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NRF2/ARE signaling pathway is a principal regulator of cellular redox homoeostasis. The stress-induced transcription factor, NRF2, can shield cells from the oxidative damages via binding to the consensus antioxidant-responsive element (ARE) and driving several cyto-protective genes expression. Increasing evidence indicated that aberrant activation of NRF2 in malignant cells may support their survival through various pathways to detoxify chemotherapy drugs, attenuate drug-induced oxidative stress, or induce drug efflux, all of which are crucial in developing drug resistance. Accordingly, NRF2 is a potential drug target for improving the effectiveness of chemotherapy and to reverse drug resistance in cancer cells. A stable ARE-driven reporter human head and neck squamous cell carcinoma (HNSCC) cell line, HSC3-ARE9, was established and utilized to screen novel NRF2 inhibitors from a compound library. The cotton plant derived phenolic aldehyde-gossypol was selected for further analyses. The effects of gossypol in cancer cells were determined by western blotting, RT-qPCR, clonogenic assay, and cell viability assays. The gossypol-responsive gene expression levels were assessed in the Oncomine database. The effects of gossypol on conferring chemo-sensitization were evaluated in etoposide-resistant and cisplatin-resistant cancer cells. Our study is the first to identify that gossypol is effective to reduce both basal and NRF2 activator tert-butylhydroquinone (t-BHQ)-induced ARE-luciferase activity. Gossypol diminishes NRF2 protein stability and thereby leads to the suppression of NRF2/ARE pathway, which resulted in decreasing the expression levels of NRF2 downstream genes in both time- and dose-dependent manners. Inhibition of NRF2 by gossypol significantly decreases cell viabilities in human cancer cells. In addition, we find that gossypol re-sensitizes topoisomerase II poison treatment in etoposide-resistant cancer cells via suppression of NRF2/ABCC1 axis. Moreover, gossypol suppresses NRF2-mediated G6PD expression thereby leads to induce synthetic lethality with cisplatin not only in parental cancer cells but also in cisplatin-resistant cancer cells. These findings suggest that gossypol is a novel NRF2/ARE inhibitor, and can be a potential adjuvant chemotherapeutic agent for treatment of chemo-refractory tumor.

Rationale: NRF2, a redox sensitive transcription factor, is up-regulated in head and neck squamous cell carcinoma (HNSCC), however, the associated impact and regulatory mechanisms remain unclear. Methods: The protein expression of NRF2 in HNSCC specimens was examined by IHC. The regulatory effect of c-MYC on NRF2 was validated by ChIP-qPCR, RT-qPCR and western blot. The impacts of NRF2 on malignant progression of HNSCC were determined through genetic manipulation and pharmacological inhibition in vitro and in vivo. The gene-set enrichment analysis (GSEA) on expression data of cDNA microarray combined with ChIP-qPCR, RT-qPCR, western blot, transwell migration/ invasion, cell proliferation and soft agar colony formation assays were used to investigate the regulatory mechanisms of NRF2. Results: NRF2 expression is positively correlated with malignant features of HNSCC. In addition, carcinogens, such as nicotine and arecoline, trigger c-MYC-directed NRF2 activation in HNSCC cells. NRF2 reprograms a wide range of cancer metabolic pathways and the most notable is the pentose phosphate pathway (PPP). Furthermore, glucose-6-phosphate dehydrogenase (G6PD) and transketolase (TKT) are critical downstream effectors of NRF2 that drive malignant progression of HNSCC; the coherently expressed signature NRF2/G6PD/TKT gene set is a potential prognostic biomarker for prediction of patient overall survival. Notably, G6PD- and TKT-regulated nucleotide biosynthesis is more important than redox regulation in determining malignant progression of HNSCC. Conclusions: Carcinogens trigger c-MYC-directed NRF2 activation. Over-activation of NRF2 promotes malignant progression of HNSCC through reprogramming G6PD- and TKT-mediated nucleotide biosynthesis. Targeting NRF2-directed cellular metabolism is an effective strategy for development of novel treatments for head and neck cancer.

Nuclear factor erythroid-2-related factor 2 (NRF2) mainly regulates transcriptional activation through antioxidant-responsive elements (AREs) present in the promoters of NRF2 target genes. Recently, we found that NRF2 was overexpressed in a KB-derived drug-resistant cancer cell panel. In this panel, KB-7D cells, which show acquired resistance to topoisomerase II (Top II) poisons, exhibited the highest NRF2 activation. To investigate whether NRF2 directly contributed to acquired resistance against Top II poisons, we manipulated NRF2 by genetic and pharmacological approaches. The result demonstrated that silencing of NRF2 by RNA interference increased the sensitivity and treatment with NRF2 activator decreased the sensitivity of KB and KB-7D cells toward Top II poisons. Further, increased B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation activated NRF2 signaling in KB-7D cells. Moreover, increased binding of NRF2 to an ARE in the promoter of ATP-binding cassette subfamily C member 1 (ABCC1) directly contributed to Top II poison resistance. In addition, activation of NRF2 increased glutathione level and antioxidant capacity in KB-7D cells compared with that in KB cells; moreover, high glutathione level provided survival advantage to KB-7D cells. Our study is the first to show that aberrant NRF2 activation is via increased B-Raf-mediated NRF2 gene transcription and HATs-mediated NRF2 protein acetylation, which increases the acquired resistance and promote the survival of Top II poison-resistant cancer cells. Importantly, NRF2 downstream effectors ABCC1 and glutathione directly contribute to acquired resistance and survival, respectively. These results suggest that blockade of NRF2 signaling may enhance therapeutic efficacy and reduce the survival of Top II poison-refractory tumors in clinical.

A major challenge in personalized cancer medicine is to establish a systematic approach to translate huge oncogenomic datasets to clinical situations and facilitate drug discovery for cancers such as endometrial carcinoma. We performed a genome-wide somatic mutation-expression association study in a total of 219 endometrial cancer patients from TCGA database, by evaluating the correlation between ∼5,800 somatic mutations to ∼13,500 gene expression levels (in total, ∼78, 500, 000 pairs). A bioinformatics pipeline was devised to identify expression-associated single nucleotide variations (eSNVs) which are crucial for endometrial cancer progression and patient prognoses. We further prioritized 394 biologically risky mutational candidates which mapped to 275 gene loci and demonstrated that these genes collaborated with expression features were significantly enriched in targets of drugs approved for solid tumors, suggesting the plausibility of drug repurposing. Taken together, we integrated a fundamental endometrial cancer genomic profile into clinical circumstances, further shedding light for clinical implementation of genomic-based therapies and guidance for drug discovery.

We have designed and synthesized certain novel oxime- and amide-bearing coumarin derivatives as nuclear factor erythroid 2 p45-related factor 2 (Nrf2) activators. The potency of these compounds was measured by antioxidant responsive element (ARE)-driven luciferase activity, level of Nrf2-related cytoprotective genes and proteins, and antioxidant activity. Among them, (Z)-3-(2-(hydroxyimino)-2-phenylethoxy)-2H-chromen-2-one (17a) was the most active, and more potent than the positive t-BHQ in the induction of ARE-driven luciferase activity. Exposure of HSC-3 cells to various concentrations of 17a strongly increased Nrf2 nuclear translocation and the expression level of Nrf2-mediated cytoprotective proteins in a concentration-dependent manner. HSC-3 cells pretreated with 17a significantly reduced t-BOOH-induced oxidative stress. In the animal experiment, Nrf2-mediated cytoprotective proteins, such as aldo-keto reductase 1 subunit C-1 (AKR1C1), glutathione reductase (GR), and heme oxygenase (HO-1), were obviously elevated in the liver of 17a-treated mice than that of control. These results suggested that novel oxime-bearing coumarin 17a is able to activate Nrf2/ARE pathway in vivo and are therefore seen as a promising candidate for further investigation.

Objective We report a rare case of vaginal cuff dehiscence with small bowel evisceration at 7 months post robotic-staging surgery. Case Report A 41-year-old woman was sent to the emergency room with sudden onset of abdominal pain, vaginal bleeding, and vaginal protruding mass after sexual activity. She had a history of synchronous uterine and ovarian cancer treated with robotic-staging surgery 7 months before. Then she received six courses of postoperative adjuvant chemotherapy, and the last chemotherapy ended 1 month ago. At the operation room, some small bowel loops were noted in the vaginal tip with cuff dehiscence and bleeding. After repositioning of the small bowel, a 2.5-cm vaginal cuff dehiscence was repaired transvaginally. The patient recovered well, and is free of disease and has normal sexual activity 2 months after repairs. Conclusion Unusual delayed-type vaginal cuff dehiscence hints the possibility that a combination of robotic surgery and postoperative chemotherapy might result in delayed healing of the vaginal cuff.

Background The purpose of this study was to evaluate the feasibility of robotic surgery and compare its surgical outcomes with those of laparoscopic surgery and laparotomy, with regard to performing staging surgery to manage ovarian cancer. Methods One hundred and thirty-eight women who received surgical staging procedures for treatment of stage IA–IIIC epithelial ovarian cancer and borderline tumours were retrospectively included in the study. All enrolled cases were reviewed for patient demographics, peri-operative parameters, complications and survival. Results The operation time and blood loss was significantly reduced in the robotic and laparoscopic groups. Moreover, robotic surgery was associated with decreased postoperative pain score. The length of hospital stay and time to full diet resumption were also shortened for those who underwent robotic and laparoscopic procedures. Survival analysis and complication rates were similar between the two groups. Conclusion Robotic surgery is a feasible alternative in managing ovarian cancer as long as there is careful consideration given to patient selection. Copyright © 2015 John Wiley & Sons, Ltd.

OBJECTIVE: To review and evaluate the potential adverse effects of these oral contraceptives (OCP) to overweight women. CASE REPORT: A 19-year-old college student with a body mass index (BMI) of 35.2 kg/m(2) who received 2 months of OCP containing cyproterone and ethinyl estradiol for polycystic ovary syndrome (PCOS)-related menstrual problems was complicated with a thromboembolism-related life-threatened disease. After intensive care including the use of an extracorporeal membrane oxygenation system thrombolytic treatment anticoagulant and inferior vena filter she recovered well without significant sequelae. CONCLUSION: This case illustrates the risk of using OCPs especially for those containing cyproterone and ethinyl estradiol components as a treatment for menstrual problems in young women with PCOS and a high BMI. Copyright (c) 2016. Published by Elsevier B.V.

To summarize available evidence from randomized-controlled trials which have evaluated triggering of final oocyte maturation with concomitant GnRH agonists and hCG in patients undergoing IVF, and to analyze whether dual triggering is as efficacious as hCG triggering in terms of oocyte and pregnancy outcomes. A comprehensive literature search was performed to identify randomized-controlled trials comparing IVF outcomes between women receiving combined administration of hCG with GnRH agonists and those receiving hCG alone for triggering of final oocyte maturation. Four studies including 527 patients eligible for inclusion in meta-analysis were identified. No significant difference in the number of mature oocytes or fertilized oocytes retrieved was found between groups. Clinical pregnancy rate with dual triggering was significantly higher as compared with hCG-alone triggering (pooled OR = 0.48, 95% CI 0.31–0.77, P = 0.002), but there was no significant difference in the ongoing pregnancy rate between groups. Results of meta-analysis indicate comparable or significantly improved outcomes with the use of GnRH agonists plus hCG as compared with hCG alone for triggering of final oocyte maturation.

Background: To explore the relationship between overactive bladder (OAB) symptoms and paravaginal defects (PVDs), and to identify the necessity of PVD repair by transvaginal mesh (TVM) for the treatment of OAB symptoms. Methods: A retrospective clinical study of 30 women with advanced cystocele with limited apical and posterior vaginal wall prolapse was conducted to identify any changes in OAB symptoms following a single Perigee procedure. Prolapse was assessed using the pelvic organ prolapse quantification (POP-Q) system, and paravaginal defects were identified by sonography. Complete urodynamic examination was performed prior to and one year after operation. All patients completed the overactive bladder questionnaire pre- and postoperatively for a quantitative assessment of OAB symptoms. Results: All patients showed a significant improvement at points Aa and Ba in the POP-Q system. The results of the administered questionnaire revealed statistically significant improvement postoperatively. The difference of OAB symptoms between the group with PVDs and that with central defects was not statistically significant (p = 0.67). Moreover, no statistically significant improvement of OAB symptoms in the group with repaired PVDs was observed postoperatively (p = 0.42). Conclusion: Statistical improvements of symptoms exist after Aa and Ba points recovery as evaluated by POP-Q system regardless of PVD existence identified by sonography. Repairing PVD did not show significantly improve the severity of OAB symptoms in objective urodynamic data or subjective questionnaire data. The superiority of TVM in PVD repair to manage OAB symptoms seems not manifest. Keywords: Cystocele, Overactive bladder, Overactive detrusor, Surgical mesh, Urinary bladder prolapse, Vaginal prolapse

Oxaliplatin treatment is a mainstay of treatment for advanced gastrointestinal tract cancer, but the underlying mechanisms of acquired oxaliplatin resistance remain largely obscured. We previously demonstrated that increased DNA repair capacity and copper-transporting ATPase 1 (ATP7A) level contributed to oxaliplatin resistance in the human gastric carcinoma cell line TSGH-S3 (S3). In the present study, we applied gene array technology to identify additional resistance factors in S3 cells. We found that interleukin-6 (IL-6), aldo-keto reductase 1C1 (AKR1C1), and AKR1C3 are the top 3 upregulated genes in S3 cells when compared with parent TSGH cells. Despite a higher level of endogenous IL-6 in S3, IL-6 receptor (IR-6R, gp-80, and gp-130) levels were similar between TSGH and S3 cells. The addition of exogenous IL-6, IL-6 targeted siRNA, or neutralizing antibodies neither affected Stat3 activation, a downstream target of IL-6, nor changed oxaliplatin sensitivity in S3 cells. However, manipulation of AKR1C activity with siRNA or AKR1C inhibitors significantly reversed oxaliplatin resistance. AKR1Cs are classical antioxidant response element (ARE) genes that can be transcriptionally upregulated by nuclear factor erythroid 2-related factor 2 (Nrf2). Knockdown of Nrf2 not only decreased the levels of AKR1C1, AKR1C2, and AKR1C3 mRNA and protein but also reversed oxaliplatin resistance in S3 cells. Taken together, these results suggest that activation of the Nrf2/AKR1C axis may contribute to oxaliplatin resistance in S3 cells but that the IL-6 signaling pathway did not contribute to resistance. Manipulation of Nrf2/AKR1Cs activity may be useful for management of oxaliplatin-refractory gastric cancers.

The induction of detoxifying enzymes and antioxidant proteins by chemopreventive agents protects cells from oxidizing substances capable of damaging DNA integrity and initiating carcinogenesis. Coniferyl aldehyde, a naturally occurring substance, has been found in many foods and edible plants. We and others previously demonstrated that trans-coniferylaldehyde (t-CA) has potential antimutagenic and antioxidant properties. However, the mechanism underlying its Nrf2-mediated antioxidant effect remains largely unknown. In the present study, we demonstrated that t-CA significantly stimulated antioxidant-responsive element (ARE)-driven luciferase activity in a cell model and increased the expression of ARE-dependent detoxifying/antioxidant genes and their protein products in vitro and in vivo. The detoxifying/antioxidant genes activated by t-CA, especially heme oxygenase-1 (HO-1), were found to be involved in its cytoprotective effects against oxidative stress and cell injuries elicited by carcinogens tert-butylhydroperoxide and arecoline. Furthermore, the t-CA-induced phosphorylation and nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2) played a crucial role in this ARE-mediated cellular defense. Moreover, we found that p38 MAPK and protein kinase C (PKC) signaling pathways participated in the t-CA-induced, Nrf2-mediated cytoprotective effect. Among them, p38α/MAPKAPK-2 and an atypical PKC, PK-N3, were critical for the activation of the Nrf2/HO-1 axis by t-CA. In conclusion, we demonstrated for the first time that t-CA attenuates carcinogen-induced oxidative stress by activating Nrf2 via p38α/MAPKAPK-2- and PK-N3-dependent signaling pathways. In addition, t-CA increased the level of Nrf2-mediated detoxifying/antioxidant proteins in vivo, suggesting that t-CA may have potential for use in the management of carcinogenesis and meriting further investigation.

We have previously reported a gene Tdpoz1 (previously called 2cpoz56) that is temporally expressed in unfertilized eggs and in early embryos of the mouse. The putative TDPOZ1 protein carries a tumor necrosis factor receptor-associated factor (TRAF) domain (TD) and a POZ/BTB domain. On the analysis of nine bacterial artificial chromosome (BAC) clones, we have uncovered four more Tdpoz1 homologs in the mouse genome, designated Tdpoz2 through Tdpoz5. Tdpoz1 and Tdpoz2 are found 30 kb apart in a fully sequenced BAC clone (GenBank accession number AF545858 ). The genes are intronless in the coding region and each carries an intron in the 5′-untranslated region as in other early embryonic genes. The Tdpoz gene cluster is mapped on chromosome 3 at 3F2.1–2.2. RT-PCR experiments and a search of expressed sequence tag (EST) databases show that the Tdpoz1–5 genes are transcribed in early embryos, particularly at the two-cell stage. Exhaustive database searches have further uncovered three more mouse Tdpoz homologs in chromosomes 3 and 11 and 25 other Tdpoz-like orthologs in the genomes of other animal and plant species including human, rat, C. elegans, Drosophila, Arabidopsis and rice. In the rat genome, eight rat Tdpoz genes are found as a cluster in chromosome 2. Hence, TDPOZ proteins form a new protein family on the basis of similar protein domain organization. Based on reported characteristics of known TD- and POZ-bearing proteins, we speculate that TDPOZ proteins may be nuclear scaffold proteins probably involved in transcription regulation in early development and other cellular processes.

Molecular events involved in gene expression in unfertilized eggs and pre-implantation embryos are beginning to be understood. In this work, we investigated the transcription and processing of two structurally juxtaposed mouse RING finger protein genes, Rnf33 and Rnf35. Transcripts of these genes are detected only in eggs and in pre-implantation embryos. Both genes are intronless except for a solitary intron in the 5'-untranslated region. Here, we showed by rapid amplification of cDNA ends (RACE) and reverse transcription experiments that Rnf35 transcription uses a single promoter and a terminating site. On the other hand, Rnf33 is transcribed using multiple promoters. At the four-cell stage, however, Rnf33 mRNA with a single transcription start site derived from the proximal promoter is detected, indicating that it is the major promoter. Sequences upstream of the Rnf35 and the major Rnf33 transcription start sites carry no TATA boxes but a putative transcription initiator (Inr) element is discernible in each case. The processing of the 3'-end of the Rnf33 mRNA is also in disarray with multiple 3'-ends, an event that may be related to the absence of the AAUAAA element and the utilization of AAUAAA-like proxies. The multiplicity of the 3'-untranslated region is partially amended at the four-cell stage when only two major 3'-ends are in use. This work demonstrates that expression of some maternal and early zygotic genes may be opportunistic until a stringent transcriptional regulation mechanism is imposed.

We have previously reported a Krupple-like finger protein gene, Zfp352, which is expressed temporarily in two- to eight-cell mouse embryos. The Zfp352 gene is intron-less in the coding region but carries a solitary 4.3-kb intron in the 5'-untranslated region. In this study, we have analyzed the Zfp352 promoter activity in early embryos and in somatic cells. We determined that the major Zfp352 promoter, designated P1 and located upstream of exon 1, is utilized in both early embryos and in somatic cells. A TATA-like box and a transcription initiator element are discernible in the P1 promoter. We uncovered an alternative promoter, designated P2, in the intron. 5'-Rapid amplification of cDNA ends and real-time RT-PCR experiments indicated that the P2 promoter is weak and is probably fortuitous in early embryos. In somatic cells, however, transfection experiments showed that P2 is as active as P1 as a promoter. Furthermore, P2 appears to be composed of two different subdomains used differentially for transcription initiation in embryos and in somatic cells. Our observations may bear relevance in explaining developmental deficiencies associated with somatic cell cloning experiments.

Rnf33 and Rnf35 are mouse RING finger protein genes that are transcribed temporally in the preimplantation mouse embryo, predominantly at the two-cell embryonic stage. The genes are juxtaposed in a 20-kb genomic region and are both intronless except for a single intron in the 5' untranslated region (5'-UTR). Based on analysis of the Rnf33/35 genomic sequence and cDNA sequences derived by in silico mining, we found that the Rnf33 and Rnf35 mRNAs are apparently transcribed from the same putative promoter and may be products of alternative splicing of the same pre-mRNA generated through differential 3' cleavage and polyadenylation. We also detected a second variant of Rnf35 in two-cell embryo generated through a second splicing event using an unconventional 5' splice junction. Our observations on the mode of transcription of Rnf33 and Rnf35 are consistent with the hypothesis that transcription of zygotic genes is promiscuous, and that the solo 5'-UTR intron may serve to facilitate efficient translation.