The research project aims at understanding the potential application of Saxifraga aizoides extract (SAE) on cancer cell models. In order to achieve this result, a scientific approach should be followed:

Materials and methods

Plant material

Saxifraga aizoides plant was collected in summer season 2015 at about 2000 meters above the sea level in Passo del Tonale (TN), Italy. Plant was identified according to Pignatti 1982. A voucher specimen number was deposited in the herbarium of the OU Endocrinology, University of Padova, with number 011SA. Plant was air-dried and powdered. 30g of powder was added to a mixture with ethanol (70%) for 24 h, then filtered. The solvent was removed by vacuum evaporator. A crude extract of Saxifraga aizoides was obtained (SAE).

GC/MS analysis of Saxifraga aizoides extract

The chemical analysis will be performed by re-solubilizing the dry extract in ethanol followed by gas-chromatography/mass spectrometry (GC/MS) analysis Cavaggioni et al. 2006).

Evalutation of anti-proliferative activity

Four different cell lines will be used: HeLa (ovarian cancer), HCT-116 (Colon cancer), PC-3 (Prostate cancer), MCF-7 (breast cancer) cell lines. Cell lines will be obtained by ATCC or will be a gift of other research labs. Cells will be cultured in DMEM/RPMI medium supplemented with 10% FBS, 100 U/ml penicillin, 100 ug/ml streptomycin in a humidified atmosphere with 5% CO₂ at 37°C. Cells will be plated on 96-well tissue culture microtiter plates at a density of 1x10⁴ cells/well and treated with SAE, at different concentrations. MTT cell viability (Sigma-Aldrich) will be tested after 72 h of treatment, as described elsewhere. For each drug, we measured the Inhibitory Concentration 50 (IC₅₀, defined as 50% of the inhibitory effect on cell viability). All experiments will be performed in quadruplicate and repeated three times.

The cytotoxic effects of SAE will be confirmed by the Trypan blue dye exclusion method. The assay will be performed at 72 h after treatment, using the single IC₅₀ dose determined by MTT assay. At the end of treatment, cells will be collected by trypsinization, centrifuged and the cell pellet will be resuspended in 1 ml of PBS. Next, 10 ul of the resulting cell suspension will be admixed with 10 ul of Trypan blue (0.4% in PBS). The numbers of non-stained viable cells (NSt cells) and stained dead cells (St cells) will be counted using a hemocytometer. Cell viability will be then calculated by the following formula: Viability (%)= ¼ (NSt cells)/(St cells + NSt cells) x 100. Experiments will be performed in triplicated and repeated three times. The results will be interpreted as the ratio of viable cells after drug treatments to that of the untreated control.

Cell morphology assessment with Wright’s staining

Cells will be cultured on coverslips for 48h, incubated overnight in 0.1% FBS, and then treated with SAE for 72h. After that, cells will be washed in PBS, fixed in methanol for 5 min and Wright’s stained for 5 min. Cell morphology will be assessed by light microscopy at x400 magnification. The experiments will be repeated twice.

Statistical analysis

Statistical analysis will be performed using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA) and Microsoft Excel software. Cell line data will be analyzed using a two-tailed paired Student’s t-test. A p<0.05 will be considered statistically significant. Data will be expressed as mean ± standard error of mean (sem).

Expected results

We expect to obtain a SAE that will be used to explore its effects on cell cancer models. We will have a GC/MS profile of SAE. We will test the 4 cancer cell lines for viability after treatment with SAE, thanks to MTT and trypan blue assays. Furthermore cells morphology changes will be assessed by light microscopy. Clonogenic assay will be provide a specific insight on cell survival after treatment with SAE.

Implementation

The study of the Saxifraga aizoides extract (SAE) will be implemented by the in vivo experiments on animal models (both mice and rats).

Acute toxicity studies

The project will analyze the acute toxicity of the plant extract, evaluating the lethal dose (LD50) in mice. The method is described in Lorke (1983). Animals (at least 8 mice) will be deprived of food over night before testing. SAE will be administred per os in increasing doses (10, 100, 1000 mg/kg). Treated animals will be observed for 8 h looking for signs of toxicity. After 24h, if no animal will die, another scaling doses of SAE will administered (1500, 3000, 5000 mg/kg). Again animals will be observed for signs of toxicity or mortality.

Anti-inflammatory studies

Anti-inflammatory study will be performed using wistar rats. The method is derived by Winter et al. (1963). Aspirin (250 mg/kg) will be a positive control in a group of animals (at least 6 rats). Another group of animals (at least 6 rats) will be administred with Saxifraga aizoides extract. The treatments will start 1 hour before carrageenan injection (the drug will induce an inflammatory reaction in the site of injection). Carrageenan will be prepared as 1% w/v solution in 0.9% w/v NaCl and 0.1 ml will be injected near rat's paw. The paw size will be measured will digital calliper in the 2 different groups and in the control group (no extract and no aspirin), at 0, 2, 4, 6 h after carrageenan injection.

Ethics

The request to use animal models will be submitted to Local Ethics Committee only after successfully completion of in vitro studies. Procedures involving animals and their care will be in conformity with institutional guidelines that comply with national and international law and policies (D.L. 116/92 and subsequent implementing circulars), and experimental protocols will be approved by the Local Ethics Committee of Padua University (CEASA).