Data Supplement from Synergistic Killing Effect between Vorinostat and Target of CD146 in Malignant Cells

The editors are publishing this note to alert readers to a concern about [this article][1] ([1][2]). In Fig. 5D, similarities exist in the mAb AA98 and Vorinostat + mIgG slides. The authors stand by the conclusions in the article. 1. 1.[↵][3]1. Ma X, 2. Liu J, 3. Wu J, 4. Yan

The authors would like to correct figure, as the error was introduced in the preparation of this figure for publication. We sincerely apologize for having this error in the article, the authors have provided corrected version of figure here.

It is still controversial whether cervical cancer patients with clinical responses after neoadjuvant chemotherapy (NACT) have a better long-term survival or not. This study was designed to investigate the effect of the clinical response on the disease-free survival (DFS) of cervical cancer patients undergoing NACT. A total of 853 patients from a retrospective study were used to evaluate whether the clinical response was an indicator for the long-term response, and 493 patients from a prospective cohort study were used for further evaluation. The survival difference was detected by log-rank test, univariate and multivariate Cox regression and a pooled analysis. The log-rank test revealed that compared with non-responders, the DFS of responders was significantly higher in the retrospective data (P = 0.007). Univariate Cox regression showed that the clinical response was an indicator of long-term survival in the retrospective study (HR 1.83, 95% CI 1.18-2.85, P = 0.007). In a multivariate Cox model, the clinical response was still retained as an independent significant prognostic factor in the retrospective study (HR 1.59, 95% CI 1.01-2.50, P = 0.046). The result was also validated in the prospective data with similar results. These findings implied that the clinical response can be regarded as an independent predictor of DFS.

Background: The increasing use of anti-Müllerian hormone (AMH) in clinic has raised concerns regarding the reliable reference range for this test. However, the reference range for AMH in normal Chinese female population has not been established. Furthermore, relationship between AMH and other clinical markers such as body mass index (BMI) and antral follicle counts (AFCs) and other sex-related hormones have not been examined in normal population-based women. Objective: We aimed to determine the age-specific reference range for serum AMH in healthy Chinese women throughout reproductive age to menopause and to estimate relationship between AMH and other clinical markers in healthy women. Study Design: In this multicenter and nationwide study, advertisements were used to recruit 2055 women, aged 20 to 55 years, from 6 different regions in China; 1590 (77.37%) women met the inclusion criteria for the reference range population. We measured the baseline serum AMH levels using new Beckman Coulter Gen II assay. Serum concentration of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), testosterone (T), prolactin (PRL), progesterone (PRG), and AFCs were also determined in the follicular phase. Main Outcome Measures: The AMH-Age nomogram and AMH levels of different age-groups and the relationship between AMH and other clinical markers. Results: Serum AMH concentrations declined progressively with age. A quadratic model defined as log (AMH) = (−1.970 + 0.296 × Age − 0.006 × Age2) fitted best the decline of AMH with age. The median AMH levels were 6.23, 5.65, 4.55, 3.74, 2.78, and 1.09 ng/mL for the 20 ≤ age < 25, 25 ≤ age < 30, 30 ≤ age < 33, 33 ≤ age < 37, 37 ≤ age < 40, and 40 ≤ age < 55 groups, respectively. The 5th to 95th percentiles of the AMH levels, as the reference range, were 2.06 to 12.66, 1.77 to 13.83, 1.48 to 11.45, 0.87 to 9.76, 0.56 to 9.49, and 0.08 to 5.70 ng/mL for each age-group. The AMH levels were positively correlated with AFCs and T, LH, PRL and PRG levels and negatively correlated with BMI and FSH levels and were not significantly correlated with E2 levels. The relationship between AMH and other variables remain unchanged except for PRL, which was not significantly correlated with AMH levels after controlling for both age and BMI. Conclusions: This study determined the normal reference ranges for serum AMH levels in a large population-based sample of healthy Chinese women.

Background The increasing use of anti-Müllerian hormone (AMH) in clinic has raised concerns regarding the reliable reference range for this test. However, the reference range for AMH in normal Chinese female population has not been established. Furthermore, relationship between AMH and other clinical markers such as body mass index (BMI) and antral follicle counts (AFCs) and other sex-related hormones have not been examined in normal population-based women. Objective We aimed to determine the age-specific reference range for serum AMH in healthy Chinese women throughout reproductive age to menopause and to estimate relationship between AMH and other clinical markers in healthy women. Study Design In this multicenter and nationwide study, advertisements were used to recruit 2055 women, aged 20 to 55 years, from 6 different regions in China; 1590 (77.37%) women met the inclusion criteria for the reference range population. We measured the baseline serum AMH levels using new Beckman Coulter Gen II assay. Serum concentration of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E 2 ), testosterone (T), prolactin (PRL), progesterone (PRG), and AFCs were also determined in the follicular phase. Main Outcome Measures The AMH-Age nomogram and AMH levels of different age-groups and the relationship between AMH and other clinical markers. Results Serum AMH concentrations declined progressively with age. A quadratic model defined as log (AMH) = (−1.970 + 0.296 × Age − 0.006 × Age 2 ) fitted best the decline of AMH with age. The median AMH levels were 6.23, 5.65, 4.55, 3.74, 2.78, and 1.09 ng/mL for the 20 ≤ age < 25, 25 ≤ age < 30, 30 ≤ age < 33, 33 ≤ age < 37, 37 ≤ age < 40, and 40 ≤ age < 55 groups, respectively. The 5th to 95th percentiles of the AMH levels, as the reference range, were 2.06 to 12.66, 1.77 to 13.83, 1.48 to 11.45, 0.87 to 9.76, 0.56 to 9.49, and 0.08 to 5.70 ng/mL for each age-group. The AMH levels were positively correlated with AFCs and T, LH, PRL and PRG levels and negatively correlated with BMI and FSH levels and were not significantly correlated with E 2 levels. The relationship between AMH and other variables remain unchanged except for PRL, which was not significantly correlated with AMH levels after controlling for both age and BMI. Conclusions This study determined the normal reference ranges for serum AMH levels in a large population-based sample of healthy Chinese women.

目的:研究miR-9在卵巢癌细胞上皮间质转化(EMT)中的作用.方法:上调或者下调miR-9后,在RNA水平上通过RT-qPCR检测卵巢癌细胞系SKOV3和A2780中上皮指标E-cadherin表达变化;在蛋白水平,通过western blotting方法检测2株细胞系中上皮指标E-cadherin和间质指标vimentin蛋白表达变化.生物信息学预测可能靶向E-cadherin 3'UTR的miRNA,双荧光素酶报告系统进一步验证miR-9靶向结合E-cadherin的3'UTR区.结果:上调miR-9后,卵巢癌细胞系中E-cadherin表达受到明显抑制,vimentin表达明显增加;反之,下调miR-9后,E-cadherin表达明显增高,vimentin表达明显降低.通过生物信息学预测发现miR-9可以直接靶向E-cadherin的3'UTR区,荧光素酶报告系统验证预测结果正确.结论:miR-9促进卵巢癌细胞上皮间质转化.

In this work, for better applications of atmospheric pressure plasma jets, the physics of plasma streamers in a glass tube with a part of it covered by a conductor is investigated. To better understand the propagation mechanism of plasma bullets in capillary tubes passing through a curved or narrow passage for some biomedical or material applications, the propagation of plasma streamers in a tube covered by a floating conductor is investigated. For a plasma streamer propagating in a tube covered by a conductor, the plasma streamer is suppressed and becomes shorter, and a secondary streamer is generated in the tube at the downstream end of the conductor. The larger the area covered by the conductor, or the thinner the tube, the stronger the plasma streamer is inhibited. The electric potential of the conductor is measured to be as high as 6 kV. On the other hand, a higher voltage applied on the HV electrode, or a higher gas flow rate will make the secondary plasma streamer longer. It is found that the capacitor formed by the conductor outside the tube and the wall of the tube plays an important role in inhibiting the original plasma streamer and generating the secondary streamer. Moreover, the active species generated by the original plasma play important role in generating a secondary plasma streamer.

目的 探讨microRNA-9(miR-9)参与调控卵巢癌细胞EMT过程,以及影响卵巢癌细胞侵袭及转移的分子机制.方法 使用TargetScan及PicTar数据库,预测可能靶向E-cadherin 3'UTR区的miRNA,双荧光素酶报告体系进行验证;上调候选miR后,用qRT-PCR和Western blot检测E-cadherin 的表达变化;细胞免疫荧光观察E-cadherin、β-Catenin和Vimentin的表达;划痕实验、Transwell实验,观察卵巢癌细胞运动和侵袭能力的改变.结果 TargetScan、PicTar预测发现miR-9是唯一可与CDH1结合的miRNA.双荧光素酶报告系统验证预测结果正确.在SKOV-3中上调miR-9后,E-cadherin的表达受到显著抑制;细胞形态向间质细胞样转变,发生EMT分子水平的特征性改变;体外实验表明,卵巢癌细胞的运动和侵袭能力得到明显促进.结论 miR-9可以通过靶向调控E-cadherin表达,促进卵巢癌细胞的EMT进程,对卵巢癌细胞的运动和侵袭能力产生重要影响.

Approximately one million hysterectomies are performed each year in China. However, national data regarding the indications and the surgical approaches for hysterectomy are lacking. The aim of this study was to examine the surgical indications for hysterectomy in different age groups and the relative merits of different surgical approaches for hysterectomy in Chinese women. Clinical data from 4653 cases of hysterectomy performed in Tongji Hospital from 2004 to 2009 were analysed. Hysterectomy was most commonly performed among women aged 40-49 years (2299; 49.4%). Overall, colporrhagia and abdominal pain were the two most common indications for hysterectomy. The most common indications by age groups were as follows: malignant ovarian tumour, < 20 years; malignant uterine tumour, 20-29 and 30-39 years; uterine myoma, 40-49 and 50-59 years; and uterine prolapse, 60-69 and > 70 years. The proportion of malignant aetiology also varied by age, being the highest in women aged < 20 years (75.0%) and the lowest in those aged 40-49 years (19.9%). Approximately 35% women who had hysterectomies also had concomitant bilateral oophorectomy. The lowest rate of oophorectomy occurred in women aged 30-39 years (15.8%), whereas the highest rate was in those aged 50-59 years (75.9%). The abdominal surgical approach was used in 84% of all hysterectomies. Surgeries using the vaginal approach required a significantly shorter operating time (118 min average) than all other approaches (P < 0.05). Both the amount of bleeding and the blood transfusion volume required were smaller in vaginal approaches, with no significant differences between the others. The surgical approaches used were also related to the scope of surgery. Both the surgical indications and the rates of bilateral oophorectomy varied by age. In terms of both operating time and the amount of bleeding and blood transfusion volume required, the vaginal approach was superior to all other surgical approaches.

目的:开展重组腺病毒-胸苷激酶基因制剂(adenovirus-mediated delivery of herpes simplex virus thymidine kinase gene,ADV-tk)局部或静脉用药后于动物体内的分布行为的研究.方法:分别采用绿色荧光蛋白(green fluorescent protein,GFP)和原位杂交法检测分析重组腺病毒及胸苷激酶于肝脏、脾脏、淋巴结、肠系膜、肾上腺、胸腺、肾脏、甲状腺、前列腺、卵巢等组织分布的情况.结果判断采用免疫组织化学评分法(immuno-histochemical scores,IHS),并结合阳性细胞百分比及阳性细胞染色强弱2个方面进行评价.结果:(1)3种不同给药途径的组织分布特征具有相似性,ADV或tk阳性表达的组织脏器从强到弱分布依次为:肝脏、脾脏、淋巴结、肠系膜、肾上腺、胸腺、肾脏、甲状腺、前列腺、卵巢.ADV或tk mRNA的丰度具有一致性.但不同给药途径在组织分布丰度上有明显区别,由强至弱分别为:局部注射＞腹腔注射＞静脉注射,在组织分布特征上具有一致性.(2)小鼠腹腔注射后,ADV及tk的各组织分布动态变化上具有一致性,腹腔注射后1 d ADV及tk即具有高表达,7d表达最强,14 d下降60％左右,21 d残留20％左右,32 d基本消失.(3)随着ADV-GFP给药剂量的增强,ADV的组织分布表现为剂量依赖性逐步增强.剂量为病毒颗粒数每1 kg为6.7×108时,ADV主要分布于肝脏、脾脏、淋巴结和肠系膜,分布较低.当剂量达到病毒颗粒数每1 kg为6×109时,ADV在10个器官的分布以肝脏、脾脏、淋巴结、肠系膜和肾上腺分布增加最为明显.当剂量增加到病毒颗粒数每1 kg为1×1012时,ADV在10个器官的分布继续增加;在病毒颗粒数每1 kg为1×1013的大剂量病毒剂量情况下,肝脏的转染效率增加不太显著,但在其他阳性器官的分布增加.结论:本实验提示,采用每周用药1次,病毒颗粒数每1 kg为1×1012,局部或局部血管用药的给药方案可以得到肝脏有效转染强度,有望为今后指导制定临床的最佳给药方案提供借鉴与参考.

doi: 10.1016/j.canlet.2013.08.022 * Corresponding author. Cancer Biology Research Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, PR China. Fax: +86 27 83662681. E-mail address: qlgao@tjh.tjmu.edu.cn (Q. Gao). 1 Note: Tengji and Danni Gong contributed equally to this work. Fig. 1. The sensitivity to cisplatin and the expression of Stat3 was compared between OV2008 and C13* ovarian cancer cells. OV2008 and C13* cells were exposed to increasing concentrations of cisplatin for 24 or 48 h. (A) Relative cell viability was determined by MTT assay after treatment with cisplatin for 48 h. (B) Apoptotic ratios increased in a dose-dependent and time-dependent manner, both in two cell lines by flow cytometry. The results represent data from three independent experiments after treatment for 24 h (C) or 48 h (D). (E) Expression of Stat3 mRNA (124 bp PCR product) was detected with RT-PCR. GAPDH (197 bp PCR product) was co-amplified as the internal control. (F) Expression of Stat3 and p-Stat3 protein was detected with Western blot analysis in total cell extracts. GAPDH was reprobed to confirm equal protein loading.

The initiation of primordial follicle development is essential for female fertility, but the signals that trigger this process are poorly understood. Given the potentially important roles of microRNAs (miRNAs) in the ovary, we aimed to study the expression patterns and regulatory functions of miRNAs during the initiation of primordial follicle development. Expression patterns of miRNA in the neonatal mouse ovary were profiled by microarray, and 24 miRNAs whose abundances differed significantly between ovaries from 3- and 5-day-old mice were identified. Pathway enrichment analysis revealed that 48 signal transduction pathways are modulated by the up-regulated miRNAs and 29 pathways are modulated by the down-regulated miRNAs (P-value and false discovery rate < 0.001). A miRNA-mRNA regulatory network was established for TGF-beta signaling pathway-related genes. Among the miRNAs involved in this pathway, miR-145 was chosen for further analysis. Down-regulation of miR-145 using an antagomir (AT) decreased the proportion and number of the primordial follicles and increased that of the growing follicles in the cultured ovaries (P < 0.05). The mean oocyte diameter in the primordial follicles was significantly greater in the AT group relative to the AT-negative control group (P < 0.05), whereas the mean oocyte diameter in growing follicles was smaller in the AT group than in the AT-negative control group. In addition, we confirmed that miR-145 targets Tgfbr2. The miR-145 AT caused an increase in TGFBR2 expression and activation of Smad signaling but did not affect the p38 MAPK or JNK pathway. These data suggest that miRNAs and the signaling pathways they modulate are involved in the initiation of primordial follicle development, and miR-145 targets Tgfbr2 to regulate the initiation of primordial follicle development and maintain primordial follicle quiescence.

The aim of the present study was to investigate the role of Stat3 in cisplatin resistant ovarian cancer. It was first demonstrated that higher activated Stat3 was detected in cisplatin-resistant ovarian cancer cell lines. To provide evidence that supported the hypothesis that phosphorylated-Stat3 expression may promote cisplatin resistance, ectopic Stat3 was expressed by IL-6 stimulation that partially abrogates Stat3, as opposed to the knock-down of Stat3 by specific siRNA that restores cisplatin sensitivity against ovarian cancer cells. This hypothesis was further confirmed by clinical tumor specimens of ovarian cancer obtained from patients with cisplatin-resistance. Based on these premises, Stattic [1], an effective small molecular inhibitor of Stat3, was used to inhibit Stat3 activation. The data presented here show that Stattic restored the sensitivity to cisplatin in chemoresistant ovarian cancer by significant reductions in the expression of the anti-apoptosis protein Bcl-2, Bcl-XL, Survivin protein and phosphorylated-Akt levels. Consistent with these observations, this experiment demonstrated the first evidence of Stattic circumvented cisplatin resistance of orthotopic xenograft ovarian cancer in vivo. Altogether, these findings emphasize the importance of Stat3 in cisplatin resistance in ovarian cancer and provide a further impetus to clinically evaluate biological modifiers that may circumvent cisplatin resistance in patients with chemoresistant ovarian cancer.

microRNAs (miRNAs/miRs) may have a crucial function in tumor metastasis through the regulation of a plethora of signaling pathways. Increasing evidence has shown that miR-199a is important in regulating the tumor metastasis of ovarian cancer, although the precise biological function of miR-199a is unclear at present. In the current study, it was observed that the expression levels of miR-199a were higher in OV2008 cells compared with C13* cells. However, lower levels of mammalian target of rapamycin (mTOR) protein were detected by western blotting in the OV2008 cells compared with the C13* cells. The miR-199a levels were increased in the C13* cells using miR-199a mimics and the mTOR levels were observed to decrease. This may have resulted in a reversal of cisplatin resistance in the C13* cells. To test this hypothesis, the Renilla luciferase reporter gene system was used to analyze the mTOR levels. The results indicated that the expression levels of mTOR were significantly blocked by the increased miR-199a levels. When the miR-199a inhibitor was applied to decrease the miR-199a levels, it was observed that the mTOR expression levels were increased, while cisplatin-induced apoptosis was decreased in the OV2008 cells. The study concludes that miR-199a is able to reverse cisplatin resistance in human ovarian cancer cells through the inhibition of mTOR and that mTOR may be the target of miR-199a during this process.

Objective: To demonstrate the changes of ovarian aging markers across the Stages of Reproductive Aging Workshop (STRAW) stages and modify it with subclassification of mid reproductive age stage (MR). Design: Healthy females were classified according to the STRAW system. Serum basal FSH, LH, E2, and anti-Müllerian hormone (AMH) were detected, FSH/LH ratio calculated, and antral follicle counts (AFCs) determined in follicular phase. Results: Progression through the whole STRAW stages under MR stage subdivided is associated with elevations in FSH, LH, FSH/LH ratio and decreases in E2, AMH and AFCs (p < 0.001). Both serum AMH and AFCs decreased early (after 25 years) and significantly (p < 0.01) with chronological age in MR stage. 0.982 ng/ml AMH and 3 antral follicles (low level of MR 25–30 years) were set as cutoffs to distinguish MR stage into early mid reproductive age (EMR) and late mid reproductive age (LMR) stages. The women in EMR stage compared with LMR could retrieve more oocytes in IVF treatment (p < 0.05) and has a higher pregnancy chance (57.9%) though not significant. Conclusion(s): The early and marked fall in serum AMH levels and AFCs suggest fine markers to further categorize and define the MR stage, demonstrating disparate reproductive aging period with reduced ovarian reserve in young age across the STRAW stages.

Peptide-based therapies have emerged as one of the most promising therapeutics strategy in cancer-targeted therapy. Using our laboratory newly identified peptide TMTP1 and diphtheria toxin, we developed a new fusion protein that showed remarkable ability to target highly metastatic tumors. Fusion protein toxins were generated by fusing the first 390 amino acids of diphtheria toxin [truncated diphtheria toxin (DT390)] to different repeats of peptide TMTP1 (DT390-TMTP1, DT390-biTMTP1, and DT390-triTMTP1). Efficacies of the recombinant fusion proteins on tumor growth and metastasis were evaluated in vitro and in vivo. DT390-triTMTP1 showed the most powerful toxicity against cancer, which led to tumor growth retardation or regression and prolonged survival of human prostate cancer PC-3M-1E8 subcutaneously bearing or gastric cancer MKN-45 orthotopic nude mice. Increased TUNEL and caspase-3 staining and reduced ki67 staining in tumor cells suggested that the anticancer effects of DT390-triTMTP1 were through selectively inducing apoptosis and inhibiting proliferation of cancer cells. In a murine model of human orthotopic gastric carcinoma, DT390-biTMTP1 significantly inhibited metastases to liver and spleen, while DT390-triTMTP1 not only totally suppressed metastasis but also reduced primary tumors by 66.6%. In the biodistribution test, DT390-triTMTP1 was observed to home to tumor tissue rapidly and lasted over 48 h, with only a transient appearance in liver and kidney immediately after injection. Thus, our present study provided a novel recombinant fusion protein DT390-triTMTP1 with preferential targeting and high cytotoxicity, which may be a promising strategy for the targeted therapy of cancer metastasis.

Objective Due to their lower risk for induction of resistance,antimicrobial peptides with selective anticancer effect could be developed into a new generation of anticancer drugs.We conjugated an antimicrobial peptide with tumor-targeting peptides （TMTP1） to explore whether it has inhibiting effect on the progression and metastasis of transplanted prostate cancer and gastric cancer in nude mice.Methods Subcutaneously transplanted human prostate cancer and orthotopically transplanted human gastric cancer in nude mice were prepared.50