Inhibition of the sodium potassium adenosine triphosphatase pump sensitizes cancer cells to anoikis and prevents distant tumor formation.

Normal epithelial cells undergo apoptosis upon detachment from the extracellular matrix, a process termed "anoikis." However, malignant epithelial cells with metastatic potential resist anoikis and can survive in an anchorage-independent fashion. Molecules that sensitize resistant cells to anoikis will be useful chemical probes to understand this pathway. To identify novel anoikis sensitizers in anoikis-resistant PPC-1 prostate adenocarcinoma cells, a library of 2,000 off-patent drugs and natural products was screened for their ability to preferentially induce cell death in suspension over adherent culture conditions. This screen identified five members of the family of cardiac glycosides as anoikis sensitizers, including ouabain, peruvoside, digoxin, digitoxin, and strophanthidin. We conducted further studies with ouabain to discern the mechanism of cardiac glycoside-induced anoikis sensitization. Ouabain initiated anoikis through the mitochondrial pathway of caspase activation. In addition, ouabain sensitized cells to anoikis by inhibiting its known target, the Na(+)/K(+) ATPase pump, and inducing hypoosmotic stress. Resistance to anoikis permits cancer cells to survive in the circulation and facilitates their metastasis to distant organs, so we tested the effects of Na(+)/K(+) ATPase inhibition on distant tumor formation in mouse models. In these mouse models, ouabain inhibited tumor metastases but did not alter the growth of subcutaneous tumors. Thus, we have identified a novel mechanism to sensitize resistant cells to anoikis and decrease tumor metastasis. These results suggest a potential mechanism for the observed clinical reduction in metastasis and relapse in breast cancer patients who have undergone treatments with cardiac glycosides.

A chemical screen identifies anisomycin as an anoikis sensitizer that functions by decreasing FLIP protein synthesis.

Malignant epithelial cells with metastatic potential resist apoptosis that normally occurs upon loss of anchorage from the extracellular matrix, a process termed "anoikis." Resistance to anoikis enables malignant cells to survive in an anchorage-independent manner, which leads to the formation of distant metastases. To understand the regulation of anoikis, we designed, automated, and conducted a high-throughput chemical screen for anoikis sensitizers. PPC-1 anoikis-resistant prostate cancer cells were seeded in hydrogel-coated ultralow binding plates for suspension conditions and standard tissue culture plates to promote adhesion. After seeding, cells were treated with aliquots from a library of previously characterized small molecules, and viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt, assay. From this chemical screen, we identified anisomycin that induced apoptosis in suspension conditions, but was not toxic to these cells grown under adherent conditions. Anisomycin sensitized cells to anoikis by decreasing levels of the caspase-8 inhibitor FLIP and subsequently activating the death receptor pathway of caspase activation. Although anisomycin activated c-Jun-NH(2)-kinase and p38, these kinases were not functionally important for the effect of anisomycin on anoikis and FLIP. Rather, anisomycin decreased FLIP and sensitized cells to anoikis by inhibiting its protein synthesis. Finally, we showed that anisomycin decreased distal tumor formation in a mouse model of prostate cancer metastases. Thus, a novel chemical screen identified anisomycin as an anoikis sensitizer that acts by decreasing FLIP protein synthesis. Our results suggest that FLIP is a suppressor of anoikis and inhibiting FLIP protein synthesis may be a useful antimetastatic strategy.

Critical role for Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein in anoikis resistance and distant tumor formation.

BACKGROUND: Normal epithelial cells undergo anoikis, or apoptosis on loss of anchorage to the extracellular matrix, by initiating the death receptor pathway of caspase activation. However, malignant epithelial cells with metastatic potential resist anoikis and can survive in an anchorage-independent fashion. We hypothesized that c-Fas-associated death domain-like interleukin-1-converting enzyme-like inhibitory protein (FLIP), an endogenous inhibitor of death receptor signaling, may suppress anoikis. METHODS: We assessed viability and apoptosis of PPC-1 prostate cancer cells cultured in adherent and suspension conditions using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt and Annexin V staining assays. Expression of the death receptor Fas and activation of caspase 8 were measured using flow cytometry. Expression of Fas ligand was measured by reverse transcription-polymerase chain reaction. FLIP protein expression was measured by immunoblotting. Small-molecule inhibitors of FLIP (including the death receptor sensitizer 5809354) and small-interfering (si) RNA directed against FLIP were used to assess the effects of FLIP inhibition on anoikis of prostate cancer cells in vitro and in vivo. All statistical tests were two-sided. RESULTS: PPC-1 cells cultured in suspension resisted anoikis, despite increased expression of Fas (0 versus 8 hours, mean relative percent expression = 100% versus 135%, difference = 35%, 95% confidence interval [CI] = 10% to 61%; P = .02) and Fas L (0 versus 24 hours, mean relative percent expression = 100% versus 208%, difference = 108%, 95% CI = 18% to 197%; P = .02). Knockdown of FLIP expression by siRNA or treatment with 5809354 sensitized prostate cancer cells to anoikis (control siRNA versus FLIP siRNA at 10 nM, mean relative percent viability = 95% versus 51%, difference = 44%, 95% CI = 34% to 54%; P<.001; control versus 5809354 at 20 microM, mean relative percent viability = 96% versus 52%, difference = 44%, 95% CI = 13% to 75%; P = .015). Inhibition of FLIP expression specifically activated caspase 8 in PPC-1 cells grown in suspension but not adherent conditions and decreased the metastatic potential of circulating PPC-1 cells in vivo. CONCLUSIONS: FLIP may be a suppressor of anoikis and therefore a possible target for antimetastatic therapeutic strategies.

Wheelchair accessibility of public buildings in the central business district of Harare, Zimbabwe.

PURPOSE: To evaluate accessibility of wheelchairs into public buildings in the central business district of Harare, Zimbabwe and to identify architectural barriers faced by wheelchair users in public buildings. METHODS: This was a descriptive study of 20 public buildings in the Harare business district of Zimbabwe. Wheelchair accessibility was determined on a point scale based on specifications provided. Descriptive statistics of simple percentages and means were used to determine the level of compliance to the guidelines of the instrument, and wheelchair accessibility to the surveyed buildings. RESULTS: Of the various items surveyed, elevators recorded the highest average accessibility (83% compliance), while parking areas recorded the lowest average of 18%. The compliance score of ramps was 39%, while entrances were 71% compliant. Wheelchair accessibility to toilets was 51%. CONCLUSIONS: The poor compliance score for items such as parking areas, and ramps for wheelchair users is probably because the physically challenged persons and experts in the area of physical ability management are not consulted and involved in the design and construction of these buildings. High compliance of entrances and toilets may be purely coincidental and not for meeting the needs of wheelchair users. To improve on the wheelchair accessibility in public buildings, there should be liaison between the users the disabled, and various professionals (the occupational therapists, physiotherapists, politicians, engineers and architects). The findings of this study indicate a great challenge to the aforementioned professionals. Recommendations were made to increase wheelchair accessibility to public buildings.

Comparison of wheelchair accessibility of public buildings pre and post the International Year of Disabled Persons (IYDP) in Harare, Zimbabwe: a case study.

OBJECTIVE: To compare the accessibility to wheelchair users of public buildings built pre and post the International Year of the Disabled Persons (1981) in Harare central business district in Zimbabwe. DESIGN: A case study. SETTING: Harare central business district. SUBJECTS: 20 public buildings with at least two floors open to all users in the central business district of Harare, Zimbabwe. MAIN OUTCOME MEASURES: Wheelchair accessibility of public buildings. RESULTS: There were no significant differences in the median percentage compliance of parking areas (p = 0.546), ramps (p = 0.155) and toilets (p = 0.648) between the buildings built before the International Year of the Disabled Persons (IYDP) and those built after the IYDP. The overall median compliance of parking areas, ramps and toilets was 14% (Q1 = 14, Q3 = 21), 54% (Q1 = 43, Q3 = 62) and 45% (Q1 = 31.5, Q3 = 70), respectively. However, there was some evidence (p = 0.067) to suggest that compliance of entrances was better IYDP [median = 100% (Q1 = 90, Q3 = 100)] than pre IYDP [median = 80% (Q1 = 70, Q3 = 100)]. Meanwhile there was a significant difference in the median percentage compliance of elevators (p = 0.014) between pre[median = 90% (Q1 = 80, Q3 = 90)] and post [median = 100% (Q1 = 100, Q3 = 100)] IYDP. CONCLUSION: Overall compliance was poor. It is recommended that people with disabilities and the experts in the area of physical ability management be consulted and involved in the design and construction of these buildings.

Haematologic features of the human immunodeficiency virus (HIV) infection in adult Zimbabweans.

OBJECTIVE: To describe the haematologic features of the HIV infection in adult Zimbabweans and compare the features in the different clinical stages of the disease. DESIGN: Descriptive cross sectional study. SETTING: Parirenyatwa Hospital, a tertiary and referral medical centre in Harare, and the blood donor clinics of the Blood Transfusion Service in Harare. SUBJECTS: Patients attending HIV outpatients clinics or receiving inpatient care at Parirenyatwa Hospital and asymptomatic persons donating blood at the BTS Harare. MAIN OUTCOME MEASURES: Full blood counts and bone marrow cell counts and morphology. RESULTS: Blood cytopenia was found in 47.5% of adults with HIV infection. The most frequent abnormalities were lymphopenia (31.5%); anaemia (30.8%); neutropenia (29.6%); thrombocytopenia (24.7%); eosinophilia (23.5%) and leucopenia (11.7%). Frequency of anaemia in the AIDS and symptomatic groups (43.4% and 24.5% respectively) was greater than in the carriers (6.7%), while the frequency of other cytopenias and of eosinophilia was about the same in all groups. There was also a general lack of association between the severity of haematologic abnormalities and the clinical stage of the disease. CONCLUSION: Severe haematologic changes occur frequently in HIV infection and AIDS but routine full blood count may not be helpful in the monitoring of the disease or the prediction of onset of AIDS.

Pancytopenia in Zimbabwe.

BACKGROUND: There has been little systematic study of the clinical spectrum of pancytopenia, and the optimal diagnostic approach to pancytopenia remains undefined. METHODS: The authors studied 134 hospitalized pancytopenic patients in Zimbabwe in both consecutive and nonconsecutive fashion. RESULTS: The most common cause of pancytopenia was megaloblastic anemia, followed by aplastic anemia, acute leukemia, acquired immunodeficiency syndrome (AIDS), and hypersplenism. Severe pancytopenia was usually due to aplastic anemia. Patients with aplastic anemia and acute leukemia were usually children, whereas those with megaloblastic anemia were adults. Moderate to severe anemia was noted throughout the series, but was most striking in patients with megaloblastic anemia, aplastic anemia, and acute leukemia. The mean corpuscular volume (MCV) was elevated in most patients with megaloblastic hematopoiesis, aplastic anemia, and acute nonlymphocytic leukemia. Normal or low MCV values were noted in almost one third of patients with megaloblastic anemia. Anisocytosis, poikilocytosis, macroovalocytosis, microcytosis, fragmentation, and teardrop erythrocytes were more prominent on the blood films of patients with megaloblastic anemia. CONCLUSIONS: Megaloblastic anemia, aplastic anemia, and AIDS are the most common causes of pancytopenia in Zimbabwe. Aplasia is the most frequent cause of severe pancytopenia. The authors have formulated tentative guidelines for the evaluation of pancytopenic patients in this setting.

Appreciation of community-based rehabilitation by caregivers of children with a disability.

Evaluations of community-based rehabilitation (CBR) programmes generally focus on quantitative data. To gain insight into the determinants of the outcomes, process-oriented data are needed. From the literature six variables have been identified that possibly correlate with the evaluation of CBR by caregivers of children with a disability. These variables are: (1) traditional beliefs, (2) impact of a child with a disability on the caregiver, (3) community involvement, (4) perceived ability to teach the child, (5) attitude towards various health services, and (6) expectations for the future of a disabled child. The study was done in the CBR projects in Zimbabwe. Seventy-five caregivers were interviewed. Background, perceived abilities to teach and expectations of the caregivers were very different. This study revealed a significant correlation between the appreciation of CBR and the attitude towards various health services, and between the perceived ability to teach and the expectations for the future of the child.

Vitamin B12 deficiency is the primary cause of megaloblastic anaemia in Zimbabwe.

In a study of the pathogenesis and clinical features of megaloblastic anaemia in southern Africa, we evaluated 144 consecutive Zimbabwean patients with megaloblastic haemopoiesis. Vitamin B12 deficiency was diagnosed in 86.1% of patients and was usually due to pernicious anaemia; isolated folate deficiency accounted for only 5.5% of cases. Anaemia was present in 95.8% of patients; the haemoglobin (Hb) was < or = 6 g/dl in 63.9%. Neurological dysfunction was noted in 70.2% of vitamin B12-deficient patients and was most striking in those with Hb values > 6 g/dl. Serum levels of methylmalonic acid, homocysteine, or both, were increased in 98.5% of patients. Vitamin B12 deficiency is the primary cause of megaloblastic anaemia in Zimbabwe and, contrary to textbook statements, is often due to pernicious anaemia. Isolated folate deficiency is less common. As reported in industrialized countries 75 years ago, anaemia is almost always present and often severe. Neurological dysfunction due to vitamin B12 deficiency is most prominent in patients with mild to moderate anaemia.