Analysis of HPV genotype-specific concordance between EUROArray HPV and HPV 3.5 LCD-Array Kit in cervical samples of 163 patients.

PURPOSE: Human papilloma virus (HPV) as the most common viral infection of the anogenital tract is highly associated with intraepithelial neoplasia and cancer of the cervix and other anogential regions. To date, 15 high-risk (HR-) HPV and 3 probably/possibly HR-HPVs have been found to be associated with cervical cancer. Therefore, a screening especially for HR-HPV by appropriate tests is important for detection of precancerous lesions to prevent cancer. The purpose of this study was to analyze prospectively the concordance of the EUROArray HPV genotyping assay (Euroimmun; EUROArray) and the HPV 3.5 LCD-Array Kit (Chipron; LCD-Array). METHODS: Liquid-based, clinician-collected cervical cytology samples (n = 163) from women undergoing cervical inspection at the dysplasia consultation in the colposcopy clinic at the Medical Center-University of Freiburg, Germany were analyzed. Genotype-specific agreement was assessed by Cohen's kappa statistic and McNemar's P value of significance between proportions. RESULTS: Seventeen of the HR-HPV genotypes included in both assays were detected in 42.3% and 38% of samples by EUROArray and by LCD-Array, respectively; i.e. an agreement of 92.0% and a kappa value of 0.83 could be proven between the EUROArray and the LCD-Array. In 50 of 72 samples, identical HR-HPV genotypes were analyzed (81.9%, κ = 0.47) and genotyping for HPV 16 and/or 18 was highly concordant in both tests (relative agreement 96.3%, κ = 0.88). Detection of any HR-HPV was not significantly different after comparison of EUROArray with LCD-Array. CONCLUSION: Both of the tests showed comparable results for the detection of HPV in cervical specimens and permit these assays to be suitable for routine diagnostics.

Slow-freezing versus vitrification for human ovarian tissue cryopreservation.

PURPOSE: Ovarian tissue can be cryopreserved prior to chemotherapy using either the slow-freezing or the vitrification method; however, the data on the equality of the procedures are still conflicting. In this study, a comparison of the cryo-damage of human ovarian tissue induced by either vitrification or slow-freezing was performed. METHODS: Ovarian tissue from 23 pre-menopausal patients was cryopreserved with either slow-freezing or vitrification. After thawing/warming, the tissue was histologically and immunohistochemically analyzed and cultured in vitro. During tissue culture the estradiol release was assessed. RESULTS: No significant difference was found in the proportion of high-quality follicles after thawing/warming in the slow-freezing and vitrification group, respectively (72.7 versus 66.7 %, p = 0.733). Estradiol secretion by the ovarian tissue was similar between groups during 18 days in vitro culture (area-under-the-curve 5,411 versus 13,102, p = 0.11). Addition of Sphingosine-1-Phosphate or Activin A to the culture medium did not alter estradiol release in both groups. The proportion of Activated Caspase-3 or 'Proliferating-Cell-Nuclear-Antigen' positive follicles at the end of the culture period was similar between slow-freezing and vitrification. CONCLUSION(S): Slow-freezing and vitrification result in similar morphological integrity after cryopreservation, a similar estradiol release in culture, and similar rates of follicular proliferation and apoptosis after culture.

Prevalence of PD-L1 in Cervical Cancer Patients and the Potential for Combining an Immune Checkpoint Inhibitor With Lenvatinib.

BACKGROUND/AIM: Cervical cancer is the fourth most common cause of cancer-related deaths in women worldwide. The potential for targeted therapy against the immune checkpoint programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) and receptor tyrosine kinases was examined in cervical cancer patients and cell lines. MATERIALS AND METHODS: On tissue microarrays, PD-L1 was analyzed in 123 samples of patients with cervical cancer using immunohistochemistry. In SiHa, HeLa, and CaSki cervical cancer cell lines we examined the combination of lenvatinib with a PD-1/PD-L1 inhibitor using cell viability assays, the activation of cell signaling pathway proteins using western blots and gene expression using quantitative reverse transcriptase-PCR. RESULTS: Of 113 evaluable samples, 90 (79.6%) had more than 1% PD-L1 positive cells. The single treatment with the PD-1/PD-L1 inhibitor resulted in the greatest reduction in growth for CaSki and lenvatinib in HeLa cells. In contrast, the combined treatment of lenvatinib with the PD-1/PD-L1 inhibitor demonstrated a significantly stronger impeded proliferation compared to the single treatment in all three cell lines. Moreover, the combined treatment caused significantly less phosphorylation of the signaling molecules ERK and S6 in SiHa and of S6 and STAT3 in HeLa cells but not in CaSki. All treatments diminished the mRNA levels of PD-L1, Il-8, and FGFR in SiHa cells. CONCLUSION: PD1 is frequently expressed in cervical cancer samples. Combining lenvatinib with a PD-1/PD-L1 inhibitor diminished proliferation of cervical cancer cell lines. Consequently, this combination might be a promising option to treat cervical cancer. Signaling pathways involved in tumor cell growth are blocked by the combined treatment but still a model of the underlying mechanism cannot be drawn.

Targeting triple-negative breast cancer through the somatostatin receptor with the new cytotoxic somatostatin analogue AN-162 [AEZS-124].

Previously, we have shown that the targeted cytotoxic somatostatin (sst) analogue AN-162 [AZSE-124] inhibits the growth of MDA-MB-231 human breast cancers xenografted into nude mice. In this study, we examined the trafficking of AN-162 into the cell, the expression of the somatostatin receptors (sstr) in specimens of human triple-negative breast cancers (TNBC), and the effect of AN-162 on HCC 1806 human TNBC xenografts. The expression of sstr in TNBC tumor samples was investigated by immunohistochemical staining. The expression of sstr in HCC 1806 was evaluated by reverse transcription PCR. Internalization studies with I-labeled AN-162 were carried out and the autofluorescence sign of doxorubicin moiety in the cell nucleus after incubation with AN-162 was measured using a fluorescence assay. The effects of AN-162 on the growth of HCC 1806 xenografted into nude mice were studied. A fluorescence microscopy cytotoxicity assay in vitro to detect cell death after treatment with AN-162 was also carried out. About 28% of TNBC tumor specimens showed a positive staining for sstr subtype 2a. HCC 1806 expresses all five subtypes of sstr. In the fluorescence cytotoxicity assay, dead HCC 1806 cells were found 24 h after incubation with AN-162. The growth of HCC 1806 tumors in nude mice was significantly inhibited by treatment with AN-162. AN-162 was internalized into the HCC 1806 cells and doxorubicin moiety was detected in the cell nuclei. This study is the first to show that the trafficking of the cytotoxic sst analogue AN-162 into the cell is mediated by sstr. Our work shows that the growth of xenografted HCC 1806 TNBCs can be effectively inhibited in vivo with AN-162. This investigation provides information on the mechanism of action and efficacy of this new targeted cytotoxic sst analogue and identifies in this relation the sstr as a favorable therapeutic target in TNBC.

Distribution of HPV Subtypes in Diverse Anogenital and Oral Samples from Women and Correlation of Infections with Neoplasia of the Cervix.

BACKGROUND: Cancers and intraepithelial lesions of different anogenital areas as well as oral cancer are associated with human papilloma virus (HPV) infections. METHODS: In this study cervical, vaginal, vulvar, anal, and oral samples were taken from 509 patients visiting our dysplasia consultation clinic. HPV genotyping was performed using the EUROArray HPV test. RESULTS: Positivity of HR HPV was found in 60.4-64.3% of anogenital and 14.6% of oral samples. HPV 16 showed the highest incidence in all investigated areas. In cervical and vaginal samples HPV 31 was detected second most, while in vulvar, anal, and oral samples HPV 53 was the second most common subtype. HPV 18 was found lower in all areas, while HPV 51, HPV 52, and HPV 73 were detected higher than expected from published data. A good concordance between cervical, vaginal and vulvar samples was examined for most of the HPV. HR HPV infection was higher in cervical cancer (CC; 91.7%) and high-grade intraepithelial squamous lesions (HSIL; 93.9%) compared to low-grade SIL (LSIL; 69.6%) and normal samples (44.8%). CONCLUSION: In addition to the well described HPV subtypes, we found others with high incidences in the investigated areas which may be evident for HSIL and CC of those areas.

Heparanase expression in term placentas of diabetic patients and healthy controls.

PURPOSE: The prevalence of diabetic disorders in pregnancy is rising, which goes along with increased risks for maternal and foetal complications during pregnancy and delivery. The expression of the endo-ß-D: -glucuronidase, heparanase (HPSE), may increase under hyperglycaemic conditions, is believed to play an important role in diabetes associated morbidity outside the female reproductive tract and is expressed in the placenta throughout gestation. However, the placental expression of HPSE has not been investigated in diabetic patients. MATERIALS AND METHODS: Placental biopsies of 30 patients with pre-existing or gestational diabetes and 30 healthy controls were analysed by real-time PCR and immunohistochemistry with regard to the presence of HPSE at term. RESULTS: Patients and controls were comparable with respect to foetal outcome and maternal characteristics except for maternal body mass index. We were unable to show significant differences in placental HPSE expression between diabetic patients and healthy controls. DISCUSSION: This study suggests that HPSE expression in term placentas is not affected by maternal diabetes and thus does not contribute to pathological processes in diabetic pregnancies with deliveries at term.

Induction of programmed cell death by inhibition of AKT with the alkylphosphocholine perifosine in in vitro models of platinum sensitive and resistant ovarian cancers.

PURPOSE: We analyzed the anti-tumor effect and the mechanism of action of perifosine, an orally active alkylphospholipid AKT inhibitor using in vitro models of human ovarian cancer. METHODS: Ovarian cancer cells OAW42, PA-1, SKOV3, and A2780 as well as platinum resistant A2780cis cells were incubated with increasing concentrations of perifosine, with and without multi-caspase inhibitor zVAD-FMK. The effect of a combined treatment with cisplatin and perifosine was investigated in OAW42, SKOV3, A2780 and A2780cis cells. Cytotoxic effects of perifosine were analyzed using crystal violet staining, FACS analysis of DNA content as well as Annexin V/propidium iodide-double staining. The effect of perifosine on AKT phosphorylation was determined by Western blotting. RESULTS: Perifosine displayed anti-tumor activity in all five cell lines, which increased time-dependently. While IC(50) values at 24 h were >40 µM, IC(50) values after 72 h decreased to 10 µM in OAW42 and 25 µM in PA-1 and 30 µm in SKOV3 cells. In platinum resistant A2780cis cells perifosine showed good antiproliferative activity (IC(50) = 3 µm). At adequate doses, perifosine increased cytotoxic effects of cisplatin in OAW42, A2780 and A2780cis cell. Anti-tumor activity of perifosine was not confined to a specific phase of the cell cycle and could not be decreased by the pan-caspase inhibitor zVAD-FMK. AnnexinV/propidium iodide-double staining after treatment with perifosine was not indicative of classical apoptosis. AKT phosphorylation was dose-dependently inhibited by perifosine. CONCLUSIONS: Perifosine showed substantial cytotoxic effects in various in vitro models of ovarian cancer. Since anti-tumor effects were not confined to platinum-sensitive cells perifosine seems to be a good candidate for clinical studies in patients especially with platinum resistant ovarian cancer.

Inhibitors of PD-1/PD-L1 and ERK1/2 impede the proliferation of receptor positive and triple-negative breast cancer cell lines.

PURPOSE: Triple-negative breast cancer (TNBC) is characterized by an unfavorable prognosis and missing systemic therapeutic approaches beside chemotherapy. Targeting the immune checkpoint PD-1/PD-L1 showed promising results in breast cancer and especially in TNBC. The extracellular signal-regulated kinase 1/2 (ERK1/2) is an important driver of carcinogenesis. Here, the effect of combined PD-1/PD-L1 and ERK1/2 inhibitor treatment is investigated of cell growth and intracellular impact of breast cancer cell lines. METHODS: The IC50 values of each inhibitor and the effect of combined treatment were determined in three TNBC cell lines of different subtypes and one non-TNBC cell line. Phospho-specific antibodies were used in western blot analyses to investigate an effect on ERK1/2 activation. Expressions of immune modulatory and cell cycle-associated genes were examined by quantitative reverse transcription PCR. RESULTS: Both inhibitors PD-1/PD-L1 and ERK1/2 impeded the proliferation of TNBC to a higher extent than of non-TNBC. By combined treatment, cell lines were inhibited either synergistically or additively. ERK1/2 and S6 phosphorylation were reduced and expressions of c-Fos and FosL were diminished after ERK1/2 inhibitor as single and combined treatment. Between genes involved in immune modulation, IL-8 was upregulated in TNBC cells after combined treatment. CONCLUSION: In conclusion, combination of PD-1/PD-L1 and ERK1/2 inhibitors showed favorable effects for a new therapy strategy, with better results in TNBC cell lines than in non-TNBC cells. The effects have to be validated in models that can reflect the interaction between immune and tumor cells like the situation in the tumor micro-environment.

Concentrations of inflammatory cytokines and the outcome in ICSI cycles.

UNLABELLED: The aim of this study was to measure circulating and intrafollicular concentrations of three inflammatory cytokines from women undergoing ovarian stimulation in order to determine their prognostic value in the outcome of intracytoplasmic sperm injection/embryo transfer cycles. MATERIALS AND METHODS: A total of 72 women following ovarian stimulation and intracytoplasmic sperm injection were included. Blood serum samples were drawn at the day of chorionic gonadotropin administration. Follicular fluids were collected at the day of oocyte retrieval. The total fractions of tumor necrosis factor alpha, interleukin (IL)-1beta and IL-6 were measured with commercially available immunoassays. RESULTS: The concentrations of IL-1beta, both in serum and follicular fluids, were significantly different between ICSI cycles that resulted in pregnancy and those that failed. The concentrations of the other two cytokines did not significantly differ between successful and unsuccessful cycles. CONCLUSION: The circulating and intrafollicular concentrations of IL-1beta seem to be related to the pregnancy outcome in ICSI cycles of healthy women.

PSMD9 expression correlates with recurrence after radiotherapy in patients with cervical cancer.

In the current retrospective cohort study, the expression of the Proteasome 26S non-ATPase Subunit 9 (PSMD9) was investigated in 102 patients with cervical cancer. The rat homologue of PSMD9, Bridge-1, was identified as a binding protein of the transcription factors PDX-1 and E-12 via its PDZ-domain. The aim of the current study was to evaluate the prognostic or predictive value of PSMD9 expression as a biomarker for patients with cervical cancer. Tissue microarrays were constructed from formalin-fixed paraffin-embedded tissue specimens of cervical cancer and peritumoral stroma after hysterectomy and a Bridge-1 antibody was used to perform immunohistochemistry. The immunoreactions were analyzed using an immunoreactive score, which evaluated the number of positive cells as well as their intensity of PSMD9 expression. A misinterpretation of statistically significant results after multiple testing was controlled by the false discovery rate correction using the algorithm of Benjamini and Hochberg. All tumor tissues and almost all peritumoral stroma tissues expressed PSMD9. The PSMD9 expression in tumor tissues was significantly higher compared with the peritumoral stroma. PSMD9 expression correlated significantly with the expression of the proliferation marker MIB-1. Patients with stronger PSMD9 expression tended to exhibit a higher odds ratio for the recurrence of the disease in all patients (n=102) as well as in the subgroup of 47 patients having received a combined chemoradiotherapy following hysterectomy. In the group of 62 patients having that received radiotherapy following hysterectomy, which included the chemoradiotherapy patients, a higher PSMD9 expression significantly increased the odds for a recurrence to 1.983-fold even after FDR correction (P=0.0304). In conclusion, PSMD9 was indicated to be overexpressed in tumor tissues and associated with tumor cell proliferation. Therefore, PSMD9 may be useful as a tumor marker. Furthermore, increased PSMD9 overexpression may be used to predict resistance against radiation.

Triple-negative breast cancers express receptors for growth hormone-releasing hormone (GHRH) and respond to GHRH antagonists with growth inhibition.

Triple-negative breast cancers do not express receptors for estrogen or progesterone and do not overexpress HER2. These tumors have an unfavorable prognosis and at present chemotherapy is the only treatment option. Because the antagonists of growth hormone-releasing hormone (GHRH) have been shown to inhibit growth of a variety of cancers by endocrine and paracrine/autocrine mechanisms, we evaluated the expression of GHRH receptors in human specimens of triple-negative breast cancers and the response to GHRH by in vitro models. In samples of triple-negative breast cancers we found mRNA expression for the GHRH receptor and its functional splice variant SV1 in 25 and 70% of the cases, respectively and for GHRH in 80% of the samples. Immunoreaction of SV1 was detected in the human triple-negative breast cancer cell line HCC1806 while HCC1937 was negative. The growth of HCC1806 was stimulated by GHRH(1-44)NH(2) and inhibited by GHRH antagonist MZ-J-7-118. In addition, in HCC1806 MAP-kinases ERK-1/2 were activated by GHRH. Our findings suggest the existence of an autocrine loop consisting of GHRH and GHRH receptors in triple-negative breast cancers. Our in vitro studies demonstrate that targeting the GHRH receptor may be a therapeutic option which should be evaluated in studies in vivo.

Inhibition of estrogen receptor positive and negative breast cancer cell lines with a growth hormone-releasing hormone antagonist.

GHRH antagonists have been shown to inhibit growth of various human cancer cell lines xenografted into nude mice including estrogen receptor negative human breast cancers. Previous observations also suggest that GHRH locally produced in diverse neoplasms including breast cancer might directly affect proliferation of tumor cells. In the present study we demonstrate that a novel highly potent GHRH antagonist JMR-132 strongly inhibits the proliferation of both estrogen receptor negative SKBR 3 and estrogen receptor positive ZR 75 human breast cancer cell lines in vitro. The proliferation in vitro of ZR 75 and SKBR 3 was increased after direct stimulation with GHRH(1-29)NH2. The GHRH antagonist JMR-132 had a significant antiproliferative activity in the absence of GHRH and nullified the proliferative effect of GHRH in these cell lines. SKBR 3 and ZR 75 expressed the GHRH ligand as well as the pituitary type of GHRH-receptor, which likely appears to mediate the antiproliferative mechanisms in these cell lines. These in vitro results suggest that JMR-132 is a potent inhibitor of breast cancer growth, independent of the estrogen receptor status. Further investigations on the combination treatment with endocrine agents affecting the estrogen pathway and GRHR antagonists are needed in order to improve the treatment of breast cancer.

Effects of Combined Treatment with Vitamin D and COX2 Inhibitors on Breast Cancer Cell Lines.

BACKGROUND: Vitamin D is known for its anticancer potential. Prostaglandin E2 (PGE2) is a proliferative and inflammation-activating agent. The production of PGE2 is dependent on the activity of cyclooxygenase-2 (COX2). A link between vitamin D and PGE2 metabolism was shown recently. MATERIALS AND METHODS: In MDA-MB-231 and MCF-7 breast cancer cell lines, we investigated the influence of calcitriol and the COX2 inhibitor celecoxib on cell growth via the MTT test, as well as on the protein and mRNA expression of COX2 using western blot and quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The proliferation of MCF-7 and MDA-MB-231 was inhibited by both calcitriol and the COX2 inhibitor celecoxib and even more strongly by their combination. Moreover, calcitriol inhibited COX2 protein expression in MDA-MB-231 cells, as well as COX2 mRNA expression in both cell lines. CONCLUSION: The combination of calcitriol and celecoxib demonstrated a synergistic growth-inhibitory effect in breast cancer cell lines.

Immunohistochemistry of proteins for DNA mismatch repair in correlation to prognostic factors of mammarian cancer.

Mutations in genes of the DNA mismatch repair system (MMR) are strongly linked to the development of hereditary non-polyposis colorectal cancer and play a significant role in sporadic cancer too. Besides the repair of chromosomal mismatches produced during replication, the MMR is the linkage of DNA mismatches to cell cycle control. Proteins of the MMR are necessary for the induction of apoptosis in response to non-tolerable amounts of DNA damage. We correlated the immunoreactivity of the MMR proteins hMSH2, hMLH1 and PMS2 to the immunoreaction of p53, the proliferation marker Ki67 and clinical prognosis factors such as tumor grading and staging, steroid receptor expression and hemangiosis carcinomatosa or lymphangiosis carcinomatosa in 200 samples from patients with diagnosed breast cancer. No correlation could be detected among the expression of the three MMR-proteins hMSH2, hMLH1 and PMS2. The expression of hMSH2 correlated positively with the expression of p53, with the appearance of distant metastases, low differentiation and the appearance of hemangiosis carcinomatosa and lymphangiosis carcinomatosa, while it negatively correlated with the expression of the estrogen receptor. No correlation was detected between hMLH1 or PMS2 and any of the investigated factors. The expression of hMSH2 seems to be related with predictors of an unfavorable course of disease in breast cancer.

Correlation of DNA mismatch repair protein hMSH2 immunohistochemistry with p53 and apoptosis in cervical carcinoma.

BACKGROUND: Mutations in genes of the DNA mismatch repair system (MMR) are linked to hereditary non-polyposis colorectal cancer and also play a role in sporadic cancer. Besides its repair function, the MMR is the linkage of DNA mismatch recognition to the cell cycle control. MATERIALS AND METHODS: The correlation between the immunoreactivity of the MMR protein hMSH2 and p53, apoptosis, clinical prognosis factors and the survival rate in 102 samples of cervical carcinoma was determined. RESULTS: hMSH2 immunoreactivity was correlated with p53 and weakly correlated with apoptosis. hMSH2 immunoreactivity could not be correlated to any tumour markers, while apoptosis correlated significantly with T stage, FIGO classification and the relative risk of death from cervical cancer. CONCLUSION: In cervical cancer, the processes of DNA mismatch repair, cell cycle control and apoptosis seemingly act in concert. Decreased expression of the hMSH2 mismatch repair protein might lead to a failure in the induction of apoptosis.

Semen DNA fragmentation index, evaluated with both TUNEL and Comet assay, and the ICSI outcome.

BACKGROUND: In intracytoplasmic sperm injection (ICSI), there is always a risk of using spermatozoa with damaged DNA. The purpose of this study was to evaluate the percentage of spermatozoa with DNA fragmentation in processed semen samples used in ICSI cycles and to investigate the relationship between the DNA fragmentation index (DFI) and the ICSI outcome. PATIENTS AND METHODS: Fifty-six couples undergoing ICSI treatment were included. DFI was evaluated, by both terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) and single cell gel electrophoresis (Comet) assays, in the processed semen samples used for ICSI. RESULTS: Of the processed semen samples 17.85% had > or =10% spermatozoa with fragmented DNA. There was no correlation between DFI and the ICSI outcome. DFI assessed by the TUNEL assay was negatively correlated with sperm concentration, progressive motility and sperm morphology. CONCLUSION: A considerable proportion of processed semen samples used for ICSI have a high DFI. However, DFI of the processed semen samples does not seem to be related to the ICSI outcome.

Effects of an Antagonistic Analog of Growth Hormone-Releasing Hormone on Endometriosis in a Mouse Model and In Vitro.

Endometriosis is a benign gynecologic disorder causing dysmenorrhea, pelvic pain, and subfertility. Receptors for the growth hormone-releasing hormone (GHRH) were found in endometriotic tissues. Antagonists of GHRH have been used to inhibit the growth of endometriotic endometrial stromal cells. In this study, the GHRH receptor splice variant (SV) 1 was detected in human endometrial tissue samples by Western blots and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The highest messenger RNA (mRNA) and protein levels of SV1 were found in eutopic endometrium from patients with endometriosis compared to ectopic endometriotic tissues and endometrium from normal patients. The highest expression for GHRH mRNA was found by qRT-PCR in ectopic endometriosis lesions. In an in vivo mouse model with human endometrial explants from patients with endometriosis, 10 µg MIA-602 per day resulted in significantly smaller human endometrial xenotransplants after 4 weeks compared to mice treated with vehicle. The endometrial tissues expressed SV1 before and after xenotransplantation. The proliferation of endometrial stromal cells as well as the endometriosis cell lines 12-Z and 49-Z was decreased by exposure to 1 µM MIA-602 after 72 hours. The protein levels of epithelial growth factor receptors in 12-Z and 49-Z cell lines were reduced 48 and 72 hours after the administration of 1 µM MIA-602. MIA-602 decreased the activation of the MAP-kinases ERK-1/2. Our study demonstrates the presence of SV1 receptor as a target for treatment with GHRH antagonist in endometriosis. Endometrial tissues respond to MIA-602 with inhibition of proliferation in vitro and in vivo. The use of MIA-602 could be an effective supplement to the treatment strategies in endometriosis.

Disrupting Y-Box-Binding Protein 1 Function Using OSU-03012 Prevents Endometriosis Progression in In Vitro and In Vivo Models.

The objective of the present study was to test the ability of OSU-03012 (2-amino-N-[4-[5-phenanthren-2-yl-3-(trifluoromethyl)pyrazol-1-yl]phenyl]acetamide), a novel and potent celecoxib-derivative, to impair endometriosis progression in in vitro and in vivo models based on its ability to indirectly block Y-box-binding protein 1 (YB-1) function. 12Z human endometriotic epithelial cells and sexually mature female C57BL/6J mice were treated with OSU-03012. Cellular proliferation was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid assay. Expression of YB-1 and phosphorylated YB-1 in 12Z cells and endometriotic lesions was evaluated by Western blotting and immunohistochemistry (IHC). The IHC for proliferating cell nuclear antigen was performed. OSU-03012 treatment resulted in decreased YB-1 and its phosphorylated form in both in vitro and in vivo models. Endometriotic lesion size was significantly reduced in OSU-03012-treated mice (27.6 ± 4.0 mm3) compared to those from the control group (50.5 ± 6.9 mm3, P < .0001). A significant reduction in endometriotic epithelial cell proliferation was observed in endometriotic lesions exposed to OSU-03012 treatment ( P = .0346). In conclusion, targeting YB-1 via OSU-03012 showed a potent antiproliferative effect on endometriotic epithelial cells in vitro and in a mouse model of disease.

Claudin-1 expression in cervical cancer.

Claudin-1 is a tight junction protein that has been demonstrated to be involved in tumorigenesis and tumor progression in various types of solid tumors. In the present study, the protein expression of claudin-1 in squamous cervical cancer tissues obtained from 106 patients was analyzed by immunohistochemistry. In addition, the grade of claudin-1 expression was analyzed for associations with certain clinicopathological parameters. A significant overexpression of claudin-1 was detected in the tumor cells, when compared with that in the peritumoral stroma. There was no significant association between claudin-1 expression and FIGO stage, tumor size, grading or the appearance of distant metastases. Cervical cancer patients scoring positive for claudin-1 protein expression tended to exhibit more lymph node metastasis (28.3%), compared with claudin-1-negative patients (7.1%). Regarding overall survival, the results of the present study suggest a better prognosis for claudin-1-negative patients. In order to elucidate whether claudin-1 overexpression has a significant prognostic impact on squamous cervical cancer, further studies are required.

Cell-specific enhancement of liposomal transfection by steroids in steroid receptor expressing cells.

The DNA transfer efficiency in liposomal versus viral transfection is very low, mainly due to an insufficient nuclear transport of the delivered DNA after its endocytotic uptake to the cell. Ligand activation of intracellular steroid receptors and their subsequent mobilization to the nucleus could result in a co-transport of DNA to the nucleus. The augmentation of nuclear transport of DNA after steroid addition might cause enhanced transfection efficiency. We used cell lines from gynecologic malignoma expressing steroid receptors, such as T47D and Mcf-7 breast cancer cell lines, as well as receptor-negative cell lines, such as Hec1A from endometrium carcinoma or the breast cancer cell line MDA-MB-231. The cells were transfected by the liposomal transfection agent Dotap with the gene for firefly-luciferase as a reporter gene and transfection efficiencies were determined in the luciferase assay. We compared the effect of the addition of cholesterol and steroids in different cell lines on the transfection efficiency. The addition of cholesterol to transfection agents led to an enhancement of the luciferase activity in all cell lines. Steroids enhanced the transfection efficiency only in receptor-positive cell lines. The transfection efficiencies of HEC-1A or MDA-MB-231 cells were not enhanced by steroids. A progesterone preincubation of receptor-positive T47D cells resulted in a decrease of progesterone receptors and afterwards the progesterone enhanced transfection was dramatically diminished. We presume that the transfection enhancement by steroids is dependent on increased nuclear import of the delivered DNA only in the presence of steroid receptors. Steroid enhancement of transfection is different from the benefit of cholesterol for transfection that acts on general cellular properties or the transfection complex as such. Liposomal transfection in combination with steroids might be useful for a cell-specific enhancement of gene transfer for example in gynecological malignoma where subgroups are expressing high levels of steroid receptors.