Objective To investigate the effect of neoadjuvant chemotherapy on the proportion of G0-G1 phase cells and the expressions of ATP-binding cassette sub-family G member 2 (ABCG2)and CD44 +CD24 -/low in breast cancer patients.Methods Sixty untreated cases with breast invasive ductal carcinoma from May 201 3 to March 201 4 were chosen.All patients were tested by core needle biopsy and pathological diagno-sis,then treated by neoadjuvant chemotherapy.The proportion of G0-G1 phase cells and the contents of ABCG2 and CD44 +CD24 -/low before and after therapy were compared.Results After chemotherapy,the contents of ABCG2 and CD44 +CD24 -/low were (25.1 0 ±1 .50)% and (36.40 ±3.80)/1 05 ,and the proportion of G0-G1 phase cells was (70.50 ±1 .50)%,which were higher than those before treatment [(1 5.88 ±1 .22)%, (25.00 ±3.40)/1 05 ,(60.65 ±1 .30)%](t =8.685,P <0.05;t =9.226,P <0.05;t =8.898,P <0.05).All patients completed four courses of chemotherapy,and the cCR rate,cPR rate,SD rate were respec-tively 1 8.3% (1 1 /60),73.3% (44 /60),8.3%(5 /60).Conclusion Cell cycle can be arrested and the proportion of stem cells can be raised after neoadjuvant chemotherapy which has exact curative effect and clini-cal popularization value.

OBJECTIVETo study the effect of targeted Sox4 gene-knock-down on the growth of xenografts of Xuanwei female lung cancer cell line XWLC-05 cells in nude mice.

METHODSRecombinant plasmid pGFP-V-RS-Sox4 shRNA was constructed and transfected into XWLC-05 cells. Real-time quantitative PCR and Western blot were applied to confirm the effect of Sox4 gene-knock-down. XWLC-05 cells stably transfected with the plasmids were inoculated into nude mice to establish the xenograft model. The nude mouse status, tumor formation and tumor growth were observed, and the tumor inhibition rate was calculated. CT scan was performed to assess the metastasis of xenografts. Immunohistochemical staining was applied to detect Sox4 and ki-67 protein expression.

RESULTSRecombinant plasmid pGFP-V-RS-A-Sox4 shRNA which can effectively knocking-down Sox4 gene was successfully constructed and the stable transfected cells were selected by puromycin-screening. The success rate of tumor cell inoculation was 100% in the mice of all groups except those inoculated with saline. The body weight of all mice inoculated with parental XWLC-05 cells (blank control), pGFP-V-RS-scram shRNA trsfected XWLC-05 cells (negative control), and pGFP-V-RS-Sox4 shRNA transfected XWLC-05 cells was increased to a varying degree, but there was no significant difference among the groups (P > 0.05 ). The growth of xenografts was significantly inhibited after silencing the Sox4 gene expression when compared with that of the blank and negative controls (P < 0.05) . The volume of removed tumors of the Sox4 gene-inhibited mice was (2.30 ± 0.34) cm(3) , significantly smaller than that of the negative control (3.99 ± 0.45) cm(3) and the blank control (4.03 ± 0.42) cm(3) (P < 0.05) . The weight of removed tumors of Sox4 gene-inhibited mice was (0.86 ± 0.14) g, significantly lower than that of the negative control (1.84 ± 0.27) g and blank control (1.86 ± 0.22) g, (P < 0.05). Immunohistochemical staining showed that Sox4 and ki-67 proteins mainly expressed in cell nuclei. The staining was significantly decreased in xenografts of Sox4-inhibited mice when compared with the negative and blank controls (P < 0.05). No distant metastasis was found in any mouse by CT imaging and pathological examination during the observation period.

CONCLUSIONSThe xenograft model of Xuanwei female lung cancer cell line XWLC-05 cells in nude mice is successfully established. Knocking-down of Sox4 gene can suppress the xenograft tumor growth.

Animals ; Female ; Gene Knockdown Techniques ; Heterografts ; Humans ; Lung Neoplasms ; genetics ; pathology ; Mice ; Mice, Nude ; SOXC Transcription Factors ; metabolism

AIM:To study the epigenetic mechanisms involved in the evolution of prostate cancer from an an-drogen-dependent state to an androgen-independent state , and the global difference of histone H 3 methylation between an-drogen-dependent and -independent prostate cancer cells .METHODS:The methylation sites and patterns of histone H 3 in androgen-dependent prostate cancer cell line LNCaP and androgen-independent prostate cancer cell line DU 145 were ana-lyzed by heavy methyl stable isotope labeling with amino acids in cell culture ( SILAC) coupled with 2D LC-MS/MS.West-ern blotting was used to verify the results from MS .The differential expression of related methylases and demethylases was tested by real-time PCR.RESULTS:Five methylation sites on histone H3 were found in both cell lines, the patterns of which were as follows: H3K14me2, H3R17me1, H3K36me1, H3K36me2, H3K36me3, H3R72me2, H3K79me1 and H3K79me2.There were 2 different peptides both containing methylated H 3K36,“KSAPATGGVKKPHR” and“KSAPSTG-GVKKPHR”, which were different from the 31th amino acid residue “A” and “S”.The former peptide belonging to his-tone H3 variants, H31T, H31 and H32, was mainly identified in DU145 cells, the total peptide counts of which were much more than that of the latter peptide belonging to histone H 3 variant H31T, suggesting that these 2 cell lines expressed differ-ent histone H3 variants.Mono-and dimethylation of H3K36 were not different between these 2 cell lines, but the trimethyl-ation was significantly higher in DU 145 cells than that in LNCaP cells .Many H3K36 demethyltransferases were decreased in DU145 cells compared with LNCaP cells .CONCLUSION: The differential expression of histone H 3 variants and H3K36 demethyltransferases may result in up-regulation of H3K36 tri-methylation during the evolution of prostate cancer from an androgen-dependent state to an androgen-independent state .

Objective To explore the inhibitory effect of Oldenlandia diffusa extract on colorectal cancer angiogenesis in BALB/c mice. Methods Thirty-two BALB/c mice with subcutaneous CT26 colon cancer animal model were randomly equally divided into four groups,including the control group (groupⅠ,saline 0.1 mL/(10. d), O. diffusa ethanol extract of 90 mg/(kg.d) (groupⅡ), O. diffusa ethanol extract of 180 mg/(kg.d) (groupⅢ) and O. diffusa ethanol extract of 360 mg/(kg.d) (group Ⅳ) . Each group of mice were treated with intragastric administration of law administration 12 days after vaccination, then stopped and continue fed to 32 days, and the mice were killed. Micro-vascular dense ( MVD) was observed and countered under the microscopy by immunohistory chemistry. Results The murine colon tumor volumes of GroupⅡ,ⅢandⅣwere significantly less than that of groupⅠ,with significant difference ( <0.05) . The tumor microvessel density values of four groups was (7.83±2.87), (5.32±1.27), (1.77±0.70) and (1.87±0.68),respectively. The number of tumor blood vessels in GroupⅡ,Ⅲ and Ⅳ were significantly less than that of Ⅰ group, with significant difference ( <0.05) . Conclusion Within a certain dose range, the ethanol extract of O. diffusa can significantly inhibit the mouse colon cancer and the mechanism may be realated to inhibiting tumor angiogenesis.

Objective To explore the inhibitory effect of the ethanol extract of Oldenlandia diffusa on the proliferation of CT-26 colon cancer cells which come from BALB/c mice. Method We determined the inhibitory effect of different concentrations of ethanol extract of Oldenlandia diffusa on CT-26 cells' proliferation by using methyl thiazolyl tetrazolium (MTT), and calculated the 50% inhibiting concentration (IC50) . Results As to the same concentration, the inhibitory effect of the ethanol extract of Oldenlandia diffusa on CT-26 cells was increased with time, for exsample:after treated with 0.08 mg/mL of ethanol extract of Oldenlandia diffusa for 24 h, 48 h and 72 h, the inhibitory rates of CT-26 cells were (16.67 ±9.35)%, (34.66 ±9.23)%and (40.07 ±9.16)%, respectively. After treating CT-26 cancer cells for 24 h, 48 h and 72 h, the IC50 values of the ethanol extract of Oldenlandia diffusa were 0.315,0.155 and 0.115 mg/mL, respectively. In the same treatment time, the inhibitory effect of the ethanol extract of Oldenlandia diffusa on CT-26 cells was increased with the increase of concentration:after treatment for 72 h with different concentrations (0.06 mg/mL,0.08 mg/mL,0.10 mg/mL,0.12 mg/mL, 0.14 mg/mL,0.16 mg/mL,0.18 mg/mL and 0.20 mg/mL) of the ethanol extract of Oldenlandia diffusa,the inhibitory rates of CT-26 cells were (35.46 ±3.59)%, (40.07 ±9.16)%, (40.77 ±6.92)%, (52.81 ±1.87)%, (54.22±2.35)%, (68.72±3.71)%, (70.04±8.03)%and (71.84±3.12)%, respectively. Conclusion The ethanol extract of olenlandia diffusa can inhibit the proliferation of CT-26 colon cancer cells from BALB/c mice in a time and dose dependent manner.

BACKGROUND:Side population(SP)cells,with varied contents,are widely distributed in adult tissues,embryos,and certain tumor cells.Haematological tumor is one of the main pathological conditions,which endangers human life.Thus,it is important to recognize SP cells in haematological tumor cell lines.OBJECTIVE:To identify whether hematologic tumor cell lines contain SP cells,and to explore a stable detection and separation methods.METHODS:Cells with the characteristics of stem cells being capable of fluorescent dye Hoechst33342 were isolated by flow cytometry;whether there were SP cells in logarithmic growth period of NB4,Raji,K562/ADM and K562 or not were detected.After sorting K562 subpopulations,the purity of sorted cells was detected.RESULTS AND CONCLUSION:After Hoechst33342 staining,flow cytometry results showed that the SP cells appeared in the haematological tumor NB4,Raji,K562/ADM and K562 cell lines,which accounted for 0.8%,2.7%,1.3% and 2.7%,respectively.These cells could be blocked by Verapamil.The purity was greater after a second detection of SP and Non SP cells in K562 cells.

Objective To study the clinical and prognostic value of CK19 mRNA-positive circulating tumor cells in early breast cancer patients. Methods We analyzed the peripheral blood in 50 patients with early breast cancer after surgery and before the initiation of any adjuvant treatment for the presence of CK19 mRNA-positive circulating tumor cells using a nest reverse polymerase chain reaction assay. All patients were followed up. Results CK19 mRNA-positive cells were detected in 40.0 %(20/50) of patients with early breast cancer, 12.5 %(3/24) of patients with breast benign lesions, but 5 %(1/20) in healthy individuals (P =0.017,P =0.004); 11 to 20 of them relapsed during the follow-up period (P =0.002). There was no significant association between the detection of CK19 mRNA-positive cell and the patients' menstrual status, tumor stage, tumor size, etc (P ＞0.05). Detection of peripheral-blood CK19 mRNA-positive cells was associated with reduced median relapse-free interval in early breast cancer patients (P =0.007). Conclusion CK19 mRNA is one of the molecular markers for the detection of circulating tumor cells in early breast cancer. Detection of peripheral blood CK19 mRNA-positive cells might be an important predictive value as a marker of relapse in early breast cancer patients.

Objective To study the effect of histone deacetylase inhibitors on cell cycle of breast cancer cell line MCF-7.Methods The breast cancer cell line MCF-7 was treated by histone deacetylase inhibitors (TSA、SAHA、CS055、MS-275)respectively and observed by MTT assay after 24 hours,48 hours,72 hours,96 hours.The chosen inhibitor was used to treat MCF-7 cell in different concentration.The flow cytometry was used to detect the Sphase cell and Cyclin A2,Cyclin D1.SPSS 10.0 was used to analyze the data.Results Within the four inhibitors,SAHA showed the most powerful effect of depression of cell growth and time-effect relation.The percentage of Sphase cells and level of Cyclin A2 decreased,the level of Cyclin D1 increased.Conclusions SAHA is the most powerful histone deacetylase inhibitors for breast cancer cell MCF-7,the effect of depression of cell growth shows time-effect relation.Cyclin A2 and Cyclin D1 were involed in this regulation of cell cycle.

Objective To investigate the expression of cellular chemokine CCL22 and its receptor CCR4, as well as its clinical significance in systemic lupus erythematosus (SLE), along with its roles in the pathogenesis of this disease. Methods Forty-eight patients with SLE and 26 normal human controls were recruited into this study. The patient cohort included 2 males and 46 females with an average age of 33.98± 12.73 years and disease course of 1 month to 20 years. Blood samples were collected from the subjects. ELISA and flow cytometry were used to examine the plasma concentration of CCL22 together with the CCR4 expression on peripheral blood cells. SLEDAI was applied to evaluate the severity of SLE patients. Results The plasma concentration of CCL22 was 227.03±122.84 ng/L in SLE group, 369.53±79.10 ng/L in the control group, 168.09±61.83 ng/L in patients with lupus nephritis and 292.77±163.45 ng/L in patients without lupus nephritis; there was a significant difference between the SLE patients and normal con-trols (P < 0.05) as well as between patients with lupus nephritis and those without (P < 0.05). Increased percentage of CCR4-expressing cells were observed in the peripheral blood of patients with SLE compared with the controls (20.24%±13.86% vs 10.44%±3.07%, P < 0.01), and the percentage of CD3+CCR4+ cells was significantly higher than that of CD3-CCR4+ cells. Moreover, a decrease was noted in the plasma con-centration of CCL22 in severe patients (P < 0.05). In SLE patients, the percentage of CCR4 increased with the rise in SLEDAI score, whereas the plasma concentration of CCL22 negatively correlated with SLEDAI score (r = -0.308, P < 0.05). Conclusion CCL22/CCR4 may play a certain role in the development, pro-gression and organ involvement in SLE.

BACKGROUNDIt has been known that vascular endothelial growth factor (VEGF) and its receptor (VEGFR2) play important roles in tumor angiogenesis. The aim of this study is to investigate whether an oral DNA vaccine against VEGFR2 has the inhibition effect on tumor growth and angiogenesis, and explore its mechanism in vivo.

METHODSC57BL/6 mice were respectively given the DNA vaccine encoding VEGFR2 (vaccine group), pcDNA3.1 (plasmid group) and saline (saline group). All the mice were then inoculated with Lewis lung carcinoma 3LL cells. Weight, size and microvessel density (MVD) of transplanted tumors were observed. The levels of CD3+ and CD8+ T cells in peripheral blood of mice were detected by flow cytometry.

RESULTSWeight of transplanted tumors in vaccine group was significantly smaller than those in plasmid and saline groups (P < 0.05), and MVD was significantly lower in vaccine group than that in plasmid and saline groups (P < 0.05). After inoculated with 3LL cells, CD3+ and CD8+ T cell levels of vaccine group were markedly higher than those of plasmid and saline groups (P < 0.05).

CONCLUSIONSThe oral DNA vaccine can significantly inhibit angiogenesis and growth of transplanted tumor in mice. It may act through killing endothelial cells of tumor.