Madan M. Godbole

Molecular iodine induces caspase-independent apoptosis in human breast carcinoma cells involving the mitochondria-mediated pathway.

Molecular iodine (I2) is known to inhibit the induction and promotion of N-methyl-n-nitrosourea-induced mammary carcinogenesis, to regress 7,12-dimethylbenz(a)anthracene-induced breast tumors in rat, and has also been shown to have beneficial effects in fibrocystic human breast disease. Cytotoxicity of iodine on cultured human breast cancer cell lines, namely MCF-7, MDA-MB-231, MDA-MB-453, ZR-75-1, and T-47D, is reported in this communication. Iodine induced apoptosis in all of the cell lines tested, except MDA-MB-231, shown by sub-G1 peak analysis using flow cytometry. Iodine inhibited proliferation of normal human peripheral blood mononuclear cells; however, it did not induce apoptosis in these cells. The iodine-induced apoptotic mechanism was studied in MCF-7 cells. DNA fragmentation analysis confirmed internucleosomal DNA degradation. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling established that iodine induced apoptosis in a time- and dose-dependent manner in MCF-7 cells. Iodine-induced apoptosis was independent of caspases. Iodine dissipated mitochondrial membrane potential, exhibited antioxidant activity, and caused depletion in total cellular thiol content. Western blot results showed a decrease in Bcl-2 and up-regulation of Bax. Immunofluorescence studies confirmed the activation and mitochondrial membrane localization of Bax. Ectopic Bcl-2 overexpression did not rescue iodine-induced cell death. Iodine treatment induces the translocation of apoptosis-inducing factor from mitochondria to the nucleus, and treatment of N-acetyl-l-cysteine prior to iodine exposure restored basal thiol content, ROS levels, and completely inhibited nuclear translocation of apoptosis-inducing factor and subsequently cell death, indicating that thiol depletion may play an important role in iodine-induced cell death. These results demonstrate that iodine treatment activates a caspase-independent and mitochondria-mediated apoptotic pathway.

Expression of peroxisome proliferator-activated receptors (PPARS) in human astrocytic cells: PPAR? agonists as inducers of apoptosis

We report the isolation by RT‐PCR of partial cDNAs encoding the human peroxisome proliferator‐activated receptor (PPAR) isoforms PPARβ and ‐γ in human primary astrocytes (HPA) as well as in the human malignant astrocytoma cell line T98G. In contrast, we failed to detect PPARα mRNA in either of these two cell types. Because PPARβ is ubiquitously expressed but has, as yet, no known function, we pursued our functional studies of these cells with regard to PPARγ. To that end, we showed that PPARγ protein is abundantly expressed in both cell types, having a molecular weight of approximately 50 kDa. Immunocytochemistry revealed a predominantly nuclear localization of this receptor. Moreover, incubation of the two cell types with 1–12 μM 15‐deoxy PGJ2 or 1–12 μM ciglitazone, both of which are agonists of PPARγ, induced loss of cellular viability as assessed by the MTT assay after a 4 hr incubation. Reduced cellular viability as a consequence of exposure to PGJ2 or ciglitazone resulted from induction of apoptosis, as assessed by DNA fragmentation and Hoechst staining, and involves activation of the CPP32 (caspase‐3) protease. These data show that modulation of the process of apoptosis is one function of PPARγ in cells derived from the human astrocytic lineage. J. Neurosci. Res. 61:67–74, 2000.

Are children with idiopathic nephrotic syndrome at risk for metabolic bone disease

BACKGROUND Children with idiopathic nephrotic syndrome (INS) may be at risk for metabolic bone disease (MBD) because of biochemical derangements caused by the renal disease, as well as steroid therapy. No large study to date has shown conclusively that these children are prone to MBD. METHODS We prospectively studied 100 consecutive children with INS for clinical, biochemical, and radiological evidence of MBD. These children were treated with prednisone as follows: initial episode, prednisone, 60 mg/m2/d for 6 weeks, followed by 40 mg/m2 on alternate days for 6 weeks. Relapses were treated with 60 mg/m2/d until remission for 3 days, followed by 40 mg on alternate days for 4 weeks and tapered by 10 mg/m2/wk. Osteoporosis is defined as a bone mineral density (BMD) value evaluated by dual-energy X-linked absorptiometry of the lumbar spine of a z score of 2.5 SDs less than the mean. Univariate and multivariate analyses were performed to analyze for factors predictive of low BMD z score. Children were divided into two groups: those who had received repeated courses of steroid therapy (group II: frequent relapsers (FRs), steroid dependent (SD), or steroid nonresponders (SNRs) versus those who had received infrequent courses (group I: infrequent relapsers). RESULTS Twenty-two of 100 children (22%) had osteoporosis. Comparing clinical features, we observed that 6 of 70 children in group II were symptomatic (hypocalcemic signs) compared with none of 30 children in group I (P = 0.10). However, children in group II had significantly lower mean BMD z scores compared with group I (-1.65 +/- 1.35 versus -1.08 +/- 1.0; P = 0.01). Also, 20 of 70 children in group II had osteoporosis compared with 2 of 30 children in group I (P = 0.012). Children in group II had been administered significantly greater doses of steroids compared with group I (P < 0.00001). On multivariate analysis, factors predictive of a low BMD score were older age at onset (P = 0.000), lower total calcium intake (P = 0.000), and greater cumulative steroid dose (P = 0.005). CONCLUSION Children with INS are at risk for low bone mass, especially those administered higher doses of steroids (FRs, SD, or SNRs). These children should undergo regular BMD evaluations, and appropriate therapeutic interventions should be planned.

Reduction in oxidative stress and cell death explains hypothyroidism induced neuroprotection subsequent to ischemia/reperfusion insult.

Hypometabolic state following hypothermia is known to protect tissues from ischemic injury. Hypothyroidism produces a hypometabolic state. The present study was undertaken to investigate the protective effects of hypothyroidism following cerebral ischemia and to ascertain the underlying mechanism. Euthyroid (E) and hypothyroid (H) animals were exposed to a 2 h of middle cerebral artery occlusion followed by 24 h of reperfusion (I/R). Specific enzymatic methods and flowcytometry were used to assess the quantitative changes of molecules involved in neuronal damage as well as in protection. As compared to euthyroid ischemic reperfused (E + I/R) rats, H + I/R rats had insignificant neurological deficit, and smaller area of infarct. H + I/R rats had significantly lower markers of oxidative stress, and lactate dehydrogenase (LDH) activity (a marker for necrosis). Natural antioxidant activity (particularly superoxide dismutase) and integrity of mitochondria (membrane potential) were maintained in H + I/R group but not in E + I/R group. The number of neurons undergoing apoptosis significantly lower in hypothyroid ischemic rats as compared to euthyroid ones. These results suggest that hypothyroid animals face ischemia and reperfusion much better compared to euthyroid animals. A possible explanation could be the decreased oxidative stress and maintained antioxidant activity that finally leads to a decrease in necrosis and apoptosis. These observations may suggest strategies to induce brain-specific downregulation of metabolism that may have implications in the management of strokes in human beings.

Inhibition of N-(4-hydroxyphenyl)retinamide-induced autophagy at a lower dose enhances cell death in malignant glioma cells

The question whether chemotherapy-induced autophagy is causative to the demise of the cells or a part of the survival mechanism activated during cellular distress is unclear. Others and we have previously demonstrated apoptosis-inducing capacity of N-(4-hydroxyphenyl)retinamide (4-HPR) in malignant glioma cells. We provide evidences of 4-HPR-induced autophagy at a lower concentration (5 microM). Suboptimal dose of 4-HPR treatment of malignant glioma cell lines increased G(2)/M arrest, whereas cell accumulated in S phase at a higher concentration. 4-HPR-induced autophagy was associated with acidic vacuole [acidic vesicular organelle (AVO)] formation and recruitment of microtubule-associated protein light chain 3 (LC3). At a higher concentration of 10 microM of 4-HPR, glioma cells undergoing apoptosis manifested autophagic features indicated by autophagosome formation, AVO development and LC3 localization. Autophagy inhibition at an early stage by 3-methyl adenine inhibited the AVO formation and LC3 localization with an enhancement in cell death. Bafilomycin A1, a specific inhibitor of vacuolar type Hthorn-ATPase also prevented AVO formation without effecting LC-3 localization pattern and also enhanced the extent of 4-HPR-induced cell death. 4-HPR activated c-jun and P38(MAPK) at both 5 and 10 microM concentrations, whereas increased activation of extracellular signal-regulated kinase 1/2 and NF-kappaB was seen only at lower dose. Inhibiting phosphoinositide 3-kinase and mitogen-activated protein kinases pathways modulated 4-HPR-induced cell death. This is the first report that provides evidences that besides apoptosis induction 4-HPR can also induce autophagy. These results indicate that 4-HPR-induced autophagy in glioma cell may provide survival advantage and inhibition of autophagy may enhance the cytotoxicity to 4-HPR.

Severe hyperthyroidism induces mitochondria‐mediated apoptosis in rat liver

Thyrotoxicosis may be associated with a variety of abnormalities of liver function. The pathogenesis of hepatic dysfunction in thyrotoxicosis is unknown, but has been attributed to mitochondrial dysfunction. We studied the effect of altered thyroid function on the apoptotic index in rat liver. Extensive DNA fragmentation and significantly increased caspase‐3 activity (P < .001) and caspase‐9 activation (P < .005) were observed in hyperthyroid rat liver; cell death by apoptosis was confirmed. In hyperthyroid rat liver, 60% of mitochondria exhibited disruption of their outer membranes and a decrease in the number of cristae. These findings, along with significant translocation of cytochrome c and second mitochondria‐derived activator of caspases to cytosol (P < .005), suggest activation of a mitochondrial‐mediated pathway. However, no change in the expression levels of Bcl‐2, Bax, and Bcl‐xL were found in hyperthyroidism. For in vitro experiments, rat liver mitochondria were isolated and purified in sucrose density gradients and were treated with triiodothyronine (T3; 2–8 μM). T3 treatment resulted in an abrupt increase in mitochondrial permeability transition. Using a cell‐free apoptosis system, the apoptogenic nature of proteins released from mitochondria was confirmed by observing changes in nuclear morphologic features and DNA fragmentation. Proteins released by 6 μM T3 contained significantly increased amounts of cytochrome c (P < .01) and induced apoptotic changes in 67% of nuclei. In conclusion, using in vivo and in vitro approaches, we provide evidence that excess T3 causes liver dysfunction by inducing apoptosis, as a result of activation of a mitochondria‐dependent pathway. Thus, the results of this study provide an explanation for liver dysfunction associated with hyperthyroidism. (HEPATOLOGY 2004;39:1120–1130.)

Wilm's tumor-1 protein levels in urinary exosomes from diabetic patients with or without proteinuria.

Background Podocyte injury is an early feature of diabetic nephropathy (DN). Recently, urinary exosomal Wilms tumor-1 protein (WT1), shed by renal epithelial cells, has been proposed as a novel biomarker for podocyte injury. However, its usefulness as biomarker for early diabetic nephropathy has not been verified yet. We investigated urinary exosomal WT1 in type-1 diabetic patients to confirm its role as a non-invasive biomarker for predicting early renal function decline. Methods The expression of WT1 protein in urinary exosomes from spot urine samples of type-1 diabetes mellitus patients (n = 48) and healthy controls (n = 25) were analyzed. Patients were divided based on their urinary albumin excretion, ACR (mg/g creatinine) into non- proteinuria group (ACR<30 mg/g, n = 30) and proteinuria group (ACR>30 mg/g, n = 18). Regression analysis was used to assess the association between urinary exosomal levels of WT1 with parameters for renal function. Receiver Operating Characteristic (ROC) curve analysis was used to determine the diagnostic performance of exosomal WT-1. Results WT1 protein was detected in 33 out of 48 diabetic patients and in only 1 healthy control. The levels of urinary exosomal WT1 protein is significantly higher (p = 0.001) in patients with proteinuria than in those without proteinuria. In addition, all the patients with proteinuria but only half of the patients without proteinuria were positive for exosomal WT1. We found that the level of exosomal WT1 were associated with a significant increase in urine protein-to-creatinine ratio, albumin-to-creatinine ratio, and serum creatinine as well as a decline in eGFR. Furthermore, patients exhibiting WT1-positive urinary exosomes had decreased renal function compared to WT1-negative patients. ROC analysis shows that WT-1 effectively predict GFR<60 ml. min-1/1.73 m2. Conclusion The predominant presence of WT1 protein in urinary exosomes of diabetic patients and increase in its expression level with decline in renal function suggest that it could be useful as early non-invasive marker for diabetic nephropathy.

Maternal Thyroid Hormone before the Onset of Fetal Thyroid Function Regulates Reelin and Downstream Signaling Cascade Affecting Neocortical Neuronal Migration

Though aberrant neuronal migration in response to maternal thyroid hormone (TH) deficiency before the onset of fetal thyroid function (embryonic day [E] 17.5) in rat cerebral cortex has been described, molecular events mediating morphogenic actions have remained elusive. To investigate the effect of maternal TH deficiency on neocortical development, rat dams were maintained on methimazole from gestational day 6 until sacrifice. Decreased number and length of radial glia, loss of neuronal bipolarity, and impaired neuronal migration were correctible with early (E13-15) TH replacement. Reelin downregulation under hypothyroidism is neither due to enhanced apoptosis in Cajal-Retzius cells nor mediated through brain-derived neurotrophic factor-tyrosine receptor kinase B alterations. Results based on gel shift and chromatin immunoprecipitation assays show the transcriptional control of reelin by TH through the presence of intronic TH response element. Furthermore, hypothyroidism significantly increased TH receptor α1 with decreased reelin, apolipoprotein E receptor 2, very low-density lipoprotein receptor expression, and activation of cytosolic adapter protein disabled 1 that compromised the reelin signaling. Integrins (α(v) and β₁) are significantly decreased without alteration of α₃ indicating intact neuroglial recognition but disrupted adhesion and glial end-feet attachment. Results provide mechanistic basis of essentiality of adequate maternal TH levels to ensue proper fetal neocortical cytoarchitecture and importance of early thyroxine replacement.

Maternal thyroid hormone deficiency affects the fetal neocorticogenesis by reducing the proliferating pool, rate of neurogenesis and indirect neurogenesis.

Neuronal progenitor cell proliferation and their optimum number are indispensable for neurogenesis, which is determined by cell cycle length and cell cycle quitting rate of the dividing progenitors. These processes are tightly orchestrated by transcription factors like Tbr2, Pax6, and E2f-1. Radial glia and intermediate progenitor cells (IPC) through direct and indirect neurogenesis maintain surface area and neocortical thickness during development. Here we show that fetal neurogenesis is maternal thyroid hormone (MTH) dependent with differential effect on direct and indirect neurogenesis. MTH deficiency (MTHD) impairs direct neurogenesis through initial down-regulation of Pax6 and diminished progenitor pool with recovery even before the onset of fetal thyroid function (FTF). However, persistent decrease in Tbr2 positive IPCs, diminished NeuN positivity in layers I-III of neocortex, and reduced cortical thickness indicate a non-compensatory impairment in indirect neurogenesis. TH deficiency causes disrupted cell cycle kinetics and deranged neurogenesis. It specifically affects indirect neurogenesis governed by intermediate progenitor cells (IPCs). TH replacement in hypothyroid dams partially restored the rate of neurogenesis in the fetal neocortex. Taken together we describe a novel role of maternal TH in promoting IPCs derived neuronal differentiation in developing neo-cortex. We have also shown for the first time that ventricular zone progenitors are TH responsive as they express its receptor, TR alpha-1, transporters (MCT8) and deiodinases. This study highlights the importance of maternal thyroid hormone (TH) even before the start of the fetal thyroid function.

Anti-apoptotic role of omega-3-fatty acids in developing brain: perinatal hypothyroid rat cerebellum as apoptotic model.

Inadequate maternal intake of omega‐3‐fatty acids (ω3 FAs) causes adverse neurodevelopmental outcome in the progeny; however, their molecular mechanism of action is obscure. Since ω3 FAs are known to inhibit neuronal apoptosis during neuro‐degeneration, we investigated their possible contribution in regulating neuronal apoptosis during brain development. Using rat model of hypothyroidism‐induced neuronal apoptosis, we provide evidence for anti‐apoptotic role of ω3 FAs during cerebellar development. ω3 FAs were supplemented as a mixture of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to pregnant and lactating rats, and primary hypothyroidism was induced by administering methimazole. The cerebella from postnatal day 16 (d16) pups were isolated, and studies on apoptosis were conducted. We observed that ω3 FA‐supplementation significantly reduced DNA fragmentation and caspase‐3 activation in developing cerebellum of hypothyroid pups. The protection provided by ω3 FAs was associated with their ability to prevent increases in the level of pro‐apoptotic basal cell lymphoma protein‐2 (Bcl‐2)‐associated X protein (Bax) in the cerebellum during thyroid hormone (TH) deficiency. ω3 FAs increased the levels of anti‐apoptotic proteins like Bcl‐2 and Bcl‐extra large (Bcl‐xL), known to be repressed in hypothyroidism. ω3 FAs also restored levels of cerebellar phospho (p)‐AKT, phospho‐extracellular regulated kinase (p‐ERK) and phospho‐c‐Jun N‐terminal kinase (p‐JNK), which were altered by hypothyroid insults, without interfering with the expression of TH responsive gene, myelin basic protein (mbp). Taken together, these results supplement an insight into the molecular mechanism of action of ω3 FAs in developing brain that involves regulation of apoptotic signaling pathways under stress.