Effects of combined treatment with rapamycin and cotylenin A, a novel differentiation-inducing agent, on human breast carcinoma MCF-7 cells and xenografts

INTRODUCTION: Rapamycin, an inhibitor of the serine/threonine kinase target of rapamycin, induces G(1 )arrest and/or apoptosis. Although rapamycin and its analogues are attractive candidates for cancer therapy, their sensitivities with respect to growth inhibition differ markedly among various cancer cells. Using human breast carcinoma cell line MCF-7 as an experimental model system, we examined the growth-inhibitory effects of combinations of various agents and rapamycin to find the agent that most potently enhances the growth-inhibitory effect of rapamycin. METHOD: We evaluated the growth-inhibitory effect of rapamycin plus various agents, including cotylenin A (a novel inducer of differentiation of myeloid leukaemia cells) to MCF-7 cells, using either MTT assay or trypan blue dye exclusion test. The cell cycle was analyzed using propidium iodide-stained nuclei. Expressions of several genes in MCF-7 cells with rapamycin plus cotylenin A were studied using cDNA microarray analysis and RT-PCR. The in vitro results of MCF-7 cells treated with rapamycin plus cotylenin A were further confirmed in vivo in a mouse xenograft model. RESULTS: We found that the sensitivity of rapamycin to MCF-7 cells was markedly affected by cotylenin A. This treatment induced growth arrest of the cells at the G(1 )phase, rather than apoptosis, and induced senescence-associated β-galactosidase activity. We examined the gene expression profiles associated with exposure to rapamycin and cotylenin A using cDNA microarrays. We found that expressions of cyclin G(2), transforming growth factor-β-induced 68 kDa protein, BCL2-interacting killer, and growth factor receptor-bound 7 were markedly induced in MCF-7 cells treated with rapamycin plus cotylenin A. Furthermore, combined treatment with rapamycin and cotylenin A significantly inhibited the growth of MCF-7 cells as xenografts, without apparent adverse effects. CONCLUSION: Rapamycin and cotylenin A cooperatively induced growth arrest in breast carcinoma MCF-7 cells in vitro, and treatment with rapamycin and cotylenin A combined more strongly inhibited the growth of MCF-7 cells as xenografts in vivo than treatment with rapamycin or cotylenin A alone, suggesting that this combination may have therapeutic value in treating breast cancer. We also identified several genes that were markedly modulated in MCF-7 cells treated with rapamycin plus cotylenin A

Tissue-Specific Increases in 11β-Hydroxysteroid Dehydrogenase Type 1 in Normal Weight Postmenopausal Women

With age and menopause there is a shift in adipose distribution from gluteo-femoral to abdominal depots in women. Associated with this redistribution of fat are increased risks of type 2 diabetes and cardiovascular disease. Glucocorticoids influence body composition, and 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) which converts inert cortisone to active cortisol is a putative key mediator of metabolic complications in obesity. Increased 11βHSD1 in adipose tissue may contribute to postmenopausal central obesity. We hypothesized that tissue-specific 11βHSD1 gene expression and activity are up-regulated in the older, postmenopausal women compared to young, premenopausal women. Twenty-three pre- and 23 postmenopausal, healthy, normal weight women were recruited. The participants underwent a urine collection, a subcutaneous adipose tissue biopsy and the hepatic 11βHSD1 activity was estimated by the serum cortisol response after an oral dose of cortisone. Urinary (5α-tetrahydrocortisol+5β-tetrahydrocortisol)/tetrahydrocortisone ratios were higher in postmenopausal women versus premenopausal women in luteal phase (P<0.05), indicating an increased whole-body 11βHSD1 activity. Postmenopausal women had higher 11βHSD1 gene expression in subcutaneous fat (P<0.05). Hepatic first pass conversion of oral cortisone to cortisol was also increased in postmenopausal women versus premenopausal women in follicular phase of the menstrual cycle (P<0.01, at 30 min post cortisone ingestion), suggesting higher hepatic 11βHSD1 activity. In conclusion, our results indicate that postmenopausal normal weight women have increased 11βHSD1 activity in adipose tissue and liver. This may contribute to metabolic dysfunctions with menopause and ageing in women

Transforming growth factor beta-1 (TGFB1) and peak bone mass: association between intragenic polymorphisms and quantitative ultrasound of the heel

BACKGROUND: Variance of peak bone mass has a substantial genetic component, as has been shown with twin studies examining quantitative measures such as bone mineral density (BMD) and quantitative ultrasound (QUS). Evidence implicating single nucleotide polymorphisms (SNPs) of the transforming growth factor beta-1 (TGFB1) gene is steadily accumulating. However, a comprehensive look at multiple SNPs at this locus for their association with indices of peak bone mass has not been reported. METHODS: A cohort of 653 healthy Caucasian females 18 to 35 years old was genotyped for seven TGFB1 SNPs. Polymorphisms were detected by restriction endonuclease digestion of amplified DNA segments. RESULTS: The frequencies of the least common allele at G-800A, C-509T, codon 10 (L10P), codon 25 (R25P), codon 263 (T263I), C861-20T, and 713-8 delC loci were 0.07, 0.33, 0.41, 0.08, 0.04, 0.25 and 0.01, respectively. A significant association was seen between QUS Stiffness Index (QUS-SI) and the SNP at codon 10 and the linked promoter SNP, C-509T. This association remained significant after multiple regression was used to incorporate important clinical covariates – age, BMI, level of activity, family history, and caffeine intake – into the model. CONCLUSION: The association of QUS-SI with -509T is consistent with a gene-dose effect, while only individuals homozygous for the codon 10P allele showed a significant increase. In this cohort of young healthy Caucasian females, the T allele at position -509 is associated with greater bone mass as measured by calcaneal ultrasound

Acquisition of Complement Inhibitor Serine Protease Factor I and Its Cofactors C4b-Binding Protein and Factor H by Prevotella intermedia

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with 125I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases

Molecular control of HIV-1 postintegration latency: implications for the development of new therapeutic strategies

The persistence of HIV-1 latent reservoirs represents a major barrier to virus eradication in infected patients under HAART since interruption of the treatment inevitably leads to a rebound of plasma viremia. Latency establishes early after infection notably (but not only) in resting memory CD4+ T cells and involves numerous host and viral trans-acting proteins, as well as processes such as transcriptional interference, RNA silencing, epigenetic modifications and chromatin organization. In order to eliminate latent reservoirs, new strategies are envisaged and consist of reactivating HIV-1 transcription in latently-infected cells, while maintaining HAART in order to prevent de novo infection. The difficulty lies in the fact that a single residual latently-infected cell can in theory rekindle the infection. Here, we review our current understanding of the molecular mechanisms involved in the establishment and maintenance of HIV-1 latency and in the transcriptional reactivation from latency. We highlight the potential of new therapeutic strategies based on this understanding of latency. Combinations of various compounds used simultaneously allow for the targeting of transcriptional repression at multiple levels and can facilitate the escape from latency and the clearance of viral reservoirs. We describe the current advantages and limitations of immune T-cell activators, inducers of the NF-κB signaling pathway, and inhibitors of deacetylases and histone- and DNA- methyltransferases, used alone or in combinations. While a solution will not be achieved by tomorrow, the battle against HIV-1 latent reservoirs is well- underway

Prevention of multidrug resistance (MDR) in osteosarcoma by NSC23925

Background: The major limitation to the success of chemotherapy in osteosarcoma is the development of multidrug resistance (MDR). Preventing the emergence of MDR during chemotherapy treatment has been a high priority of clinical and investigational oncology, but it remains an elusive goal. The NSC23925 has recently been identified as a novel and potent MDR reversal agent. However, whether NSC23925 can prevent the development of MDR in cancer is unknown. Therefore, this study aims to evaluate the effects of NSC23925 on prevention of the development of MDR in osteosarcoma. Methods: Human osteosarcoma cell lines U-2OS and Saos were exposed to increasing concentrations of paclitaxel alone or in combination with NSC23925 for 6 months. Cell sublines selected at different time points were evaluated for their drug sensitivity, drug transporter P-glycoprotein (Pgp) expression and activity. Results: We observed that tumour cells selected with increasing concentrations of paclitaxel alone developed MDR with resistance to paclitaxel and other Pgp substrates, whereas cells cultured with paclitaxel–NSC23925 did not develop MDR and cells remained sensitive to chemotherapeutic agents. Paclitaxel-resistant cells showed high expression and activity of the Pgp, whereas paclitaxel–NSC23925-treated cells did not express Pgp. No changes in IC50 and Pgp expression and activity were observed in cells grown with the NSC23925 alone. Conclusions: Our findings suggest that NSC23925 may prevent the development of MDR by specifically preventing the overexpression of Pgp. Given the significant incidence of MDR in osteosarcoma and the lack of effective agents for prevention of MDR, NSC23925 and derivatives hold the potential to improve the outcome of cancer patients with poor prognosis due to drug resistance

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Background: Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. // Methods: We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung's disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. // Findings: We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung's disease) from 264 hospitals (89 in high-income countries, 166 in middle-income countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in low-income countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. // Interpretation: Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between low-income, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030