Identification of new fusion genes and their clinical significance in endometrial cancer / 中华医学杂志(英文版)

BACKGROUND@#Fusion genes may play an important role in tumorigenesis, prognosis, and drug resistance; however, studies on fusion genes in endometrial cancer (EC) are rare. This study aimed to identify new fusion genes and to explore their clinical significance in EC.@*METHODS@#A total of 28 patients diagnosed with EC were enrolled in this study. RNA sequencing was used to obtain entire genomes and transcriptomes. STAR-comparison and STAR-fusion prediction were applied to predict the fusion genes. Chi-square tests and Student t tests were used to verify the clinical significance with SPSS 13.0 software.@*RESULTS@#New fusion genes were found, and the number of fusion genes varied from 3 to 110 among all patients with EC. The type of fusion genes varied and included messenger RNA (mRNA)-mRNA, long non-coding RNA (lncRNA)-lncRNA, and lncRNA-mRNA. There were six fusion genes with high fusion rates, namely, RP11-123O10.4-GRIP1, RP11-444D3.1-SOX5, RP11-680G10.1-GSE1, NRIP1-AF127936.7, RP11-96H19.1-RP11-446N19.1, and DPH7-PTP4A3. Further studies showed that these fusion genes are related to stage, grade, and recurrence, in which NRIP1-AF127936.7 and DPH7-PTP4A3 were found only in stage III patients with EC. DPH7-PTP4A3 was found in grades 2 and 3, and recurrent patients with EC.@*CONCLUSION@#Fusion genes play an essential role in EC. Six genes that are overexpressed with high fusion rates are identified. NRIP1-AF127936.7 and DPH7-PTP4A3 might be related to stage, and DPH7-PTP4A3 be related to grade and recurrence.

Intra-tumor heterogeneity for endometrial cancer and its clinical significance / 中华医学杂志(英文版)

BACKGROUND@#Management of tumors has become more complex owing to tumor heterogeneity. Fewer studies have been performed on intra-tumor heterogeneity of endometrial cancer (EC) until now. Therefore, it is of great clinical value to explore the intra-tumor heterogeneity of EC based on clinical features and gene expression profiles.@*METHODS@#A total of 1688 patients with EC were screened and 114 patients were finally selected, including specimens from 84 patients with primary EC without relapse (PE) and the paired metastases (P-M) specimens, as well as specimens from 30 patients with primary EC with relapse (RPE) and the paired relapsed EC (P-RE) specimens. Microarray and RNA-seq were used to detect gene expression of EC samples. Clinicopathological characteristics and molecular data were compared between PE and P-M groups and between RPE and P-RE groups to explore the intra-tumor heterogeneity of EC.@*RESULTS@#The clinical intra-tumor spatial heterogeneity of pathological type, grade, ER status, and PR status between PE and P-M were 17.9%, 13.1%, 28.6%, and 28.6%, respectively. The clinical intra-tumor spatiotemporal heterogeneity of pathological type, grade, ER status, and PR status between RPE and P-RE were 16.7%, 33.3%, 25.0%, and 37.5%, respectively. Cluster analysis sorts EC samples based on progression type of lesion and their pathological type. There were differentially expressed genes between PE and P-M and between RPE and P-RE, of which gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis were mainly enriched in cell proliferation, the p53 signaling pathway, etc. CONCLUSIONS:: Clinical and molecular data showed that there was spatiotemporal heterogeneity in intra-tumor of EC, which may add to the complexity of diagnosis and therapeutics for EC. Considering the intra-tumor heterogeneity, sequential chemotherapy and precision medicine may be a more suitable treatment plan for EC.

Intra-tumor heterogeneity for endometrial cancer and its clinical significance / 中华医学杂志(英文版)

Background@#Management of tumors has become more complex owing to tumor heterogeneity. Fewer studies have been performed on intra-tumor heterogeneity of endometrial cancer (EC) until now. Therefore, it is of great clinical value to explore the intra-tumor heterogeneity of EC based on clinical features and gene expression profiles.@*Methods@#A total of 1688 patients with EC were screened and 114 patients were finally selected, including specimens from 84 patients with primary EC without relapse (PE) and the paired metastases (P-M) specimens, as well as specimens from 30 patients with primary EC with relapse (RPE) and the paired relapsed EC (P-RE) specimens. Microarray and RNA-seq were used to detect gene expression of EC samples. Clinicopathological characteristics and molecular data were compared between PE and P-M groups and between RPE and P-RE groups to explore the intra-tumor heterogeneity of EC.@*Results@#The clinical intra-tumor spatial heterogeneity of pathological type, grade, ER status, and PR status between PE and P-M were 17.9%, 13.1%, 28.6%, and 28.6%, respectively. The clinical intra-tumor spatiotemporal heterogeneity of pathological type, grade, ER status, and PR status between RPE and P-RE were 16.7%, 33.3%, 25.0%, and 37.5%, respectively. Cluster analysis sorts EC samples based on progression type of lesion and their pathological type. There were differentially expressed genes between PE and P-M and between RPE and P-RE, of which gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis were mainly enriched in cell proliferation, the p53 signaling pathway, etc.@*Conclusions@#Clinical and molecular data showed that there was spatiotemporal heterogeneity in intra-tumor of EC, which may add to the complexity of diagnosis and therapeutics for EC. Considering the intra-tumor heterogeneity, sequential chemotherapy and precision medicine may be a more suitable treatment plan for EC.

Identification of new fusion genes and their clinical significance in endometrial cancer / 中华医学杂志(英文版)

Background@#Fusion genes may play an important role in tumorigenesis, prognosis, and drug resistance; however, studies on fusion genes in endometrial cancer (EC) are rare. This study aimed to identify new fusion genes and to explore their clinical significance in EC.@*Methods@#A total of 28 patients diagnosed with EC were enrolled in this study. RNA sequencing was used to obtain entire genomes and transcriptomes. STAR-comparison and STAR-fusion prediction were applied to predict the fusion genes. Chi-square tests and Student t tests were used to verify the clinical significance with SPSS 13.0 software.@*Results@#New fusion genes were found, and the number of fusion genes varied from 3 to 110 among all patients with EC. The type of fusion genes varied and included messenger RNA (mRNA)-mRNA, long non-coding RNA (lncRNA)-lncRNA, and lncRNA-mRNA. There were six fusion genes with high fusion rates, namely, RP11–123O10.4–GRIP1, RP11–444D3.1–SOX5, RP11– 680G10.1–GSE1, NRIP1–AF127936.7, RP11–96H19.1–RP11–446N19.1, and DPH7–PTP4A3. Further studies showed that these fusion genes are related to stage, grade, and recurrence, in which NRIP1–AF127936.7 and DPH7–PTP4A3 were found only in stage III patients with EC. DPH7–PTP4A3 was found in grades 2 and 3, and recurrent patients with EC.@*Conclusion@#Fusion genes play an essential role in EC. Six genes that are overexpressed with high fusion rates are identified. NRIP1– AF127936.7 and DPH7–PTP4A3 might be related to stage, and DPH7–PTP4A3 be related to grade and recurrence.

Scutellaria barbata D. Don inhibits colorectal cancer growth via suppression of Wnt/β-catenin signaling pathway / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the effect of the ethanol extract of Scutellaria barbata D. Don (EESB) on colorectal cancer (CRC) growth and Wnt/β-catenin signaling pathway in vivo and in vitro.</p><p><b>METHODS</b>In vivo experiment, CRC xenograft mouse model was constructed with injection of HT-29 cells. Following xenograft implantation, twenty mice were randomly divided into EESB-treated group (n=10) and control group (n=10) by a random number table, and were given with intra-gastric administration of 2 g/kg EESB or saline, 5 days a week for 16 days, respectively. At the end of experiment, tumors were removed and weighed by electronic scales. The proliferation biomarker Ki-67 of tumor was evaluated by immunohistochemistry (IHC) assay. In vitro study, HT-29 cells were treated with 0, 0.5, 1.5, 2.5 mg/mL EESB for 24 h. At the end of the treatment, the viability and survival of HT-29 cells were determined by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and colony formation assay, respectively. The mRNA expression of c-Myc, Survivin and adenomatous polyposis coli (APC) was examined by reverse transcription-polymerase chain reaction (RT-PCR) both in tumor tissues of CRC xenograft mice and HT-29 cells. Protein expression of c-Myc, Survivin, APC, and β-catenin as well as β-catenin phosphorylation level were evaluated by IHC assay or Western blotting.</p><p><b>RESULTS</b>EESB significantly reduced tumor weight in CRC xenografts mice, compared with the control group (P<0.05). IHC assay showed that EESB significantly inhibited protein expression of Ki-67 in tumor tissues (P<0.05). MTT assay showed that EESB significantly reduced HT-29 cell viability in a dose-dependent manner (P<0.05). Colony formation assay showed that EESB dose-dependently decreased the survival of HT-29 cells (P<0.05). In addition, RT-PCR assay showed that EESB decreased the mRNA expression of c-Myc and Survivin and increased APC expression, both in tumor tissues of CRC xenograft mice and HT-29 cells (P<0.05). IHC assay or Western blotting showed that EESB decreased protein expression of β-catenin, c-Myc and Survivin, as well as increased APC expression and β-catenin phosphorylation in tumor tissues or HT-29 cells (P<0.05).</p><p><b>CONCLUSIONS</b>EESB significantly reduced tumor growth in CRC xenografts mice, and inhibited the viability and survival of HT-29 cells. EESB could suppress the activation of the Wnt/β-catenin pathway, which might be one of the mechanisms whereby Scutellaria barbata D. Don exerts its anticancer activity.</p>

Low programmed cell death 5 expression is a prognostic factor in ovarian cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Ovarian cancer is a leading gynecological malignancy. We investigated the prognostic value of programmed cell death 5 (PDCD5) in patients with ovarian cancer.</p><p><b>METHODS</b>Expression levels of PDCD5 mRNA and protein were examined in six ovarian cancer cell lines (SKOV3, CAOV3, ES2, OV1, 3AO, and HOC1A) and one normal ovarian epithelial cell line (T29) using reverse transcription polymerase chain reaction, Western blotting, and flow cytometry. After inducing PDCD5 induction in SKOV3 cells or treating this cell line with taxol or doxorubicin (either alone or combined), apoptosis was measured by Annexin V-FITC/propidium iodide staining. Correlations between PDCD5 protein expression and pathological features, histological grade, FIGO stage, effective cytoreductive surgery, and serum cancer antigen-125 values were evaluated in patients with ovarian cancer.</p><p><b>RESULTS</b>PDCD5 mRNA and protein expression were downregulated in ovarian cancer cells. Recombinant human PDCD5 increased doxorubicin-induced apoptosis in SKOV3 cells (15.96 ± 2.07%, vs. 3.17 ± 1.45% in controls). In patients with ovarian cancer, PDCD5 expression was inversely correlated with FIGO stage, pathological grade, and patient survival (P < 0.05, R = 0.7139 for survival).</p><p><b>CONCLUSIONS</b>PDCD5 expression is negatively correlated with disease progression and stage in ovarian cancer. Therefore, measuring PDCD5 expression may be a good method of determining the prognosis of ovarian cancer patients.</p>

The Number of Positive Pelvic Lymph Nodes and Multiple Groups of Pelvic Lymph Node Metastasis Influence Prognosis in Stage IA-IIB Cervical Squamous Cell Carcinoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Pelvic lymph node metastasis (LNM) is an important prognostic factor in cervical cancer. Cervical squamous cell carcinoma accounts for approximately 75-80% of all cervical cancers. Analyses of the effects of the number of positive lymph nodes (LNs), unilateral versus bilateral pelvic LNM and a single group versus multiple groups of pelvic LNM on survival and recurrence of cervical squamous cell carcinoma are still lacking. The study aimed to analyze the effects of the number of positive pelvic LNs and a single group versus multiple groups of pelvic LNM on survival and recurrence.</p><p><b>METHODS</b>We performed a retrospective review of 296 patients diagnosed with Stage IA-IIB cervical squamous cell carcinoma who received extensive/sub-extensive hysterectomy with pelvic lymphadenectomy/pelvic LN sampling at Peking University People's Hospital from November 2004 to July 2013. Ten clinicopathological variables were evaluated as risk factors for pelvic LNM: Age at diagnosis, gravidity, clinical stage, histological grade, tumor diameter, lymph-vascular space involvement (LVSI), depth of cervical stromal invasion, uterine invasion, parametrial invasion, and neoadjuvant chemotherapy.</p><p><b>RESULTS</b>The incidence of pelvic LNM was 20.27% (60/296 cases). Pelvic LNM (P = 0.00) was significantly correlated with recurrence. Pelvic LNM (P = 0.00), the number of positive pelvic LNs (P = 0.04) and a single group versus multiple groups of pelvic LNM (P = 0.03) had a significant influence on survival. Multivariate analysis revealed that LVSI (P = 0.00), depth of cervical stromal invasion (P = 0.00) and parametrial invasion (P = 0.03) were independently associated with pelvic LNM.</p><p><b>CONCLUSIONS</b>Patients with pelvic LNM had a higher recurrence rate and poor survival outcomes. Furthermore, more than 2 positive pelvic LNs and multiple groups of pelvic LNM appeared to identify patients with worse survival outcomes in node-positive IA-IIB cervical squamous cell carcinoma. LVSI, parametrial invasion, and depth of cervical stromal invasion were identified as independent clinicopathological risk factors for pelvic LNM.</p>

Molecular subtypes identified by gene expression profiling in early stage endometrioid endometrial adenocarcinoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Early stage (FIGO stage I-II) endometrioid endometrial adenocarcinoma (EEA) is very common in clinical practice. However, patients with the early stage EEA show various clinical behaviors due to biological heterogeneity. Hence, we aimed to discover distinct classes of tumors based on gene expression profiling, and analyze whether the molecular classification correlated with the histopathological stages or other clinical parameters.</p><p><b>METHODS</b>Hierarchical clustering was performed for class discovery in 28 early stage EEA samples using a special cDNA microarray chip containing 492 genes designed for endometrial cancer. Correlations between clinicopathologic parameters and our classification were analyzed. And the significance analysis of microarrays (SAM) array was used to identify the signature genes according to the tumor grade and myometrial invasion.</p><p><b>RESULTS</b>Three tumor subtypes (subtypes I, II and III) were identified by hierarchical clustering, each subtype had different clinicopathological factors, such as tumor grade, myometrial invasion status, and FIGO stage. Moreover, SAM analysis showed 34 up-regulated genes in high grade tumors, and 38 up-regulated genes and 1 down-regulated in deep myometrial invasive tumors. The overlap genes between these two high-risk factors were markedly up-regulated in subtype I, but down-regulated in subtype III.</p><p><b>CONCLUSION</b>We have identified novel molecular subtypes in early stage EEA. Differential gene signatures characterize each tumor subtype, which could be used for recognizing the tumor risk and providing a basis for further treatment stratification.</p>

Value of T cell receptor gamma alternate reading frame protein and keratin 5 in endometrial carcinoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Tumors with different gene expression develop and progress in different ways. To deepen our understanding of the progression in endometrial cancer, and provide a useful tool for accurate diagnosis and prognosis assessment, we identified the new molecular prognostic markers in endometrial carcinoma and analyzed the relationship of them with clinical and pathological features of endometrial carcinoma.</p><p><b>METHODS</b>Ninety-four cases of endometrial endometrioid adenocarcinoma with complete data from the Peking University People's Hospital from 2000 to 2008 and 40 cases of normal endometrium were enrolled. Among these, 30 endometrial endometrioid adenocarcinoma samples of different International Federation of Gynecology and Obstetrics (FIGO) stage were selected for further Agilent genome-wide microarray analysis. Significance analysis of microarrays (SAM) was used to identify genes that are significantly associated with tumor progress. Immunohistochemistry was utilized to identify the genes of interest in endometrial carcinoma and normal endometrium. The relationship between the genes and the age, clinical stage, histological grade, myometrium invaded depth, lymph node metastasis status, and the expression of ER, PR, P53, and PTEN were analyzed by χ(2) test.</p><p><b>RESULTS</b>Analysis between FIGO 1988 stage I and stage III identified a 362-gene "progress signature"; 171 down-regulated and 191 up-regulated genes. Among the alterative genes, TARP (T cell receptor gamma alternate reading frame protein) and KRT5 (keratin 5) decreased 3.57 fold and 5.8 fold in FIGO stage III patients. The expression of TARP in endometrial carcinoma increased compared to normal endometrium, while that of KRT5 decreased (P < 0.05). The expression of TARP and KRT5 decreased when stage, histological grading, myometrium invaded depth increased (P < 0.05). In the cases with lymph node metastasis, the expression of TARP decreased, while the expression of KRT5 did not differ (both P < 0.05) both. The expression of P53 had a negative relationship with the expression of KRT5 (P < 0.05), but not with the expression of TARP (P > 0.05). There was no correlation between the expression of TARP and KRT5 and the expression of ER, PR, PTEN (all P > 0.05). There was no significant difference in TARP and KRT5 expression in patients aged 50 or younger and patients older than 50 (P > 0.05).</p><p><b>CONCLUSION</b>The expression of TARP and KRT5 was correlated with the progress of endometrial cancer and their role needs further study.</p>

Oxaliplatin-based combination chemotherapy is still effective for the treatment of recurrent and platinum-resistant epithelial ovarian cancer: results from a single center / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Combination paclitaxel and carboplatin is currently a first-line regimen for ovarian cancer. However, many patients develop tumor recurrence or drug resistance to this regimen. The study aims to investigate the effectiveness and safety of an oxaliplatin + epirubicin + ifosfamide regimen for the treatment of recurrent and drug-resistant epithelial ovarian cancer.</p><p><b>METHODS</b>A retrospective analysis of 73 patients with recurrent and drug-resistant ovarian cancer was performed; 38 cases of them received oxaliplatin + epirubicin + ifosfamide regimens (IAP group), 35 patients received non-oxaliplatinbased chemotherapy regimens (control group). The therapeutic effects and side effects of the oxaliplatin + epirubicin + ifosfamide regimen were analyzed and summarized. Kaplan-Meier survival curves and Cox proportional hazards regression were used to compare progression-free and overall survival between the two groups.</p><p><b>RESULTS</b>Of the 38 patients in the IAP group, 14 patients (36.84%) achieved complete remission, 12 (31.58%) achieved partial remission, 2 (5.26%) achieved stable disease and 10 (26.32%) developed progressive disease. The overall effective rate (complete or partial remission) of the IAP regime was 68.42%. While, of the 35 patients in the control group, 12 patients (34.29%) achieved complete remission, 3 (8.57%) achieved partial remission, 5 (14.29%) achieved stable disease and 15 (42.86%) developed progressive disease. The overall effective rate was 42.86%, which was lower than that in the IAP group (P = 0.035, χ(2) = 4.836). Progression-free survival was 9.5 months (0-64 months) in the IAP group vs. 3 months (0-74 months) in the non-oxaliplatin group (P = 0.014 by Kaplan-Meier survival curves; HR = 2.260; 95%CI 1.117-4.573; P = 0.023 by Cox proportional hazards regression). Median overall survival was 46 months (9-124 months) in the IAP group vs. 35 months (9-108 months) in non-oxaliplatin group (P = 0.018 by Kaplan-Meier survival curves; HR = 2.272; 95%CI 1.123-4.598; P = 0.022 by Cox proportional hazards regression). In IAP group, 15.79% (6/38) of the patients suffered grade III-IV bone marrow arrest. The main non-hematological side effects of the IAP regimen included nausea and vomiting (21.05%, 8/38), peripheral neurotoxicity (15.79%, 6/38) and hepatic or renal lesions (2.63%, 1/38). The main side effects of the two chemotherapy regimens showed no statistical difference.</p><p><b>CONCLUSION</b>The oxaliplatin-based IAP regimen is potentially effective for salvage chemotherapy in patients with recurrent and drug-resistant ovarian cancer, with a better therapeutic effect and tolerable side effects.</p>

Gonadotropin-releasing hormone agonists cotreatment during chemotherapy in borderline ovarian tumor and ovarian cancer patients / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Recently, conservative surgery is acceptable in young patients with borderline ovarian tumor and ovarian cancer. The preservation of these patients' future fertility has been the focus of recent interest. This study aimed to observe the effect of gonadotropin-releasing hormone agonists (GnRHa) cotreatment during chemotherapy in borderline ovarian tumor and ovarian cancer patients.</p><p><b>METHODS</b>Sixteen patients who were treated with fertility preservation surgery for borderline ovarian tumor and ovarian cancer and then administered GnRHa during chemotherapy in Peking University People's Hospital from January 2006 to July 2010 were retrospectively analyzed. This group was compared with a control group of 16 women who were treated concurrently with similar chemotherapy (n = 5) without GnRHa or were historical controls (n = 11). The disease recurrence, the menstruation status and reproductive outcome were followed up and compared between the two groups.</p><p><b>RESULTS</b>There were no significant differences between both groups regarding age, body weight, height, marriage status, classification of the tumors, stage of the disease, as were the cumulative doses of each chemotherapeutic agent. One (1/16) patient in the study group while 2 (2/16) patients in the control group relapsed 2 years after conclusion of the primary treatment (P > 0.05). All of the 16 women in the study group compared with 11 of the 16 patients in the control group resumed normal menses 6 months after the termination of the treatment (P < 0.05). There were 4 spontaneous pregnancies in the study group while 2 in the control group, all of the neonates were healthy.</p><p><b>CONCLUSIONS</b>GnRHa administration before and during chemotherapy in borderline ovarian tumor and ovarian cancer patients who had undergone fertility preservation operation may bring up higher rates of spontaneous resumption of menses and a better pregnancy rate. Long-term follow up and large scale clinical studies are required.</p>

Mammalian target of rapamycin inhibitor abrogates abnormal osteoclastogenesis in neurofibromatosis type 1 / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Neurofibromatosis type 1 (NF1) is the most common genetic syndrome predisposing patients to various tumors due to dysregulation of the Ras signaling pathway. Recent research has shown NF1 patients also suffer a spectrum of bone pathologies. The pathogenesis of NF1 bone diseases is largely unknown. There is no current treatment. By Nf1 heterozygote (Nf1+/-) mice and Nf1 conditional knockout mice, we and other groups demonstrated abnormal osteoblast and osteoclast function due to dysregulation of Ras signaling. However, the specific downstream effector pathways linked to NF1 abnormal osteoblastogenesis and osteoclastogenesis have not been defined. In this study, we investigated the Ras downstream effector related with NF1 bone disease.</p><p><b>METHODS</b>We used Nf1+/+ and Nf1+/- mice as normal and NF1 models. Bone stromal cells extracted from Nf1+/+ and Nf1+/- mice were induced osteoclasts. The osteoclast cell was stained by tartrate resistant acid phosphatase staining. The osteoclast cell number was counted and the surface area of osteoclast cells was calculated under the microscope. The mRNA of mammalian target of rapamycin (mTOR) was determined by quantitative reverse-transcription-polymerase chain reaction. The presence of ribosomal protein S6 kinase was determined by Western blotting.</p><p><b>RESULTS</b>Compared with Nf1+/+ mice, Nf1+/- mice had about 20% more of osteoclast cells. These osteoclast cells were larger in size with more nuclei. Hyperactive mTOR was detected in Nf1+/- osteoclast cells. Inhibition of mTOR signaling by rapamycin in Nf1+/- osteoclasts abrogated abnormalities in cellular size and number.</p><p><b>CONCLUSION</b>mTOR pathway inhibition may represent a viable therapy for NF1 bone diseases.</p>

Effects of insulin, insulin-like growth factor-I and -II on proliferation and intracellular signaling in endometrial carcinoma cells with different expression levels of insulin receptor isoform A / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hyperinsulinemia, insulin-like growth factor (IGF)-I and -II (IGF-II) are associated with increased risk of endometrial carcinoma. Insulin receptor isoform A (IR-A) is more frequently expressed in endometrial carcinoma than in normal endometrial tissues. To better understand their roles in endometrial carcinoma, we investigated the effects of insulin, IGF-I, and IGF-II in endometrial carcinomas cells with different IR-A expression levels.</p><p><b>METHODS</b>To explore the role of IR-A in mediating the activity of IGF-I, IGF-II, and insulin, we investigate the cellular proliferation of endometrial carcinoma cell lines RL95-2 and RL95-2-IR-A by MTS assays. Then we examined the protein kinase Akt phosphorylation and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in both cell lines by Western blotting. The effect of IGF-II and AG1024 on cell cycle progression and apoptosis was assessed by flowcytometry. To examine whether the effects of IGFs were mediated by IR-A, we blocked IGF-I receptor (IGF-IR) in both cell lines using AG1024, an IGF-IR-specific inhibitor.</p><p><b>RESULTS</b>IGF-I and IGF-II significantly enhanced proliferation of both cell lines (P < 0.05). By contrast, insulin significantly increased proliferation of RL95-2-IR-A cells only (P < 0.05). IGF-I and IGF-II significantly increased pAkt levels in RL95-2 cells and pERK1/2 levels in RL95-2-IR-A cells (all, P < 0.05). Insulin increased pERK1/2 levels in RL95-2-IR-A cells only (P < 0.05). LY294002 and PD98059 inhibited the specific signaling activities and cellular proliferation. After AG1024 pretreatment, neither IGF-I nor IGF-II affected pAkt levels in RL95-2 cells. IGF-II, but not IGF-I, increased pERK1/2 levels in RL95-2-IR-A cells. After AG1024 pretreatment, the proliferation rate and DNA content corresponding to the S phase increased and apoptosis decreased significantly in IGF-II-treated RL95-2-IR-A cells only (P < 0.05).</p><p><b>CONCLUSIONS</b>The proliferation effect of insulin is mediated by IR-A. When IR-A dominates in a cell line, IGF-II activated cell proliferation mainly through the ERK1/2 pathway. On the other hand, IGF-II activated cell proliferation mainly through the Akt pathway. IR-A can at least partly mediate the proliferative and anti-apoptotic effects of IGF-II through the ERK1/2 pathway.</p>

Nifedipine induced autophagy through Beclin1 and mTOR pathway in endometrial carcinoma cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Endometrial carcinoma is one of the most common female tract genital malignant tumors. Nifedipine, an L-type calcium channel antagonist can inhibit cell proliferation of carcinomas. Recent studies indicated that a rise in the free cytosolic calcium ([Ca(2+)](c)) was a potent inducer of autophagy. Here, we investigated the relationship between nifedipine and autophagy in Hec-1A cells.</p><p><b>METHODS</b>Cells were cultured with nifedipine (10 µmol/L) and harvested at different times for counting cell number. MTT assay was applied to evaluate the cell viability and transwell assay to reveal cell migration. Apoptotic cells were detected with annexin V/PI assay. Then cells were treated with 3-methyladenine (3-MA) (2.5 mmol/L) for 0, 5, 15, 30, 60, and 120 minutes and the expression of the L-type calcium channel alpha1D (Cav1.3) protein was detected. At last, cells were cultured and assigned to four groups with different treatment: untreated (control group), 10 µmol/L nifedipine (N group), 2.5 mmol/L 3-MA (3-MA group), and 10 µmol/L nifedipine plus 2.5 mmol/L 3-MA (N+3MA group). Autophagy was detected with GFP-LC3 modulation by fluorescent microscopy, and expression of the autophagy-associated proteins (LC3, Beclin1 and P70s6K) by Western blotting and monodansylcadaverine (MDC) labeled visualization.</p><p><b>RESULTS</b>Proliferation of Hec-1A cells was obviously suppressed by nifedipine compared with that of the untreated cells for 24, 48, and 96 hours (P = 0.000 for each day). The suppression of migration ability of the nifedipine-treated cells (94.0 ± 8.2) was significantly different from that of the untreated cells (160.00 ± 9.50, P = 0.021). The level of early period cell apoptosis induced by nifedipine was (2.21 ± 0.19)%, which was (2.90 ± 0.13)% in control group (P = 0.052), whereas the late period apoptosis level reached (10.38 ± 0.96)% and (4.40 ± 0.60)% (P = 0.020), respectively. The 3-MA group induced a slight increase in the Cav1.3 levels within 15 minutes, but significantly attenuated the Cav1.3 levels after 30 minutes. There were more autophagic vacuoles labeled by MDC in the N group (20.63 ± 3.36) than the control group (6.29 ± 0.16, P = 0.015). GFP-LC3 localization revealed that the LC3 levels of cells in 3-MA group, N+3MA group, 3-MA group were 2.80 ± 0.29, 2.30 ± 0.17, and 1.80 ± 0.21, respectively. Cells in the N group showed significant augmentation of autophagy (P < 0.05). Western blotting analysis confirmed the down-regulation of LC3 levels in 3-MA group (0.85 ± 0.21) and N+3MA group (1.21 ± 0.12) compared with nifedipine treatment (2.64 ± 0.15, P < 0.05). The annexin-V-FITC/PI assay showed that the level of early period cell apoptosis induced in the N+3-MA group ((11.22 ± 0.91)%) differed significantly from that of the control group ((2.51 ± 0.70)%) and N group ((3.47 ± 0.39)%). Similarly, the late period level of the N+3-MA group ((55.19 ± 2.51)%) differed significantly from that of the control group ((15.81 ± 1.36)%) and the N group ((22.09 ± 2.48)%, P < 0.05). The down-regulated expression of P70s6k and up-regulated expression of the Beclin1 revealed significant differences between the N+3-MA group and control group (P = 0.025; Beclin1: P = 0.015).</p><p><b>CONCLUSIONS</b>Proliferation and migration in vitro of endometrial carcinoma Hec-1A cells are significantly suppressed by nifedipine. The nifedipine leads autophagy to oppose Hec-1A cells apoptosis. Autophagy inhibition by 3-MA leads down-regulation of Cav1.3 and enhances nifedipine-induced cell death. The nifedipine-induced autophagy is linked to Beclin1 and mTOR pathways.</p>

Histone deacetylase inhibitors inducing human cervical cancer cell apoptosis by decreasing DNA-methyltransferase 3B / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Histone deacetylase (HDAC) inhibitors are a group of small chemical molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have multiple biological effects such as cell cycle arrest, apoptosis, cell differentiation and auotophagy. At molecular level, HDAC inhibitors cause histone and nonhistone acetylation and induce gene expression. HDAC inhibitors are widely used in cancer therapy because of its function of inducing apoptosis. However, the mechanisms of apoptosis effect are not fully understood. TSA is a classical HDAC inhibitor and widely used in epigenetic and anti-cancer research. In this study, we selected Trichostatin A (TSA) to investigate the mechanisms of HDAC inhibitors apoptotic effect on cancer cells.</p><p><b>METHODS</b>Cervical cancer cell lines such as Hela, Caski and normal human keratinocyte line HaCaT were treated with various concentrations of TSA. Crystal violent assay and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were performed to determine cell number. PARP cleavage and FITC-AnexinV were performed to determine apoptosis. DNA-methyltransferase (DNMT)1, DNMT3A and DNMT3B were determined by regular PCR, qPCR and Western Blotting. Small interfering RNA (SiRNAi) was used to knock down DNMT3B.</p><p><b>RESULTS</b>HDAC inhibitors only induce cervical cancer cell apoptosis. At 1 µmol/L of TSA, 86% of Hela cell and 76% of Caski went apoptosis. For normal cells, HDAC inhibitors have no cytotoxic effect at therapeutic dosage, (90.0 ± 8.4)% of normal cell survive after treated with 1 µmol/L of TSA. We compared 1 µmol/L group with untreated control with t-test. There was no significance between 1 µmol/L group and untreated control for normal cell (P > 0.05). HDAC inhibitors decreased DNMT3B in cancer cell but not in normal cell. Manually knock-down of DNMT3B induced Hela and Caski cell apoptosis. More than 99% of Hela and Caski cell went apoptosis after deprived of DNMT3B.</p><p><b>CONCLUSIONS</b>DNMT3B was essential to cervical cancer cell survival. Down-regulated DNMT3B by HDAC inhibitors may play an important role in the toxicity of HDAC inhibitors on cervical cancer cells.</p>

Evaluation of whether serum tumor markers in patients with epithelial ovarian carcinoma change following chemotherapy / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Phenotypic and genotypic heterogeneity is a known feature of many cancers. Whether serum tumor marker kinds vary and change following chemotherapy is still unclear. The aim of this study was to investigate whether there is a change in the expression of serum tumor markers following chemotherapy, and the potential clinical significance in patients with epithelial ovarian carcinoma (EOC) or primary serous peritoneal carcinoma (PSPC).</p><p><b>METHODS</b>Samples were collected before surgery, during chemotherapy and during follow-up for enzyme-linked immunosorbent assay (ELISA)-based evaluation of serum CA-125, CA19-9 and CP2 levels in patients with EOC or PSPC who had received primary debulking surgery followed by adjuvant chemotherapy. In total, 72 patients were examined, including 37 patients with recurrent lesions and 35 patients receiving first-line chemotherapy.</p><p><b>RESULTS</b>In 35 de novo patients, 20% (7/35) demonstrated a significant changed serum tumor marker kinds among whom the patients with mucinous carcinoma (57.1%, 4/7) showed resistance to chemotherapy. In the 37 recurrent patients, 51.4% (19/37) had changed serum tumor markers, of whom 57.9% (11/19) presented with serous carcinoma. There was no significant difference in median progression-free survival or overall survival in patients with drug-sensitive or drug-resistant recurrence in patients with changed tumor marker kinds relative to those with unchanged markers. However, for patients with changed serum tumor markers there was a trend towards prolonged survival compared with the unchanged serum tumor marker group. In the 17 patients with secondary recurrence, 37.5% (6/17) had changed tumor marker levels. The ratios of CA-125/CP2 and CA-125/CA19-9 were significantly different after either chemotherapy or recurrence.</p><p><b>CONCLUSIONS</b>Serum tumor marker expression in patients with EOC or PSPC may change after chemotherapy or recurrence, indicating that in addition to the markers that are abnormal before surgery, those markers that are normal should also be monitored during chemotherapy and follow-up.</p>

A live birth of activated one-day-old unfertilized oocyte for a patient who experienced repeatedly near-total fertilization failure after intracytoplasmic sperm injection / 中华医学杂志(英文版)

Total or near-total fertilization failure after intracytoplasmic sperm injection (ICSI) is a rare event, but it occurs repeatedly because of sperm defects in activating oocyte. The case presents a successful pregnancy and live birth after calcium ionophore A23187 (A23187) activation on one-day-old unfertilized oocytes in a patient whose husband suffered oligoasthenoteratozoospermia, and who had experienced repeated near-total fertilization failure after ICSI. In the second ICSI cycle, only one oocyte was fertilized while nine were unfertilized. Oocyte activation with A23187 were performed on the one-day-old unfertilized oocytes after ICSI and resulted in fertilization and embryo transfer. A clinical pregnancy was achieved and a healthy baby was born. To our knowledge, this is the first reported case of a healthy birth after oocyte activation on the one-day-old unfertilized oocyte. This indicates that "rescue oocyte activation" on one-day-old unfertilized oocytes after ICSI may be helpful for preventing total or near-total fertilization failure after ICSI.

Neoadjuvant chemotherapy for locally advanced cervical cancer reduces surgical risks and lymph-vascular space involvement / 癌症

Neoadjuvant chemotherapy (NACT), which can reduce the size and therefore increase the resectability of tumors, has recently evolved as a treatment for locally advanced cervical cancer. NACT has been reported to decrease the risk of pathologic factors related to prognosis of cervical cancer. To further assess the effects of NACT on surgery and the pathologic characteristics of cervical cancer, we reviewed 110 cases of locally advanced cervical cancer treated with radical hysterectomy with or without NACT at the People's Hospital of Peking University between January 2006 and December 2010. Of 110 patients, 68 underwent platinum-based NACT prior to surgery (NACT group) and 42 underwent primary surgery treatment (PST group). Our results showed 48 of 68 (70.6%) patients achieved a complete response or partial response to NACT. Estimated blood loss, operation time, and number of removed lymph nodes during surgery, as well as complication rates during and after surgery were not significantly different between the NACT group and the PST group. The rates of deep stromal invasion, positive parametria, positive surgical vaginal margins, and lymph node metastasis were not significantly different between the two groups. However, the rate of lymph-vascular space involvement (LVSI) was significantly lower in the NACT group than in the PST group (P = 0.021). In addition, the response rate of NACT was significantly higher in the patients with chemotherapeutic drugs administrated via artery than via vein. Our results suggest that NACT is a safe and effective treatment for locally advanced cervical cancer and significantly decreases the rate of LVSI.

Pien Tze Huang induced apoptosis in human colon cancer HT-29 cells is associated with regulation of the Bcl-2 family and activation of caspase 3 / 中国结合医学杂志

<p><b>OBJECTIVE</b>To investigate the cellular effects of Pien Tze Huang (PZH) in the HT-29 human colon carcinoma cell line.</p><p><b>METHODS</b>The viability of HT-29 cells was determined by MTT assay. A fluorescence-activated cell sorting (FACS) analysis with annexin-V/propidium iodide (PI) and JC-1 staining were performed to determine cell apoptosis and the loss of mitochondrial membrane potential, respectively. Activation of caspase 3 was evaluated by a colorimetric assay. The mRNA expression levels of Bcl-2 and Bax were measured by reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULTS</b>PZH, in a dose- and time-dependent manner, reduced viability and induced apoptosis of HT-29 cells. Moreover, PZH treatment resulted in the collapse of the mitochondrial membrane potential, activation of caspase 3, and an increase in the Bax/Bcl-2 ratio.</p><p><b>CONCLUSION</b>PZH inhibits the growth of HT-29 cells by inducing cancer cell apoptosis via regulation of the Bcl-2 family and activation of caspase 3, which may, in part, explain its anticancer activity.</p>

Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background, which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use, especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability.</p><p><b>METHODS</b>Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propagate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line's ability to directly differentiate into pancreatic and hepatic cells.</p><p><b>RESULTS</b>We generated a new Chinese human embryonic stem cells line, CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines: normal morphology, karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line.</p><p><b>CONCLUSIONS</b>This newly established Chinese cell line, CH1, which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells, will provide a useful tool for embryo development research, along with clinical treatments for diabetes and some hepatic diseases.</p>