DICKKOPF-4 activates the noncanonical c-Jun-NH2 kinase signaling pathway while inhibiting the Wnt-canonical pathway in human renal cell carcinoma

BACKGROUND:

To the authors' knowledge, the functional significance of the Wnt antagonist dickkopf homolog 4 (DKK4) has not been investigated previously in renal cancer.

METHODS:

The authors initially observed that the expression of DKK4 was significantly higher in renal cancer tissues compared with adjacent normal kidney tissues. To assess the function of DKK4, stable DKK4‐transfected cells were established, and functional analyses were performed, including a T‐cell factor/lymphoid enhancer factor (TCF/LEF) reporter assay and tests for cell viability, colony formation, apoptosis, cell cycle, invasive capability, wound‐healing capability, and in vivo tumor growth.

RESULTS:

The relative TCF/LEF activity was significantly lower in DKK4‐transfected cells compared with empty vector, and nuclear β‐catenin expression was decreased in DKK4 transfectants. In addition, expression levels of the β‐catenin downstream effector proteins cyclin D1 and c‐Myc were decreased in DKK4 transfectants. However, greater invasiveness and migration were observed in stably transfected DKK4 cells. Increased growth of DKK4‐transfected tumors also was observed in nude mice. Members of the Wnt noncanonical/c‐Jun‐NH2 kinase (JNK) signaling pathway also were effected, such as c‐Jun, which had significantly increased expression and phosphorylation in DKK4‐stable transfectants, and matrix metalloproteinase‐2, which had significantly increased expression in DKK4‐stable transfectants.

CONCLUSIONS:

This is the first study to indicate that DKK4 expression is increased in renal cancer tissues and that DKK4 activates the noncanonical JNK signaling pathway while inhibiting the Wnt‐canonical pathway. Cancer 2011. © 2010 American Cancer Society.     

Insulin-like growth factor-binding protein-3 suppresses tumor growth via activation of caspase-dependent apoptosis and cross-talk with NF-κB signaling

Nuclear factor-kappaB (NF-κB) is constitutively activated in a variety of human cancers including prostate cancer and involved in tumorigenesis, tumor progression and chemo-resistance. Insulin-like growth factor-binding protein-3 (IGFBP-3) is a potent tumor suppressor and is significantly suppressed in a variety of cancers. Diverse biological effects of IGFBP-3 have been reported to be both dependent and independent of the IGF/IGF-I receptor (IGF-IR) axis. The precise underlying mechanisms of IGF/IGF-IR-independent, antiproliferative actions of IGFBP-3 are yet to be elucidated. We found an inverse correlation between NF-κB activity and IGFBP-3 expression during prostate cancer progression using an in vitro prostate cancer progression model. Restoration of IGFBP-3 resulted in significant inhibition of constitutively elevated NF-κB activity in prostate cancer cells. IGFBP-3 further inhibited the expression of NF-κB-regulated angiogenic factors such as VEGF and IL-8, and cell adhesion molecules, ICAM-1 and VCAM-1. This inhibitory action of IGFBP-3 was IGF/IGF-IR-independent since IGFBP-3 mutant devoid of IGF binding affinity had a similar inhibitory effect. We identified that IGFBP-3 degrades the key NF-κB regulatory molecules-IκBα and p65-NF-κB proteins through activation of caspase-8 and -3/-7, thereby inhibiting elevated NF-κB activity in prostate cancer. Finally intratumoral administration of IGFBP-3 resulted in significant tumor suppression as well as sensitization of antitumor effect of doxorubicin. Our findings indicate that IGFBP-3 exerts antitumor effects via IGF-independent mechanisms which involve activation of caspase-dependent apoptosis and cross-talk with NF-κB signaling. The use of IGFBP-3 as a cancer therapeutic with this distinctive suppression mechanism may offer alternate means to treat chemotherapy resistant tumors.     

In vitro anti-hepatitis B and SARS virus activities of a titanium-substituted-heteropolytungstate

A structural determined heteropolytungstate, [K₄(H₂O)₈Cl][K₄(H₂O)₄PTi₂W₁₀O₄₀]·NH₂OH 1, has been synthesized and evaluated for in vitro antiviral activities against hepatitis B (HBV) and SARS virus. The identity and high purity of compound 1 were confirmed by elemental analysis, NMR, IR analysis and single-crystal X-ray diffraction. The compound 1, evaluated in HepG 2.2.15 cells expressing permanently HBV, significantly reduced the levels of HBV antigens and HBV DNA in a dose-dependent and time-dependent manner. EC₅₀ values were determined to be 54 μM for HBeAg, 61 μM for HBsAg and 2.66 μM for supernatant HBV DNA, as compared to 1671, 1570, 169 μM, respectively, for the commercially-available hepatitis B drug adefovir dipivoxil (ADV). Intracellular cccDNA, pgRNA and HBcAg were also found to be decreased by compound 1 in a concentration-dependent manner. Cytotoxicity results showed that compound 1 has low toxicity in HepG 2 cells with CC₅₀ value of 515.20 μM. The results indicate that compound 1 can efficiently inhibit HBV replication in HepG 2.2.15 cells line in vitro. Additionally, compound 1 also shows high anti-SARS activity at an EC₅₀ of 7.08 μM and toxicity with a CC₅₀ of 118.6 μM against MDCK cells.     

腓骨肌萎缩症2L热休克蛋白B8过表达对细胞相对活力的影响

目的 探讨轴索型腓骨肌萎缩症2L(axonal Charcot-Marie-Tooth disease type 2L,CMT2L)K141N突变的热休克蛋白B8(heat shock protein B8,HSPB8)对细胞相对活力的影响.方法 应用脂质体转染技术,将野生型HSPB8(wt HSPB8)和K141N突变型HSPB8( K141N HSPB8)分别转染SHSY-5Y细胞,使其过量表达上述蛋白,以表达绿色荧光蛋白(green fluorescent protein,GFP)为阴性对照,转染24 h后用44℃致死性热休克处理40 min,用四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)比色法检测SHSY-5Y细胞的相对活力.结果 未处理组pEGFP-wt HSPB8与pEGFP-K141N HSPB8组间吸光度值比较P＞0.05,而热休克处理组pEGFP-wt HSPB8与pEGFP-K141N HSPB8组间吸光度值比较P＜0.05,表明热休克处理后各组细胞相对活力差异有统计学意义,热休克处理后过表达wt HSPB8的SHSY-5Y细胞活力最高,过表达K141N HSPB8的细胞次之,过表达GFP空载体的细胞活力最低.结论 证实了HSPB8在细胞受到致死性热休克刺激时对于保护细胞活力方面起着重要作用,K141N突变型HSPB8对细胞相对活力的保护作用较野生型HSPB8减弱.     

Post-ischemic administration of nimodipine following focal cerebral ischemic-reperfusion injury in rats alleviated excitotoxicity, neurobehavioural alterations and partially the bioenergetics

The present study focuses on the temporal calcium significance in middle cerebral artery occluded (2 h ischemia)-reperfused (70 h reperfusion) rats treated with nimodipine (NM) through concurrent measurements of excitotoxicity, bioenergetics and neurobehavioural paradigms. Further, the suitable therapeutic time window of calcium channel antagonism in stroke was also ascertained. NM (5 mg/kg, i.p.) was administered at pre (30 min before the induction of ischemia), during (1 h following occlusion of MCA) and post-ischemic (3 h after begin of reperfusion) states. The magnitude of neuroprotection in terms of excitotoxicity (glutamate, glutamine synthetase, Na⁺K⁺ATPase), bioenergetics (ATP, NAD⁺) and neurobehavioural paradigms (neurological score and open field exploratory behaviour) were measured and compared to ensure the therapeutic time-window of NM in stroke. Middle cerebral artery occlusion-reperfusion (MCAO/R) was found to elevate glutamate, glutamine synthetase levels and deplete Na⁺K⁺ATPase activity in the vehicle treated group (IR group). Significant decrease in bioenergetics such as ATP and NAD⁺ levels was also observed. Further, IR group demonstrated grievous oxidative stress (increase in lipid peroxidation, protein carbonyl content, nitrite/nitrate levels and decrease in superoxide dismutase and glutathione levels) along with anxiogenic behaviour, neurological deficits and neuronal damage and decreased nuclear to cytoplasm ratio in CA1 hippocampal region. Post-ischemic NM administration reversed the excitotoxicity, neurobehavioural and histopathological alterations significantly, but it restored bioenergetics level in MCAO/R rats only partially.These findings were further confirmed with the combination treatment (CT) of post-ischemic NM and pre-ischemic memantine (MN) administration, since MN showed protective effect in the pre-ischemic administration (Babu and Ramanathan, 2009). The failure of NM to forefend the neurodegeneration on pre- and during-ischemic administration suggests that the initial phase damages in ischemic-reperfusion (IR) might be mediated through other mechanism(s) such as glutamergic overstimulation or reverse operation of glutamate transporters. From the present study, it is concluded that calcium plays a crucial role in post-ischemic status and the suitable therapeutic time window of calcium antagonism is the post-ischemic state.     

Cardioprotective effects of saponins from Panax japonicus on acute myocardial ischemia against oxidative stress-triggered damage and cardiac cell death in rats

Aim

To study the cardioprotective effects of saponins from Panax japonicus (SPJ) on acute myocardial ischemia injury rats induced by ligating of the left anterior descending branch (LAD), on the basis of this investigation, the possible mechanism of SPJ was elucidated.

Materials and methods

SPJ was identified by high performance liquid chromatography-evaporative light scattering detection. Male Sprague-Dawley rats (200–220 g) were randomly divided into four groups: sham-operated, LAD, LAD + l-SPJ (SPJ, 50 mg/kg/day, orally) and LAD + h-SPJ (SPJ, 100 mg/kg/day, orally). Before operation, the foregoing groups were pretreated with homologous drug once a day for 7 days, respectively. After twelve hours in LAD, the cardioprotective effects of SPJ were evaluated by infarct size, biochemical values, hemodynamic, and histopathological observations and the antioxidative and antiapoptotic relative gene expressions.

Results

SPJ significantly improved heart function and decreased infarct size; remarkably decreased levels of serum lactate dehydrogenase, creatine kinase, xanthine oxide and malondialdehyde content, increased contents of serum total antioxidant capacity, superoxide dismutase (SOD), glutathione peroxidase, catalase; quantitative real-time PCR results showed that SPJ might markedly reverse the down-regulated mRNA expressions of the SOD1, SOD2 and SOD3, ameliorate the increased Bax and caspase-3 mRNA expressions and decreased Bcl-2 mRNA expression and ratios of Bcl-2 to Bax. Histopathological observations provided supportive evidence for biochemical analyses, and with the dose of SPJ increasing, the aforesaid improvement became more and more strong.

Conclusions

The studies demonstrated that in ischemic myocardium, oxidative stress caused the overgeneration and accumulation of reactive oxygen species (ROS), which was central of cardiac ischemic injury. SPJ exerted beneficially cardioprotective effects on myocardial ischemia injury rats, mainly scavenging oxidative stress-triggered overgeneration and accumulation of ROS, alleviating myocardial ischemia injury and cardiac cell death.     

Immunomodulatory active compounds from Tinospora cordifolia

Ethnopharmacological relevance

Tinospora cordifolia mentioned as “Rasayana” is extensively used in various herbal preparations for the treatment of different ailments for its general tonic, antiperiodic, antispasmodic, antiinflammatory, antiarthritic, antiallergic and antidiabetic properties. It is extensively used in Ayurveda due to its potential in improving the immune system and the body resistance against infections.

Aim of the study

The aim of the study was to isolate and characterise the immunomodulatory active compounds of Tinospora cordifolia.

Materials and methods

The immunomodulatory activity of different extracts, fractions and isolated compounds in relation to phagocytosis and reactive oxygen species production in human neutrophil cells have been investigated using the PMN phagocytic function studies, NBT, NO and chemiluminescence assay.

Results

The results obtained indicate that ethyl acetate, water fractions and hot water extract exhibited significant immunomodulatory activity with an increase in percentage phagocyctosis. Chromatographic purification of these fraction led to the isolation of a mixture of two compounds 2, 3 isolated for the first time from natural source and five known compounds 1, 4–7 which were characterized as 11-hydroxymustakone (2), N-methyl-2-pyrrolidone (3), N-formylannonain (1), cordifolioside A (4), magnoflorine (5), tinocordiside (6), syringin (7) by nuclear magnetic resonance (NMR) and mass spectrometry (MS) and comparing the spectral data with reported one. Cordifolioside A and syringin have been reported to possess immunomodulatory activity. Other five compounds showed significant enhancement in phagocytic activity and increase in nitric oxide and reactive oxygen species generation at concentration 0.1–2.5 μg/ml.

Conclusions

Seven immunomodulatory active compounds belonging to different classes have been isolated and characterised indicating that the immunomodulatory activity of Tinospora cordifolia may be attributed to the synergistic effect of group of compounds.     

Standardized flavonoid-rich fraction of Artemisia princeps Pampanini cv. Sajabal induces apoptosis via mitochondrial pathway in human cervical cancer HeLa cells

Ethnopharmacological relevance

Artemisia princeps Pampanini is widely used in Eastern traditional medicine for the treatment of circulatory disorders, such as, dysmenorrhea, hematuria, hemorrhoids, and inflammation, and is also used to treat chronic conditions, such as, cancers, ulcers, and digestive disorders.

Aim of the study

The purpose of this study is to investigate the effect of a standardized flavonoid-rich fraction of Artemisia princeps Pampanini cv. Sajabal (FRAP) on the induction of apoptosis and the molecular mechanism involved in human cervical cancer HeLa cells.

Materials and methods

Human cervical cancer HeLa cells were treated with FRAP and apoptosis was detected by cell morphologic observation, annexin-V-PI staning and western blot analysis on the expression of protein associated with cell death.

Results

FRAP led to the cleavages of caspase-3, -8, and -9 and the cleavage of poly (ADP-ribose) polymerase (PARP) in HeLa cells. Caspase-3 inhibitor (z-DEVD-fmk), caspase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (z-LEHD), and broad caspase inhibitor (z-VAD-fmk) significantly suppressed the FRAP-induced accumulation of annexin V positive cells. Furthermore, it was found that FRAP caused a loss of mitochondrial membrane potential (MMP) and the release of cytochrome c to the cytosol. Furthermore, the overexpression of Bcl-xL significantly prevented FRAP-induced apoptosis, MMP changes, and the activations of caspase-3, -8, and -9. Interestingly, pretreatment with caspase-8 inhibitor significantly reduced the FRAP-induced activation of caspase-3 but not that of caspase-9, whereas the caspase-3 inhibitor, z-DEVD-fmk, markedly attenuated the FRAP-induced activation of caspase-8. In BALB/c^nu/nu mice bearing a HeLa xenograft, FRAP dosed at 25 or 50 mg/kg significantly inhibited tumor growth.

Conclusion

Our results indicate caspase-mediated activation of the mitochondrial death pathway plays a critical role in the FRAP-induced apoptosis of HeLa cells and that FRAP inhibits the in vivo tumor growth of HeLa xenograft mice.     

The antimicrobial,antioxidative, anti-inflammatory activity and cytotoxicity of different fractions of four South African Bauhinia species used traditionally to treat diarrhoea

Ethnopharmacological importance

Many Bauhinia species, including those indigenous to South Africa, are used in traditional medicine across the world for treating ailments such as gastrointestinal tract (GIT) disorders, diabetes, infectious diseases and inflammation.

Aims

Several relevant aspects of different fractions of leaf extracts of Bauhinia bowkeri (BAB), Bauhinia galpinii (BAG), Bauhinia petersiana (BAP), and Bauhinia variegata (BAV) used in South African traditional medicine to alleviate diarrhoea related symptoms were evaluated.

Materials and Methods

The antioxidative activities of the extracts were determined using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS⁺) radical scavenging and ferric reducing antioxidant power (FRAP) methods. In vitro antimicrobial activities of the extracts were determined against bacterial strains (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis) and clinical isolates of the opportunistic fungal strains (Aspergillus fumigatus, Candida albicans, and Cryptococcus neoformans) using a serial dilution microplate method. The polyphenolic contents were quantified using standard methods, and anti-inflammatory activities of the crude extracts were determined using the cyclooxygenase and soybean 15-lipoxygenase enzyme inhibitory assays. The safety of the extracts was evaluated by determining the cytotoxicity against Vero cell lines.

Results

The acidified 70% acetone crude extract and their fractions had good antiradical potency against the DPPH and ABTS radicals. The methanol soluble portions of the butanol fractions were more potent (EC₅₀ ranges from 0.64±0.05 to 1.51±0.07 and 0.88±0.18 to 1.49±0.09 μg/ml against DPPH and ABTS radical respectively) compared to the standard, trolox and ascorbic acid (EC₅₀ ranges from 1.47±0.24 to 1.70±0.27 μg/ml) for both DPPH and ABTS. The crude extracts contained variable quantities of phenolic content. The crude extracts and their fractions had weak to good antimicrobial activities, inhibiting the growth of the organisms at concentrations ranging from 39 to 2500 μg/ml. The BAG crude extract and its fractions were the most active against the fungi (MICs ranging from 39 to 625 μg/ml) while the BAB extract and its fractions were the least active with the MICs ranging between 39 and 2500 μg/ml. Aspergillus fumigatus was the least susceptible fungus while Cryptococcus neoformans was the most susceptible.The phenolic-rich crude extracts of BAB, BAG, and BAP had moderate to good dose-dependent cyclooxygenase-1 enzyme inhibitory activity with inhibitions between 22.8% and 71.4%. The extracts were however, inactive against cyclooxygenase-2. The extracts had some level of cytotoxicity towards Vero cell lines, reducing cell viability to less than 10% at concentrations more than 50 μg/ml.

Conclusion

The biological activities observed in Bauhinia species provide a scientific basis for the use of the plants in traditional medicines to treat diseases with multi-factorial pathogenesis such as diarrhoea, with each aspect of activity contributing to the ultimate therapeutic benefit of the plants. However, the use of the phenolic-rich extracts of these plants to treat diarrhoea or any other ailments in traditional medicine needs to be monitored closely because of potential toxic effects and selective inhibition of COX-1 with the associated GIT injury.