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Fusion toxins are an emerging class of targeted therapeutics for the treatment of cancer. Diphtheria toxin-stem cell factor (DT-SCF) is one such novel fusion toxin designed to target malignancies expressing c-kit. Since, c-kit overexpression has been reported on many types of cancers, it appeared to be a reasonably good molecule to target. In the present study, we report construction, expression, purification, and characterization of DT-SCF. DT-SCF gene coding for 1-387 amino acids of diphtheria toxin, His-Ala linker, 2-141 amino acids of SCF was cloned into expression vector with C terminal His tag. The induced DT-SCF protein was exclusively expressed in insoluble fraction. Purification of DT-SCF was achieved by inclusion body isolation and metal affinity chromatography under denaturing and reducing conditions. Purified DT-SCF was renatured partially on-column by gradually reducing denaturant concentration followed by complete refolding through rapid dilution technique. Cell viability assay provided the evidence that DT-SCF is a potent cytotoxic agent selective to cells expressing c-kit. The novelty of this study lies in employing SCF as a ligand in construction of fusion toxin to target wide range of malignancies expressing c-kit. Efficacy of DT-SCF fusion toxin was demonstrated over a range of malignancies such as chronic myeloid leukemia (K562), acute lymphoblastic leukemia (MOLT4), pancreatic carcinoma (PANC-1), and cervical carcinoma (HeLa 229). This is the first study reporting specificity and efficacy of DT-SCF against tumor cells expressing c-kit. There was significant correlation (P = 0.007) between c-kit expression on cells and their sensitivity to DT-SCF fusion toxin.

Delonix elata, Enicostemma axillare, Merremia tridentata, Mollugo cerviana and Solanum incanum are medicinal plants used in traditional Indian medicine for the treatment of various ailments. These plants were selected to evaluate their potential antibacterial activity. To determine antibacterial activity and phytochemicals in the crude extracts of five medicinal plants used in traditional Indian medicine for the treatment of various ailments like rheumatism, piles fever, skin diseases and snake bite. The antibacterial activity of organic solvent extracts of these plants were determined by disc diffusion and broth dilution techniques against grampositive bacterial strains (Bacillus subtilis, Staphylococcus aureus) and gram-negative bacterial strains (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). Results revealed that the chloroform and methanol extracts of D. elata and methanol extracts of M. cerviana exhibited significant antibacterial activity against gram-positive and gram-negative strains with minimum bactericidal concentration (MBC) ranging from 1.5 to 100 mg/ml. Methanol extracts of M. tridentata exhibited activity only against gram-positive bacterial strains with MBC ranging from 12.5 to 100 mg/ml. Extracts of E. axillare and S. incanum showed activity only against B. subtilis and were not bactericidal at 100 mg/ml. The most susceptible organism to the organic extracts from all the studied plants was B. subtilis and the most resistant organism was P. aeruginosa. The presence of phytochemicals such as alkaloids, tannins, triterpenoids, steroids and glycosides in the extracts of these plants supports their traditional uses as medicinal plants for the treatment of various ailments. The present study reveals potential use of these plants for developing new antibacterial compounds against pathogenic microorganisms.

No abstract

The current treatment strategies, chemotherapy and radiation therapy being used for the management of cancer are deficient in targeted approach leading to treatment related toxicities and relapse. Contrarily, fusion toxins exhibit remarkable tumor specificity thus emerging as an alternative therapy for the treatment of cancer. Diphtheria toxin-HN-1 peptide (DT/HN-1) is a fusion toxin designed to target the head and neck squamous cell carcinoma (HNSCC). The aim of this study was to construct, characterize, and evaluate the cytotoxicity and specificity of DT/HN-1 fusion toxin against the HNSCC cells. The purified DT/HN-1 fusion toxin was characterized by SDS-PAGE and western blotting. Refolding of purified fusion toxins was monitored by fluorescence spectra and circular dichroism spectra. The activity of DT/HN-1 fusion toxin was demonstrated on various HNSCC cell lines by cell viability assay, cell proliferation assay, protein synthesis inhibition assay, apoptosis and cell cycle analysis. The fusion toxin DT/HN-1 demonstrated remarkably high degree of cytotoxicity specific to the HNSCC cells. The IC(50) of DT/HN-1 fusion toxin was ~1 to 5 nM in all the three HNSCC cell lines. The percentage apoptotic cells in DT/HN-1 treated UMB-SCC-745 cells are 16% compared to 4% in untreated. To further demonstrate the specific toxicity of DT/HN-1 fusion toxin towards the HNSCC cells we constructed, characterized and evaluated the efficacy of DT protein. The DT protein coding for only a fragment of diphtheria toxin without its native receptor binding domain failed to exhibit any cytotoxicity on all the cell lines used in this study thus establishing the importance of a ligand in achieving targeted toxicity. To evaluate the translocation ability of HN-1 peptide, an additional construct DTΔT/HN-1 was constructed, characterized and evaluated for its cytotoxic activity. The fusion toxin DTΔT/HN-1 deficient of the translocation domain of diphtheria toxin showed no cytotoxicity on all the cell lines clearly indicating the inability of HN-1 peptide to translocate catalytic domain of the toxin into the cytosol.