AKT inhibition mitigates GRP78 (glucose-regulated protein) expression and contribution to chemoresistance in endometrial cancers.

Overexpression of the unfolded protein response master regulator GRP78 is associated with poor prognosis and therapeutic resistance in numerous human cancers, yet its role in endometrial cancers (EC) is undefined. To better understand the contribution of GRP78 to EC, we examined its expression levels in EC patient samples and EC cell lines. We demonstrate that GRP78 overexpression occurs more frequently in EC tissues compared with that found in normal endometrium, and that GRP78 expression occurs in most EC cell lines examined. Functional analysis demonstrated that GRP78 is inducible by cisplatin in EC cells, and siRNA knockdown of GRP78 augments chemotherapy-mediated cell death. Examination of AKT and GRP78 expression demonstrated that inhibition of AKT activity by MK2206 blocks GRP78 expression in EC cells. SiRNA studies also revealed that knockdown of GRP78 reduces but does not abrogate AKT activity, demonstrating that GRP78 is required for optimal AKT activity. In the presence of MK2206, siRNA knockdown of GRP78 does not augment AKT mediated survival in response to cisplatin treatment, suggesting that GRP78's antiapoptosis functions are part of the AKT survival pathway. Targeted therapies that reduce GRP78 expression or activity in cancers may serve to increase the effectiveness of current therapies for EC patients.

Subcutaneous device-free islet transplantation.

Diabetes mellitus is a chronic metabolic disease, characterized by high blood sugar levels; it affects more than 500 million individuals worldwide. Type 1 diabetes mellitus (T1DM) is results from insufficient insulin secretion by islets; its treatment requires lifelong use of insulin injections, which leads to a large economic burden on patients. Islet transplantation may be a promising effective treatment for T1DM. Clinically, this process currently involves directly infusing islet cells into the hepatic portal vein; however, transplantation at this site often elicits immediate blood-mediated inflammatory and acute immune responses. Subcutaneous islet transplantation is an attractive alternative to islet transplantation because it is simpler, demonstrates lower surgical complication risks, and enables graft monitoring and removal. In this article, we review the current methods of subcutaneous device-free islet transplantation. Recent subcutaneous islet transplantation techniques with high success rate have involved the use of bioengineering technology and biomaterial cotransplantation-including cell and cell growth factor co-transplantation and hydrogel- or simulated extracellular matrix-wrapped subcutaneous co-transplantation. In general, current subcutaneous device-free islet transplantation modalities can simplify the surgical process and improve the posttransplantation graft survival rate, thus aiding effective T1DM management.

Germline PRKACA amplification-associated primary pigmented nodular adrenocortical disease: a case report and literature review.

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare adrenocorticotropin hormone (ACTH)-independent Cushing's syndrome (CS). Pediatric patients with PPNAD typically have unusual skin lesions and slow growth with unknown causes. We present a case of a female Chinese patient with PPNAD caused by the germline PRKACA gene copy number gain of chromosome 19. The patient initially presented with kidney stones, short stature, and obesity. After further testing, it was discovered that the patient had diabetes, mild hypertension, low bone mass, a low ACTH level, and hypercortisolemia, and neither the low-dose or high-dose dexamethasone suppression test was able to inhibit hematuric cortisol, which paradoxically increased. PPNAD was pathologically diagnosed after unilateral adrenalectomy. Chromosome microarrays and whole exon sequencing analyses of the peripheral blood, as well as testing of sectioned adrenal tissue, showed a rise in the copy number of the duplication-containing PRKACA gene on chromosome 19p13.13p13.12, a de novo but not heritable gene defect that causes disease. The clinical signs and symptoms supported the diagnosis of Carney complex (CNC). One significant mechanism of CNC pathogenesis may be the rise in germline PRKACA copy number of chromosome 19. When assessing PPNAD patients for CNC, the possibility of PRKACA gene amplification should be considered. The effect of PRKACA gene amplification on the clinical manifestations of CNC needs to be confirmed by more cases.

Germline PRKACA amplification-associated primary pigmented nodular adrenocortical disease: a case report and literature review

SUMMARY Primary pigmented nodular adrenocortical disease (PPNAD) is a rare adrenocorticotropin hormone (ACTH)-independent Cushing's syndrome (CS). Pediatric patients with PPNAD typically have unusual skin lesions and slow growth with unknown causes. We present a case of a female Chinese patient with PPNAD caused by the germline PRKACA gene copy number gain of chromosome 19. The patient initially presented with kidney stones, short stature, and obesity. After further testing, it was discovered that the patient had diabetes, mild hypertension, low bone mass, a low ACTH level, and hypercortisolemia, and neither the low-dose or high-dose dexamethasone suppression test was able to inhibit hematuric cortisol, which paradoxically increased. PPNAD was pathologically diagnosed after unilateral adrenalectomy. Chromosome microarrays and whole exon sequencing analyses of the peripheral blood, as well as testing of sectioned adrenal tissue, showed a rise in the copy number of the duplication-containing PRKACA gene on chromosome 19p13.13p13.12, a de novo but not heritable gene defect that causes disease. The clinical signs and symptoms supported the diagnosis of Carney complex (CNC). One significant mechanism of CNC pathogenesis may be the rise in germline PRKACA copy number of chromosome 19. When assessing PPNAD patients for CNC, the possibility of PRKACA gene amplification should be considered. The effect of PRKACA gene amplification on the clinical manifestations of CNC needs to be confirmed by more cases.

Effect of mifepristone on endometrial matrix metalloproteinase expression and leukocyte abundance in new medroxyprogesterone acetate users.

PURPOSE: This double-blind, placebo-controlled study was conducted to evaluate the molecular mechanism of mifepristone controlling breakthrough bleeding (BTB) in new depot-medroxyprogesterone acetate (DMPA) users. METHOD: A total of 50 regularly cycling women who were new starters of DMPA were randomized to receive 50 mg of mifepristone or placebo once every 14 days for six cycles. Endometrial biopsies were obtained on each patient before, during and after treatment. Endometrial matrix metalloproteinase 1 (MMP-1) and MMP-9 protein and mRNA were determined by immunohistochemistry and real-time PCR, respectively. The number of T lymphocytes (CD3-positive) and mast cells (mast tryptase-positive) was evaluated by immunohistochemistry. RESULTS: MMP-1, MMP-9, CD3-positive and mast tryptase-positive cells increased following the DMPA treatment. Addition of mifepristone to DMPA-exposed endometrium for 1 week significantly decreased stromal MMP-9 expression and numbers of CD3-positive and mast tryptase-positive cells. CONCLUSION: The decreased rates of BTB in new users of DMPA by mifepristone are associated with decreased MMP-1 and MMP-9 expression and fewer mast and T cells.

Surface chemical immobilization of parylene C with thermosensitive block copolymer brushes based on N-isopropylacrylamide and N-tert-butylacrylamide: synthesis, characterization, and cell adhesion/detachment.

Poly(N-isopropylacrylamide) (pNIPAM), poly(N-tert-butylacrylamide) (pNTBAM), and their copolymer brushes were covalently immobilized onto parylene C (PC) surfaces via surface initiated atom transfer radical polymerization (ATRP). Contact angle measurement between 13 and 40°C showed that the hydrophobicity of the modified PC surfaces was thermally sensitive. Among these samples, PC grafted with pNIPAM (PC-NI), PC grafted with pNTBAM (PC-NT) and PC grafted with copolymer brushes containing pNTBAM and pNIPAM (PC-NT-NI) exhibited the lower critical solution temperature (LCST) at 29, 22, and 24°C, respectively. Cytocompatibility study for the modified surfaces was performed by 5 days human skin fibroblast culture at 37°C. Data showed that only a very small amount of cells adhered on the PC and PC-NI surfaces, while a significantly higher amount of cell adhesion and growth was observed on PC-NT and PC-NT-NI surfaces. Furthermore, cell detachment at the temperatures of 24 and 6°C were studied after the substrates were cultured with cells at 37°C for 24 h. The results showed that the cells on PC-NI formed the aggregations and loosely attached on the substrate after 30-min culture at 24°C, while no significant cell detachment was observed for PC-NT and PC-NT-NI samples at this temperature. By continuing the cell culture for additional 100 min at 6°C for PC-NT and PC-NT-NI, about 10 and 35% of the cells were found detached respectively, and the unattached cells aggregated on the substrate. In comparison, cells cultured on the tissue culture petri dish (TCP) exhibited no quantity and morphology changes at the culture temperatures of 37, 24, and 6°C. This study showed that: (1) immobilization of PC with nonthermal sensitive pNTBAM could provide PC surface thermal sensitive hydrophilicity; (2) the chlorines on the polymer brushes of PC-NT could be used to further initiate the ATRP pNIPAM and form block copolymer brushes; (3) the incorporation of pNTBAM into pNIPAM on PC-NT-NI could change the surface thermal hydrophilicity property, and be further applied to decrease the LCST of the modified PC surface; (4) grafted pNIPAM brushes on PC-NI by ATRP showed very low cell adhesion and proliferation in 5 days fibroblast culture at 37°C, and cell detached at 24°C; (5) the incorporation of pNTBAM into pNIPAM on PC-NT-NI decreased the thermal sensitivity of cell adhesion/detachment, cell detached at 6°C, but the cell adhesion and proliferation were significantly improved at a wide temperature range.

Cutaneous papilloma and squamous cell carcinoma therapy utilizing nanosecond pulsed electric fields (nsPEF).

Nanosecond pulsed electric fields (nsPEF) induce apoptotic pathways in human cancer cells. The potential therapeutic effective of nsPEF has been reported in cell lines and in xenograft animal tumor model. The present study investigated the ability of nsPEF to cause cancer cell death in vivo using carcinogen-induced animal tumor model, and the pulse duration of nsPEF was only 7 and 14 nano second (ns). An nsPEF generator as a prototype medical device was used in our studies, which is capable of delivering 7-30 nanosecond pulses at various programmable amplitudes and frequencies. Seven cutaneous squamous cell carcinoma cell lines and five other types of cancer cell lines were used to detect the effect of nsPEF in vitro. Rate of cell death in these 12 different cancer cell lines was dependent on nsPEF voltage and pulse number. To examine the effect of nsPEF in vivo, carcinogen-induced cutaneous papillomas and squamous cell carcinomas in mice were exposed to nsPEF with three pulse numbers (50, 200, and 400 pulses), two nominal electric fields (40 KV/cm and 31 KV/cm), and two pulse durations (7 ns and 14 ns). Carcinogen-induced cutaneous papillomas and squamous carcinomas were eliminated efficiently using one treatment of nsPEF with 14 ns duration pulses (33/39 = 85%), and all remaining lesions were eliminated after a 2nd treatment (6/39 = 15%). 13.5% of carcinogen-induced tumors (5 of 37) were eliminated using 7 ns duration pulses after one treatment of nsPEF. Associated with tumor lysis, expression of the anti-apoptotic proteins Bcl-xl and Bcl-2 were markedly reduced and apoptosis increased (TUNEL assay) after nsPEF treatment. nsPEF efficiently causes cell death in vitro and removes papillomas and squamous cell carcinoma in vivo from skin of mice. nsPEF has the therapeutic potential to remove human squamous carcinoma.

Quantitative detection of EP3-II, III and VI messenger RNA in gravid and non-gravid human myometrium using real-time RT-PCR.

OBJECTIVE: To examine mRNA expression of prostaglandin E2 receptor isoforms EP3-II, III and VI in lower uterine segment myometrium in the non-pregnant and pregnant state using quantitative real-time RT-PCR. METHODS: Myometrial samples were obtained at the time of cesarean delivery or hysterectomy. Pregnant subjects were categorised based on the presence or absence of labour. Labour was defined as regular uterine contractions resulting in cervical change. Quantification for EP3 isoforms II, III and VI mRNA was performed using real-time RT-PCR. RESULTS: There were no differences between non-pregnant and non-labouring pregnant subjects in mRNA expression of EP3-II, III and VI. However, when compared to pregnant subjects not in labour, labouring subjects had a 3.8-fold reduction in EP3-II expression (p < 0.001) and 5.3-fold reduction in EP3-VI expression (p = 0.022). CONCLUSIONS: In human parturition, there is decreased mRNA expression of lower-uterine segment EP3 receptor isoforms II and VI during labour. This may reflect differential relaxation of the lower segment of the uterus allowing dilatation and descent of the fetus.

Effect of mifepristone on the expression of endometrial secretory leukocyte protease inhibitor in new medroxyprogesterone acetate users.

The effect of mifepristone on the expression of secretory leukocyte protease inhibitor (SLPI) in the endometrium of women using depot medroxyprogesterone acetate (DMPA) was investigated in this randomized, placebo-controlled trial. The study showed that the administration of DMPA led to a substantial inhibition of endometrial SLPI protein and mRNA, and that the addition of mifepristone to DMPA-exposed endometrium partially restored the expression of glandular SLPI.

Mifepristone alters expression of endometrial steroid receptors and their cofactors in new users of medroxyprogesterone acetate.

OBJECTIVE: To evaluate the effect of mifepristone on the expression of endometrial steroid receptors and their co-factors in depot medroxyprogesterone acetate (DMPA) users. DESIGN: A prospective, randomized, placebo-controlled trial. SETTING: Reproductive research center. PATIENT(S): Fifty healthy women with regular menstrual cycle. INTERVENTION(S): One hundred fifty milligrams of DMPA were given every 3 months. Two pills (25 mg each) of placebo or mifepristone were administered every 14 days during the DMPA therapy. Four endometrial biopsy specimens were obtained from each patient. MAIN OUTCOME MEASURE(S): The expression of estrogen receptor subtypes alpha and beta (ERalpha and ERbeta), progesterone receptors A and B (PRAB and PRB), and androgen receptor messenger RNA and protein was detected by real-time polymerase chain reaction and immunohistochemistry, respectively. Steroid receptor coactivator 1 (SRC-1), silencing mediator for retinoid and thyroid-hormone receptors, and cell proliferation were evaluated by immunohistochemistry. RESULT(S): The expression of endometrial ERalpha, PRAB, PRB, and SRC-1 was increased significantly after 1 week of mifepristone, but the increase was no longer seen after 10 weeks. A positive correlation between endometrial ERalpha, PRAB, PRB, and SRC-1 production and proliferation was demonstrated. CONCLUSION(S): Short-term exposure of mifepristone in new starters of DMPA increases the expression of endometrial ERalpha, PRAB, PRB, and SRC-1 and promotes cell proliferation. Prolonged exposure to mifepristone does not alter the suppression of these receptors that are caused by DMPA and continues to result in endometrial atrophy.

Effects of mifepristone on proliferation and apoptosis of human endometrium in new users of medroxyprogesterone acetate.

BACKGROUND: Mifepristone has been demonstrated to decrease breakthrough bleeding (BTB) in users of progestin-only contraceptives. METHODS: Endometrial biopsies were collected from 50 normal cycling women who were new users of depot medroxyprogesterone acetate (DMPA) randomized to receive either mifepristone or placebo before, during and after treatment. Proliferation, apoptosis and sex steroid receptors were evaluated by either immunohistochemistry or TUNEL assay. RESULTS: Administration of mifepristone to DMPA-exposed endometrium for 1 week significantly increased endometrial expression of Ki-67 (MKI67), estrogen receptor (ER)alpha and progesterone receptors A and B (PRAB) and decreased the number of TUNEL-positive and caspase-3 (CASP3)-active cells in the endometrial stroma. However, after 10 weeks of mifepristone treatment, no significant difference in proliferation, apoptosis and the expression of ERalpha or PRAB could be detected between the endometrium treated with DMPA alone and endometrium treated with mifepristone and DMPA. CONCLUSIONS: Administration of mifepristone to DMPA users significantly increases endometrial proliferation and decreases endometrial stromal apoptosis in the short term. Prolonged exposure to mifepristone does not counteract the inhibitory effects of progestin therapy on endometrial proliferation. Estrogen and progesterone receptors may play an important role in these effects.

TetX is a flavin-dependent monooxygenase conferring resistance to tetracycline antibiotics.

The tetracycline antibiotics block microbial translation and constitute an important group of antimicrobial agents that find broad clinical utility. Resistance to this class of antibiotics is primarily the result of active efflux or ribosomal protection; however, a novel mechanism of resistance has been reported to be oxygen-dependent destruction of the drugs catalyzed by the enzyme TetX. Paradoxically, the tetX genes have been identified on transposable elements found in anaerobic bacteria of the genus Bacteroides. Overexpression of recombinant TetX in Escherichia coli followed by protein purification revealed a stoichiometric complex with flavin adenine dinucleotide. Reconstitution of in vitro enzyme activity demonstrated a broad tetracycline antibiotic spectrum and a requirement for molecular oxygen and NADPH in antibiotic degradation. The tetracycline products of TetX activity were unstable at neutral pH, but mass spectral and NMR characterization under acidic conditions supported initial monohydroxylation at position 11a followed by intramolecular cyclization and non-enzymatic breakdown to other undefined products. TetX is therefore a FAD-dependent monooxygenase. The enzyme not only catalyzed efficient degradation of a broad range of tetracycline analogues but also conferred resistance to these antibiotics in vivo. This is the first molecular characterization of an antibiotic-inactivating monooxygenase, the origins of which may lie in environmental bacteria.