Neuroprotective effects of dimerumic acid and deferricoprogen from Monascus purpureus NTU 568-fermented rice against 6-hydroxydopamine-induced oxidative stress and apoptosis in differentiated pheochromocytoma PC-12 cells

Context Oxidative stress plays a key role in neurodegenerative disorders, including Parkinson’s disease (PD). Rice fermented with Monascus purpureus Went (Monascaceae) NTU 568 (red mould rice) was found to contain antioxidants, including dimerumic acid (DMA) and deferricoprogen (DFC).

Objective The effects of DMA and DFC on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity and potential protective mechanisms in differentiated PC-12 pheochromocytoma cells were investigated.

Materials and methods DMA (0–60?μM) or DFC (0–10?μM) was co-treated with 6-OHDA (200?μM, 24?h exposure) in differentiated PC-12 cells. Cell viability and intercellular reactive oxygen species (ROS) were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) assays, respectively. Cell apoptosis was determined by DNA fragmentation analysis and propidium iodide staining by flow cytometry. Western blot analysis was used to measure the levels of cell protein expression.

Results DMA and DFC significantly increased cell viability to 72% and 81% in 6-OHDA-induced differentiated PC-12 cell cultures, respectively. Furthermore, DMA and DFC reduced 6-OHDA-induced formation of extracellular and intercellular ROS by 25% and 20%, respectively, and decreased NADPH oxidase-2 expression in differentiated PC-12 cells. DMA and DFC inhibited 6-OHDA-induced apoptosis and decreased activation of caspase-3 via regulation of Bcl-2-associated X protein (Bax) and Bcl-2 protein expression in differentiated PC-12 cells.

Conclusion DMA and DFC may protect against 6-OHDA toxicity by inhibiting ROS formation and apoptosis. These results showed that the metabolites from M. purpureus NTU 568 fermentation were potential therapeutic agents for PD induced by oxidative damage and should be encouraged for further research.     

Dopaminergic neurotoxicant 6-OHDA induces oxidative damage through proteolytic activation of PKCδ in cell culture and animal models of Parkinson's disease

The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 μM) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 μM) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKCδ) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 μM). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKCδ^D327A and kinase dead PKCδ^K376R or siRNA-mediated knockdown of PKCδ protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKCδ promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKCδ expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKCδ cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKCδ^D327A protein protected against 6-OHDA-induced PKCδ activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKCδ is a key downstream event in dopaminergic degeneration, and these results may have important translational value for development of novel treatment strategies for PD.     

Effects of asarinin on dopamine biosynthesis and 6-hydroxydopamine-induced cytotoxicity in PC12 cells

This study investigated the effects of asarinin on dopamine biosynthesis and 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in rat adrenal pheochromocytoma (PC12) cells. Treatment with asarinin (25–50 μM) increased intracellular dopamine levels and enhanced L-DOPA-induced increases in dopamine levels. Asarinin (25 μM) induced cyclic AMP-dependent protein kinase A (PKA) signaling, leading to increased cyclic AMP-response element binding protein (CREB) and tyrosine hydroxylase (TH) phosphorylation, which in turn stimulated dopamine production. Asarinin (25 μM) also activated transient phosphorylation of extracellular signal-regulated kinase (ERK1/2) and Bad phosphorylation at Ser 112, both of which have been shown to promote cell survival. In contrast, asarinin (25 μM) inhibited sustained ERK1/2, Bax, c-Jun N-terminal kinase (JNK1/2) and p38 mitogen-activated protein kinase (p38MAPK) phosphorylation and caspase-3 activity, which were induced by 6-OHDA (100 μM). These results suggest that asarinin induces dopamine biosynthesis via activation of the PKA-CREB-TH system and protects against 6-OHDA-induced cytotoxicity by inhibiting the sustained activation of the ERK-p38MAPK-JNK1/2-caspase-3 system in PC12 cells.     

Antioxidant and neuroprotector effect of Lepidium meyenii (maca) methanol leaf extract against 6-hydroxy dopamine (6-OHDA)-induced toxicity in PC12 cells

Reactive oxygen species (ROS) are normally produced during cell metabolism, there is strong evidence to suggest that ROS produced in excess impair the cell and may be etiologically related to various neurodegenerative diseases. This study was undertaken to examine the effects of Lepidium meyenii (MACA) methanol leaf extract on neurotoxicity in PC12 cell exposed to 6-hydroxydopamine (6-OHDA). Fresh samples of “maca” leaves were processed in order to obtain foliar extracts and to evaluate the neurobiological activity on PC12 cells, subjected to the cytotoxic effect of 6-OHDA through the determination of the capacity antioxidant, cell viability and cytotoxicity assays on PC12 cells. The results of the tests of antioxidant activity, showed maximum values of 2262.37 and 1305.36 expressed in Trolox equivalents (TEAC), for the methanolic and aqueous fractions respectively. Cell viability assays at a dose of 10?μg extract showed an increase of 31% and 60% at 6 and 12?h of pretreatment, respectively. Cytotoxicity assays at the same dose and exposure time showed a 31.4% and 47.8% reduction in lactate dehydrogenase (LDH) activity and an increase in superoxide dismutase (SOD) activity. The results allow us to affirm that the methanolic foliar extract of “maca” presents in vitro neurobiological activity of antioxidant protection, increase in cell viability and reduction of cytotoxicity against oxidative stress generated by 6-OHDA. In conclusion, the present study shows a protective role for Lepidium meyenii leaf extract on 6-OHDA-induced toxicity by an antioxidant effect.     

Antioxidant and cytotoxic potential of a new thienyl derivative from Tagetes erecta roots

Context: The search for newer compounds against pathogenic species continues unabated due to drug resistance. Traditionally, Tagetes erecta Linn. (Compositae) has been used for the treatment of various parasitic and microbial diseases.

Objective: To evaluate the antioxidant activity of the ethanol extract of Tagetes erecta roots and its cytotoxicity against prostate and HeLa cancer cell lines followed by activity-guided isolation.

Materials and Methods: The antioxidant screening was carried out using diphenylpicrylhydrazyl (DPPH) radical scavenging assay with serial concentrations ranging from 2 to 100 µg/mL, and cytotoxicity was evaluated against prostate (PC-3) and HeLa cell lines using microculture tetrazolium test (MTT) assay with concentrations ranging from 500 to 1.89 µg/mL. Isolation of the ethanol extract was carried out using column chromatography whereby 21 isolates were obtained (T₁-T₂₁), and the most active isolate was subjected for characterization using ultraviolet (UV), infrared (IR), nuclear magnetic resonance (NMR), and mass spectroscopic techniques.

Results: The ethanol extract scavenged DPPH free radicals thereby exhibiting antioxidant activity with an IC₅₀ of 35.9 µg/mL. In addition, the extract conferred noticeable cytotoxicity against the HeLa (LD₅₀ of 164.28 µg/mL) and PC-3 cell lines (LD₅₀ of 407.3 µg/mL). Among all the isolates, T₃ showed antioxidant activity with IC₅₀ of 11.56 µg/mL and cytotoxicity with LD₅₀ of 12.5 µg/mL against HeLa and 30.25 µg/mL against PC-3 cell lines and was characterized as 2-ethynyl-5-(thiophen-2-yl) thiophene.

Discussion: The new thienyl compound (T₃) exhibited profound antioxidant activity and cytotoxicity at relatively lower concentrations than the extract.

Conclusion: The observations provide support for the ethnobotanical use of the plant.     

Vasorelaxant effects of the extracts and some flavonoids from the buds of Coreopsis tinctoria

Abstract

Context: The buds of Coreopsis tinctoria Nutt (Compositae) are used in the treatment of hypertension in the Uyghur folk medicine in China.

Objective: To investigate vasorelaxant properties of extracts and some flavonoids from C. tinctoria (CT) and their underlying mechanisms in isolated rat thoracic aortic rings.

Materials and methods: Vasorelaxant effects of ethanol extracts of CT (CTA) and its flavonoids as well as water–ethanol eluates from CTA by AB-8 resins (CTAA～CTAF) were evaluated on rat aortic rings pre-contracted with phenylephrine (PE, 1?µM) or high KCl (60?µM). We evaluated the effect of CTA, CTAD and CTAE on PE-induced contraction in a Ca²⁺-free medium and a dose–effect curve of Ca²⁺ in pre-contracted ring with high KCl.

Results: Endothelial removal did not modify the effect of CTAD and CTAE (3.00?g/L) neither on PE-pre-contracted rings (164.78?±?21.44 and 191.47?±?16.75%) nor on KCl-pre-contracted rings (75.68?±?10.76 and 125.14?±?17.41%) compared with intact-endothelium rings pre-contracted with high KCl (100.49?±?17.30 and 110.81?±?16.33%). CTAD and CTAE (3.00?g/L) down-regulated the dose–effect curve of Ca²⁺ in pre-contraction with high KCl, and inhibited the pre-contraction with PE in a Ca²⁺-free medium (p?<?0.05). Seven flavonoids were obtained from CTAD, of which luteolin (5) and quercetin (6) were found to be the most effective relaxation in rings precontracted with PE (EC₅₀: 0.006 and 0.039?g/L, p?<?0.05) or high KCl (EC₅₀: 0.023 and 0.045?g/L, p?<?0.05).

Discussion and conclusion: These data demonstrated the vasorelaxant effect of CT, and its mechanism is likely due to an inhibitory effect on calcium movements through cell membranes.     

Three new C-alkylated flavonoids from Desmodium oblongum

Three new C-alkylated flavonoids, 4′-hydroxy-8-isobutyryl-7-methoxy-6-methyl-flavone (1), 4′,7-dimethoxy-8-isobutyryl-6-methyl-flavone (2), and 4′,7-dimethoxy-8-isobutyryl-flavone (3), together with three know ones luteolin (4), kaemferol (5), and quercetin (6), were isolated from Desmodium oblongum. Their structures were elucidated by spectroscopic methods, including extensive 1D- and 2D-NMR techniques. Compound 1 showed cytotoxicity against NB4, SHSY5Y, and MCF7 cell lines with IC₅₀ values of 8.5, 6.5, and 7.8 μM; compound 2 showed cytotoxicity against SHSY5Y and PC3 cell lines with IC₅₀ values of 5.0 and 6.8 μM; compound 3 displayed cytotoxicity against SHSY5Y and MCF7 cell lines with IC₅₀ values of 6.4 and 9.4 μM, respectively.     

Mangiferin ameliorates 6-hydroxydopamineinduced cytotoxicity and oxidative stress in ketamine model of schizophrenia

BackgroundAccumulating evidence indicates that mangiferin (MGF), a natural xanthone, by virtue of its antioxidant and anti-inflammatory properties is neuroprotective. Here we sought to verify the cytoprotective role of MGF on cultured rat primary mesencephalic cells exposed to 6-hydroxydopamine (6-OHDA) in vitro, and the MGFs anti-inflammatory potential in mouse model of ketamine-induced schizophrenia in vivo.Methods3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay was performed to measure cell viability in mesencephalic cell cultures exposed to neurotoxin (6-OHDA, 40 µM). Schizophrenia was induced in mice by ketamine (50 mg/kg, ip, twice a day, for 7 days). The treatment effects of MGF (50 mg/kg, po, for 7 days) were verified on locomotor behavioral changes in open-field test, and on the oxidant stress-related increase in lipid-peroxidation (malondialdehyde) and interleukin-6 (IL-6) levels in brain tissues.ResultsMGF (10–100 µM) produced no per se effect on cell viability as measured by MTT assay, but significantly prevented the 6-OHDA-induced cell death in a concentration-dependent manner. Acridine orange/ethidium bromide (AO/EtBr) staining confirmed the absence of 6-OHDA-induced morphological changes characteristic of apoptosis/necrosis. In open-field test, ketamineinduced impaired locomotor activity and behavioral changes such as grooming and stereotyped but not rearing were effectively ameliorated by MGF pretreatment. Also, ketamine-associated increase in brain tissue levels of IL-6 and MDA were significantly lowered in MGF-pretreated mice.ConclusionMangiferin has a neurocytoprotective role related, at least in part, to an antioxidant and anti-inflammatory mechanism, which could be explored for more effective therapies of schizophrenia and other neurodegenerative diseases.     

Assessment of luteolin isolated from Eclipta alba leaves in animal models of epilepsy

Context: Eclipta alba (Linn) Hassk. (Asteraceae) has been reported to be a nerve tonic and has been used to treat epilepsy in folk medicine.

Objective: The present study isolates and characterizes luteolin from E. alba and evaluates its antiepileptic potential in chemically induced acute and chronic models in mice.

Materials and methods: The methanol extract (16.85% w/w) of E. alba leaves was subjected to fractionation for isolation of luteolin. In acute pentylenetetrazole (PTZ) model, luteolin (5, 10, 20?mg/kg, i.p.) was administered 30?min prior to PTZ injection (100?mg/kg) in Swiss albino mice. Kindling was induced by chronic administration of PTZ (35?mg/kg) on every alternate day (48 days). Luteolin was investigated on the course of kindling development and oxidative stress markers [reduced glutathione (GSH) and malondialdehyde (MDA)] in kindled mice.

Results: Single-dose pretreatment with luteolin (10 and 20?mg/kg, i.p.) was found to be effective in an acute PTZ model (100% protection from mortality) and it did not exhibit any effect on motor coordination at the same doses. PTZ-induced kindling was significantly (p?p?p?Discussion and conclusion: The results of the present study demonstrated that luteolin had an anticonvulsant effect in an acute PTZ model. Luteolin exhibited and inhibitory effect on the course of kindling and associated oxidative stress and hence could be a potential molecule in the treatment of epilepsy.     

Bioactivity-guided isolation of cytotoxic constituents from three medicinal plants

Abstract

Context: The ethanol extracts and their fractions of three Indian medicinal plants, Ervatamia coronaria (Jacq.) Stapf, (Apocynaceae), Mimosa pudica L. (Mimosaceae) and Caesalpinia bonduc (L.) Roxb. (Caesalpiniaceae) were tested for their cytotoxic activity in the brine shrimp lethality (BSL) bioassay and in various cancer cell lines. The plants were selected based on their traditional use in the treatment of cancer/tumors.

Objectives: To investigate the in vitro cytotoxicity of Ervatamia coronaria, Mimosa pudica and Caesalpinia bonduc.

Materials and methods: Ethanolic extracts and their fractions of E. coronaria, M. pudica and C. bonduc were subjected to cytotoxicity studies using BSL bioassay method with concentrations of 10, 50, 100, 500 and 1000?µg/ml. The alkaloid fraction of E. coronaria with significant cytotoxicity in BSL bioassay was subjected to in vitro cytotoxicity studies with HT-29, A-549, HepG-2, MCF-7 and L-6 cell lines at concentrations of 12.5, 25, 50, 100 and 200?µg/ml and a DNA fragmentation study using the HT-29 cell line.

Results: The alkaloid fractions of E. coronaria and M. pudica showed significant cytotoxicity with LC₅₀ values of 65.83 and 85.10?µg/ml in the BSL bioassay, respectively. The purified alkaloid fraction of E. coronaria exhibited highest cytotoxicity in HT-29, A-549 and MCF-7 cell lines with IC₅₀ values of 32.5, 47.5 and 72.5?µg/ml, respectively, and induced DNA fragmentation in the HT-29 cell line at a concentration of 65?µg/ml.

Conclusion: The alkaloid fraction of E. coronaria exhibited significant cytotoxicity. Alkaloids such as ervatamine, apparicine and coronaridine that were earlier reported may be responsible for this activity.     

Subcutaneous antifungal screening of Latin American plant extracts against Sporothrix schenckii and Fonsecaea pedrosoi

Context: Subcutaneous mycoses are chronic infections caused by slow growing environmental fungi. Latin American plants are used in folk medicine to treat these afflictions. Moreover, the potential of the rich Latin American biodiversity for this purpose has not been fully explored.

Objectives: The aim of the study was to screen Latin American plant extracts against two species of subcutaneous fungi: Sporothrix schenckii and Fonsecaea pedrosoi.

Materials and methods: One hundred ninety-five organic extracts from 151 Latin American plants were screened against two subcutaneous fungi by the agar dilution method at a concentration of 100 µg/mL, and minimum inhibitory concentrations (MICs) of active extracts were determined. Positive (amphothericin B) and negative (50% ethanol) controls were used.

Results and discussion: Twenty eight extracts showed activity at ≤100 µg/mL. Of these, four extracts from Gnaphalium gaudichaudianum DC (Asteraceae), Plumeria rubra L (Apocynaceae), Tecoma stans (L.) Juss. ex Kunth. (Bignoniaceae), and Trichostigma octandum (L.), H. Walter showed activity against F. pedrosoi at MIC 12.5 µg/mL; and, four extracts from Bourreria huanita (Lex.) Hemsl. (Boraginaceae), Phytolacca bogotensis Kunth (Phytolaccaceae), Monnina xalapensis Kunth (Polygalaceae) and Crataegus pubescens (C. Presl) C. Presl (Rosaceae) against S. schenckii. This is the first report on antifungal activity of the Latin American plants against these two subcutaneous fungi.

Conclusion: S. schenkii and F. pedrosoi were inhibited by B. huanita (MIC: 12.5 and 25 µg/mL), G. gaudichaudianum (MIC: 50 and 12.5 µg/mL) and T. triflora (MIC: 25 µg/mL).     

Antiproliferative and apoptotic activities of extracts of Asclepias subulata

Abstract

Context: Asclepias subulata Decne. (Apocynaceae) is a shrub used in the Mexican traditional medicine for the treatment of cancer.

Objective: The objective of this study was to evaluate the antiproliferative activity of methanol extract of aerial parts of A. subulata and its fractions against different cancer cell lines. Additionally, we analyzed the mechanism of action of the active fractions.

Materials and methods: Methanol extract fractions were prepared by serial extraction with n-hexane, ethyl acetate, and ethanol. The antiproliferative activity of methanol extract and its fractions was evaluated, against several murine (M12.C3.F6, RAW 264.7, and L929) and human (HeLa, A549, PC-3, LS 180, and ARPE-19) cell lines by the MTT assay, using concentrations of 0.4–400?µg/mL for 48?h. Ethanol and residual fractions were separated using silica gel column. Apoptosis induction of cancer cells was evaluated by Annexin and JC-1 staining using flow cytometry.

Results: Methanol extract and its fractions showed antiproliferative activity against all human cancer cell lines tested. Methanol extract had the highest antiproliferative activity on A549 and HeLa cells (IC₅₀ values?<?0.4 and 8.7?µg/mL, respectively). Ethanol and residual fractions exerted significant antiproliferative effect on A549 (IC₅₀?<?0.4?µg/mL) and PC3 cells (IC₅₀ 1.4 and 5.1?µg/mL). Apoptotic assays showed that CEF7, CEF9, CRF6, and CRF5 fractions induced mitochondrial depolarization in A549 cells, 70, 73, 77, and 80%, respectively. Those fractions triggered the apoptosis mitochondrial pathway.

Conclusion: Our data show that A. subulata extracts have potent antiproliferative properties on human cancer cell lines. This plant should be considered an important source of potent anticancer compounds.     

Protective effect of hydrogen sulphide against 6-OHDA-induced cell injury in SH-SY5Y cells involves PKC/PI3K/Akt pathway

BACKGROUND AND PURPOSE

Hydrogen sulphide (H₂S) is a novel neuromodulator. The present study aimed to investigate the protective effect of H₂S against cell injury induced by 6-hydroxydopamine (6-OHDA), a selective dopaminergic neurotoxin often used to establish a model of Parkinson''s disease for studying the underlying mechanisms of this condition.

EXPERIMENTAL APPROACH

Cell viability in SH-SY5Y cells was measured using MTT assay. Western blot analysis and pharmacological manipulation were employed to study the signalling mechanisms.

KEY RESULTS

Treatment of SH-SY5Y cells with 6-OHDA (50–200 µM) for 12 h decreased cell viability. Exogenous application of NaHS (an H₂S donor, 100–1000 µM) or overexpression of cystathionine β-synthase (a predominant enzyme to produce endogenous H₂S in SH-SY5Y cells) protected cells against 6-OHDA-induced cell apoptosis and death. Furthermore, NaHS reversed 6-OHDA-induced loss of tyrosine hydroxylase. Western blot analysis showed that NaHS reversed the down-regulation of PKCα, ε and Akt and the up-regulation of PKCδ in 6-OHDA-treated cells. Blockade of PKCα with Gö6976 (2 µM), PKCε with EAVSLKPT (200 µM) or PI3K with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (20 µM) reduced the protective effects of H₂S. However, inhibition of PKCδ with rottlerin (5 µM) failed to affect 6-OHDA-induced cell injury. These data suggest that the protective effects of NaHS mainly resulted from activation of PKCα, ε and PI3K/Akt pathway. In addition, NaHS-induced Akt phosphorylation was significantly attenuated by Gö6976 and EAVSLKPT, suggesting that the activation of Akt by NaHS is PKCα, ε-dependent.

CONCLUSIONS AND IMPLICATIONS

H₂S protects SH-SY5Y cells against 6-OHDA-induced cell injury by activating the PKCα, ε/PI3K/Akt pathway.     

Parkin represses 6-hydroxydopamine-induced apoptosis via stabilizing scaffold protein p62 in PC12 cells

Aim:

Parkin has been shown to exert protective effects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in different models of Parkinson disease. In the present study we investigated the molecular mechanisms underlying the neuroprotective action of parkin in vitro.

Methods:

HEK293, HeLa and PC12 cells were transfected with parkin, parkin mutants, p62 or si-p62. Protein expression and ubiquitination were assessed using immunoblot analysis. Immunoprecipitation assay was performed to identify the interaction between parkin and scaffold protein p62. PC12 and SH-SY5Y cells were treated with 6-OHDA (200 μmol/L), and cell apoptosis was detected using PI and Hoechst staining.

Results:

In HEK293 cells co-transfected with parkin and p62, parkin was co-immunoprecipitated with p62, and parkin overexpression increased p62 protein levels. In parkin-deficient HeLa cells, transfection with wild-type pakin, but not with ligase activity-deficient pakin mutants, significantly increased p62 levels, suggesting that parkin stabilized p62 through its E3 ligase activity. Transfection with parkin or p62 significantly repressed ERK1/2 phosphorylation in HeLa cells, but transfection with parkin did not repress ERK1/2 phosphorylation in p62-knockdown HeLa cells, suggesting that p62 was involved in parkin-induced inhibition on ERK1/2 phosphorylation. Overexpression of parkin or p62 significantly repressed 6-OHDA-induced ERK1/2 phosphorylation in PC12 cells, and parkin overexpression inhibited 6-OHDA-induced apoptosis in PC12 and SH-SY5Y cells.

Conclusion:

Parkin protects PC12 cells against 6-OHDA-induced apoptosis via ubiquitinating and stabilizing scaffold protein p62, and repressing ERK1/2 activation.     

In vitro demonstration of enhanced prostate cancer toxicity: pretargeting with Bombesin bispecific complexes and targeting with polymer-drug-conjugates

Abstract

Background: Bombesin has been used to target Bombesin receptor, a growth receptor, which is over-expressed in many cancers, including prostate cancer. Polymer-anti-neoplastic-drug-conjugates (PDC) were also developed to reduce non-specific toxicity and increase tumor toxicity utilizing the enhanced permeability and retention effect, benefitting treatment of large tumors with well-established vasculature.

Purpose: If PDCs were delivered by targeted delivery to cancer cells, tumor toxicity would be enhanced and non-specific toxicity decreased.

Methods: Cardiocyte toxicity was assessed in H9c2 cardiocytes with doxorubicin (Dox) or N-terminal DTPA-modified-Doxorubicin-loaded-polyglutamic acid polymers (D-Dox-PGA). Therapeutic efficacy of targeted D-Dox-PGA after pretargeting with Bombesin-conjugated anti-DTPA-antibody Bispecific Complexes (Bom-BiSpCx) was compared to that of Dox in PC3 cells. Bom-BiSpCx was generated by thioether bond between Bombesin to Anti-DTPA antibody.

Results: D-Dox-PGA was demonstrated to have less cardiocyte toxicity (IC50?=?20?µg/ml) than free Dox (1.55?µg/ml, p?<?0.001). However, after pre-targeting of human prostate cancer PC3 cells with Bom-BiSpCx and targeting with D-Dox-PGA, IC₅₀ (13.2?µg/ml) was about two times less than that of Dox (28.5?µg/ml, p?<?0.0001).

Discussion: Targeted delivery of PDCs having lower cardiocyte toxicity enabled higher efficiency cancer cell therapy.

Conclusion: This study may allow development of very efficient targeted prostate cancer pro-drug therapy.     

6-羟基多巴胺诱导PC12细胞释放谷氨酸及死亡不受Ⅰ组亲代谢型谷氨酸受体配基的影响(英文)

目的:探讨Ⅰ组亲代谢型谷氨酸受体(mGluR)配基对6-羟基多巴胺(6-OHDA)诱导的PC12细胞死亡及谷氨酸(Glu)释放的影响。方法:培养PC12细胞,以100μmol/LⅠ组mGluR激动剂(RS)-3,5-dihydroxyphenylglycine(DHPG)和拮抗剂DL-2-amino-3-phosphonopropionic acid(DL-AP3)预先剌激细胞1h,再加入6-OHDA100μmol/L共孵育24h,显微镜下观察细胞形态变化,用TUNEL法检测凋亡细胞,用噻唑蓝(MTT)法检测细胞存活率,并用高效液相色谱检测Glu的释放量。结果:6-OHDA 降低PC12细胞存活率(P<0.01),其诱导的Glu释放呈浓度和时间依赖性。Ⅰ组mGluR配基不能减少6-OHDA引起的PC12细胞死亡,也不影响6OHDA引起的Glu释放量。结论:Ⅰ组mGluR配基对6-OHDA引起死亡的PC12细胞无保护作用。     

Inhibitory effects of phenolic compounds from Artocarpus styracifolius on respiratory burst of rat neutrophils

Context: Searching for polymorphonuclear neutrophils (PMNs) respiratory burst inhibitors is an important topic in the treatment of human diseases associated with inflammation.

Objective: To investigate the inhibitory effects of phenolics isolated from Artocarpus styracifolius Pierre (Moraceae) on respiratory burst induced by phorbol myristate acetate (PMA).

Materials and methods: The anti-respiratory burst activities of eight phenolics (20?µM) were assessed by determining luminol-dependent chemiluminiscence in rat PMNs. Cytotoxicity of active compounds (1–1000?µM) was assayed by Trypan blue dye exclusion method. Cell-free models were employed to evaluate scavenging capacity of active compounds (20?µM) against reactive oxygen species.

Results: The PMA-induced respiratory burst was significantly inhibited (p?1–6) at the concentration of 20?µM (below the toxic concentration) with the inhibition rate ranging from 25.0 to 99.6%. The inhibitory potency estimated by IC₅₀ was in the order of AS1 (3.1?µM) >AS6 (5.9?µM) >AS2 (9.1?µM) >AS3 (10.0?µM) >AS5 (29.7?µM) >AS4 (57.7?µM). AS1–4, four isoprenylated flavones, potently quenched superoxide anion, hydroxyl radical, and hydrogen peroxide at the concentration of 20?µM with their scavenging rates in the range of 30.1–78.1%, 35.4–69.7%, and 65.5–86.3%, respectively. In contrast, AS5–6, two isoprenylated 2-arylbenzofurans, showed less effect than that exhibited by AS1–4.

Conclusion and discussion: The isoprenylated phenolics from A. styracifolius can potently inhibit PMA-induced respiratory burst in rat neutrophils without showing cytotoxicity. The inhibitory effects of these isoprenylated phenolics on the respiratory burst might depend on their different types of structure.     

In vitro 6-hydroxydopamine-induced toxicity in striatal,cerebrocortical and hippocampal slices is attenuated by atorvastatin and MK-801

Parkinson's disease (PD) involves the loss of striatal dopaminergic neurons, although other neurotransmitters and brain areas are also involved in its pathophysiology. In rodent models to PD it has been shown statins improve cognitive and motor deficits and attenuate inflammatory responses evoked by PD-related toxins. Statins are the drugs most prescribed to hypercholesterolemia, but neuroprotective effects have also been attributed to statins treatment in humans and in animal models. This study aimed to establish an in vitro model of 6-hydroxydopamine (6-OHDA)-induced toxicity, used as an initial screening test to identify effective drugs against neural degeneration related to PD. The putative neuroprotective effect of atorvastatin against 6-OHDA-induced toxicity in rat striatal, cerebrocortical and hippocampal slices was also evaluated. 6-OHDA (100 μM) decreased cellular viability in slices obtained from rat cerebral cortex, hippocampus and striatum. 6-OHDA also induced an increased reactive oxygen species (ROS) production and mitochondrial dysfunction. Co-incubation of 6-OHDA with atorvastatin (10 μM) or MK-801 (50 μM) an N-methyl-d-aspartate (NMDA) receptor antagonist, partially attenuated the cellular damage evoked by 6-OHDA in the three brain areas. Atorvastatin partially reduced ROS production in the hippocampus and striatum and disturbances of mitochondria membrane potential in cortex and striatum. 6-OHDA-induced toxicity in vitro displays differences among the brain structures, but it is also observed in cerebrocortical and hippocampal slices, besides striatum.     

Design,synthesis, and antitumor evaluation of quinoline‐imidazole derivatives

A series of compounds bearing quinoline‐imidazole ( 8a–e , 9a–e , 10a–e , 11a–e , and 12a–e ) not reported previously were designed and synthesized. The target compounds were evaluated for antitumor activity against A549, PC‐3, HepG2, and MCF‐7 cells by the MTT method, with NVP‐BEZ235 being the positive control. Most compounds showed moderate activity and compound 12a showed the best activity against HepG2, A549, and PC‐3 cells, with half‐maximal inhibitory concentration (IC₅₀) values of 2.42 ± 1.02 µM, 6.29 ± 0.99 µM, and 5.11 ± 1.00 µM, respectively, which was equal to NVP‐BEZ235 (0.54 ± 0.13 µM, 0.36 ± 0.06 µM, 0.20 ± 0.01 µM). Besides, the IC₅₀ value of 12a against the cell line WI‐38 (human fetal lung fibroblasts) was 32.8 ± 1.23 µM, indicating that the target compounds were selective for cancer cells. So, 11a and 12a were evaluated against PI3Kα and mTOR to find out if the compounds acted through the PI3K‐Akt‐mTOR signal transduction pathway. The inhibition ratios to PI3Kα and mTOR were slightly lower than that of NVP‐BEZ235, suggesting there may be some other mechanisms of action. The structure–activity relationships and docking study of 11a and 12a revealed that the latter was superior. Moreover, the target compounds showed better in vitro anticancer activity when the C‐6 of the quinoline ring was replaced by a bromine atom.