Synergistic effects of Ferula gummosa and radiotherapy on induction of cytotoxicity in HeLa cell line

Objective: Cervical cancer is the second most common type of cancer among women, worldwide; and for treatment of this type of cancer radiotherapy is commonly used. Ferula gummosa Boiss(“Barije” in Persian, from the family Apiaceae), (F. gummosa), is an extremely precious medicinal plant which naturally grows throughout the Mediterranean and Central Asia and is a native plant in Iran. The present study examined the cytotoxic effects of F. gummosa in terms of induction of apoptosis and radiosensitivity in HeLa cells. Materials and Methods: In order to determine F. gummosa cytotoxicity in HeLa cells, the cells were incubated with different concentrations of the plant resin(0-1000 µg/ml) for 24, 48 and 72 hr. Cytotoxicity was determined by MTT assay. The role of apoptosis in F. gummosa cytotoxicity was investigated using flow cytometry following propidium iodide (PI) staining of DNA. For radiosensitivity assessment, F. gummosa-treated cells were exposed to 2 Gy γ-rays, and cytotoxicity was determined in irradiated and non-irradiated (control) groups by MTT and the synergism factor was calculated. Results: F. gummosa decreased cell viability in HeLa cells in a concentration- and time-dependent manner. Flow cytometryanalysisindicated that apoptosis is involved in F. gummosa-induced cytotoxicity. Co-administration of F. gummosa and radiotherapy, showed that this plantat non-toxic low doses, could result in almost 5-fold increment in sensitization of cells towards radiation-induced toxicity. Conclusion: The concurrent use of F. gummosa and radiation increases radiosensitivity and cell death. Therefore, F. gummosa can be considered as a potential radiosensitizer agent against cervical cancer

Amino acid-mPEGs: Promising excipients to stabilize human growth hormone against aggregation

Objective(s): Today, the non-covalent PEGylation methods of protein pharmaceuticals attract more attention and possess several advantages over the covalent approach. In the present study, Amino Acid-mPEGs (aa-mPEGs) were synthesized, and the human Growth Hormone (hGH) stability profile was assessed in their presence and absence.Materials and Methods: aa-mPEGs were synthesized with different amino acids (Trp, Glu, Arg, Cys, and Leu) and molecular weights of polymers (2 and 5 KDa). The aa-mPEGs were analyzed with different methods. The physical and structural stabilities of hGH were analyzed by SEC and CD spectroscopy methods. Physical stability was assayed at different temperatures within certain intervals. Molecular dynamics (MD) simulation was used to realize the possible mode of interaction between protein and aa-mPEGs. The cell-based method was used to evaluate the cytotoxicity.Results: HNMR and FTIR spectroscopy indicated that aa-mPEGs were successfully synthesized. hGH as a control group is known to be stable at 4 °C; a pronounced change in monomer degradation is observed when stored at 25 °C and 37 °C. hGH:Glu-mPEG 2 kDa with a molar ratio of 1:1 to the protein solution can significantly increase the physical stability. The CD spectroscopy method showed that the secondary structure of the protein was preserved during storage. aa-mPEGs did not show any cytotoxicity activities. The results of MD simulations were in line with experimental results.Conclusion: This paper showed that aa-mPEGs are potent excipients in decreasing the aggregation of hGH. Glu-mPEG exhibited the best-stabilizing properties in a harsh environment among other aa-mPEGs

Identification of structural features of condensed tannins that affect protein aggregation

A diverse panel of condensed tannins was used to resolve the confounding effects of size and subunit composition seen previously in tannin-protein interactions. Turbidimetry revealed that size in terms of mean degree of polymerisation (mDP) or average molecular weight (amw) was the most important tannin parameter. The smallest tannin with the relatively largest effect on protein aggregation had an mDP of ~7. The average size was significantly correlated with aggregation of bovine serum albumin, BSA (mDP: r=-0.916; amw: r=-0.925; p<0.01; df=27), and gelatin (mDP: r=-0.961; amw: r=-0.981; p<0.01; df=12). The procyanidin/prodelphinidin and cis-/trans-flavan-3-ol ratios gave no significant correlations. Tryptophan fluorescence quenching indicated that procyanidins and cis-flavan-3-ol units contributed most to the tannin interactions on the BSA surface and in the hydrophobic binding pocket (r=0.677; p<0.05; df=9 and r=0.887; p<0.01; df=9, respectively). Circular dichroism revealed that higher proportions of prodelphinidins decreased the apparent α-helix content (r=-0.941; p<0.01; df=5) and increased the apparent β-sheet content (r=0.916; p<0.05; df=5) of BSA

Determination of LMF binding site on a HSA-PPIX complex in the presence of human holo transferrin from the viewpoint of drug loading on proteins.

Holo transferrin (TF) and the natural complex of human serum albumin and protoporphyrin IX (HSA-PPIX) are two serum carrier proteins that can interact with each other. Such an interaction may alter their binding sites. In this study, fluorescence spectroscopy, as well as zeta potential and molecular modeling techniques, have been used to compare the complexes (HSA-PPIX)-LMF and [(HSA-PPIX)-TF]-LMF. The Ka1, Ka2, values of (HSA-PPIX)-LMF and [(HSA-PPIX)-TF]-LMF were 1.1×10(5) M(-1), 9.7×10(6) M(-1), and 2.0×10(4) M(-1), 1.8×10(5) M(-1), respectively, and the n1, n2 values were respectively 1.19, 1.53 and 1.17, 1.65. The second derivative of the Trp emission scan of (HSA-PPIX)-LMF exhibited one negative band at 310 nm, whereas for the [(HSA-PPIX)-TF]-LMF system, we observed one negative band at 316 nm indicating an increase in polarity around Trp. The effect of TF on the conformation of (HSA-PPIX)-TF was analyzed using three-dimensional fluorescence spectroscopy. The phase diagram indicated that the presence of a second binding site on HSA and TF was due to the existence of intermediate structures. Zeta potential analysis showed that the presence of TF increased the positive charges of the HSA-PPIX system. Site marker experiments revealed that the binding site of LMF to HSA-PPIX changed from Sudlow's site IIA to Sudlow's site IIIB in the presence of TF. Moreover, molecular modeling studies suggested the sub-domain IIIB in HSA as the candidate place for the formation of the binding site of LMF on the (HSA-PPIX)-TF complex

Time-resolved fluorescence data of the (HSA-PPIX) TF and (HSA-PPIX) TF/drug complexes (λ_ex = 295 nm, λ_em = 345 nm, pH 7.4, T = 298 K).

<p>Time-resolved fluorescence data of the (HSA-PPIX) TF and (HSA-PPIX) TF/drug complexes (λ_ex = 295 nm, λ_em = 345 nm, pH 7.4, T = 298 K).</p

Three-dimensional fluorescence spectra characteristic of the interaction of (HSA-PPIX)-TF in the absence and presence of LMF.

<p>Three-dimensional fluorescence spectra characteristic of the interaction of (HSA-PPIX)-TF in the absence and presence of LMF.</p

Molecular modeling of the interaction of LMF with the [(HSA-PPIX)-TF] complex, (A) and the second site of the interaction of LMF with [(HSA-PPIX)-TF] (B), represented as a solid ribbon, colored by secondary structure, LMF represented as sticks.

<p>The docking position of LMF to the protein is highlighted. LMF was docked in sub-domain IIIB of (HSA-PPIX). The distance between the binding site candidates of LMF to Trp is also illustrated. The hydrogen bonds between LMF and (HSA-PPIX) are represented as green dashed lines.</p

Investigation on the Interaction between Cyclophosphamide and Lysozyme in the Presence of Three Different Kind of Cyclodextrins: Determination of the Binding Mechanism by Spectroscopic and Molecular Modeling Techniques

molecule

Comparison of the binding constants of the (HSA-PPIX) LMF and [(HSA-PPIX)-TF] LMF systems before and after the addition of the site probe.

<p>Comparison of the binding constants of the (HSA-PPIX) LMF and [(HSA-PPIX)-TF] LMF systems before and after the addition of the site probe.</p

Stern-Volmer quenching constants of the various complexes with LMF at λ_ex = 280 nm.

<p>Stern-Volmer quenching constants of the various complexes with LMF at λ_ex = 280 nm.</p