Cannabidiol, a non-psychoactive cannabinoid compound, inhibits proliferation and invasion in U87-MG and T98G glioma cells through a multitarget effect.

In the present study, we found that CBD inhibited U87-MG and T98G cell proliferation and invasiveness in vitro and caused a decrease in the expression of a set of proteins specifically involved in growth, invasion and angiogenesis. In addition, CBD treatment caused a dose-related down-regulation of ERK and Akt prosurvival signaling pathways in U87-MG and T98G cells and decreased hypoxia inducible factor HIF-1α expression in U87-MG cells. Taken together, these results provide new insights into the antitumor action of CBD, showing that this cannabinoid affects multiple tumoral features and molecular pathways. As CBD is a non-psychoactive phytocannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anti-cancer drug in the management of gliomas

Tephrochronology and chronostratigraphy of the Miocene Chilcatay and Pisco formations (East Pisco Basin, Peru)

Strata of Chilcatay and Pisco formations exposed in the Ica Desert (East Pisco Basin, southern Peru) preserve one of the most complete and rich records of Miocene marine vertebrates of the world. Despite its exceptional importance, the chronostratigraphy of these fossil-bearing deposits has been only sporadically studied in the literature until recently. This work presents a detailed reconstruction of the chronostratigraphic framework, achieved by mapping and logging of seven sections of the Miocene Chilcatay and Pisco formations along the western side of the Ica River. The Chilcatay Formation consists of two allomembers, namely Ct1 and Ct2, bounded at the base by unconformities CE0.1 and CE0.2, respectively. Similarly, the immediately overlying Pisco Formation is divided into allomembers P0, P1, and P2, bounded at the base by unconformities PE0.0, PE0.1 and PE0.2, respectively. The new 39Ar–40Ar results presented here, combined with ages of previous work, provide precise constraints on the age of several stratigraphically referenced volcanic ash layers intercalated in the studied fossil-bearing succession, placing its vertebrate fossil fauna within a refined temporal framework and laying the solid ground for its detailed regional and global comparison. The ages of the allomembers, and thus their associated faunas, can be reliably estimated by the combination of 39Ar–40Ar dating on tephra layers with diatom biostratigraphy. In the study area, the two methods are mutually consistent and constrain the deposition of the Chilcatay Formation between 19.2 and 18.0 Ma, that of P1 between 9.5 and 8.6 Ma, and that of P2 between 8.4 and 6.7 Ma. In the absence of direct dating of the P0 allomember, which lacks both preserved tephra suitable for 39Ar–40Ar dating and microfossils, its age can be constrained to the temporal gap between the youngest age available from the underlying Chilcatay strata (18.0 Ma) and the oldest age available from the overlying P1 strata (9.5 Ma)

Effect of increasing CBD concentrations on Akt phosphorylation in U87-MG and T98G glioma cells.

<p>U87-MG and T98G cells were treated with CBD for 24 h and lysates from cells were used to assess Akt protein levels. (A,C) Western blot analysis of phospho- and pan-Akt. A representative western blot is shown. (B,D) Densitometric analysis of Akt signal bands from three independent experiments is reported. *p<0.05, **p<0.01 <i>vs</i> Control [C], Dunnett’s t test.</p

Effect of increasing CBD concentrations on the protein expression profile of U87-MG glioma cells.

<p>U87-MG cells were treated with CBD for 24 h and supernatants were used to determine different protein levels through a human array kit/proteome profiler. (A) Representative proteomic membrane analysis with indication of the modified proteins. (B) Densitometric analysis of the membrane spots reported as percentage of the untreated control. Data represent the mean ± S.E.M. of three independent experiments. **p<0.01, ***p<0.001 <i>vs</i> Control, Dunnett’s t test. U87-MG cells were treated with CBD for 24 h and lysates from cells were used to assess MMP-9 and TIMP-4 protein levels. (C) Western blot analysis of MMP-9 and TIMP-4. A representative western blot is shown. (D) Densitometric analysis of MMP-9 and TIMP-4 signal bands from three independent experiments is reported. **p<0.01 <i>vs</i> Control [C], Dunnett’s t test.</p

Effect of increasing CBD concentrations on U87-MG (A) and T98G (C) glioma cell invasion.

<p>U87-MG and T98G cells were treated with CBD, seeded onto a filter coated with matrigel and incubated for 24 h at 37°C. Cells that invaded the lower surface of the filter were quantified. The invasion was expressed as percentage of the untreated control. Data represent the mean ± S.E.M. of three independent experiments. **p<0.01 <i>vs</i> Control [C], Dunnett’s t test. Effect of increasing CBD concentrations on U87-MG (B) and T98G (D) glioma cell viability. U87-MG and T98G glioma cells were cultured in serum-free medium with increasing concentrations of CBD. Cell viability was determined by MTT assay after 24 h of treatment. The viability was expressed as percentage of the untreated control. Data represent the mean ± S.E.M. of at least three independent experiments.</p

Effect of CBD on HIF-1α level in glioma cells grown in normoxic and hypoxic conditions.

<p>U87-MG and T98G glioma cells were grown under normoxia and two different hypoxic conditions (see Materials and methods). Cells were treated with CBD for 24 h and lysates from the cells were used to assess HIF-1α protein levels. (A–C) Western blot analysis of HIF-1α and β-actin. A representative western blot is shown. (B–D) Densitometric analysis of HIF-1α signal bands from three independent experiments is reported. *p<0.05, **p<0.01 <i>vs</i> Control [C], ° p<0.05, °° p<0.01, °°° p<0.001, °°°° p<0.0001 <i>vs</i> normoxia Control [C], Dunnett’s t test.</p

Effect of increasing CBD concentrations on ERK phosphorylation in U87-MG and T98G glioma cells.

<p>U87-MG and T98G cells were treated with CBD for 24 h and lysates from cells were used to assess ERK protein levels. (A,C) Western blot analysis of phospho- and total-ERK. A representative western blot is shown. (B,D) Densitometric analysis of ERK signal bands from three independent experiments is reported. *p<0.05, **p<0.01 <i>vs</i> Control [C], Dunnett’s t test.</p