Breast cancer is the most common malignancy in women and is the leading cause of death among women world-wide [1]. Despite many advances in the diagnosis and treatment of breast cancer, metastasis remains an insurmountable challenge. Although the molecular mechanisms are not completely understood, it is well established that the formation and growth of new blood vessels is critical for sustained tumor growth and metastasis [2]. Studies have found that benign lesions associated with high vascular density are correlated with an increased risk of developing breast cancer. Traditionally, the mechanism(s) controlling the development of tumor vasculature and perfusion were thought to be endothelial cell-lined vascular networks [3]. Thus, efforts to reduce the growth and spread of breast cancer focused on the mechanism(s) of angiogenesis by which tumors establish a blood supply for survival, growth, and metastasis [4].

Plant Cell. 2008 January; 20(1): 75–87

Guard cells play a critical role in plant growth and development by optimizing gas exchange under variable environments.

A 51-year-old man with a diagnosis of myelodysplasia and non-Hodgkin's lymphoma underwent an unmatched allogenic bone marrow transplantation and was treated posttransplant with chronic immunosuppressive medication. Eight months following transplantation, he presented with progressive dysarthria, cognitive and visual decline. Evaluation included brain magnetic resonance (MR) imaging demonstrating multifocal areas of increased T2 and FLAIR (fluid attenuated inversion recovery) signals involving the left frontal, parietal, and occipital lobes. The MR lesions demonstrated diffuse increased signal on DWI (diffusion-weighted images) and normal to low signal on ADC (apparent diffusion coefficients). Contrast-enhanced T1 images were unremarkable. Lumbar puncture revealed a mild elevation in cerebrospinal fluid (CSF) protein. CSF PCR assay for viral DNA fragments were negative on two occasions. Serum serology for HIV was negative as well. A brain biopsy was subsequently performed. The clinical and neuroimaging differential diagnoses as well as neuropathologic correlation are presented.,

Data for this study were collected using …Five works will be examined, all of which …This investigation takes the form of a case-study of the …This study was exploratory and interpretative in nature.This study uses a qualitative case study approach to investigate …The research data in this thesis is drawn from four main sources: …The approach to empirical research adopted for this study was one of …This dissertation follows a case-study design, with in-depth analysis of …By employing qualitative modes of enquiry, I attempt to illuminate the …Qualitative and quantitative research designs were adopted to provide …Both qualitative and quantitative methods were used in this investigation.A holistic approach is utilised, integrating X, Y and Z material to establish …The study was conducted in the form of a survey, with data being gathered via …The methodological approach taken in this study is a mixed methodology based on …A combination of quantitative and qualitative approaches was used in the data analysis.

Zaid Al-Wahab. Oncotarget. 2015 Mar 12

Metformin is a member of the biguanide class of antihyperglycemic agents and has been recently revealed to have anti-tumorogenic effects [30, 31]. Metformin decreases hepatic glucogenesis, increases insulin sensitivity, enhances peripheral glucose uptake, and decreases glucose absorption from the gastrointestinal tract [32]. On the cellular level, metformin inhibits mitochondrial complex 1, which interferes with oxidative phosphorylation, resulting in decreased adenosine triphosphate (ATP) production and energetic stress [33]. Epidemiologic studies have shown that metformin lowers cancer risk and improves cancer outcomes in diabetic patients when compared with patients treated with other types of antihyperglycemic agents [34, 35]. Therefore, metformin has been repurposed as therapy in gynecologic and non-gynecologic cancers [36] and is currently being evaluated in various clinical trials [36]. The most evaluated mechanism of metformin’s antihyperglycemia and anti-tumor activity is the activation of AMPK [36]. Metformin has also been demonstrated to induce SIRT1 levels in hepatic cancer lines [24].

Zaid Al-Wahab. Oncotarget. 2015 Mar 12

Mouse diet We purchased various mouse diets from Bioserv (Frenchtown, NJ). The mice groups were fed on purified RD or nutritionally balanced HED with 60% kilocalories from fat (35.7% carbohydrate; 20.5% protein) or 30% nutritionally supplemented CRD as described before [91, 92].

Zaid Al-Wahab. Oncotarget. 2015 Mar 12

Tumor generation Six-week-old female C57B6 mice were weighed and randomized into the 3 dietary treatment groups as described above: (1) RD (n = 20), (2) HED (n = 20) and (3) CRD (n = 10). Mice were weighed twice a week. After 30 days of respective diet, 5 × 106 ID-8 mouse ovarian cancer cells in 200 μl phosphate-buffered saline were injected into the peritoneal cavity of the mice [13]. Post-7 days of tumor implantation, 1 set of mice from the RD and HED groups (n = 10) was given 200 mg/body weight metformin in water [93] daily, till the end of the study. The mice were monitored daily for any discomfort and weighed twice a week. The diet regimens were continued for another 60 days, after which the mice were sacrificed and autopsied. Ascitic fluid, blood, tumor tissue and vital organs were collected from each mouse. Tumor score Tumor nodules morphology and count were identified grossly at the liver, spleen, kidneys, bowel, peritoneum and diaphragm. A scoring system to identify the tumor burden in every organ was used as 0: no nodule observed; 1: 1 nodule observed; 2: 2 to 5 nodules and 3: more than 5 nodules observed per organ [13]. Scoring was performed in a blinded manner by 2 individuals including a gynecology oncology fellow (ZW).

Zaid Al-Wahab. Oncotarget. 2015 Mar 12

Enzyme-linked immunosorbent assay (ELISA) Levels of leptin, adiponectin, insulin, IGF-1, IL-6, VEGF and MCP-1 were estimated by enzyme-linked immunosorbent assay (ELISA) in plasma and ascitic fluid. The insulin ELISA was purchased from Millipore (Billerica, MA) and all other kits were from R&D System (Minneapolis, MN). The ELISAs were carried out according to the manufacturers’ instructions.

Zaid Al-Wahab. Oncotarget. 2015 Mar 12

Immunohistochemistry After excising the tumor tissues and other organ tissue from mice, specimens were fixed in 10% formaldehyde for 48 hours and paraffin-embedded. Consecutive sections of 4 micron thick were cut and processed for hematoxylin and eosin staining; immunohistochemistry for pACC (cat. no. 3661, used at 1:100), p-mTOR (cat. No: 2976, used at 1:50), pAkt (Ser473, cat. No: 4060 used at 1:50), SIRT1 (cat no: 15404, used at 1:100) and Ki-67 (cat. No: ab15580, used at 1:100), ɣH2A.X (cat no:9718, used at 1:100). Antibodies to pACC, p-mTOR, pAkt, and ɣH2A.X were from Cell Signaling Technology (Denver, MA). Ki-67 was from Abcam (Cambridge, MA). p16 was from Proteintech (cat. No: 10883–1-AP, used at 1:200; Proteintech Group, Chicago, IL). SIRT1 was from Santa Cruz Biotech (Santa Cruz, CA). Solutions obtained from Dako Cytomation (Carpinteria, CA) were used for performing immunostaining as described before [13, 93]. The slides were examined under a light microscope, and representative pictures were taken from a minimum of 3 slides of each group [93]. The quantification of the stain intensity was performed by assigning a score of 0–1 for no or weak stain; 2 for moderate stain and 3 for strong stain. All slides were examined in a blinded manner by 2 individuals, including a pathologist (RA).

Zaid Al-Wahab. Oncotarget. 2015 Mar 12

pAkt and p-mTOR expression were inhibited by metformin Phosphorylated protein kinase B (pAkt) and phosphorylated mTOR (p-mTOR), are the 2 common downstream signaling molecules for action of CRD, AMPK and SIRT1 [21, 36, 39–41]. CRD mice had the lowest and HED and RD mice had the highest expression of pAkt (Figure 7A, 7B) and p-mTOR (Figure 7C, 7D) as reported previously [13]. Metformin treatment reduced both pAkt and p-mTOR expression in HED and RD groups in the peritoneal and adipose tissue, similar to CRD (Figure 7A–7D). The quantification of the staining intensity is represented as bar graphs. This demonstrates that metformin modulated common oncogenic factors with CR, even under HED conditions.

Zaid Al-Wahab. Oncotarget. 2015 Mar 12

Metformin induced AMPK and SIRT1 AMPK and SIRT are 2 enzymes involved in regulation of energy metabolism and reported to mediate the positive effects of CR [16, 25]. The CRD group showed the strongest activation of phosphorylated acetyl-CoA carboxylase (pACC), a surrogate marker for AMPK activation, while HED groups demonstrated almost no phosphorylation of ACC as reported before [13] (Figure 6A, 6B). Metformin increased the pACC expression significantly in RD and HED groups in both peritoneal and adipose tumor tissue. A similar pattern was also observed for SIRT1 expression in the tumor tissues (Figure 6D, 6E). Metformin treatment also activated AMPK and SIRT1 in the liver, which was also associated with the amelioration of the hepatic steatosis observed in the HED group (Figure 6C, 6F). The quantification of the staining intensity is represented as bar graphs (0–1: no or weak stain; 2: moderate stain and 3: strong stain), respective of each panel. Taken together, these results suggest that metformin activated AMPK and increased the expression of SIRT1 significantly, parallel to CRD both in the tumor and in the host tissue.

Zaid Al-Wahab. Oncotarget. 2015 Mar 12

Metformin decreased the inflammatory markers and angiogenic factors The role of inflammatory molecules (monocyte chemoattractant protein-1 [MCP-1] and interleukin 6 [IL-6]) and angiogenic factors (vascular endothelial growth factor [VEGF]) in ovarian tumorogenesis is well established [37, 38], and the inhibition of these markers by CRD has been recently demonstrated [13]. As observed with growth factor levels, metformin had more pronounced inhibition of these factors in HED group compared to the RD group. Excluding plasma VEGF (Figure 5Ci), the levels of MCP-1, IL-6 and VEGF were significantly reduced by metformin (Figure 5A, 5B, 5Cii). Metformin did not reduce IL-6 and VEGF in the plasma of RD mice but significantly reduced MCP-1 (Figure 5Ai, Bi, 5Ci), while all 3 were significantly inhibited in the ascitic fluid (Figure 5Aii, 5Bii, 5Cii). The CRD group had lower levels of MCP-1, IL-6 and VEGF compared to the HED and RD groups. Interestingly, metformin decreased MCP-1 and VEGF levels in the ascites of the RD and HED groups more significantly than CR (Figure 5Aii, 5Cii). Based on these results, it can be suggested that metformin significantly alters the inflammatory and angiogenic armamentarium of ovarian cancer cells, even under the conditions of rich nutrition.

Zaid Al-Wahab. Oncotarget. 2015 Mar 12

Metformin regulates the levels of hormones controlling the energy balance Growth hormones including insulin, IGF-1, leptin and adinopectin that regulate energy metabolism were estimated under metformin treatments in plasma and ascites fluid of the mice. Metformin was most efficient in reducing the levels of insulin, IGF-1 and leptin in both the plasma and ascites of the HED group (Figure 4A–4C). Adiponectin was significantly increased in the plasma but not in the ascites by metformin in the HED mice (Figure 4D). Metformin decreased IGF-1 and leptin levels significantly in both the plasma and ascites of the RD group, while insulin was reduced only in the ascites (Figure 4A–4C). Adiponectin was significantly increased in the plasma but not in the ascites by metformin in the RD mice (Figure 4D). CRD mice still had the lowest levels of IGF-1, insulin and leptin and increased adiponectin as observed previously [13]. In general, HED fed mice showed a tumor promoting environment while mice on CRD showed the inverse profile. Metformin reversed most of these tumor promoting effects of diet in HED and RD, similar to the CRD group; however, CRD was the most effective in maintaining the lowest levels of all growth factors and hormones.

Zaid Al-Wahab. Oncotarget. 2015 Mar 12

Metformin decreases the tumor burden and ascites volume To investigate if metformin can reduce tumor progression similar to CR, a set of HED and regular diet (RD) fed mice were given metformin daily in drinking water 7 days after tumor implantation. As previously reported [13], mice on HED had highest weight gain while the CR diet (CRD) mice maintained their weight (Figure 1A). Metformin intake did not significantly affect the weight gain of HED or RD mice (Figure 1A), which was also reflected in the end weights at the time of the sacrifice (Figure 1B). Metformin treatment significantly reduced the ascites accumulation in both RD and HED groups, but the highest reduction was observed in the CRD group (Figure 1C). Metformin treatment of the HED group was most effective in reducing the tumor progression with significant reduction of tumor burden in the peritoneum, diaphragm, bowel, liver, kidney and spleen (Figure 2A–2F). In the RD group, metformin reduced the tumor burden at the bowel, liver, kidney and spleen. CRD group had the lowest tumor burden at all organ sites (Figure 2A–2F). The hemotoxylin and eosin evaluation of the tumor sections also showed reduced tumor nodules at the diaphragm, peritoneum and adipose tissue (Figure 3A–3C) with metformin treatment in both HED and RD groups, akin to CRD group. This was also reflected in the decreased number of positive Ki-67 stained cells observed in tumors from CRD and metformin treated groups, quantified as Ki-67 index (Figure 3D). Overall, our data showed that metformin decreased the ascites and tumor burden in both the RD and HED groups significantly, similar to CRD. However, the tumor reduction by metformin in HED group was more pronounced than the tumor reduction observed in the RD treatment group.

Qing Gu. Oncotarget. 2015 Mar 12

Non-small cell lung cancer (NSCLC) remains a major cause of cancer-related deaths worldwide [1]. Although surgical resection is the main treatment for early-stage NSCLC, radiotherapy plays an important role in patients who cannot not tolerate curative surgery because of advanced age or pulmonary or cardiovascular disease [2,3]. However, local recurrence and distant metastasis frequently occur after radiation therapy, which have become the greatest barrier in the management of cancer [3, 4]. The mechanism of local recurrence and distant metastasis remains a complex issue. Multiple factors have been reported to be involved in cancer metastasis, including tumor-stromal interactions and inflammatory and immune reactions [5-8]. The pro-metastatic effect of radiation has been reported for several types of cancer, including thyroid tumors, Lewis lung cancer, prostate small-cell carcinoma and melanoma [9-12]. The interactions between cancer cells and the surrounding stromal fibroblasts modulated by irradiation have been suggested to be involved in tumor invasion and metastasis [13-15]. However, the mechanism that mediates radiation-induced invasiveness remains largely unknown.