Improved laparoscopic transanal pull-through technique for low-rectal cancer resection

Objective： We described the applicability and evaluated the advantages of improved laparoscopic transanal pull-through （ILTPT） for low-rectal cancer resection. Materials： ILTPT was performed in 4 patients. Five or 4 ports were used. After isolation and section of the inferior mesenteric vessels, the rectum and sigmoid colon was mobilized. Total mesorectal excision and dissection of the distal rectum from the puborectalis muscle was carried out under laparoscopic guidance. The sigmoid colon and rectum were exteriorized via the anus. The rectum was divided proximally. Next, a purse-string suture was placed in the proximal segment, and the distal end of the sigmoid colon was returned to the pelvic cavity. The distal rectum was divided with Curved cutter staplermade by Jonson-Jonson company. Dislodging specimen, the continuity of the intestinal tract was restored using PROXIMATE ILS Curved and Straight Intraluminal Staplers CDH29/33 （Ethicon） through the rectum. Results： None of the cases were converted to open surgery. Average operation time was 180 min （range, 160-210 min）. No blood loss or any other complications were noted. Average postoperative stay was 9 days. Complications such as necrosis, anastomotic leakage and stricture, and genitoudnary disorders were not found in any of the patients at the 1 m follow-up. Conclusion： This report suggests that ILTPT is feasible and safe in Anus-Conserving Operation for low Rectal Cancer without auxiliary incision. If only we hold the applicability of ILTPT less trauma, more beautiful.

Cytarabine and paclitaxel exhibit different cell-cycle specificities in different cell growing status

Objective： To investigate the cell-cycle specificities of cytarabine and paclitaxel in different growing status of target cell. Methods： Using flow cytometry, we tested the cell-cycle specificities of cytarabine and paclitaxel on acute lymphocyte leukemia cell line Molt-4 in different growing status and on clinical acute lymphocyte leukemia specimens in vitro as well as in leukemia patients in vivo. Results： Cytarabine induced S phase specific cebcyde blockage and apoptosis in exponentially growing Molt-4, but showed G0/G1 phase specificity in high-density cultured Molt-4 and in clinical specimens. Paclitaxel induced G2/M phase specific cell-cycle blockage and apoptosis in exponential Molt-4, but showed G0/G1 phase specificity in high-density cultured Molt-4 and S phase specificity in clinical specimens. In the first day of clinical chemotherapy, cytarabine induced G0/G1 with a little S phase apoptosis in leukemia cells of acute lymphocyte leukemia patient in vivo. Cytarabine plus paclitaxel together had almost the same effect in the second day. Conclusion： The cell-cycle effects of cytarabine and paditaxel were different in different target cell growing status. It should be noted that the in vivo effect of these agents may be different from people usually anticipated during clinical chemotherapy. So the combined chemotherapeutic regimens may need to be redesigned.

TNF-α induces apoptosis of Molt-4 cells and cell cycle specificity of Bcl-2 phosphorylation

Objective： The aim of the study was to observe the expression of Bcl-2 and its phosphorylation in Molt-4 cells induced by tumor necrosis factor-α （TNF-α）, and to investigate the possible mechanism of cell cycle specificity of apoptosis. Methods： Exponentially growing Molt-4 cells were treated with TNF-α. Apoptosis was detected by DNA fragmentation assay. API method was applied to illustrate the cell cycle specificity of apoptotic cells. Cells of sub-phases were sorted by FACSvan- tage flow cytometer and then submitted to immunoblot. Results： Molt-4 cells which were treated with TNF-α went to apoptosis and showed a DNA ladder pattern. Most apoptosis happened in Gl-phase of cell cycle. Bcl-2 expression increased for the Molt-4 cells treated with TNF-α. The phosphorylation state of Bcl-2 was only presented in Gl-phase cells, in accordance with the specified time and cell cycle phase of apoptosis. Conclusion： The phosphorylation of Bcl-2 in the Molt-4 cells treated with TNF-α happened with the same cell cycle specificity as cell apoptosis. The cell cycle specificity of Bcl-2 phosphorylation was one of the mechanisms of receptor-mediated apoptosis. The cell cycle machine can trigger the apoptosis program.

Clinical significance of the negative lymph node count after the axillary dissection of breast cancer patients

Objective： The purpose of this study was to evaluate the impact of the negative lymph node （LN） count on the survival of the breast cancer patients in early stage after the axillary dissection. Methods： The breast cancer patients with T1-2N0-1M0 stage between January 2001 and December 2005 in Jiangsu Cancer Hospital, who underwent the axillary LNs dissection, were enrolled in this study. We analyzed the data of these patients including information of follow-up and postop- erative pathological results. All patients were divided into two groups according to the axillary LN status and each group was divided into four subgroups according to the negative LN count. Cox regression analysis was performed to screen the patho- logical factor including the negative LN count on the survival and to compare the different negative LN count on the survival. Results： COX proportional hazard regression model showed that the survival of the breast cancer was significantly associ- ated with the negative LN count. In T1 2N0 group, when the negative LN count was 3 or less, 4 to 5, 6 to 9 and 10 or more, the median survival time was （82.6 ±4.1） months, （101.5 ± 1.3） months, （104.7 ±1.0） months, and （110.5 ±0.9） months respectively （P 〈 0.05）. In T1-2N0 group, when the negative LN count was 6 or less, 7 to 8, 9 to 10 and 11 or more, the median survival time was （95.4 ± 1.9） months, （101.8 ± 1.1） months, （104.9 ± 1.0） months, and （106.5 ± 0.9） months respectively （P 〈 0.05）. Conclusion： The negative LN count can reflect the adequacy of the axillary dissection. Increasing negative LN count is independently associated with improved survival in pT1-2N0M0 or pT1-2N0M0 staging breast cancer patients. The negative LN count should be considered for incorporation into staging for breast cancer with the axillary LN dissection.

Study of sensitivity of different chemotherapeutic agents on colon cancer cell line SW480

Objective:The aim of this study was to investigate the sensitivity of chemotherapeutic agents 5-FU,cisplatin(DDP) or TAXOL on colon cancer cell line SW480 with different methods,to find out the best examine time period for further study of chemotherapeutic sensitivities.Methods:The SW480 cell was treated with 5-FU(200μg/mL),DDP(150μg/mL) or TAXOL(50μg/mL) respectively for 4h,8h or 12h.MTT assay was used to examine the cell survival rate,Annexin V/PI assay was used to analysis the apoptosis rate,Western Blot assay was applied to examine the expression of apoptotic protein.Results:(1) Results of MTT assay showed that the survival rate of SW480 cells at 4h,8h or 12h was:5-FU(200μg/mL)96.0%±8.2%,85.4%±7.8%,74.4% ±10.2%(P<0.05);DDP(150μg/mL) 99.0%±6.4%,88.7%±4.7%(P<0.05),46.9%±2.6%(P<0.01);TAXOL(50μg/mL) 51.5%±4.2%(P<0.01),31.9%±3.1%,17.6%±2.3%,or blank group 97.2%±5.8%,98.7%±7.2%,97.5%±7.5% respectively.(2) The apoptosis rate of cancer cells at 4h,8h or 12h was:control group:3.4%±0.2%,6.2%±0.4%,7.0%±0.5%;5-FU(200μg/mL) 4.0%±0.3%,4.8%±0.4%,7.7%±0.7%;DDP(150μg/mL) 8.5%±0.9%,18.6%±1.6%(P<0.05),67.0%±6.2%(P<0.01);or TAXOL(50μg/mL) 32.0%±5.2%(P<0.01),76.6%±8.5%,94.0% ±8.2%,respectively.(3) Western Blot assay showed that the expression of apoptosis associated protein.PARP,X-linked inhibitor of apoptasis(XIAP),Caspase-9 and Bcl-xL were changed.Conclusion:The sensitivity of chemotherapy could be assessed by MTT assay,Annexin V/PI assay and Western Blot.The best examine time of the three chemotherapuc agents was 5-FU(200μg/mL):>12h,DDP(150μg/mL):8-12h,or TAXOL(50μg/mL):<4h.

Epidermal growth factor enhances chemosensitivityof colon cancer by inducing cancer stem cells toenter the cell cycle

Objective The aim of the study was to investigate whether colon cancer stem cells induced by epidermal growth factor(EGF) to enter the cell cycle enhanced the chemosensitivity of colon cancer.Methods In vitro,EGF was used to stimulate the entry of human colon cancer HCT116 cells into the cell cycle.Before and after treatment with EGF,CD133+ HCT116 cells were collected and flow cytometry was conducted to determine the apoptosis rate based on the 5-Fu and Ki-67 expression rates.The cell cycle distribution of the two groups was also determined.In vivo,a subcutaneous xenograft model of HCT116 human colon cancer cell lines in nude mice was established.The nude mice were divided into two groups and treated with EGF and 5-Fu,respectively.Differences in the growth of implanted tumors revealed the efficiency of cycle-induction combined chemotherapy.Results(1) After EGF stimulation,the S-G2/M proportion of CD133+ HCT116 cells and Ki67 expression were increased,indicating that more cancer stem cells entered the cell cycle and promoted proliferation;(2) After EGF stimulation,CD133+ HCT116 cells showed a higher apoptosis rate induced by 5-Fu.(3) Animal experiments showed that the group subjected to combined treatment with EGF and 5-Fu had smaller tumor sizes compared to the group treated with 5-Fu alone.Conclusion EGF enhanced tumor sensitivity to chemotherapeutic drugs,likely by promoting tumor stem cells to enter the cell cycle.

Therapeutic potential of stem cell in liver regeneration

Liver transplantation is the only life-saving procedure for patients with end-stage liver disease.However,its potential benefits are hampered by many disadvantages,such as the relative shortage of donors,operative risks,and high costs.These issues have prompted the search for new alternative therapies for irreversible liver disease.Stem cell therapy,with the ability for self-renewal and potential for multilineage differentiation,is a promising alternative approach.Several studies have demonstrated that transplantation of hepatic stem/progenitor cells or hepatocyte-like cells derived from multipotent stem cells leads to donor cell-mediated repopulation of the liver and improved survival rates in experimental models of liver disease.However,a registered clinical application based on stem cell technology will take at least an additional 5 to 10 years because of some limitations;e.g.the lack of suitable cell sources and risk of teratoma formation.This review summarizes the general understanding of the therapeutic potentials of stem cells in liver disease,including the sources,mechanisms,and delivery methods of hepatic stem cells in liver regeneration,and discusses some challenges for their therapeutic application.

Effect of pEGFP-survivin on GBC-SD cell growth and chemotherapy sensitivity

This paper is aimed to investigate the effect of survivin shRNA on chemotherapy resistance in human gallbladder carcinoma GBC-SD cells.The viability of human gallbladder carcinoma GBC-SD,GBC-SD/enhanced greenfluorescent protein(EGFP),and GBC-SD/survivin cells was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,and mRNA and protein of survivin were tested by reverse transcription-polymerase chain reaction(RT-PCR)and Western blot.After the cells were treated with cisplatin(DDP)(3.0μg/mL)for the same time,cell survival rate and IC50 was detected with MTT,cell apoptosis was detected withfluorescence-activated cell sorting(FACS),and the nuclear alteration was observed by TdT-mediated deox-yuridine triphosphate nick end labeling(TUNEL).In addition,caspase-3 activity was detected by using colorimetric method.Cell viability was decreased sig-nificantly in GBC-SD/survivin cells,and survivin expres-sion was decreased significantly(mRNA and protein of survivin were decreased by 74.7%and 71.5%,respec-tively).After treatment with DDP,cell survival rate and IC50 was decreased significantly(2.03�0.24μg/mL)in GBC-SD/survivin cells,while apoptotic rate(84.3%)was elevated significantly as compared with the other two groups.There were brown apoptotic nuclei in all the cells.Caspase-3 activity in all the cells was increased atfirst and then decreased,but the caspase-3 activity in GBC-SD/survivin cells was significantly higher than the other two groups.The survivin shRNA could down-regulate the expression of survivin in GBC-SD cells significantly and improve the sensibility to chemotherapy.

The role of CDK1 siRNA interference in cell cycle and cell apoptosis

In the present report,cyclin-dependent kinase1(CDK1)siRNA was transfected into cells to silence the CDK1 gene expression and study its role in the cell cycle and cell apoptosis.The siRNA targeting CDK1 gene was chemically synthesized and transfected into Hela cells by lipofectamine 2000.The expression levels of CDK1 gene and protein were examined by real-time quantitative polymerase chain reaction(PCR)and Western blot,respectively.The cell cycle was analyzed by using DNA content analysis byflow cytometry.Cell apoptosis was detected by the Annexin V/PI method.The morphological changes of transfected cells were examined under the microscopy by Wright-Giemsa stain.CDK1 gene was successfully silenced by its siRNA,and the CDK1 protein expression level was decreased significantly,especially from 48th h to 60th h after transfection.The DNA content analysis showed that transfection of CDK1 siRNA led to cells accumulating in G2/M phase.There was no significant difference in the apoptotic rate between transfected cells and the control cells after transfection of CDK1 siRNA for 48 or 60 h.More double nucleus or multinucleus cells could be seen under the microscopy among the transfected cells.The decreased CDK1 expression by siRNA silencing gave rise to cell cycle arrest in G2/M phase but did not induce apoptosis.

Novel applications of next-generation sequencing in breast cancer research

With the rapid development of medicine,the studies of genes have become increasingly concerned by more people and being the contend of a great of researches.The next generation sequencing with its own advantages has been widely used in gene research nowadays.It has almost replaced the traditional sequencing methods(such as Sanger sequencing method),and played an important role in a variety of complex disease researches,including breast cancer.The next generation sequencing technology has the advantages of high speed,high throughput and high accuracy.It has been widely used in various cancers(such as prostate cancer,lung cancer,pancreatic cancer,liver cancer,etc.),especially in breast cancer.Moreover,the use of the next generation sequencing technology to make DNA sequence analysis and risk prediction has made a great contribution to the research of breast cancer.We will focus on the application of whole genome sequencing,exon sequencing and targeted gene sequencing in breast cancer gene research.