New Insights in Routine Procedure for Mathematical Evaluation of in vitro Cytotoxicity Data from Cancer Cell Lines

In oncopharmacology, the common procedure to evaluate median-effect concentrations (IC50) on experimental data is based on the use of well-established kinetic models representing inhibition effects of drugs on human cancer cell lines. Several widespread software programs, such as GraphPad Prism and CompuSyn offer possibilities for calculation of IC50 through the model of Chou. In recent study, we analyzed the results from those two software programs and compared them with the non-linear programming procedure written by us in the MAPLE symbolic software. The last evaluated IC50 more precisely and the correlation coefficient R value was better in all trails. We demonstrated the efficiency of non-linear programming procedures in examples of two cancer cell lines treated with three different drugs. The response surface analysis showed the potential of the applied kinetic model. As a result, we were able to define better the IC50 values and to use them in planning further experiments in human cancer cell lines related to single drug influence and drug-drug interference

Reduced Expression of the Retinoblastoma Protein Shows That the Related Signaling Pathway Is Essential for Mediating the Antineoplastic Activity of Erufosine

<div><p>Erufosine is a new antineoplastic agent of the group of alkylphosphocholines, which interferes with signal transduction and induces apoptosis in various leukemic and tumor cell lines. The present study was designed to examine for the first time the mechanism of resistance to erufosine in malignant cells with permanently reduced expression of the retinoblastoma (Rb) protein. Bearing in mind the high number of malignancies with reduced level of this tumor-suppressor, this investigation was deemed important for using erufosine, alone or in combination, in patients with compromised RB1 gene expression. For this purpose, clones of the leukemic T-cell line SKW-3 were used, which had been engineered to constantly express differently low Rb levels. The alkylphosphocholine induced apoptosis, stimulated the expression of the cyclin dependent kinase inhibitor p27^Kip1 and inhibited the synthesis of cyclin D3, thereby causing a G₂ phase cell cycle arrest and death of cells with wild type Rb expression. In contrast, Rb-deficiency impeded the changes induced by eru-fosine in the expression of these proteins and abrogated the induction of G₂ arrest, which was correlated with reduced antiproliferative and anticlonogenic activities of the compound. In conclusion, analysis of our results showed for the first time that the Rb signaling pathway is essential for mediating the antineoplastic activity of erufosine and its efficacy in patients with malignant diseases may be predicted by determining the Rb status.</p></div

Efficacy of the Rb-knockdown.

<p>(A) Fluorescence imaging of SKW-3 cells at 72 h after viral transduction with three different pLL 3.7-constructs (nonsense shRNA – NSO, antisense shRNAs–21 bp, 1 or 27 bp, 2) and Cell Sorting using the eGFP signal. (B) The efficacy of the Rb-knockdown estimated by Western blot before and after selection. The Rb-knockdown on protein level was calculated as percentage of the respective nonsense-control cells after densitometric analysis of the protein bands using the Quantity One 4.6.6 Program (Bio-Rad Laboratories).</p

Survival of cells with stable Rb-knockdown after 48

<p> <b>h treatment with 5-floururacil, cytosine arabinoside, doxorubicin and cisplatin.</b> SKW-3 cell clones (nonsense control – NSO, cells with 99% Rb-knockdown – shRNA 1 and cells with 83% Rb-knockdown – shRNA 2) were treated with five different concentrations of the four cytostatics. A significant difference versus the respective nonsense control is marked by an asterisk (Student’s t-test; p<0.05). Bars denote standard deviation. The table under the graphs gives the IC₅₀ values of the drugs after 48 h of treatment with the respective 95% confidence intervals.</p

Cytotoxicity and clonogenic activity of erufosine in Rb-deficient cells.

<p>The figure depicts the survival (A) or clonogenicity (B) of SKW-3 cell clones with 99% (shRNA1) or 83% (shRNA 2) stable Rb-knockdown after treatment with 2, 4, 8, 16 or 32 µM erufosine for 48 h. A significant difference versus the respective nonsense control is marked by an asterisk (Student’s t-test; p<0.05). Bars denote standard deviation. The table under the graphs gives the IC₅₀ values of erufosine after 48 h of treatment as well as the respective 95% confidence intervals.</p

Erufosine-induced apoptosis is inhibited by Rb-loss.

<p>The expression of proteins is shown, which are involved in induction of the intrinsic apoptotic pathway, respectively, for nonsense or antisense-transduced cell clones with 99% or 83% Rb-knockdown after exposure to erufosine (16 µM, 48 h). The values under the protein bands denote their intensity compared to the untreated control and are calculated after densitometric analysis with the Quantity One 4.6.6 Program (Bio-Rad).</p

Rb-loss inhibits the erufosine-induced G2 cell cycle arrest.

<p>The flow cytometry histograms present the distribution of nonsense- or antisense transduced SKW-3 cell clones with 99% (shRNA 1) and 83% (shRNA 2) Rb-knockdown in G1-, S- and G2-phases of the cell cycle before and after exposure to 16 µM erufosine for 24 and 48 h. The percentage of the cell fractions is calculated with the ModFit LT software and given in the table below the graphs.</p

In Vitro Antineoplastic and Antiviral Activity and In Vivo Toxicity of Geum urbanum L. Extracts

This study evaluated the in vitro antineoplastic and antiviral potential and in vivo toxicity of twelve extracts with different polarity obtained from the herbaceous perennial plant Geum urbanum L. (Rosaceae). In vitro cytotoxicity was determined by ISO 10993-5/2009 on bladder cancer, (T-24 and BC-3C), liver carcinoma (HEP-G2) and normal embryonic kidney (HEK-293) cell lines. The antineoplastic activity was elucidated through assays of cell clonogenicity, apoptosis induction, nuclear factor kappa B p65 (NF&kappa;B p65) activation and total glutathione levels. Neutral red uptake study was applied for antiviral activity. The most promising G. urbanum extract was analyzed by UHPLC&ndash;HRMS. The acute in vivo toxicity analysis was carried out following OEDC 423. The ethyl acetate extract of aerial parts (EtOAc-AP) exhibited the strongest antineoplastic activity on bladder cancer cell lines (IC50 = 21.33&ndash;25.28 &micro;g/mL) by inducing apoptosis and inhibiting NF&kappa;B p65 and cell clonogenicity. EtOAc and n-butanol extracts showed moderate antiviral activity against human adenovirus type 5 and human simplex virus type I. Seventy four secondary metabolites (gallic and ellagic acid derivatives, phenolic acids, flavonoids, etc.) were identified in EtOAc-AP by UHPLC&ndash;HRMS. This extract induced no signs of acute toxicity in liver and kidney specimens of H-albino mice in doses up to 210 mg/kg. In conclusion, our study contributes substantially to the detailed pharmacological characterization of G. urbanum, thus helping the development of health-promoting phytopreparations