Shifting the Soil: Metformin Treatment Decreases the Protumorigenic Tumor Microenvironment in Epithelial Ovarian Cancer.

Controversy persists regarding metformin's role in cancer therapy. Our recent work suggested metformin acts by impacting the tumor microenvironment (TME), normalizing the epigenetic profile of cancer-associated mesenchymal stem cells (CA-MSC). As CA-MSC can negatively impact tumor immune infiltrates, we evaluated metformin's impact on the human TME, focusing on the interplay of stroma and immune infiltrates. Tumor samples from (i) 38 patients treated with metformin and chemotherapy and (ii) 44 non-metformin matched controls were included in a tissue microarray (TMA). The TMA was used to compare the presence of CA-MSC, desmoplasia and immune infiltrates in the TME. In vitro and in vivo models examined metformin's role in alteration of the CA-MSC phenotype. The average percentage of CA-MSC was significantly lower in metformin-treated than in chemotherapy alone-treated tumors (p = 0.006). There were fewer regulatory T-cells in metformin-treated tumors (p = 0.043). Consistent with CA-MSC's role in excluding T-cells from tumor islets, the T-cells were primarily present within the tumor stroma. Evaluation of metformin's impact in vitro suggested that metformin cannot reverse a CA-MSC phenotype; however, the in vivo model where metformin was introduced prior to the establishment of the CA-MSC phenotype supported that metformin can partially prevent the reprogramming of normal MSC into CA-MSC. Metformin treatment led to a decrease in both the presence of protumorigenic CA-MSC and in immune exclusion of T cells, leading to a more immune-permissive environment. This suggests clinical utility in prevention and in treatment for early-stage disease and putatively in immune therapy.

Phase II clinical trial of metformin as a cancer stem cell-targeting agent in ovarian cancer.

BACKGROUNDEpidemiologic studies suggest that metformin has antitumor effects. Laboratory studies indicate metformin impacts cancer stem-like cells (CSCs). As part of a phase II trial, we evaluated the impact of metformin on CSC number and on carcinoma-associated mesenchymal stem cells (CA-MSCs) and clinical outcomes in nondiabetic patients with advanced-stage epithelial ovarian cancer (EOC).METHODSThirty-eight patients with stage IIC (n = 1)/III (n = 25)/IV (n = 12) EOC were treated with either (a) neoadjuvant metformin, debulking surgery, and adjuvant chemotherapy plus metformin or (b) neoadjuvant chemotherapy and metformin, interval debulking surgery, and adjuvant chemotherapy plus metformin. Metformin-treated tumors, compared with historical controls, were evaluated for CSC number and chemotherapy response. Primary endpoints were (a) a 2-fold or greater reduction in aldehyde dehydrogenase-positive (ALDH+) CD133+ CSCs and (b) a relapse-free survival at 18 months of more than 50%.RESULTSMetformin was well tolerated. Median progression-free survival was 18.0 months (95% CI 14.0-21.6) with relapse-free survival at 18 months of 59.3% (95% CI 38.6-70.5). Median overall survival was 57.9 months (95% CI 28.0-not estimable). Tumors treated with metformin had a 2.4-fold decrease in ALDH+CD133+ CSCs and increased sensitivity to cisplatin ex vivo. Furthermore, metformin altered the methylation signature in CA-MSCs, which prevented CA-MSC-driven chemoresistance in vitro.CONCLUSIONTranslational studies confirm an impact of metformin on EOC CSCs and suggest epigenetic change in the tumor stroma may drive the platinum sensitivity ex vivo. Consistent with this, metformin therapy was associated with better-than-expected overall survival, supporting the use of metformin in phase III studies.TRIAL REGISTRATIONClinicalTrials.gov NCT01579812.

The Precursor to Glutathione (GSH), γ-Glutamylcysteine (GGC), Can Ameliorate Oxidative Damage and Neuroinflammation Induced by Aß40 Oligomers in Human Astrocytes.

Glutathione (GSH) is one of the most abundant thiol antioxidants in cells. Many chronic and age-related diseases are associated with a decline in cellular GSH levels or impairment in the catalytic activity of the GSH biosynthetic enzyme glutamate cysteine ligase (GCL). γ-glutamylcysteine (GGC), a precursor to glutathione (GSH), can replenish depleted GSH levels under oxidative stress conditions, by circumventing the regulation of GSH biosynthesis and providing the limiting substrate. Soluble amyloid-ß (Aß) oligomers have been shown to induce oxidative stress, synaptic dysfunction and memory deficits which have been reported in Alzheimer's disease (AD). Calcium ions, which are increased with age and in AD, have been previously reported to enhance the formation of Aß40 oligomers, which have been casually associated with the pathogenesis of the underlying neurodegenerative condition. In this study, we examined the potential beneficial effects of GGC against exogenous Aß40 oligomers on biomarkers of apoptosis and cell death, oxidative stress, and neuroinflammation, in human astrocytes. Treatment with Aß40 oligomers significantly reduced the cell viability and apoptosis of astrocyte brain cultures and increased oxidative modifications of DNA, lipids, and protein, enhanced pro-inflammatory cytokine release and increased the activity of the proteolytic matrix metalloproteinase enzyme, matric metalloproteinase (MMP)-2 and reduced the activity of MMP-9 after 24 h. Co-treatment of Aß40 oligomers with GGC at 200 µM increased the activity of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) and led to significant increases in the levels of the total antioxidant capacity (TAC) and GSH and reduced the GSSG/GSH ratio. GGC also upregulated the level of the anti-inflammatory cytokine IL-10 and reduced the levels of the pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) and attenuated the changes in metalloproteinase activity in oligomeric Aß40-treated astrocytes. Our data provides renewed insight on the beneficial effects of increased GSH levels by GGC in human astrocytes, and identifies yet another potential therapeutic strategy to attenuate the cytotoxic effects of Aß oligomers in AD.

Comparative diagnostic performance of volumetric laser endomicroscopy and confocal laser endomicroscopy in the detection of dysplasia associated with Barrett's esophagus.

BACKGROUND AND AIMS: Probe-based confocal laser endomicroscopy (pCLE) and volumetric laser endomicroscopy (VLE) (also known as frequency domain optical coherence tomography) are advanced endoscopic imaging modalities that may be useful in the diagnosis of dysplasia associated with Barrett's esophagus (BE). We performed pCLE examination in ex-vivo EMR specimens and compared the diagnostic performance of using the current VLE scoring index (previously established as OCT-SI) and a novel VLE diagnostic algorithm (VLE-DA) for the detection of dysplasia. METHODS: A total of 27 patients with BE enrolled in a surveillance program at a tertiary-care center underwent 50 clinically indicated EMRs that were imaged with VLE and pCLE and classified into neoplastic (N = 34; high-grade dysplasia, intramucosal adenocarcinoma) and nonneoplastic (N = 16; low-grade dysplasia, nondysplastic BE), based on histology. Image datasets (VLE, N = 50; pCLE, N = 50) were rated by 3 gastroenterologists trained in the established diagnostic criteria for each imaging modality as well as a new diagnostic algorithm for VLE derived from a training set that demonstrated association of specific VLE features with neoplasia. Sensitivity, specificity, and diagnostic accuracy were assessed for each imaging modality and diagnostic criteria. RESULTS: The sensitivity, specificity, and diagnostic accuracy of pCLE for detection of BE dysplasia was 76% (95% confidence interval [CI], 59-88), 79% (95% CI, 53-92), and 77% (95% CI, 72-82), respectively. The optimal diagnostic performance of OCT-SI showed a sensitivity of 70% (95% CI, 52-84), specificity of 60% (95% CI, 36-79), and diagnostic accuracy of 67%; (95% CI, 58-78). The use of the novel VLE-DA showed a sensitivity of 86% (95% CI, 69-96), specificity of 88% (95% CI, 60-99), and diagnostic accuracy of 87% (95% CI, 86-88). The diagnostic accuracy of using the new VLE-DA criteria was significantly superior to the current OCT-SI (P < .01). CONCLUSION: The use of a new VLE-DA showed enhanced diagnostic performance for detecting BE dysplasia ex vivo compared with the current OCT-SI. Further validation of this algorithm in vivo is warranted.

Effective treatment of dyssynergic defecation using sacral neuromodulation in a patient with cerebral palsy.

BACKGROUND: Dyssynergic defecation is a complex bowel problem that leads to chronic constipation and abdominal pain. Management is often challenging owing to the incoordination of multiple pelvic floor muscles involved in normal defecation. CASE: We report a case of dyssynergic defecatory dysfunction in a patient with cerebral palsy treated with sacral neuromodulation. At presentation, Sitz marker study and magnetic resonance defecography showed evidence of chronic functional constipation. Anorectal manometry, rectal anal inhibitory reflex, and rectal sensation study showed intact reflex and decreased first sensation of lower canal at 50 mL. After stage 2 of InterStim implant placement, bowel habits improved to once- to twice-daily soft solid bowel movements from no regular solid bowel movements. Fecal incontinence improved from daily liquid and small solid loss to no stool leakage. CONCLUSIONS: In patients with systemic medical problems contributing to defecatory dysfunction and bowel incontinence, such as cerebral palsy, sacral neuromodulation was found to provide significant relief of bowel symptoms in addition to associated abdominal pain. As a result of intervention, the patient reported significant improvement in quality of life and less limitations due to dyssynergic defecation.

Mechanisms by which low glucose enhances the cytotoxicity of metformin to cancer cells both in vitro and in vivo.

Different cancer cells exhibit altered sensitivity to metformin treatment. Recent studies suggest these findings may be due in part to the common cell culture practice of utilizing high glucose, and when glucose is lowered, metformin becomes increasingly cytotoxic to cancer cells. In low glucose conditions ranging from 0 to 5 mM, metformin was cytotoxic to breast cancer cell lines MCF7, MDAMB231 and SKBR3, and ovarian cancer cell lines OVCAR3, and PA-1. MDAMB231 and SKBR3 were previously shown to be resistant to metformin in normal high glucose medium. When glucose was increased to 10 mM or above, all of these cell lines become less responsive to metformin treatment. Metformin treatment significantly reduced ATP levels in cells incubated in media with low glucose (2.5 mM), high fructose (25 mM) or galactose (25 mM). Reductions in ATP levels were not observed with high glucose (25 mM). This was compensated by enhanced glycolysis through activation of AMPK when oxidative phosphorylation was inhibited by metformin. However, enhanced glycolysis was either diminished or abolished by replacing 25 mM glucose with 2.5 mM glucose, 25 mM fructose or 25 mM galactose. These findings suggest that lowering glucose potentiates metformin induced cell death by reducing metformin stimulated glycolysis. Additionally, under low glucose conditions metformin significantly decreased phosphorylation of AKT and various targets of mTOR, while phospho-AMPK was not significantly altered. Thus inhibition of mTOR signaling appears to be independent of AMPK activation. Further in vivo studies using the 4T1 breast cancer mouse model confirmed that metformin inhibition of tumor growth was enhanced when serum glucose levels were reduced via low carbohydrate ketogenic diets. The data support a model in which metformin treatment of cancer cells in low glucose medium leads to cell death by decreasing ATP production and inhibition of survival signaling pathways. The enhanced cytotoxicity of metformin against cancer cells was observed both in vitro and in vivo.

Shining a new narrow band of light on old problems.

Improvements in narrow band imaging (NBI) may provide an improved view of colonic mucosa for detection of polyps and adenomas. In this issue, Leung et al. report findings to suggest that this next-generation NBI technology is superior to conventional high-definition white light endoscopy in polyp detection. These findings are based on brighter illumination, which has been a problem with older generations of NBI, which did not increase polyp detection but were useful for polyp characterization. Although these findings are very promising for this new role of second-generation NBI in polyp detection, the study must be viewed with consideration of the history of the older NBI system, the analysis of which through multiple positive and negative studies ultimately led to the conclusion that it was not beneficial for detection.

Metformin and phenethyl isothiocyanate combined treatment in vitro is cytotoxic to ovarian cancer cultures.

BACKGROUND: High mortality rates in ovarian cancer are largely a result of resistance to currently used chemotherapies. Expanding therapies with a variety of drugs has the potential to reduce this high mortality rate. Metformin and phenethyl isothiocyanate (PEITC) are both potentially useful in ovarian cancer, and they are particularly attractive because of their safety. METHODS: Cell proliferation of each drug and drug combination was evaluated by hemacytometry with Trypan blue exclusion or Sytox green staining for cell death. Levels of total and cleaved PARP were measured by Western blot. General cellular and mitochondrial reactive oxygen species were measured by flow cytometry and live cell confocal microscopy with the fluorescent dyes dihydroethidine and MitoSOX. RESULTS: Individually, metformin and PEITC each show inhibition of cell growth in multiple ovarian cancer cell lines. Alone, PEITC was also able to induce apoptosis, whereas metformin was primarily growth inhibitory. Both total cellular and mitochondrial reactive oxygen species were increased when treated with either metformin or PEITC. The growth inhibitory effects of metformin were reversed by methyl succinate supplementation, suggesting complex I plays a role in metformin's anti-cancer mechanism. PEITC's anti-cancer effect was reversed by N-acetyl-cysteine supplementation, suggesting PEITC relies on reactive oxygen species generation to induce apoptosis. Metformin and PEITC together showed a synergistic effect on ovarian cancer cell lines, including the cisplatin resistant A2780cis. CONCLUSIONS: Here we show that when used in combination, these drugs are effective in both slowing cancer cell growth and killing ovarian cancer cells in vitro. Furthermore, the combination of these drugs remains effective in cisplatin resistant cell lines. Novel combinations such as metformin and PEITC show promise in expanding ovarian cancer therapies and overcoming the high incidence of cisplatin resistant cancers.

Risk factor profile in Chinese-Australian stroke patients living in Sydney.

AIM: To examine and compare stroke risk factors and their management in stroke patients of Chinese descent versus English-speaking background (ESB)-Australian patients. METHODS: Cohort study. Fifty-one Chinese-Australians and 119 ESB-Australians who were admitted to hospitals within Sydney metropolitan area with a recent acute ischaemic or haemorrhagic stroke were recruited. RESULTS: Chinese-Australian patients tended to have a favourable smoking (0% current smokers vs 15%, P = 0.036) and drinking (5% current medium/heavy drinkers vs 17%, P = 0.005) pattern compared with the Australian patients. The prevalence of diabetes mellitus was higher in Chinese-Australians (31% vs 10%, P = 0.003). The management of hypertension and atrial fibrillation (AF) in Chinese-Australians was suboptimal (19% untreated hypertension vs 8%, P = 0.102; 78% AF not on Warfarin vs 51%, P = 0.264). CONCLUSION: The findings of this study suggest that targeting specific stroke prevention strategies may be useful for Chinese-Australians. Larger-scale studies need to be conducted to confirm these findings.

A prospective review of hip fracture subtypes, surgical procedure, cognitive status, and analgesia use across 4 Australian hospitals.

OBJECTIVES: To correlate analgesia use among patients with hip fracture requiring surgery with hip fracture subtype, cognitive status, and type of surgery in the postacute period. DESIGN AND PARTICIPANTS: Prospective review of patients with hip fractures requiring surgical intervention. A total of 415 patients (mean age: 81.2 ± 9.1 years, 74.3% women) presented with 195 subcapital fractures (39 undisplaced, 156 displaced) and 220 trochanteric fractures (136 stable, 84 unstable) requiring surgery. SETTING: Inpatient orthopedic units in 4 Australian hospitals. MEASUREMENTS: The primary outcome measures were mean analgesia usage (oral morphine equivalent) for 4 defined time intervals and total amount 36 hours following surgery. RESULTS: Patients with subtrochanteric fractures required more analgesia compared with displaced-subcapital, undisplaced-subcapital, basicervical, stable-pertrochanteric, and unstable-pertrochanteric fractures in the 24 to 36 hours following operation (24.7 vs 11.3 vs 8.8 vs 12.1 vs 7.6 vs 9.7, P = .001). Total analgesia requirements were higher in patients treated with an intramedullary nail, increasing by 1.3- to 3.3-fold in the 36 hours postsurgery. Patients with cognitive impairment utilized markedly less analgesia at all time periods measured. At 24 to 36 hours, higher levels of analgesia were noted in patients with higher premorbid level of mobility (P = .015) and activities of daily living function (P = .007). CONCLUSION: Important differences in utilization of analgesia following hip fracture across readily defined clinical groups exist. Proactive pain management for those with cognitive impairment, certain hip fracture subtypes, and surgical procedures may enable early functional mobility and other activities.

Risk factor, pattern, etiology and outcome in ischemic stroke patients with cancer: a nested case-control study.

BACKGROUND: Coagulation disorders are seen in cancer patients, but it is not clear whether cancer predisposes stroke patients to unique characteristics. The aim of the study was to investigate risk factors, pattern,etiology and outcome in stroke patients with cancer. METHODS: A retrospective review of all ischemic stroke (IS) patients with cancer (n = 56) admitted to Bankstown-Lidcombe Hospital, Sydney, Australia, between January 1999 and December 2004 was conducted and comparison made to age- and gender-matched noncancer IS patients admitted to the same hospital during the same period. RESULTS: Vascular risk factors and stroke pattern were comparable in cancer and noncancer groups. Post-stroke thrombotic episodes (myocardial infarction, deep vein thrombosis or pulmonary emboli) were more common in the cancer group than in the noncancer group (11 vs. 0%, p = 0.031). Depression was also more common in the cancer group than in the noncancer group (14 vs. 2%, p = 0.039). There was a tendency for more patients in the cancer group to die in hospital (30 vs. 14%, p = 0.078). CONCLUSIONS: Coagulation disorders were more likely to be seen in stroke cancer patients, and patients with cancer tended to have a higher in-hospital post-stroke mortality. Larger sample size studies may identify further differences in the characteristics of stroke patients with cancer.

Comparison of stroke risk factors and outcomes in patients with English-speaking background versus non-English-speaking background.

This study examined stroke risk factor profiles, management and outcomes for elderly patients with English-speaking background (ESB) and non-English-speaking background (NESB). This is an observational cohort study with both retrospective and prospective components. In total, 186 consecutive acute stroke patients aged > or =65 years admitted to our hospital were recruited over a 12-month period. Patient characteristics, stroke risk factors and management, in-hospital mortality, functional independence measurement scores before admission and at discharge, and discharge destination were recorded. On admission, NESB patients with atrial fibrillation (AF) were less likely to be taking warfarin than ESB patients (1 out of 19 with NESB vs. 19 out of 41 with ESB, p = 0.001). More NESB patients had a history of diabetes mellitus (DM) than ESB patients (41.4 vs. 10.2%, respectively; p = 0.001). However, ESB and NESB patients were comparable in terms of age, gender, preadmission functional status as well as other stroke risk factors (including smoking and alcohol drinking pattern, prevalence of hypertension and lipid disorder) and their management. In-hospital mortality was similar between ESB and NESB patients (10.2 vs. 8.6%). In conclusion, we found an association with our population of elderly NESB patients and an underutilization of warfarin for AF as well as a higher frequency of DM. Determination of the underlying reasons for such differences may be of value in the primary health care of NESB patients.

Identification of signal transduction pathways that modulate dibutyryl cyclic adenosine monophosphate activation of stanniocalcin gene expression in neuroblastoma cells.

Stanniocalcin (STC) is a new mammalian polypeptide hormone and appears to be a regulator of neuronal function. We have already shown that the induction of STC mRNA and protein expression by cAMP is integral to neuroblastoma cell differentiation, particularly neurite outgrowth. In this study, we examined the cAMP pathway in greater detail. Some common neuritogenic agents, euxanthone (PW1) and trans-retinoic acid (RA), were studied for possible interactions with the dibutyryl cAMP (dbcAMP)-mediated response. Our results showed that STC mRNA induction by dbcAMP was mediated by protein kinase A-cAMP response element binding protein (CREB) pathway, accompanied with phosphorylation of CREB and a reduction of p50, p65, and phosphorylated inhibitor kappaBalpha levels. Using a synthetic peptide nuclear factor-kappaB SN50, stimulation of dbcAMP-mediated STC expression was observed; indicating the nuclear translocation of nuclear factor kappaB might possibly repress STC expression. dbcAMP-induced STC mRNA expression was enhanced by PW1. In contrast, RA had highly suppressive effects. Cotreatment of cell with PW1 and cAMP provoked an increase in phosphorylated CREB (pCREB). Conversely, cotreatment with RA suppressed pCREB. The results highlighted the importance of phosphorylation of CREB in mediating STC gene expression. Taking a step further to dissect the possible regulatory pathways involved, with the aid of phorbol 12-myristate 13-acetate or ionomycin, additive effects on STC gene expression were observed. The induction was aided by further elevation of pCREB, which was completely abolished by Gö 6976, a Ca2+-dependent protein kinase C (PKC) alpha and PKCbeta1 inhibitor. Our results indicated that cross-talk with PKC and/or Ca2+ signaling pathways might sensitize cAMP-mediated effects, on CREB phosphorylation and STC gene expression.

Identifying the pattern of olfactory deficits in Parkinson disease using the brief smell identification test.

BACKGROUND: Selective olfactory deficits occur in 70% to 90% of patients with Parkinson disease, independent of disease severity and duration. Olfactory testing may be a useful diagnostic aid for Parkinson disease, but the types of odors most commonly affected need to be identified. OBJECTIVE: To determine the pattern and types of odors affected in Parkinson disease by means of the University of Pennsylvania 12-item Brief Smell Identification Test (B-SIT; Sensonics, Inc, Haddon Heights, NJ). DESIGN: Testing patients with Parkinson disease and control subjects in 5 movement disorder clinics. PARTICIPANTS: Forty-nine nondemented patients with Parkinson disease and 52 age- and sex-matched controls. MAIN OUTCOME MEASURES: Normal or abnormal olfactory function was determined in each subject according to predetermined age and sex norms. Predictive statistics and discriminant function analyses were performed to determine the pattern and types of odors best discriminating patients from controls. RESULTS: Abnormal olfactory function was present in 40 (82%) of patients compared with 12 (23%) of controls. The B-SIT score was unaffected by smoking behavior, disease duration, or severity. The sensitivity of the B-SIT for Parkinson disease was 0.82, with a specificity and predictive value of 0.82 and 0.77, respectively. Only 5 of the 12 B-SIT odors (gasoline, banana, pineapple, smoke, and cinnamon) were required to adequately discriminate patients with Parkinson disease from controls. CONCLUSIONS: With the use of the B-SIT, 5 specific odors appear primarily affected in patients with Parkinson disease. Significantly, the ability of patients to detect some odors was unimpaired compared with that of controls. Better diagnostic aids could be developed on the basis of the selective pattern of hyposmia observed in Parkinson disease.