Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells

Silymarin, a defined mixture of natural flavonoid, has recently been shown to have potent cancer chemopreventive efficacy against colon carcinogenesis in rat model; however, the mechanism of such efficacy is not elucidated. Here, using pure active agent in silymarin, namely silibinin, we show its antiproliferative and apoptotic effects, and associated molecular alterations in human colon carcinoma HT-29 cells. Silibinin treatment of cells at 50-100 microg/ml doses resulted in a moderate to very strong growth inhibition in a dose- and a time-dependent manner, which was largely due to a G0/G1 arrest in cell cycle progression; higher dose and longer treatment time also caused a G2/M arrest. In mechanistic studies related its effect on cell cycle progression, silibinin treatment resulted in an upregulation of Kip1/p27 and Cip1/p21 protein as well as mRNA levels, and decreased CDK2, CDK4, cyclin E and cyclin D1 protein levels together with an inhibition in CDK2 and CDK4 kinase activities. In other studies, we observed that G2/M arrest by silibinin was associated with a decrease in cdc25C, cdc2/p34 and cyclin B1 protein levels, as well as cdc2/p34 kinase activity. In the studies assessing biological fate of silibinin-treated cells, silibinin-induced cell cycle arrest and growth inhibition were not associated with cellular differentiation, but caused apoptotic death. The quantitative apoptosis analysis showed up to 15% apoptotic cell death after 48 h of silibinin treatment. Interestingly, silibinin-induced apoptosis in HT-29 cells was independent of caspases activation, as all caspases inhibitor did not reverse silibinin-induced apoptosis. This observation was further confirmed by the findings showing a lack in caspases activity increase and caspases and PARP cleavage as well as a lack in cytochrome c release in cytosol following silibinin treatment of HT-29 cells. Additional studies conducted in mice showed that silibinin doses found effective in HT-29 cells are achievable in plasma, which increases the significance of the present findings and their possible translation in in vivo anticancer efficacy of silibinin against colon cancer. Together, these results identify molecular mechanisms of silibinin efficacy as a cell cycle regulator and apoptosis inducer in human colon carcinoma HT-29 cells, and justify further studies to investigate potential usefulness of this nontoxic agent in colon cancer prevention and intervention.     

Mechanism of matrix accumulation and glomerulosclerosis in spontaneously hypertensive rats

BACKGROUND: Renal interstitial fibrosis and thickening of the glomerular basement membrane are associated with hypertension. However, the mechanism of matrix accumulation is unclear. Spontaneously hypertensive rats (SHR) develop hypertension at between 2 and 6 weeks of age. METHODS: To test the hypothesis that increased matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) contribute to the pathomechanisms of hypertensive nephropathy, the cortex and medulla of male SHR at 2 and 6 weeks were analyzed for MMP-2, MMP-7, and MMP-9 by gelatin and elastin gel zymography. The levels of TIMP-4 were measured by western blot analysis. The bands in blots were scanned and normalized with actin. To localize MMP-2 and TIMP-4 in situ, immuno-labeling was performed. To determine proteinuresis, urinary protein was measured by Bio-Rad dye binding assay. The mean arterial pressure (mmHg) was measured in Inactin-anesthetized rats by a PE-50 catheter in the femoral artery. Age-sex matched normotensive Wistar rats (NWR) were used as controls and grouped: (1). SHR, 2 weeks; (2). SHR, 6 weeks; (3). NWR, 2 weeks; and (4). NWR, 6 weeks (n = 6 in each group). RESULTS: Levels of cortex MMP-2 and MMP-9 were increased in 6 week SHR as compared with NWR. In the medulla, MMP-9 and MMP-7 were increased, but there was no change in MMP-2. The levels of cortex TIMP-4 tended to increase but insignificantly. In contrast, there were significant increases in the levels of TIMP-4 in the medulla of 6 week SHR as compared with 2 week SHR or NWR. In addition, there were substantial elastinolytic activity in the cortex of 6 week SHR. The in situ labeling suggested no TIMP-4 in the glomeruli. There was substantial TIMP-4 in the epithelial layer of tubules. The levels of fibrotic collagen were significantly higher in both the glomeruli and tubular interstitium. Urinary protein excretion was increased significantly in 6 week SHR when compared with other groups. The mean arterial pressure was 1.6-fold higher in 6 week SHR than in controls. CONCLUSION: These results suggest that increased MMP-2 and MMP-9 activity contributes to glomerular injury and hypertensive remodeling. The increased levels of TIMP-4 in the medulla may inhibit the collagenolytic activity of MMP but is unable to inhibit the elastinolytic activity. An important role of MMP-2, MMP-9, and TIMP-4 in hypertensive remodeling of the cortex and medulla in the SHR is demonstrated.     

Encapsulation in cationic liposomes enhances antitumour efficacy and reduces the toxicity of etoposide, a topo-isomerase II inhibitor

Etoposide exerts its antineoplastic effect by forming a ternary complex with topo-isomerase II and DNA, leading to DNA breaks and cell death. However, it causes myelosuppression and its lipophilicity poses a major limitation during administration. Liposomes have been reported to increase the efficacy and reduce the toxicity of antineoplastic agents. Recent evidence suggests that cationic liposomes bind efficiently to tumours. The present study was thus designed to encapsulate etoposide in cationic liposomes and to evaluate its antitumour efficacy and systemic toxicity in comparison with a conventional parenteral formulation. Etoposide encapsulated in liposomes was synthesised by thin film hydration followed by an extrusion method. Fibrosarcoma was induced in mice by subcutaneous administration of 20-methylcholanthrene. Chemotherapy was started when the tumour reached 200 mm(3) in volume. Liposomal etoposide (10 mg/m(2)/day for 5 days) significantly delayed tumour growth as compared to non-liposomal etoposide. The median time of death was calculated to be 19.5, 26.25 and 56 days in vehicle-treated controls, non-liposomal-etoposide- and liposomal-etoposide-treated groups, respectively. A transient reduction in body weight was seen in both the liposomal- and non-liposomal-etoposide-treated groups. The maximum tolerated dose was however significantly higher in the group treated with liposomal etoposide, which also exhibited a lesser degree of myelosuppression than the animals treated with non-liposomal etoposide. The present findings suggest that cationic liposomes could be considered as potential for delivery of etoposide to tumours.     

Functional and structural changes in the kidney in the early stages of obesity

The purpose of this study was to examine the histologic and functional changes that occur in the kidney in the early stages of obesity caused by a high-fat diet. Lean dogs (n = 8) were fed a standard kennel ration, and obese dogs (n = 8) were fed the standard kennel ration plus a supplement of cooked beef fat each day for 7 to 9 wk or 24 wk. Body weights were 58 +/- 5% greater and kidney weights were 31 +/- 7% greater in obese dogs, compared with the average values for lean dogs. Plasma renin activity and insulin concentrations were both 2.3-fold greater in obese dogs, compared with lean dogs. Obesity was associated with a mean arterial pressure increase of 12 +/- 3 mmHg, a 38 +/- 6% greater GFR, and a 61 +/- 7% higher renal plasma flow, compared with lean dogs. The glomerular Bowman's space area was significantly greater (+41 +/- 7%) in dogs fed the high-fat diet, compared with lean animals, mainly because of expansion of Bowman's capsule (+22 +/- 7%). There was also increased mesangial matrix and thickening of the glomerular and tubular basement membranes and the number of dividing cells (proliferating cell nuclear antigen-stained) per glomerulus was 36 +/- 8% greater in obese dogs, compared with lean dogs. There was also a trend for glomerular transforming growth factor-beta1 expression, as estimated by semiquantitative immunohistochemical analysis, to be elevated with the high-fat diet. Therefore, a high-fat diet caused increased arterial pressure, hyperinsulinemia, activation of the renin-angiotensin system, glomerular hyperfiltration, and structural changes in the kidney that may be the precursors of more severe glomerular injury associated with prolonged obesity.     

Remodeling of the endocrine pancreas: the central role of amylin and insulin resistance

BACKGROUND: Remodeling of the endocrine pancreas, caused by the deleterious effects of amylin as it is co-synthesized, co-packaged, and co-secreted with insulin, gives clinicians and researchers cause to ponder. METHODS: A literature search was done, and relevant publications and texts on amylin and islet amyloid polypeptide (IAPP) were reviewed. RESULTS: The mechanisms and clinical consequences attributed to the remodeling of the endocrine pancreas, along with proposals for reevaluating the methods of treating patients who have type 2 diabetes are illustrated and discussed. CONCLUSIONS: In addition to controlling the devastating effects ofglucotoxicity, lipotoxicity, and hypertension, we should consider the newer hypoglycemic agents with regard to their effects on the remodeling of the endocrine pancreas. This remodeling results in structural and subsequent functional changes, causing continued elevations of hemoglobin A1C. Studies are indicated to determine whether amylin (IAPP) may be implicated in the remodeling of the arterial vessel wall, the glomerulus of the kidney, and the cardiac interstitium.     

Changes in plasma volume during hypohydration and rehydration in subjects from the tropics

Summary Plasma volume (PV) at different levels of hypohydration was determined using radio-iodinated serum albumin-125 in 28 heat acclimated male volunteers in hot dry conditions in a climatic chamber. The heat acclimated subjects were hypohydrated to varying degrees i.e. 1%, 2%, 3% and 4% body mass deficit by moderate work in hot conditions in a climatic chamber maintained at 45°C dry bulb temperature and 30% relative humidity. A rehydration study was carried out in only those subjects who were hypohydrated to 3% and 4% body mass and they were brought back to a 2% level of hypohydration by giving a calculated amount of water. A significant decrease in PV was observed at 3% and 4% hypohydration only. The magnitude of the decrease was the same in both the groups and not related to the level of hypohydration. With partial rehydration in the 3% hypohydrated group PV was restored fully, while in the 4% hypohydrated group restoration was incomplete, indicating that at this hypohydration level some of the replenished water that entered in plasma may have moved to the intracellular compartment which may have contributed more at 4% hypohydration. It is suggested that with higher levels of thermal hypohydration significant reduction in the intracellular compartment may result in accentuated physiological strain during work in the heat.     

Cystic Mass in Interventricular Septum; A Rare Presentation of Sinus of Valsalva Aneurysm

A young male presented with progressively increasing breathlessness for one year. Echocardiography showed a cystic echolucent cavity in interventricular septum communicating with sinus of Valsalva. A diagnosis of unruptured aneurysm of Valsalva dissecting into the interventricular septum was made. This complication is extremely rare and early recognition may prevent a potential catastrophe. (Echocardiography 2010;27:E117‐E118)