cancer stem cells are highly enriched in. It is still unknown how to find the definitive research subject - "a single breast cancer stem cell" - that can cause a new tumor by itself. Since recent studies suggest that BrCSCs have a higher proportion in suspension culture and have a greater resistance to chemotherapy regimens, we propose a novel strategy to isolate and identify BrCSCs:suspension culture combined with anticancer regimens. This double sorting method by means of the unique properties of BrCSCs may be helpful to screen genuine cancer stem cells(CSCs).

[Involvement of epidermal growth factor receptor signaling pathway in tamoxifen resistance of MCF-7 cells].

BACKGROUND & OBJECTIVE: Tamoxifen (TAM) is an important endocrine therapeutic drug used in combined treatment for breast cancer. However the drug resistant effect of TAM has become a major concern in clinical use. Epidermal growth factor receptor (EGFR) is expressed in many tumors and the expression of EGFR in breast cancer tissues is associated with poor prognosis. This study was to investigate EGFR signaling pathway in the drug resistant effect of TAM in breast cancer cells. METHODS: An estrogen receptor (ER) positive breast cancer cell line, MCF-7 was incubated with TAM (10(-7) mol/L) for up to 6 months to produce drug resistant cells. mRNA and protein expression of EGFR signaling pathway related genes and ER gene was analyzed by fluorescent quantitation reverse transcription-polymerase chain reaction (fqRT-PCR) and Western blot. AG1478, an EGFR inhibitor, was also used to study the drug resistant effect of TAM. RESULTS: The proliferation of MCF-7 cells was inhibited in the early stage after TAM treatment. However the inhibitory effect vanished after continuous 6-month treatment and the cell proliferation rate returned back to normal. The tolerance of MCF-7 cells to TAM was confirmed by cell growth inhibitory test. In TAM resistant MCF-7 cells, the expression of EGFR mRNA was increased by 4.5 times and the protein expression of EGFR, phosphorylation-extracellular signal-regulated kinase (p-ERK1/2) was also increased dramatically (P<0.05). While ER protein expression remained unchanged. Moreover the drug resistant cells were identified more sensitive to AG1478. CONCLUSION: In endocrine therapy, TAM resistant breast cancer cells are likely to be generated by the activation of EGFR signaling pathway after long-term TAM treatment, and EGFR inhibitors might increase the therapeutic effects in breast cancer treatment.

[The nuclear factor kappa B activation: the key step of cell proliferation in estrogen receptor-negative breast cancer cells].

OBJECTIVE: To investigate the way of nuclear factor kappa B (NF-kappaB) activation and the mechanism of NF-kappaB-promoted proliferation in estrogen receptor (ER)-negative breast cancer cells. METHODS: The protein of IkappaB kinase alpha (IKKalpha) was measured by Western blot and the influence on cell-cycle was assayed by flow cytometry (FCM). RESULTS: The IKKalpha was tested higher in three ER-negative breast cancer cell lines than in MCF-7. The influence caused by epidermal growth factor (EGF), tumor necrosis factor (TNF)-alpha and E(2) to tumor cells' proliferation was tested. EGF could remarkably enhance cyclin D(1) expression about 83% more in EGF group than that in control group and proliferation index from 0.22 to 0.31 (P < 0.01). On the other hand, TNF-alpha inhibited cyclin D(1) expression. Protein kinase C inhibitor, Go6976, could peculiarly prevent NF-kappaB over-expression caused by EGF. The cell-cycle was assayed by FCM in phase G(0)/G(1) 69.36% and in phase S 22.77% when adding EGF and in phase G(0)/G(1) 91.54% and in phase S 7.81% when adding EGF and Go6976. The proliferation index decreased from 0.31 to 0.09 (P < 0.01). CONCLUSIONS: EGF-EGFR pathway can provide ER-negative breast cancer cells the signal for the autonomous growth. This signal promoted tumor cells' proliferation is transmitted by activating NF-kappaB and expressing more cyclin D(1). In this pathway, NF-kappaB play an important role as signal transmitting. The strategy to NF-kappaB activating may provide new way to treat ER-negative breast cancers.

[Optimal effects of liposome on antisense oligodeoxynucleotides uptake by mouse breast cancer TM40D cells and the effects of antisense oligodeoxynucleotides targeting c-erbB-2 on cell proliferation and apoptosis].

OBJECTIVE: To study the optimal effects of liposome on antisense oligodeoxynucleotides targeting c-erbB-2 uptake by mouse breast cancer TM40D cells and effects of AS-ODNs on cell proliferation and apoptosis. METHODS: Flow cytometric analysis and fluorescence microscope were used to study the transfection ratio of AS-ODNs in mouse breast cancer TM40D cells with/without liposome in 1, 2, 4, 6 hour and the distribution of AS-ODNs in cells. the effects of ODNs on cell proliferation and activation of apoptosis were examined by MTT assay and flow cytometry. RESULTS: Liposome could promote AS-ODNs uptake by mouse breast cancer TM40D cells, flow cytometric analysis revealed that the time of the optimal effects was 4 hours and the percentage of FAM-positive cells reached 72.23%; the ratio maintained at a relatively high level at 6 h. Also liposome facilitated the entrance of AS-ODNs into nucleus. AS-ODNs restrained the proliferation of mouse breast cancer TM40D cells, and restraint rate was 50.24%. Meanwhile AS-ODNs promoted cell apoptosis. Flow cytometry analysis revealed that antisense ODNs increased cell apoptosis by 38.50%, compared with cultured cells group 9.13% and liposome group 9.29%. CONCLUSIONS: Liposome could facilitate AS-ODNs to enter cells and their nucleus. AS-ODNs of C-erbB-2 restrained the proliferation of mouse breast cancer TM40D cells and promoted apoptosis.

[Preliminary study on lymphocyte subsets of sentinel lymph nodes in breast cancer patients].

OBJECTIVE: To study the lymphocyte subsets of sentinel lymph nodes (SLN) in breast cancer patients. METHODS: Flow cytometry was used to analyze the markers on the surface of lymphocytes such as CD3, CD4, CD8, CD16 and CD19 in the sentinel lymph node of breast cancer. RESULTS: When lymph node metastasis did not occur, there was no significant difference in the number of CD3(+) T, CD4(+) T, CD8(+) T, CD16 NK and CD19 B cells between SLN cells and non-SLN cells. With lymph node metastasis, the proportion of CD4(+) and CD8(+) T cells was significantly changed, CD8(+) T cells (66.15 +/- 5.97) were the predominant cells instead of CD4(+) T cells (69.07 +/- 5.02), whereas no significant difference in CD3(+) T, CD16 NK and CD19 B cells. CONCLUSION: The CD4(+) to CD8(+) T cell ratio in sentinel lymph nodes with metastasis is reversed in breast cancer patients. This might results from changes in microenvironment due to tumor cell invasion.

[Hypermethylation of syk gene in promoter region is associated with oncogenesis, metastasis of breast carcinoma].

OBJECTIVE: To investigate the relationship between methylation of syk (spleen tyrosine kinase) gene in promoter region and oncogenesis, metastasis in breast carcinoma. METHODS: Using RT-PCR technique, specimens from 40 breast cancer patients (tumor tissues, adjacent normal tissues), 15 fibroadenoma were detected for their expression of the syk gene. Meanwhile, Methylation-specific PCR was used to detect methylation of syk promoter region. RESULTS: The expression of syk gene were detected in normal breast tissues and fibroadenoma samples, 9 out of 40 breast carcinoma samples were found the expression of syk mRNA, syk expression in breast carcinoma was lower than that in adjacent normal tissues and fibroadenoma samples. No methylation of syk promoter was found in adjacent normal tissues and fibroadenoma samples, 17 cases in 40 breast carcinoma patients (42.5%) were detected hypermethylation of syk gene in promoter region, the rate of methylation of syk promoter in breast carcinoma was higher than that of in adjacent normal tissues (P < 0.05). In 18 cases with lymph node metastasis, 14 were found with syk promoter methylation. A significant difference was noted between two groups (P < 0.05). CONCLUSION: Hypermethylation leads to lose of the syk gene expression in human breast carcinoma. Methylation of syk promoter may be correlated to oncogenesis, metastasis of breast carcinoma.

[Expression of tyrosine kinase Syk in breast cancer and their clinical significance].

OBJECTIVE: To evaluate the effects of the Syk mRNA expression in human breast cancer on tumor growth and metastasis, and to study the correlation of expression of the Syk gene with ER, PR, p53 and HER2/neu. METHODS: Specimens from 40 breast cancer patients (tumor tissues, adjacent normal tissues), 15 fibroadenoma were detected for their expression of the Syk gene and level of Syk mRNA by semi-RT-PCR technique. Meanwhile, ER, PR, p53, HER2/neu were detected in 40 tumor tissues from breast cancer with immunohistochemical staining. RESULTS: All normal breast tissues were detected the expression of the Syk gene. Unlike normal breast tissue, 31 out of 40 breast cancer tissue did not show any detectable Syk mRNA expression, there were significant difference in two groups (chi(2) = 47.4, P < 0.05). The level of Syk mRNA in the primary breast cancer tissues were significantly lower than that in the adjacent non-cancerous breast tissues (t = 3.41, P < 0.05). Furthermore, only one breast cancer tissue in 18 patients with lymph node metastasis had the Syk mRNA expression, the rate and level of Syk mRNA expression in the patients with lymph node metastasis were lower than those without lymph node metastasis (chi(2) = 3.77, P < 0.05, t = 2.74, P < 0.05). Syk expression was correlated to p53 expression. CONCLUSION: The expression of the Syk gene may play an important role in suppressing growth and metastasis of breast cancer.

[Expression of vascular endothelial cell growth factor and its receptor mRNA in breast cancer tissues].

OBJECTIVE: To study the expression of vascular endothelial cell growth factor (VEGF) and its receptor FLT-1, FLK-1 mRNA in breast cancer tissues and their correlation with clinicopathological factors. METHODS: The expression of VEGF and and its receptor FLT-1, FLK-1 mRNA were analyzed by reverse transcription - polymerase chain reaction (RT-PCR) in the specimens from 47 patients with breast cancer and 11 patients with benign breast disease. RESULTS: VEGF121, 165 mRNA were all detected in malignant and benign breast tissues, with higher level, (0.420 +/- 0.133 and 0.291 +/- 0.094 respectively) in breast cancer tissues than in benign breast tissues, [0.196 +/- 0.067 (P = 0.000) and 0.206 +/- 0.058 (P = 0.001) respectively]. VEGF121 mRNA expression was stronger than VEGF165 mRNA expression (P = 0.000) in breast cancer tissues, whereas there is no significant difference in benign breast tissues (P = 0.666). FLT-1, FLK-1 mRNA were expressed in 18 of 47 (38.3%) and 12 of 47 (25.5%) breast cancer tissues respectively, but not in benign breast tissues. Moreover, no correlation was observed between the expression of VEGF121, VEGF165, FLT-1, FLK-1 mRNA in breast cancer tissues and patients' age, tumor size, lymph node metastasis, tumor stages, estrogen or progesterone receptor status. CONCLUSION: The expression of VEGF and its receptor FLT-1, FLK-1 mRNA were up-regulated in breast cancer tissues, suggesting its important role in angiogenesis of breast cancer.

Early rejection and pathological changes in combined pancreaticoduodenal and kidney allotransplantation in pigs.

OBJECTIVE: To study the markers of early rejection and pathological changes in simultaneous pancreaticoduodenal and kidney transplantation (SPKT). METHODS: Thirty hybrid pigs were used as donors and recipients. A renoportal end-to-end anastomosis between the left renal vein and the distal end of the portal vein was performed. Two vascular end-to-side anastomoses between the donor portal vein and recipient inferior vena cava, and between the donor aortic segment including the celiac and superior mesenteric, and left renal arteries and recipient abdominal aorta were carried out. Pancreas exocrine secretion drainage was established with duodenocystostomy. Ureterostomosis of the graft was performed. Urine amylase level, fasting blood glucose and urine volumes of kidney allograft were monitored, and pathological changes of graft were observed. RESULTS: Of 15 recipients, 2 died of disturbance of internal environment and anastomotic bleeding, respectively. Satisfactory results were obtained in the remaining 13 recipients. The changes of urine amylase concentration were prior to those of fasting blood glucose and urine volumes of kidney allograft. The degree of rejection of the kidney allograft was more severe than that of the pancreas and duodenum allograft. CONCLUSIONS: Urine amylase is the early marker of acute rejection in SPKT with bladder drainage of pancreatic exocrine secretion. The pathological change of kidney allograft is most significant in SPKT.