Evaluation of the Cytotoxic Effects of CAM Therapies: An In Vitro

The purpose of this current study was to justify the incorporation of complementary and alternate medicine (CAM) in current cancer treatments. The major drawback of anticancer drugs is their nonselective killing, which ultimately leads to attrition of normal cells. Keeping this as the foundation of our study, we made an effort to compare the cytotoxicity associated with a known chemotherapeutic drug 5-Fluorouracil (5-FU), with certain CAM therapies previously reported to have anticancer activity. The parameters chosen for the study were based on antiproliferative and cytotoxic effects on normal, kidney epithelial cells (NRK-52E). The MTT assay, colony formation assay, DNA fragmentation, and differential staining using AO/EB, following treatment with either 5-FU or CAM therapies, were performed. The CAM therapies under study were various extracts of wheatgrass, roots of Achyranthes aspera (AA), mushroom extracts (Pleurotus ostreatus, Macrolepiota procera, and Auricularia polytricha), and a homeopathic drug, Ruta graveolens (Ruta). The results showed that treatment of normal cells with the CAM therapies led to minimum cell damage in comparison to 5-FU. This evidence-based study will lead to greater acceptance of alternative therapies against cancer

Mechanistic Insights into the Antilithiatic Proteins from Terminalia arjuna: A Proteomic Approach in Urolithiasis.

Kidney stone formation during hyperoxaluric condition is inherently dependent on the interaction between renal epithelial cells and calcium oxalate (CaOx) crystals. Although modern medicine has progressed in terms of removal of these stones, recurrence and persistent side effects restricts their use. Strategies involving plant based agents which could be used as adjunct therapy is an area which needs to be explored. Plant proteins having antilithiatic activity is a hitherto unexplored area and therefore, we conducted a detailed identification and characterization of antilithiatic proteins from Terminalia arjuna (T. arjuna). Proteins were isolated from the dried bark of T. arjuna and those having molecular weights > 3 kDa were subjected to anion exchange chromatography followed by gel filtration chromatography. Four proteins were identified exhibiting inhibitory activity against CaOx crystallization and crystal growth kinetics The cytoprotective and anti-apoptotic efficacy of these purified proteins was further investigated on oxalate injured renal epithelial cells (MDCK and NRK-52E) wherein, injury due to oxalate was significantly attenuated and led to a dose dependent increase in viability of these cells. These proteins also prevented the interaction of the CaOx crystals to the cell surface and reduced the number of apoptotic cells. Identification of these 4 anionic proteins from the bark of T. arjuna was carried out by Matrix-assisted laser desorption/ionization-time of flight Mass spectrometry (MALDI-TOF MS). This was followed by database search with the MASCOT server and sequence similarity was found with Nuclear pore anchor, DEAD Box ATP-dependent RNA helicase 45, Lon protease homolog 1 and Heat shock protein 90-3. These novel proteins isolated from T. arjuna have the potential to inhibit CaOx crystallization and promote cell survival and therefore, offer novel avenues which need to be explored further for the medical management of urolithiasis

Peeping into human renal calcium oxalate stone matrix: characterization of novel proteins involved in the intricate mechanism of urolithiasis.

BACKGROUND: The increasing number of patients suffering from urolithiasis represents one of the major challenges which nephrologists face worldwide today. For enhancing therapeutic outcomes of this disease, the pathogenic basis for the formation of renal stones is the need of hour. Proteins are found as major component in human renal stone matrix and are considered to have a potential role in crystal-membrane interaction, crystal growth and stone formation but their role in urolithiasis still remains obscure. METHODS: Proteins were isolated from the matrix of human CaOx containing kidney stones. Proteins having MW>3 kDa were subjected to anion exchange chromatography followed by molecular-sieve chromatography. The effect of these purified proteins was tested against CaOx nucleation and growth and on oxalate injured Madin-Darby Canine Kidney (MDCK) renal epithelial cells for their activity. Proteins were identified by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF MS) followed by database search with MASCOT server. In silico molecular interaction studies with CaOx crystals were also investigated. RESULTS: Five proteins were identified from the matrix of calcium oxalate kidney stones by MALDI-TOF MS followed by database search with MASCOT server with the competence to control the stone formation process. Out of which two proteins were promoters, two were inhibitors and one protein had a dual activity of both inhibition and promotion towards CaOx nucleation and growth. Further molecular modelling calculations revealed the mode of interaction of these proteins with CaOx at the molecular level. CONCLUSIONS: We identified and characterized Ethanolamine-phosphate cytidylyltransferase, Ras GTPase-activating-like protein, UDP-glucose:glycoprotein glucosyltransferase 2, RIMS-binding protein 3A, Macrophage-capping protein as novel proteins from the matrix of human calcium oxalate stone which play a critical role in kidney stone formation. Thus, these proteins having potential to modulate calcium oxalate crystallization will throw light on understanding and controlling urolithiasis in humans

Detection of oxalate induced apoptosis in MDCK cells by Hoechst staining.

<p>Effect of >3 kDa fraction and purified proteins of <i>T</i>. <i>arjuna</i> on oxalate induced apoptosis in MDCK cells, visualized under fluorescence microscopy at magnification 20X; scale bar 100 microns.</p

Gel filtration chromatography profile of peak P2.

<p>Chromatogram of peak P2 subjected to Bio gel^® P-100 gel packed column and proteins eluted in an isocratic 10 mM Tris-Cl buffer (pH 7.4).</p

Detection of oxalate induced apoptosis in NRK-52E cells by Hoechst staining.

<p>Effect of >3 kDa fraction and purified proteins of <i>T</i>. <i>arjuna</i> on oxalate induced apoptosis in NRK-52E cells, visualized under fluorescence microscopy at magnification 20X; scale bar 100 microns.</p

Decreased CaOx crystal adhesion to MDCK cells by proteins.

<p>Effect of >3 kDa fraction and purified proteins of <i>T</i>. <i>arjuna</i> on CaOx crystal adherence to oxalate injured MDCK cells, visualized under polarization and phase contrast at magnification 20X and scale bar 100 microns.</p

Summary of proteins identified and characterized using the Mascot search engine and ScanProsite.

<p>Summary of proteins identified and characterized using the Mascot search engine and ScanProsite.</p