Mammalian Target of Rapamycin (mTOR) Regulates Cellular Proliferation and Tumor Growth in Urothelial Carcinoma

Mammalian target of rapamycin (mTOR) signaling has been associated with aggressive tumor growth in many cancer models, although its role in urothelial carcinoma (UCC) has not been extensively explored. Expression of phosphorylated mTOR (P-mTOR) and a downstream target, ribosomal S6 protein (P-S6), was identified in 74% (90/121) and 55% (66/121) of muscle-invasive UCCs, respectively. P-mTOR intensity and %positive cells were associated with reduced disease-specific survival (P = 0.04, P = 0.08, respectively). Moreover, P-mTOR intensity corresponded to increased pathological stage (P < 0.01), and mTOR activity was associated with cell migration in vitro. In addition, mTOR inhibition via rapamycin administration reduced cell proliferation in UCC cell lines RT4, T24, J82, and UMUC3 in a dose-dependent manner to 6% of control levels and was significant at 1 nmol/L in J82, T24, and RT4 cells (P < 0.01, P < 0.01, P = 0.03, respectively) and at 10 nmol/L in UMUC3 cells (P = 0.03). Reduced proliferation corresponded with reduced P-S6 levels by Western blot, and effects were ablated by pretreatment of cells with mTOR-specific siRNA. No effects of rapamycin on apoptosis were identified by TUNEL labeling or PARP cleavage. Administration of rapamycin to T24-xenografted mice resulted in a 55% reduction in tumor volume (P = 0.03) and a 40% reduction in proliferation (P < 0.01) compared with vehicle-injected mice. These findings indicate that mTOR pathway activation frequently occurs in UCC and that mTOR inhibition may be a potential means to reduce UCC growth.Bladder cancer occurs in multiple forms, the most common of which is urothelial carcinoma (UCC), which represents >90% of all bladder cancers.¹ Approximately 30 to 50% of patients with invasive bladder cancer into the muscular wall of the bladder will develop metastatic disease and die within 2 years of diagnosis.² In addition, virtually all patients diagnosed with distant UCC metastases will succumb to disease.³ Currently, the standard treatment modality for muscle-invasive bladder cancer is radical cystectomy; systemic chemotherapy is generally reserved for patients with metastatic disease, although these treatment regimens provide only a limited long-term benefit with only rare reports of complete remission.^4,5 In light of these clinical outcomes, identification of new therapeutic targets is needed to define potential additional treatment avenues for these patients.Activation of the mammalian target of rapamycin (mTOR) signaling pathway occurs in many cancers and has recently been shown to correlate with more aggressive disease behavior,^6,7,8,9 although it has not been examined in great detail in UCC. Activation of mTOR occurs via a multistep process that includes upstream phosphoinositide-3 kinase (PI3K) and AKT activation, leading to phosphorylation and inactivation of the tuberous sclerosis complex 1 and 2 (TSC1/TSC2) heterodimer.^10,11 Inactivation of this heterodimer results in release of Rheb inhibition and subsequent mTOR activation by means of Rheb GTPase activity. Once activated, mTOR can induce increased mRNA translation or regulate the actin cytoskeleton via differential association Rictor and Raptor proteins.^10,11 Ultimately, mTOR activity regulates the effects of a number of downstream molecules important in cellular growth, including p70 S6 kinase-1 (S6K) and elongation-initiation factor 4E binding protein-1 (4E-BP1). Selective inhibition of the mTOR pathway can be achieved using rapamycin or rapamycin analogs temsirolimus (CCI-779, Wyeth Pharmaceuticals) and everolimus (RAD001, Novartis), which are currently in use in numerous clinical trials for solid tumors, with promising results in patients with advanced renal cell carcinoma.^12,13To further investigate the potential role of mTOR signaling and inhibition in UCC of the bladder, we used human cancer specimens, xenograft models, and in vitro analysis to determine the effects of mTOR on cellular proliferation, apoptosis, tumor growth, and clinical outcomes in this cancer population.     

Accidental blood exposure: risk and prevention in interventional radiology

There is a growing concern about the transmission of bloodborne pathogens during medical procedures among health care workers and patients. Over the last three decades, radiological services have undergone many changes with the introduction of new modalities. One of these new disciplines is interventional radiology (IR) which deals with procedures such as arteriography, image-guided biopsies, intravascular catheter insertions, angioplasty and stent placements. Despite these developments, the potential for accidental blood exposure and exposure to other infectious material continues to exist. Therefore, it is important for all radiologists who perform invasive procedures to observe specific recommendations for infection control. In this review, we look at the different policies for protection and universal standards on infection control.     

Antiovulatory and abortifacient effects of Areca catechu (betel nut) in female rats

Objectives:

To study the antiovulatory and abortifacient effects of ethanolic extract of Areca catechu in female rats.

Materials and Methods:

For antiovulatory effect, ethanolic extract of A. catechu at 100 and 300 mg/kg doses was administered orally for 15 days. Vaginal smears were examined daily microscopically for estrus cycle. Rats were sacrificed on 16^th day. Ovarian weight, cholesterol estimation, and histopathological studies were done. Abortifacient activity was studied in rats at 100 and 300 mg/kg doses administered orally from 6^th to 15^th day of pregnancy. Rats were laparotomised on 19^th day. The number of implantation sites and live fetuses were observed in both horns of the uterus.

Results:

The extract of A. catechu showed a significant decrease in the duration of estrus at 100 mg/kg (P = 0.015) and 300 mg/kg doses (P = 0.002) as compared with control. Metestrus phase was also significantly reduced at 100 mg/kg (P = 0.024) and 300 mg/kg doses (P = 0.002). There was a significant increase in proestrus (P < 0.001) phase. However, diestrus phase was unchanged. Histopathological study of the ovaries showed mainly primordial, primary, and secondary follicles in the test groups as compared to control. There was also a significant (P = 0.002) decrease in ovarian weight and a significant (P = 0.021) increase in ovarian cholesterol level at 100 mg/kg dose. In the study to evaluate abortifacient effect, the mean percentage of abortion with 100 and 300 mg/kg doses were 75.5% and 72.22%, respectively, which was significantly (P = 0.008 and P = 0.006, respectively) increased when compared with control.

Conclusion:

The ethanolic extract of A. catechu at doses of 100 and 300 mg/kg has antiovulatory and abortifacient effects.     

5-flourouracil cardiotoxicity - an elusive cardiopathy: case report

5-Flourouracil (5-FU) is an S-phase specific, synthetic pyrimidine antimetabolite. It is a frequently administered chemotherapeutic agent for a variety of malignant lesions, either singly or in multidrug regimens. Its adverse side effects involving bone marrow, skin, mucous membranes, GIT and CNS are well known, whereas its cardiotoxicity is relatively uncommon and occurs in 1.2-18%. Keywords: 5-Flourouracil, Cardiotoxicity, S-phase specific.     

Immunological cross-reactivity of mycobacterial topoisomerase I and divergence from other bacteria

Mycobacterium smegmatis topoisomerase I exhibits several distinctive characteristics among all topoisomerases. The enzyme is devoid of Zn²⁺fingers found typically in other bacterial type I topoisomerases and binds DNA in a site-specific manner. Using polyclonal antibodies, we demonstrate the high degree of relatedness of the enzyme across mycobacteria but not other bacteria. This absence of cross-reactivity from other bacteria indicates that mycobacterial topoisomerase I has diverged from Escherichia coli and other bacteria. We have investigated further the immunological properties of the enzyme by raising a panel of monoclonal antibodies that recognises different antigenically active regions of the enzyme and binds it with widely varied affinity. Inhibition of a C-terminal domain-specific antibody binding by enzyme-specific and non-specific oligonucleotides suggests the possibility of using these monoclonal antibodies to probe the structure, function and in vivo role of the enzyme.     

Relationship between Bone Histology and Markers of Bone and Mineral Metabolism in African-American Hemodialysis Patients

Background and objectives: Racial differences in mineral metabolism exist in the chronic kidney disease population, especially as it relates to intact parathyroid hormone (iPTH) levels. Few data exist on the relationship of these markers to bone biopsy findings in African-American (AA) hemodialysis patients across the spectrum of renal osteodystrophy (ROD).Design, setting, participants, & measurements: In prevalent AA hemodialysis subjects, we prospectively evaluated subjects by performing transiliac bone biopsy and correlating biochemical and clinical data to bone histology.Results: Study patients (n = 43) had an average age of 53.7 (±11.6) yr, with dialysis vintage of 40.4 (±24.5) mo, 30% with diabetes, and 51% male. Bone histology revealed adynamic bone disease (ABD) (16%), mild to moderate hyperparathyroidism (HPT) (72%), severe (12%) HPT, and no osteomalacia or mixed uremic osteodystrophy. At the time of biopsy, mean corrected calcium was 9.1, 8.9, and 9.4 mg/dl (P = 0.344); calcium-phosphorus (Ca × PO₄) product was 42, 55, and 62 mg²/dl² (P = 0.002); phosphorus was 4.6, 6.2, and 6.7 mg/dl (P = 0.005); and iPTH was 225, 566, and 975 pg/ml (P = 0.006), respectively. Median values for bone-specific alkaline phosphatase (BS-AP) were 16, 34, and 64 ng/ml (P < 0.0001) among the three groups.Conclusions: These data demonstrate that across the spectrum of ROD, iPTH levels are higher than expected in AA hemodialysis subjects. iPTH, PTH peptides, and bone-specific alkaline phosphatase correlated directly with histomorphometric measurements of bone turnover and when subjects were grouped by histologic diagnosis. Only 9.5% of subjects were simultaneously within suggested Kidney Disease Outcomes Quality Initiative (K/DOQI) ranges for Ca × PO₄, phosphorus, and iPTH, of which 75% demonstrated ABD on biopsy.Abnormalities in bone and mineral metabolism occur frequently in chronic kidney disease (CKD) patients, leading to bone lesions. This disorder has more recently been termed chronic kidney disease-related mineral and bone disorder (CKD-MBD) by the Kidney Disease Improving Global Outcomes (KDIGO) foundation (1). Bone and mineral disorders in CKD patients have been associated with significant morbidity and mortality (2–5). The bone lesions, coined renal osteodystrophy (ROD), in this population can manifest symptoms such as intense pruritus, bone pain, myopathies, muscle tendon rupture, and increased incidence of fractures. Beyond symptoms, however, ROD manifests in derangements in bone histologic findings such as abnormalities in bone turnover, mineralization, and volume (6), a classification system that has been adopted by the KDIGO committee (1). Bone histology remains the “gold standard” for definitive diagnosis of the osteodystrophic lesion. However, in routine clinical practice nephrologists rely on noninvasive methods for assessment of bone turnover, such as intact parathyroid hormone (iPTH) levels.The Kidney Disease Outcomes Quality Initiative (K/DOQI) Clinical Practice Guidelines (CPG) for Bone Metabolism and Disease in Chronic Kidney Disease, released in 2003, provided the first integrated clinical action plan for the treatment of CKD-MBD (7). The treatment goals provided by these CPGs were developed through a systematic review of the literature to date (January 1, 2001) in addition to a broad-based review by experts, organizations, and the public. However, it is unclear if the recommended goals of therapy in the guidelines are also applicable to African-American (AA) hemodialysis patients. Differences in mineral metabolism between AA and non-AA individuals without CKD have been demonstrated previously in relation to calcium balance (8,9) and bone histomorphometry (10,11). These differences have been seen in the nondialysis CKD population as well, with AA CKD patients demonstrating differences in their levels of bone and mineral parameters that would suggest a more severe degree of secondary hyperparathyroidism (12). A recent study involving AA hemodialysis patients suggests that a higher serum iPTH threshold may be necessary in this ethnic group to avoid the development of adynamic bone disease (ABD) (13), which has been associated with adverse outcomes such as vascular calcifications, (14,15) fractures (16,17), and increased mortality (5,18). In particular, a iPTH of less than 200 pg/ml or a combination of a iPTH of less than 178 pg/ml with a higher serum phosphorus and calcium were associated with increased mortality in hemo- and peritoneal dialysis patients (5,18).African Americans constitute a significant proportion of patients on hemodialysis in the United States, approaching 32% in 2005 (19). Therefore, it is important to explore any potential differences in the assessment and treatment of bone and mineral disorders in this population. Although the work of Sawaya et al. and Gupta et al. has led to the recognition that there are differences between AA and non-AA hemodialysis subjects in relation to iPTH, the optimal serum levels of iPTH across the histologic spectrum of bone and mineral disorders have yet to be established in this population (13,20). The purpose of this study was to evaluate the relationship of bone histology across the spectrum of ROD to classical biochemical markers of bone and mineral metabolism in prevalent AA hemodialysis patients receiving the current standard of care for CKD-MBD.