Barbara Adinolfi

Cytotoxic activity of polyacetylenes and polyenes isolated from roots of Echinacea pallida

The n‐hexane extracts of the roots of three medicinally used Echinacea species exhibited cytotoxic activity on human cancer cell lines, with Echinacea pallida found to be the most cytotoxic. Acetylenes are present in E. pallida lipophilic extracts but essentially absent in extracts from the other two species. In the present study, the cytotoxic effects of five compounds, two polyacetylenes (namely, 8‐hydroxy‐pentadeca‐(9E)‐ene‐11,13‐diyn‐2‐one (1) and pentadeca‐(9E)‐ene‐11,13‐diyne‐2,8‐dione (3)) and three polyenes (namely, 8‐hydroxy‐pentadeca‐(9E,13Z)‐dien‐11‐yn‐2‐one (2), pentadeca‐(9E,13Z)‐dien‐11‐yne‐2,8‐dione (4) and pentadeca‐(8Z,13Z)‐dien‐11‐yn‐2‐one (5)), isolated from the n‐hexane extract of E. pallida roots by bioassay‐guided fractionation, were investigated and the potential bioavailability of these compounds in the extract was studied.

Cannabinoid derivatives induce cell death in pancreatic MIA PaCa-2 cells via a receptor-independent mechanism

Cannabinoids (CBs) are implicated in the control of cell survival in different types of tumors, but little is known about the role of CB system in pancreatic cancer. Herein, we investigated the in vitro antitumor activity of CBs and the potential role of their receptors in human pancreatic cancer cells MIA PaCa‐2. Characterization tools used for this study included growth inhibition/cell viability analyses, caspase 3/7 induction, DNA fragmentation, microarray analysis and combination index‐isobologram method. Our results demonstrate that CBs produce a significant cytotoxic effect via a receptor‐independent mechanism. The CB1 antagonist N‐(piperidin‐1‐1yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide (AM251) was the most active compound with an IC50 of 8.6 ± 1.3 μM after 72 h. AM251 induces apoptosis, causes transcriptional changes of genes in janus kinase/signal transducers and activators of transcription signaling network and synergistically interacts with the pyrimidine analogue, 5‐fluorouracil. These findings exclude the involvement of CB receptors in the regulation of MIA PaCa‐2 cell growth and put AM251 forward as a candidate for the development of novel compounds worthy to be tested in this type of neoplasia.

Modulation of P-glycoprotein activity by cannabinoid molecules in HK-2 renal cells

1 Endogenous and synthetic cannabinoid molecules have been investigated as possible MDR‐1/P‐glycoprotein (P‐gp) modulators in HK‐2‐immortalized renal cells, using calcein acetoxymethylester (calcein‐AM) as a P‐gp substrate. 2 Among the endocannabinoid molecules tested, anandamide (AEA), but not 2‐arachidonoyl‐glycerol (2‐AG) or palmitoyl‐ethanolamide (PEA), increased the intracellular fluorescence emitted by calcein, a metabolic derivative of the P‐gp substrate calcein‐AM, indicative of a reduction in transport capacity. 3 All the three synthetic cannabimimetics tested, that is, R‐(+)‐methanandamide (R(+)‐MET), AM 251 and CP55,940 significantly increased calcein accumulation in the cytosol. 4 RT–PCR demonstrated that HK‐2 cells do not express CB1 or CB2 cannabinoid receptors. 5 R(+)‐MET, AM251 and CP55,940 were also evaluated as modulators of P‐gp expression, by Western blot analysis. Only AM251 weakly enhanced the protein levels (by 1.2‐fold) after a 4‐day‐long incubation with the noncytotoxic drug concentration 2 μM. 6 The present data provide the first evidence that the endocannabinoid AEA and different synthetic cannabinoids may inhibit the P‐gp activity in vitro via a cannabinoid receptor‐independent mechanism.

P-Glycoprotein inhibitory activity of lipophilic constituents of Echinacea pallida roots in a human proximal tubular cell line.

The N-hexane root extracts from Echinacea pallida, Echinacea angustifolia and Echinacea purpurea were evaluated for inhibition of the multidrug transporter P-glycoprotein (Pgp) activity, the product of the ABCB1 gene, involved in cancer multidrug resistance (MDR) and in herb-drug or drug-drug interactions. The biological assay was performed using the human proximal tubule HK-2 cell line that constitutively expresses ABCB1. The N-hexane extracts of all three species reduced the efflux of the Pgp probe calcein-AM from HK-2 cells two-fold in a concentration-dependent manner, and E. pallida was found to be the most active species. For the first time, two polyacetylenes and three polyenes, isolated from the N-hexane extract of E. pallida roots by a bioassay-guided fractionation, were found to be able to reduce Pgp activity. Pentadeca-(8 Z,13 Z)-dien-11-yn-2-one was the most efficient compound, being able to decrease the calcein-AM efflux about three-fold with respect to the control at 30 microg/mL.

Rational design, synthesis, and pharmacological properties of new 1,8-naphthyridin-2(1H)-on-3-carboxamide derivatives as highly selective cannabinoid-2 receptor agonists.

The CB(2) receptor activation can be exploited for the treatment of diseases such as chronic pain and tumors of immune origin, devoid of psychotropic activity. On the basis of our already reported 1,8-naphthyridin-4(1H)-on-3-carboxamide derivatives, new 1,8-naphthyridin-2(1H)-on-3-carboxamide derivatives were designed, synthesized, and tested for their affinities toward the human CB(1) and CB(2) cannabinoid receptors. Some of the reported compounds showed a subnanomolar CB(2) affinity with a CB(1)/CB(2) selectivity ratio greater than 200 (compounds 6, 12, cis-12, 13, and cis-13). Further studies revealed that compound 12, which presented benzyl and carboxy-4-methylcyclohexylamide substituents bound in the 1 and 3 positions, exerted a CB(2)-mediated inhibitory action on immunological human basophil activation. On the human T cell leukemia line Jurkat the same derivative induced a concentration-dependent decrease of cell viability. The obtained results suggest that 1,8-naphthyridin-2(1H)-on-3-carboxamides represent a new scaffold very suitable for the development of new promising CB(2) agonists.

Theranostic Properties of a Survivin-Directed Molecular Beacon in Human Melanoma Cells

Survivin is an inhibitor of apoptosis overexpressed in different types of tumors and undetectable in most terminally differentiated normal tissues. In the current study, we sought to evaluate the in vitro theranostic properties of a molecular beacon-oligodeoxynucleotide (MB) that targets survivin mRNA. We used laser scanning confocal microscopy to study MB delivery in living cells and real-time PCR and western blot to assess selective survivin-targeting in human malignant melanoma cells. We further assess the pro-apoptotic effect of MB by measuring internucleosomal DNA fragmentation, dissipation of mitochondrial membrane potential (MMP) and changes in nuclear morphology. Transfection of MB into A375 and 501 Mel cells generated high signal intensity from the cytoplasm, while no signal was detected in the extracellular environment and in survivin-negative cells (i.e., human melanocytes and monocytes). MB time dependently decreased survivin mRNA and protein expression in melanoma cells with the maximum effect reached at 72 h. Treatment of melanoma cells with MB induced apoptosis by significant changes in MMP, accumulation of histone-complexed DNA fragments in the cytoplasm and nuclear condensation. MB also enhanced the pro-apoptotic effect of standard chemotherapeutic drugs tested at clinically relevant concentrations. The MB tested in the current study conjugates the ability of imaging with the pharmacological silencing activity against survivin mRNA in human melanoma cells and may represent an innovative approach for cancer diagnosis and treatment.

Anticancer activity of anandamide in human cutaneous melanoma cells

Cannabinoids are implicated in the control of cell proliferation, but little is known about the role of the endocannabinoid system in human malignant melanoma. This study was aimed at characterizing the in vitro antitumor activity of anandamide (AEA) in A375 melanoma cells. The mRNA expression of genes that code for proteins involved in the metabolism and in the mechanism of AEA action was assessed by RT-PCR. Cell viability was tested using WST-1 assay and the apoptotic cell death was determined by measuring caspase 3/7 activities. A375 cells express high levels of fatty acid amide hydrolase (FAAH), cyclooxygenase (COX)-2, cannabinoid receptor 1 (CB1), transient receptor potential cation channel subfamily V member 1 (TRPV1) and G-protein-coupled receptor 55 (GPR55) genes. AEA induced a concentration-dependent cytotoxicity with an IC50 of 5.8 ± 0.7 µM and such an effect was associated to a caspase-dependent apoptotic pathway. AEA cytotoxicity was potentiated by FAAH inhibition (2-fold increase, p<0.05) and mitigated by COX-2 or lipoxygenase (LOX) inhibition (5- and 3-fold decrease, respectively; p<0.01). Blocking CB1 receptors partially decreased AEA cytotoxicity, whereas selective antagonism on the TRPV1 barely affected the mechanism of AEA action. Finally, methyl-β-cyclodextrin, a membrane cholesterol depletory, completely reversed the cytotoxicity induced by the selective GPR55 agonist, O-1602, and AEA. Overall, these findings demonstrate that AEA induces cytotoxicity against human melanoma cells in the micromolar range of concentrations through a complex mechanism, which involves COX-2 and LOX-derived product synthesis and CB1 activation. Lipid raft modulation, probably linked to GPR55 activation, might also have a role.

Anti-proliferative activity of aguerin B and a new rare nor-guaianolide lactone isolated from the aerial parts of Centaurea deflexa

The phytochemical investigation of the aerial parts of Centaurea deflexa led to the identification of 21 compounds, among which three phenolic acids, one sterol, ten flavonoids, one phenylpropanoid derivative, two lignans and four sesquiterpene lactones. One of the latter compounds was a new, rare active principle (1) having an uncommon 15-nor-guaianolide skeleton. The biological investigation was carried out through a bio-guided assay fractionation of C. deflexa extracts and highlighted an anti-proliferative activity of two sesquiterpene lactones, aguerin B and the newly identified 15-nor-guaianolide (1) against human pancreatic and colonic cancer cells. Of the two compounds, only aguerin B showed to induce apoptotic cell death, confirming the role as pro-apoptotic moiety of the α-methylene-γ-lactone ring present in aguerin B but not in 1.

Rosiglitazone reverses salbutamol-induced β2-adrenoceptor tolerance in airway smooth muscle

BACKGROUND AND PURPOSE β2‐Adrenoceptor agonists are important therapeutic agents in the treatment of asthma and chronic obstructive pulmonary disease. The regular use of these drugs has been associated with proasthmatic‐like changes that limit their efficacy and increase the risk of severe adverse reactions. We investigated whether the peroxisome‐proliferator‐activated receptor (PPAR)γ agonist rosiglitazone modulated salbutamol‐induced β2‐adrenoceptor desensitization in vivo and in vitro.

Cytotoxic activity and G1 cell cycle arrest of a Dienynone from Echinacea pallida.

In the present study, a further investigation of the cytotoxic activity of an acetylenic constituent of Echinacea pallida roots, namely, pentadeca-(8 Z,13 Z)-dien-11-yn-2-one, was performed, revealing a concentration-dependent cytotoxicity on several human cancer cell lines, including leukemia (Jurkat and HL-60), breast carcinoma (MCF-7), and melanoma (MeWo) cells. As part of its mechanism of action, the ability of this constituent to arrest the cell cycle in the G1 phase was demonstrated on HL-60 cells. Furthermore, a stability test of the target compound over 72 h was carried out, indicating that the cytotoxic activity can be attributed mainly to the genuine, not oxidized, molecule.