Neobaicalein, a flavonoid from the Scutellaria litwinowii Bornm. & Sint. ex Bornm. induced apoptosis in human leukemic cell lines.

Objectives: Neobaicalein is one of the rich plant flavonoids isolated from the roots of Scutellaria spp. In this study, we evaluated and compared cytotoxic activity and the related apoptosis mechanisms of neobaicalein from Scutellaria litwinowii Bornm. & Sint. ex Bornm on apoptosis-proficient HL-60 cells and apoptosis-resistant K562 cells. Materials and Methods: Cell viability, cell apoptosis, caspase activity, and apoptosis-related protein expression were measured using MTS assay, propidium iodide (PI) staining and flow cytometry, caspase activity assay, and western blot analysis, respectively. Results: Neobaicalein significantly reduced cell viability in a dose-dependent manner using the MTS assay (P<0.05). The IC50 values (µM) against HL-60 and K562 cells after 48 hr treatment were 40.5 and 84.8, respectively. Incubation of HL-60 and K562 cells with 25, 50, and 100 µM neobaicalein for 48 hr, significantly increased the number of apoptotic cells and showed cytotoxic effects compared with the control group. Treatment with neobaicalein significantly increased Fas (P<0.05) and the cleaved form of PARP (P<0.05), and decreased the Bcl-2 levels (P<0.05) in HL-60 cells, whereas neobaicalein significantly increased Bax (P<0.05) and the cleaved form of PARP (P<0.05), and the caspases of the extrinsic and intrinsic pathways including caspases-8 (P<0.0001), -9 (P<0.01), and effector caspase-3 (P<0.0001) levels in K562 cells compared with the control group. Conclusion: It seems neobaicalein might cause cytotoxicity and cell apoptosis through interaction with the different apoptosis-related proteins of apoptotic pathways in HL-60 and K562 cells. Neobaicalein may exert a beneficial protective effect in slowing the progression of hematological malignancies.

Novel cilostamide analogs, phosphodiesterase 3 inhibitors, produce positive inotropic but differential lusitropic and chronotropic effects on isolated rat atria.

OBJECTIVES: Recently, we showed that some new synthetic compounds structurally related to cilostamide (4-(1,2-dihydro-2-oxoquinolin-6-hydroxy)- N-cyclohexyl-N-methylbutanamide), a selective phosphodiesterase 3 (PDE3) inhibitor, produce inotropic effect comparable to that of IBMX (3-isobutyl-1-methylxanthine), a non-selective PDE inhibitor, but with differential chronotropic effect. In this investigation, we compared the pharmacological effects of these compounds as potential cardiotonic agents using the spontaneously beating atria model. MATERIALS AND METHODS: In each experiment, rats were treated with reserpine. The atrium was isolated and mounted in an organ bath. We assessed chronotropic and inotropic effects using cumulative log concentration-response curves of isoprenaline alone or in combination of each test-compound. RESULTS: Majority of test compounds augment atria contraction force (ACF) significantly but with different potencies on atrium contraction rate. Cilostamide, MCPIP ([4-(4-methyl piperazin-1-yl)-4-oxobutoxy)-4-methylquinolin-2(1H)-one]), methyl carbostyril compounds- (mc1), mc2 and mc5 increased the isoprenaline effect on ACF synergistically. But, mc6 failed to potentiate the effect of isoprenalin; mc3 and mc4 did not increase ACF, which may be because of their higher hydrophilic nature. It was interesting that mc2, alone or in combination with isoprenaline, produced the highest inotropic effect while it did not affect the basal contraction rate and almost blocked the isoprenaline chronotropic effect. CONCLUSION: Combination of mc2 with isoprenaline had synergistic effect on inotropic effect, but this combination reduced isoprenaline chronotropic effect; therefore, these effects cannot be related to reducing B-adrenergic receptors activity. These compounds showed different effects; probably all of them were not mediated via PDE3 inhibition and other mechanisms are involving.

Apoptosis Induction of Salvia chorassanica Root Extract on Human Cervical Cancer Cell Line.

Salvia chorassanica Bunge is one of the Iranian endemic species of Salvia. There is not any reported literature on S. chorassanica. This study was designed to examine the in-vitro anti-proliferative and proapoptotic effects of the methanol extract of S. chorassanica and its fractions on HeLa cell line. Cells were cultured in EX-CELL®, an animal free medium specially designed for HeLa cell line and incubated with different concentrations of plant extracts. Cell viability was quantified by MTS assay. Apoptotic cells were determined using propidium iodide (PI) staining of DNA fragmentation by flow cytometry (sub-G1 peak). Activity of caspase -3, -8 and -9 was measured by the caspase colorimetric kit assay. S. chorassanica inhibited the growth of malignant cells and the CH2Cl2 fraction was determined as the most cytotoxic fraction in comparison with other fractions. The calculated IC50 values for methanol extract, n-hexane, CH2Cl2 and EtOAc fractions were 8.841, 5.45, 2.38, and 58.03 µg/mL, respectively. S. chorassanica induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to control cells indicating that the cytotoxic mechanism is characterized by apoptosis induction. The activity of caspase-3 and 8 proteins in treated HeLa cells was significantly higher than that of the control while caspase-9 activity did not change significantly. Based on the result obtained from our study, the apoptosis pathway involved in S. chorassanica-induced cell death may be through the extrinsic pathway and it can be a novel promising candidate in the treatment of cancer.

Acetylcholinesterase inhibitors promote angiogenesis in chick chorioallantoic membrane and inhibit apoptosis of endothelial cells.

Alzheimer's disease (AD) is one of the most common causes of dementia in the elderly. Recently, a great attention has been paid to the possible role of vascular changes in the pathogenesis of AD. Reduced microvascular density and degeneration of the endothelium are of structural cerebrovascular changes in AD. Acetylcholinesterase (AChE) inhibitors are widely used for the improvement of AD symptoms. Until now, however, the effects of AChE inhibitors on vascular changes including angiogenesis and endothelial cell apoptosis are not fully understood. In the present work, the effects of three AChE inhibitors (donepezil, rivastigmine, and galantamine) were tested on H2O2-induced apoptosis in human umbilical vein endothelial cells (HUVECs) and on angiogenesis in chicken chorioallantoic membrane model. Incubation of HUVEC with H2O2 led to a significant decrease in cell viability and an increase in the percentage of apoptotic cells. The tested drugs, at concentrations of 1-100 µ M, significantly inhibited the H2O2-induced toxicity. Also, all donepezil, rivastigmine and galantamine significantly increased the number of vessels in the chorioallantoic membrane when injected into fertilized eggs. In conclusion, AChE inhibitors possess angiogenesis-accelerating properties and have antiapoptotic effects on endothelial cells. These effects of AChE inhibitors may be involved in their beneficial effects on AD.

Wogonin and neobaicalein from Scutellaria litwinowii roots are apoptotic for HeLa cells

Chemical investigation on the CH2Cl2 fraction of the Scutellaria litwinowii Bornm. & Sint., Lamiaceace, root extract for the first time resulted in the isolation of wogonin, and neobaicalein. These compounds were evaluated for their cytotoxicity towards HeLa cell lines and lymphocytes. Meanwhile, the role of apoptosis was explored in this toxicity. The cells were cultured in RPMI medium and incubated with different concentrations of isolated flavonoids. Cell viability was quantified by MTS assay. Apoptotic cells were determined using propidium iodide staining of DNA fragmentation by flow cytometry (sub-G1peak). Wogonin, and neobaicalein inhibited the growth of malignant cells in a dose-dependent manner. The IC50 values of 46.62 and 79.34 µM were, respectively, found for neobaicalein and wogonin against HeLa cells after 48 h of treatment. Neobaicalein induced a sub-G1 peak in the flow cytometry histogram of treated cells compared to control cells indicating that apoptotic cell death is involved in neobaicalein toxicity. Neobaicalein exerts cytotoxic and pro-apoptotic effects in HeLa cell lines and could be considered as a potential chemotherapeutic agent in cancer treatment.

Scutellaria litwinowii induces apoptosis through both extrinsic and intrinsic apoptotic pathways in human promyelocytic leukemia cells.

Scutellaria is a genus of Lamiaceae with known antiproliferative potentials. Scutellaria litwinowii Bornm. & Sint. ex Bornm. is one of the Iranian species of Scutellaria. Although there are widespread reports about the cytotoxic and antitumor effects of some species of this genus, research on the molecular mechanism responsible for the anticancer effects of S. litwinowii has not yet been conducted. In the current study, the apoptotic effects of S. litwinowii on 2 myeloid cell lines, apoptosis-proficient HL60 cells and apoptosis-resistant K562 cells, were analyzed. An increase in the activity of caspases-3, -8, and -9, poly (ADP ribose) polymerase (PARP) cleavage, detection of phosphatidylserine on the outer layer of cell membrane and sub-G1 peak in the flow cytometry histogram of treated cells, suggested the induction of apoptosis. S. litwinowii also increased the Bax/Bcl-2 ratio. It could be concluded that S. litwinowii induced apoptosis in both apoptosis-proficient and apoptosis-resistant leukemic cells and it might be considered as a novel candidate in the treatment of hematological malignancies.

Protective effect of Nigella sativa and thymoquinone on serum/glucose deprivation-induced DNA damage in PC12 cells.

OBJECTIVE: The discovery and development of natural products with potent antioxidant properties has been one of the most interesting and promising approaches in the search for treatment of CNS injuries. The most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability resulting cellular dysfunction. Serum/glucose deprivation (SGD) has served as an excellent in vitro model for the understanding of the molecular mechanisms of neuronal damage during ischemia and for the development of neuroprotective drugs against ischemia-induced brain injury. Nigella sativa (N. sativa) seeds and thymoquinone (TQ), its most abundant constituent, have been shown to possess anti-inflammatory, antioxidant, chemopreventive and anti-neoplastic effects both in vitro and in vivo. Therefore, in this study we investigated genoprotective effects of N. sativa and TQ on DNA damage of PC12 cells under SGD condition. MATERIALS AND METHODS: PC12 cells were cultured in DMEM medium containing 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Initially cells were pretreated with different concentrations of N. sativa extract (NSE), (10, 50, 250 µg/ml) and TQ (1, 5, 10 µg/ml) for 6 h and then deprived of serum/glucose (SGD) for 18 h. The alkaline comet assay was used to evaluate the effect of these compounds on DNA damage following ischemic insult. The amount of DNA in the comet tail (% tail DNA) was measured as an indicator of DNA damage. RESULTS: A significant increase in the % tail DNA was seen in nuclei of cells following SGD induced DNA damage (p<0.001). In the control groups, no significant difference was found in the % tail DNA between NSE- or TQ-pretreated and vehicle-pretreated PC12 cells (p>0.05). NSE and TQ pretreatment resulted in a significant decrease in DNA damage following ischemic insult (p<0.001). This suppression of DNA damage by NSE and TQ was found to be dose-dependent. CONCLUSION: These data indicate that NSE and TQ have a genoprotective property, as revealed by the comet assay, under SGD condition in PC12 cells.

Design, synthesis and biological evaluation of 6-(benzyloxy)-4-methylquinolin-2(1H)-one derivatives as PDE3 inhibitors.

Selective PDE3 (phosphodiesterase 3) inhibitors improve cardiac contractility and may be used in congestive heart failure. However, their proarrhythmic potential is the most important side effect. In this work ten new synthetic compounds (3-[(4-methyl-2-oxo-1,2-dihydro-6-quinolinyl)oxy]methylbenzamide analogs: 4a-j) were designed, synthesized and tested for the inhibitory activity against human PDE3A and PDE3B. The strategy of the design was based on the structure of vesnarinone (a selective PDE3 inhibitor) and its docking analysis results. The synthetic compounds showed better PDE3 inhibitory activity in comparison with vesnarinone. Using docking analysis, a common binding model of each compound toward PDE3 was suggested. In the next step the potential cardiotonic activity of the best PDE3A inhibitors (4b, IC(50)=0.43+/-0.04microM) was evaluated by using the spontaneously beating atria model. In the experiment, atrium of reserpine-treated rat was isolated and the contractile and chronotropic effects of the synthetic compound were assessed. That was carried out in comparison with vesnarinone. The best pharmacological profile was obtained for the compound 4b, which displayed selectivity for increasing the force of contraction (46+/-3% change over the control) rather than the frequency rate (16+/-4% change over the control) at 100microM.

Design, synthesis and pharmacological evaluation of 6-hydroxy-4-methylquinolin-2(1H)-one derivatives as inotropic agents.

Selective PDE3 inhibitors improve cardiac contractility and may be used in congestive heart failure. However, their proarrhythmic potential is the most important side effect. In this research we designed, synthesized and evaluated the potential cardiotonic activity of thirteen PDE3 inhibitors (4-[(4-methyl-2-oxo-1,2-dihydro-6-quinolinyl)oxy]butanamide analogs) using the spontaneously beating atria model. The design strategy was based on the structure of cilostamide, a selective PDE3 inhibitor. In each experiment, atrium of reserpine-treated rat was isolated and the contractile and chronotropic effects of a synthetic compounds were assessed. All experiments were carried out in comparison with IBMX, amrinone and cilostamide as standard compounds. The results showed that, among the new compounds, the best pharmacological profile was obtained with the compound 6-[4-(4-methylpiperazine-1-yl)-4-oxobutoxy]-4-methylquinolin-2(1H)-one, 4j, which displayed selectivity for increasing the force of contraction (165 +/- 4% change over the control) rather than the frequency rate (115 +/- 7% change over the control) at 100 microM and potent inhibitory activity of PDE3 with IC(50) = 0.20 microM.

Inotropic and chronotropic effects of 6-hydroxy-4-methylquinolin-2(1H)-one derivatives in isolated rat atria.

BACKGROUND: Selective phosphodiesterase (PDE3) inhibitors improve cardiac contractility and may use in congestive heart failure. However, their proarrhythmic potential is the most important side effect. METHODS: In this research, we evaluated the potential cardiotonic activity of six new synthesized selective PDE3 inhibitors (6-hydroxy-4-methylquinolin-2(1H)-one derivatives) using the spontaneously beating atria model. In each experiment, atrium of reserpine-treated rat was isolated and the contractile and chronotropic effects of a synthesized compound were assessed. The 3-isobutyl-1-methylxanthine, a non-selective PDE inhibitor, was used for comparison. RESULTS: The results showed that, among new compounds, the best pharmacological profile was obtained with the compound 6-[4-(4-methylpiperazine-1-yl)-4-oxobutoxy]-4-methylquinolin-2(1H)-one, C6, which displayed selectivity for increasing the force of contraction (168 +/- 5% change over the control) rather than the frequency rate (138 +/- 5% change over the control) at 300 microM. However, C6 at concentrations of 10 and 100 microM produced left and upward shift in the positive inotropic concentration-response curve of isoprenaline. The -log EC50 of isoprenaline was 8.843 +/- 0.171 in the absence, 9.448 +/- 0.138 and 9.456 +/- 0.107 in the presence of 10, 100 microM of C6, respectively (P<0.001, n = 6). Also, amrinone, a selective PDE3 inhibitor, shifted the isoprenaline concentration-response curve to the left and upward. The concentration of 10 and 100 microM amrinone decreased -log EC50 of isoprenaline to 9.527 +/- 0.287 and 9.423 +/- 0.243, respectively (P<0.001, n = 6). Moreover, the positive chronotropic effect of isoprenaline was not affected by amrinone or C6. CONCLUSION: This study provides functional evidence for the positive inotropic effect of C6. Considering the augmentation of isoprenaline positive inotropic concentration-response with C6 and amrinone, we conclude that C6 produces its effect via potentiation of cAMP-dependent signaling system and possibly by inhibition of PDE3 activity.

Effect of aqueous extract from Nigella sativa L. on guinea pig isolated heart.

A potent inhibitory effect of aqueous extract from N. sativa on calcium channel of guinea pig heart was found comparable and even greater than that of diltazem. The results may also indicate an opening effect for the plant on potassium channel of isolated heart.