Immunohistochemical study of nuclear factor-κB activity in colorectal cancer

Objective: The aim of the study was to explore the protein level of NF-κB change in carcinoma and different grades of tumor cells differentiation tissue in colorectal cancer patients. Methods: This was a comparative study between normal and carcinoma tissues and in different grades of tumor cells differentiation tissue in colorectal cancer patients. Ex-pression of NF-κΒ were assessed by immunohistochemical method using rabbit polyclonal antibodies against human p65 NF-κΒ proteins. Results: There was none or very little expression of NF-κB in non-neoplastic colon epithelial cells, while the expression of it's protein was significantly increased (P < 0.01) in adjacent cancerous cells. Moreover, there was a significant increase in the mean expression of NF-κB-p65 between poorly differentiated malignant epithelial cell and well-differentiated cells (P < 0.05). Conclusion: NF-κB-p65 may play an essential role in colorectal carcinogenesis and may be valuable for diagnosis, evaluating malignancy extent and prognosis.

Stage II/III Rectal Cancer Patients Benefit from Extremely Early Initiation of Postoperative Adjuvant Chemotherapy: A Retrospective Study

Background: For Stage II/III rectal cancer patients, curative resection is the primary treatment, prescribing of postoperative adjuvant chemotherapy (PAC) is regarded as a standard therapy. The interval between surgery and the initiation of PAC is usually within 8 weeks. However, the optimal cut-off is still controversial. This study aimed to explore the impact of extremely early initiation of PAC for II/III rectal cancer. Methods: Patients with Stage II/III rectal cancer treated from January 2013 to December 2015 were retrospectively collected at the Department of Tongji Hospital. According to the starting point of PAC, patients were categorized into two groups: extremely early group (The interval of PAC ≤ 2 weeks) and normal group (The interval of PAC within 3 - 5 weeks). For the sake of evaluating the effectiveness of different intervals, Overall Survival rate (OS), Progress-Free Survival rate (PFS) and Recurrence or Metastasis Rate (RMR) were analyzed, as well as the Quality of Life Score. To estimate the safety of the extremely early PAC, we evaluated the first post chemotherapy adverse reactions and defecation ability, and analyzed the variance laboratory indexes around the first postoperative adjuvant chemotherapy. Results: A total of 267 patients were included in this study. Compared to normal group (192 cases), extremely early group (75 cases) of patients attained a better tendency of OS and PFS, although there were no significant statistical differences (OS: P = 0.0930;PFS: P = 0.1058). However, the RMR was significant lower (P = 0.0452) and the Quality of Life Score was significantly higher (P = 0.0090) in extremely early group. Multivariate analysis also showed that extremely early group had better defecation ability (P = 0.0149) and less side reactions of post chemotherapy, such as vomiting (P , got a higher level of inflammatory cells (P Conclusion: For Stage II/III rectal cancer patients, extremely early to start PAC not only might be effectively prolonging the survival, but indeed decrease the tumor-related recurrence risk, increase the quality of life and decrease chemotherapy-associated adverse reactions. Meanwhile, appropriately controlling of inflammatory cells and protecting the liver function should be of concern to ensure the safety of early initial stage.

Postoperative sequential chemotherapy and radiotherapy for locally advanced gastric cancer

Objective The aim of the study was to evaluate the role of postoperative sequential chemotherapy and radiotherapy in patients with locally advanced gastric cancer.Methods From January 2003 to December 2010, 146 gastric cancer patients at our institution(Department of Oncology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China) received postoperative sequential chemotherapy and radiotherapy after radical surgery. Radiotherapy was administered as a dose of 4500 cGy in 25 fractions. For patients with positive margins, the dose was raised to 5040 cGy in 28 fractions. Three cycles of m FOLFOX or PF(cisplatin, 5-fluorouracil) chemotherapy regimen were applied before and after radiotherapy. Three-and 5-year survival rates were analyzed; any adverse effects with respect to hematology, hepatic and renal function, or the gastrointestinal tract that occurred during the treatment were evaluated.Results This cohort consisted of non-metastatic patients: 104 men and 42 women with a median age of 51.0 years. The full course of sequential chemotherapy and radiotherapy(4500–5040 cGy) was completed by 129 patients(88.4%). Seventeen regional relapses(9.8%) and 46 distant relapses(23.8%) were recorded. Fifty patients(34.2%) died during follow-up. The 3-and 5-year overall survival rates(OS) were 60% and 54%, and disease-free survival rates(DFS) were 53% and 47%, respectively. There were no significant differences in survival rate with respect to age, sex, histopathology, N stage, site of the tumor, or margin status. Multivariate analysis showed that only the depth of tumor invasion(T stage) was an independent prognostic factor for OS(P = 0.009) and DFS(P = 0.006). The rates of grades 3 and 4 neutropenia and vomiting were 9.6% and 3.4%, respectively, during the treatment.Conclusion Postoperative sequential chemotherapy with an m FOLFOX or PF regimen and radiotherapy were found to be an effective means of treating advanced gastric cancer patients with T3–T4 disease. The adverse effects of this treatment were tolerable.

Discrimination of cervical cancer cells via cognition-based features

Computer-assisted cervical screening is an effective method to save the doctors'workload and improve their work efficiency.Usually,the correct classification of cervical cells depends on the nuclear segmentation effect and the extraction of nuclear features.However,the precise nucleus segmentation remains a huge challenge,especially for densely distributed nucleus.Moreover,previous cellular classification methods are mostly based on morphological features of nucleus size or color.Those individual features can make accurate classification for severe lesions,but not for mild lesions.In this paper,we propose an accurate instance segmentation algorithm and propose cognition-based features to identify cervical cancer cells.Different from previous individual nucleus features,we also propose population features and cognition-based features according to the Bethesda System(TBS)for reporting cervical cytology and the diagnostic experience of the cytologists.The results showed that the segmentation achieves better success in complex situa-tions than that by traditional segmentation algorithms.Besides,the cell classification via cog-nition-based features also help us find out more about less severe lesions'nuclei than that based on conventional features of individual nucleus,meaning an improvement of classification accu-racy for cervical screening.

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.

Overexpressed miR-9 promotes tumor metastasis via targeting E-cadherin in serous ovarian cancer

MicroRNAs （miRNAs） play critical roles in the development and progression in various cancers. Dysfunctional miR-9 expression remains ambiguous, and no consensus on the metastatic progression of ovarian cancer has been reached. In this study, results from the bioinformatics analysis show that the 3＇-UTR of the E- cadherin mRNA was directly regulated by miR-9. Luciferase reporter assay results confirmed that miR-9 could directly target this 3＇-UTR. miR-9 and E-cadherin expression in ovarian cancer tissue was quantified by qRT- PCR. Migration and invasion were detected by wound healing and Transwell system assay in SKOV3 and A2780. qRT-PCR and Western blot were performed to detect the epithelial-mesenchymal transition-associated mRNA and proteins. Immunofluorescence technique was used to analyze the expression and subcellular localization of E- cadherin, N-cadherin, and vimentin. The results showed that miR-9 was frequently upregulated in metastatic serous ovarian cancer tissue compared with paired primary ones. Upregulation of miR-9 could downregulate the expression of E-cadherin but upregulate the expression of mesenchymal markers （N-cadherin and vimentin）. Overexpression of miR-9 could promote the cell migration and invasion in ovarian cancer, and these processes could be effectively inhibited via miR-9 inhibitor. Thus, our study demonstrates that miR-9 may promote ovarian cancer metastasis via targeting E-cadherin and a novel potential therapeutic approach to control metastasis of ovarian cancer.

Serum semaphorin 4C as a diagnostic biomarker in breast cancer:A multicenter retrospective study

Background:To date,there is no approved blood-based biomarker for breast cancer detection.Herein,we aimed to assess semaphorin 4C(SEMA4C),a pivotal protein involved in breast cancer progression,as a serum diagnostic biomarker.Methods:We included 6,213 consecutive inpatients from Tongji Hospital,Qilu Hospital,and Hubei Cancer Hospital.Training cohort and two validation cohorts were introduced for diagnostic exploration and validation.A pan-cancer cohort was used to independently explore the diagnostic potential of SEMA4C among solid tumors.Breast cancer patients who underwent mass excision prior to modified radical mastectomy were also analyzed.We hypothesized that increased pretreatment serum SEMA4C levels,measured using optimized in-house enzymelinked immunosorbent assay kits,could detect breast cancer.The endpoints were diagnostic performance,including area under the receiver operating characteristic curve(AUC),sensitivity,and specificity.Post-surgery pathological diagnosis was the reference standard and breast cancer staging followed the TNM classification.There was no restriction on disease stage for eligibilities.Results:We included 2667 inpatients with breast lesions,2378 patients with other solid tumors,and 1168 healthy participants.Specifically,118 patients with breast cancer were diagnosed with stage 0(5.71%),620 with stage I(30.00%),966 with stage II(46.73%),217 with stage III(10.50%),and 8 with stage IV(0.39%).Patients with breast cancer had significantly higher serum SEMA4C levels than benign breast tumor patients and normal controls(P<0.001).Elevated serum SEMA4C levels had AUC of 0.920(95%confidence interval[CI]:0.900–0.941)and 0.932(95%CI:0.911–0.953)for breast cancer detection in the two validation cohorts.The AUCs for detecting early-stage breast cancer(n=366)and ductal carcinoma in situ(n=85)were 0.931(95%CI:0.916–0.946)and 0.879(95%CI:0.832–0.925),respectively.Serum SEMA4C levels significantly decreased after surgery,and the reduction was more striking after modified radical mastectomy,compared with mass excision(P<0.001).The positive rate of enhanced serum SEMA4C levels was 84.77%for breast cancer and below 20.75%for the other 14 solid tumors.Conclusions:Serum SEMA4C demonstrated promising potential as a candidate biomarker for breast cancer diagnosis.However,validation in prospective settings and by other study groups is warranted.

Threonine 32 （Thr32） of FoxO3 is critical for TGF-β-induced apoptosis via Bim in hepatocarcinoma cells

Transforming growth factor-β （TGF-β） exerts apoptotic effects on various types of malignant ceils, including liver cancer cells. However, the precise mechanisms by which TGF-β induces apoptosis remain poorly known. In the present study, we have showed that threonine 32 （Thr32） residue of FoxO3 is critical for TGF-β to induce apoptosis via Bim in hepatocarcinoma Hep3B cells. Our data demonstrated that TGF-βinduced FoxO3 activation through specific de-phosphorylation at Thr32. TGF-β-activated FoxO3 cooperated with Smad2/ 3 to mediate Bim up-regulation and apoptosis. FoxO3 （de）phosphorylation at Thr32 was regulated by casein kinase I-ε （CKI-E）. CKI inhibition by small molecule D4476 could abrogate TGF-β-induced FoxO/Smad acti- vation, reverse Bim up-regulation, and block the sequential apoptosis. More importantly, the deregulated levels of CKI-ε and p32FoxO3 were found in human malignant liver tissues. Taken together, our findings suggest that there might be a CKI-FoxO/Smad-Bim engine in which Thr32 of FoxO3 is pivotal for TGF-β- induced apoptosis, making it a potential therapeutic target for liver cancer treatment.

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.