AKT is indispensable for coordinating Par-4/JNK cross talk in p21 downmodulation during ER stress.

The double-edged role of p21 to command survival and apoptosis is emerging. The current investigation highlights ER stress-mediated JNK activation that plausibly triggers cell death by attenuating endogenous p21 level. Here, we demonstrated that ER stress activator 3-AWA diminishes the p21 levels in cancer cells by averting the senescent phenotype to commence G2/M arrest. In essence, the deceleration in p21 level occurs through ER stress/JNK/Caspase-3 axis via activation/induction of proapoptotic Par-4 and inhibition of AKT. The molecular dynamics studies identified important interactions, which may be responsible for the AKT inhibition and efficacy of 3-AWA towards AKT binding pocket. Interestingly, the p21 deceleration was rescued by incubating the cells with 3-AWA in the presence of an ER stress inhibitor, Salubrinal. Furthermore, we demonstrated that p21 expression decreases solitarily in Par-4+/+ MEFs; albeit, ER stress-induced JNK activation was observed in both Par-4+/+ and Par-4-/- MEFs. Par-4 knockdown or overexpression studies established that ectopic Par-4 along with ER stress are not sufficient to downregulate p21 in PC-3 cells but are adequate for DU-145 cells and that the ER stress inflicted activation of JNK, inhibition of AKT and Par-4 induction are all crucial to p21 downmodulation by 3-AWA. By using isogenic cell lines, such as HCT-116 p53+/+ and HCT-116 p53-/-, we found that deceleration in p21 expression due to ER stress is p53 independent. Moreover, in orthotopic carcinogen-induced rat colorectal carcinoma model, we found that 3-AWA inhibits colorectal tumor growth and formation of colorectal polyps at a tolerable dose, similar to the first-line drug for colorectal cancer-5-fluorouracil.

Reprogramming of Molecular Switching Events in UPR Driven ER Stress: Scope for Development of Anticancer Therapeutics.

The incitement of unfolded protein response (UPR) during endoplasmic reticulum (ER) stress by diverse intracellular (hypoxia, nutrient deprivation, etc.) or extracellular (environmental or drug induced) stimuli is considered a major threat for perturbing cellular homeostasis leading to the aggregation of unfolded proteins inside the cell. The catastrophic UPR events emerge as a prime cellular adaptation by remodeling cancer cell signaling and restoring ER homeostasis in favor of tumor growth. The transient ER stress protects cancer cells from undergoing apoptosis, whereas the prolonged stress response further activates many cell death pathways. The present review summarizes the UPR mediated triggering of transcriptional and translational reprogramming, which will provide novel therapeutic strategies towards pro-death mechanisms rather than a cellular adaptation in tumorigenesis. Nonetheless, the current topic also points out the reprogramming of emerging molecular switching events by complex UPR-mediated signaling to trigger apoptosis. The novel agents from various natural, semi-synthetic and synthetic sources that target ER stress signaling pathway to modulate selectively the UPR phenomena with preclinical efficacy are outlined. Since major emphasis on ER stress-induced transcriptional and translational reprogramming remains to be explored, we believe that the current subject will instigate more attention from the biomedical researchers in this certain research direction.

Anticancer activity, toxicity and pharmacokinetic profile of an indanone derivative.

The present study describes anticancer effect of gallic acid based indanone derivative (1). Indanone 1 exhibited in vivo anticancer activity against Erhlich ascites carcinoma in Swiss albino mice by inhibiting tumor growth by 54.3% at 50 mg/kg b.wt. It showed antitubulin effect by inhibiting tubulin polymerase enzyme. In cell cycle analysis, it inhibited G2/M phase and induced apoptosis. It significantly suppressed VEGF-R1, VEGF-R2 and HIF-α in human breast cancer MCF-7 cells, thus exhibiting antiangiogenic activity. In acute oral toxicity, indanone 1 was well tolerated and was found to be non-toxic up to 1000 mg/kg b.wt. in Swiss albino mice. Pharmacokinetic studies in rabbits revealed rate of absorption, half life, volume of distribution with high plasma and blood clearance after i.v. administration. Indanone 1, is a safe and moderately active anticancer agent.

Expression of extracellular matrix components fibronectin and laminin in the human fetal heart.

It has been well documented that the extracellular matrix components fibronectin and laminin promote or regulate morphogenesis of the myocardial cells in mammalian heart. However, their chronological change of expression (or localization) in the human heart remains elusive. In this study, fibronectin and laminin in the left ventricle of forty-two human fetuses aged from 8 to 26 weeks gestation and left ventricular tissues obtained from a 2-week old infant and two adults were investigated by Western blot analyses and indirect immunofluorescence technique with monoclonal antibodies. In the fetal heart, fibronectins were present along the endocardium, epicardium, and linings of larger blood vessels. In 14-16 weeks gestation, fibronectin immunofluorescence became stronger but not evenly dispersed in the interstitium. After 24 weeks gestation, they were strongly positive only in the relatively larger blood vessels, as well as those in the infant and adult cardiac tissues. Laminins were strongly positive along the endocardium and basement membrane of the myocardial cells and fibroblasts during fetal life. After birth, laminins formed fine fibrillar network along the basement membrane in association with the transverse tubules of myocardial cell; these morphological characteristics remained in the adult cardiac tissues. These results indicate that fibronectin expression is relatively constant during fetal life but decreases after birth; in contrast, laminin expression is not age-dependent and constant throughout the life.

Evidence of protein kinase C translocation by ischemic preconditioning in global ischemia model.

We tested recent evidence that ischemic preconditioning (PC) involves in translocation of protein kinase C (PKC) from the cytosol to myocyte membrane. Isolated Langendorff-perfused rabbit hearts (n=96) were subjected to 60 or 45 min of ischemia (I) and 120 min of reperfusion (R) with or without PC (4 cycles of 5 min I and 5 min R; or single dose of 5 min I and 10 min R), respectively. Left ventricular function and infarct size (IS) were measured; myocardial cytosolic and membrane PKC activity were determined by 32P-gamma-ATP incorporation into PKC-specific peptide. PC enhanced improvement of functional recovery and reduced IS (26.9+/-1.4% versus 15.3+/-1.9%, p<0.01, in 60 min of I; 18.3+/-2.6% versus 8.6+/-2.5%, p<0.05, in 45 min of I); cytosolic PKC activity decreased 74% of total activity (p<0.05) both in 60 and 45 min of I; membrane PKC activity increased (1.7-fold of baseline, p<0.01, in 60 min of I; 1.8-fold, p<0.01, in 45 min of I; 1.5-fold, p<0.05, in 60 of min I and 120 min of R). From these results, it is concluded that translocation of PKC from the cytosol to myocyte membranes is an important mechanism responsible for PC effect.

Relation between ischemic preconditioning and the duration of sustained ischemia.

It has been reported that repetitive brief periods of ischemia and reperfusion (ischemic preconditioning, IP) cause a significant reduction in the extent of myocardial necrosis or in the incidence of reperfusion arrhythmias in rat heart. However, recent reports have stated that IP effect is diminished or lost in the canine or bovine heart if ischemia (mostly regional) is sustained for 40 min or longer. The main objective of this study is to assess whether IP provides myocardial protection in prolonged sustained ischemia under the condition of global ischemia in isolated rabbit heart. The hearts were subjected to 10-60 min sustained ischemia (SI) followed by 60 min reperfusion with (IP heart) or without IP (ISCH heart). IP was induced by 4 cycles of 5 min global ischemia and 5 min reperfusion. Left ventricular function (LVF), extent of infarction (EI) and ultrastructural changes were examined. As a whole, the LVF began to recover on reperfusion but there was no significant difference in the functional parameters. However, extracellular Ca2+ concentration was lower in the ISCH hearts (p < 0.05) and the EI was significantly different between the hearts which had received 60 min SI (67% in the ISCH versus 32% in the IP heart, p < 0.01). Ultrastructural changes were homogeneous in the ISCH hearts and became irreversible in accordance with increase of the duration of ischemia, while these changes were heterogeneous and restricted in the IP heart. These results suggest that IP does not attenuate the postischemic dysfunction in prolonged ischemia but it can provide an infarct size-limiting effect and delay ultrastructural changes. This cardioprotective effect may be related to calcium homeostasis.

Effect of pretreatment with diltiazem on left ventricular function and intracellular calcium distribution in postischemic reperfused guinea-pig hearts.

BACKGROUND: It has been previously demonstrated that pretreatment with diltiazem preserves mitochondrial function during postischemic reperfusion. AIM: The purpose of this study was to perform cytochemical and hemodynamical assessment to confirm this demonstration. METHODS: Isolated Langendorff-perfused guinea-pig hearts received 10 min of diltiazem (7.5 microM) treatment, were subjected to 10 min of global ischemia and to 20 min of reperfusion. Left ventricular function was monitored by connecting a balloon to a pressure transducer. Intracellular calcium was precipitated with potassium pyroantimonate and examined with a transmission electron microscope. RESULTS: Compared with the control and the ischemic hearts, the diltiazem-pretreated hearts showed a significant increase in the left ventricular developed pressure (LVDP), dP/dtmax (P < 0.01), and recovery rates of the LVDP (P < 0.01 versus ischemic hearts) and dP/dtmax (P < 0.05), and a decrease in the heart rate (P < 0.01). The left ventricular end-diastolic pressure (LVEDP) and leakage of creatine kinase were not significantly different. Calcium deposits were seen along the inner aspects of the sarcolemma and t-tubule membranes, and in the mitochondria of the control hearts. The number of these deposits was considerably reduced after ischemia. They reappeared principally in the mitochondria by reperfusion. In contrast, the calcium deposits reappeared along the sarcolemma, t-tubule membranes, and cell junctions, and in the mitochondria in the diltiazem-pretreated hearts. CONCLUSION: These results suggest that pretreatment with diltiazem may improve cardiac function during postischemic reperfusion, probably in part by maintenance of sarcolemmal integrity rather than by mitochondrial buffering function.

Quantitative study on the relation between structural and functional properties of the hearts from three different mammals.

The ultrastructural quantitative composition of left ventricular cardiac myocytes from isolated Langendorff-perfused hearts was studied in three different mammals (rabbit, guinea pig, and rat). Volume densities of mitochondria, myofibrils, and unspecified cytoplasm were determined using morphometry and were compared to functional parameters including left ventricular developed pressure (LVDP), contractility (dP/dt), heart rate, TTI (tension-time index, an index of oxygen consumption), and relative heart mass (H/B) obtained from these hearts. Each of the mammals was found to possess a very specific and characteristic quantitative composition of cardiac myocyte. Cardiac myocytes contained 26.8% mitochondria and 56.3% myofibrils in rabbits, 25.8% mitochondria and 60.9% myofibrils in guinea pigs, and 27.7% mitochondria and 58.1% myofibrils in rats. The LVDP, contractility, heart rate, and TTI were quite different among species. However, there were close correlations between the mitochondrial volume density and the LVDP (p < 0.05), and between the mitochondrial volume density and the TTI (p < 0.05), in any group of the animals. It is concluded that the mitochondrial volume density is a good indirect indicator of function of cardiac muscle related to oxidative capacity.

Human fetal heart development after mid-term: morphometry and ultrastructural study.

A total of 44 normally developed human fetal hearts aged from 17 to 40 weeks gestation were provided for routine ultrastructural and morphometric studies. For morphometric analysis, the maximal thicknesses of the anterior, lateral and posterior wall of both ventricles and that of interventricular septum were measured and the left-to-right thickness ratios calculated. The cross-sectional areas of both ventricles in the plane of the greatest heart diameter were measured with computerized image analysis system. Data were analyzed using linear regression and one-way analysis of variance. Myofibril formation occurred by attachment of thin filaments into amorphous Z materials which were presented in sarcolemmal plaques, sarcoplasmic condensations, desmosomes and in Z lines. From these Z centers, myofibrils radiated many directions and branched and anastomosed with further development. This pattern of myofibrillar development continued throughout the whole fetal period. A transverse tubule system was clearly evident in later fetal development. It occurred by invagination of sarcolemma into myocardial cells and formation of subsarcolemmal caveolae. Mitochondria, well-developed Golgi complexes, glycogen granules and well-developed microvessels were found throughout the whole fetal period. Binucleated myocytes appeared by 32 weeks gestation and this suggests that myocyte hyperplasia may cease before birth in humans. The growth of both ventricular walls, the interventricular septum and that of both ventricular cross-sectional areas showed linear regression, and the left-to-right wall thickness ratios were nearly constant. Also, there were no differences in morphometric data between the left and right ventricles. In conclusion, development of the myocyte is an ongoing process which may be continued in the post-natal period in humans, and our statistical results do not support the theory of the right ventricular dominance during the fetal period.

Effects of diltiazem on isoproterenol- or Ca-induced ventricular myocardial cell injuries in isolated perfused rabbit heart: an electron microscopic study.

The ultrastructural changes of isoproterenol- and those of Ca-induced ventricular cell injuries were compared in rabbits and the effect of diltiazem on these injuries was studied by electron microscopy. In comparison with the controls, the isoproterenol-treated (Group A), the Ca-treated (Group B), and the diltiazem-posttreated (Groups E and F) showed severe myocardial cell damage, such as sarcolemmal disruption, mitochondrial swelling, intramitochondrial electron-dense granules, membranous structures along mitochondrial cristae, thickening or close packing of the Z-lines, separation of cell junctions, frayed myofibrils, clumping of chromatin, and intracellular fluid accumulation. These ultrastructural changes were more pronounced in the Ca-treated (Groups B and F) than in the isoproterenol-treated (Groups A and E) animals. In contrast, the diltiazem-pretreated groups (Groups C and D) showed relatively intact myocardial ultrastructure. However, intramitochondrial electron-dense granules could be frequently found, and particularly the diltiazem-pretreated and Ca-treated group (Group D) showed intracellular fluid accumulation. The results of this study could suggest the following: 1) isoproterenol-induced myocardial cell damage is similar to Ca overload, 2) pretreatment with diltiazem could reduce the deleterious effects of isoproterenol-induced myocardial cell damage, but it could not prevent the effects of Ca overload completely, and 3) posttreatment with diltiazem could not provide any beneficial effect either on the isoproterenol-induced or on the Ca-overloaded myocardial cell damage, and 4) the beneficial effects of diltiazem are probably derived from the enhanced buffering function of mitochondria to cytosolic Ca or from selective inhibition of transsarcolemmal Ca influx.