Terpenoids from Zingiber officinale (Ginger) Induce Apoptosis in Endometrial Cancer Cells through the Activation of p53

Novel strategies are necessary to improve chemotherapy response in advanced and recurrent endometrial cancer. Here, we demonstrate that terpenoids present in the Steam Distilled Extract of Ginger (SDGE) are potent inhibitors of proliferation of endometrial cancer cells. SDGE, isolated from six different batches of ginger rhizomes, consistently inhibited proliferation of the endometrial cancer cell lines Ishikawa and ECC-1 at IC₅₀ of 1.25 µg/ml. SDGE also enhanced the anti-proliferative effect of radiation and cisplatin. Decreased proliferation of Ishikawa and ECC-1 cells was a direct result of SDGE-induced apoptosis as demonstrated by FITC-Annexin V staining and expression of cleaved caspase 3. GC/MS analysis identified a total of 22 different terpenoid compounds in SDGE, with the isomers neral and geranial constituting 30–40%. Citral, a mixture of neral and geranial inhibited the proliferation of Ishikawa and ECC-1 cells at an IC₅₀ 10 µM (2.3 µg/ml). Phenolic compounds such as gingerol and shogaol were not detected in SDGE and 6-gingerol was a weaker inhibitor of the proliferation of the endometrial cancer cells. SDGE was more effective in inducing cancer cell death than citral, suggesting that other terpenes present in SDGE were also contributing to endometrial cancer cell death. SDGE treatment resulted in a rapid and strong increase in intracellular calcium and a 20–40% decrease in the mitochondrial membrane potential. Ser-15 of p53 was phosphorylated after 15 min treatment of the cancer cells with SDGE. This increase in p53 was associated with 90% decrease in Bcl2 whereas no effect was observed on Bax. Inhibitor of p53, pifithrin-α, attenuated the anti-cancer effects of SDGE and apoptosis was also not observed in the p53^neg SKOV-3 cells. Our studies demonstrate that terpenoids from SDGE mediate apoptosis by activating p53 and should be therefore be investigated as agents for the treatment of endometrial cancer.     

Harmonization of the Practice of Independent Ethics Committees in Italy: Project E-Submission

Aim

The high variability of “centre-specific” documentation required by Independent Ethics Committee (IEC) plays a role in the time required for activation of participating centres of multicentre clinical trials. This study (a) provides a picture of the different activities, structural requirements and resources dedicated to the operation of the local IEC in Italy; (b) defines a detailed list of “centre-specific” documents considered as essential by the IEC for issuing its opinion and (c) suggests a “single document” to reduce the variability of the “centre-specific” documents required by the IEC.

Methodology

Two surveys were conducted through the portal of National Monitoring Centre of Clinical Trials (https://oss-sper-clin.agenziafarmaco.it/). The first survey focused on the local IEC resources and on the “centre-specific” documentation that local IEC required from the Sponsor and local Principal Investigator (PI). The second focused on “single document” required in the form of statements from the Sponsor and the PI. Answers were discussed and extended during regular scheduled teleconferences and plenary meeting.

Principal Findings

From 22/07/2009 to 15/12/2009, and from 19/04/2010 to 14/05/2010, 131 and 125 IECs responded to the first and the second surveys, respectively. 67% and 51% of IECs consider the structural requirements and the staff dedicated to the activity of the IECs as sufficient, respectively. Most of the IECs consider the “centre-specific” documentation as necessary for issuing the opinion, and a high percentage of IECs consider the proposed documentation as acceptable in substitution to any other “centre-specific” documentation already in use.

Conclusions

The harmonization of IECs practice in Italy is the first step to facilitate multicentre clinical trials. Similar efforts should be directed to reduce the total number of IECs and to standardize clinical trials approval procedures, focusing on administrative procedures as well.     

Effect of preservatives on IgG aggregation, complement-activating effect and hypotensive activity of horse polyvalent antivenom used in snakebite envenomation.

Intravenous administration of antivenoms is associated with early adverse reactions in a number of cases, but the causes of this phenomenon are still unclear. The effect of preservatives (phenol and thimerosal) on IgG aggregate and dimer formation, in vitro complement-activating effect and hypotensive activity of a whole IgG horse liquid polyvalent antivenom, produced by caprylic acid fractionation, was assessed. These parameters were studied since they have been associated with the development of early adverse reactions to the administration of antivenoms and human immunoglobulins. After a three-year storage period at 4 degrees C, antivenoms with preservatives had an increased content of IgG aggregates and dimers when compared with antivenom devoid of phenol and thimerosal. These observations correlate with a slight increment in the turbidity of preservative-containing antivenoms. The three antivenoms studied (formulation: no preservatives; with phenol and thimerosal; with thimerosal alone) activated human complement in vitro, with only minor quantitative differences among them. When antivenoms were administered as a bolus intravenous injection in rats, a rapid and prominent hypotension of short duration was observed after injection of phenol-containing antivenom, whereas such an effect was absent in antivenom free of preservative and in the one containing only thimerosal. Bolus injection of saline solution with phenol resulted in a similar hypotension, indicating that the effect is due to phenol. However, when phenol-containing antivenom was diluted 1:5 with saline solution before infusion, as occurs in the clinical use of this product, no hypotension was observed. Our results stress the need to evaluate the effects of preservatives on the physicochemical and pharmacological characteristics of antivenoms.     

A Modified Nauta-Gygax Method for Human Brain and Spinal Cord

Relatively thick frozen sections of formalin-fixed human brains are treated subsequently with an equal-parts mixture of 1% oxalic acid and 1% hydroquinone for 30-60 min, 0.005% chromic acid for 10 min, 4% hydrobromic acid for 6 min, 1% phosphotungstic acid for 15 min, 0.05% potassium permanganate for 3-10 min, equal parts of 1% oxalic acid and 1% hydroquinone for 2-5 min. After thorough washing in distilled water, the sections are then soaked in 1.5% silver nitrate for 15-30 min, Laidlaw's ammoniacal silver carbonate for 2.5 min, and then reduced in the Nauta-Gygax reducer. The sections are washed and then passed through 1% sodium thiosulfate for 1-2 min; again washed, dehydrated, cleared and covered with synthetic resin.