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

Mass Spectrometric Analysis of Differentially Expressed Proteins in an Endangered Medicinal Herb, Picrorhiza kurroa

Picrorhiza kurroa grown in the Northwestern Himalayan region is used in various herbal formulations but extensive harvesting of this plant has led it to near extinction. The active constituents responsible for the medicinal properties of P. kurroa have been identified as picroside-I and picroside-II which are present in a particular ratio (1 : 1.5) in herbal formulations like Picroliv. The biosynthetic pathway of picrosides has been partially deciphered till date and needs to be elucidated completely. Review of literature revealed that no information is available as of today on the proteome analysis of Picrorhiza kurroa w.r.t. picroside-II biosynthesis. Therefore, with the aim of identifying proteins associated with picroside biosynthesis in Picrorhiza kurroa, differential protein expression was studied under picroside accumulating versus nonaccumulating conditions using SDS-PAGE. A total of 19 differentially expressed proteins were identified using MALDI-TOF/TOF MS followed by MASCOT search. Proteins involved in diverse functions were identified amongst which the most important proteins were glyceraldehyde-3-phosphate dehydrogenase, 1-aminocyclopropane-1-carboxylate oxidase, photosystem I reaction centre subunit V, 2-oxoglutarate ferrous-dependent oxygenase and putative cytochrome P450 superfamily protein because of their role in picroside biosynthesis. These identified proteins provide an insight and a basic platform for thorough understanding of biosynthesis of secondary metabolites and various other physiological processes of P. kurroa

Effect of biomolecules from human renal matrix of calcium oxalate monohydrate (CaOx) stones on in vitro calcium phosphate crystallization

PURPOSE: Investigate the activity of high and low molecular weight biomolecules present in the matrix of human calcium oxalate (CaOx) stones not only on the initial mineral phase formation of calcium and phosphate (CaP) but also on its growth and demineralization of the preformed mineral phase. MATERIALS AND METHODS: Surgically removed renal stones were analyzed by Fourier Transform Infra Red (FTIR) spectroscopy and only CaOx stones were extracted with 0.05M EGTA, 1 mM PMSF and 1% ß-mercaptoethanol. Renal CaOx stone extract was separated into > 10 kDa and 10 kDa and 10 kDa fraction lane. CONCLUSION: Both high and low molecular weight biomolecules extracted from human renal matrix of calcium oxalate (CaOx) stones have a significant influence on calcium and phosphate (CaP) crystallization

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

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

Nephrolithiasis: Molecular Mechanism of Renal Stone Formation and the Critical Role Played by Modulators

Urinary stone disease is an ailment that has afflicted human kind for many centuries. Nephrolithiasis is a significant clinical problem in everyday practice with a subsequent burden for the health system. Nephrolithiasis remains a chronic disease and our fundamental understanding of the pathogenesis of stones as well as their prevention and cure still remains rudimentary. Regardless of the fact that supersaturation of stone-forming salts in urine is essential, abundance of these salts by itself will not always result in stone formation. The pathogenesis of calcium oxalate stone formation is a multistep process and essentially includes nucleation, crystal growth, crystal aggregation, and crystal retention. Various substances in the body have an effect on one or more of the above stone-forming processes, thereby influencing a person’s ability to promote or prevent stone formation. Promoters facilitate the stone formation while inhibitors prevent it. Besides low urine volume and low urine pH, high calcium, sodium, oxalate and urate are also known to promote calcium oxalate stone formation. Many inorganic (citrate, magnesium) and organic substances (nephrocalcin, urinary prothrombin fragment-1, osteopontin) are known to inhibit stone formation. This review presents a comprehensive account of the mechanism of renal stone formation and the role of inhibitors/promoters in calcium oxalate crystallisation

Preventive and curative effects of<i style=""> Achyranthes aspera</i> Linn. extract in experimentally induced nephrolithiasis

201-208The present study was undertaken to evaluate the efficacy of Achyranthes aspera in preventing and reducing the growth of calcium oxalate stones in ethylene glycol induced nephrolithiatic model. Hyperoxaluria was induced in rats using ethylene glycol (EG, 0.4%) and ammonium chloride (1%) for 15 days and was then replaced with EG (0.4%) only. Upon administration of cystone (750 mg/kg body wt.), aqueous extract of A. aspera (500 and 1000 mg/kg body wt.), levels of renal injury markers (lactate dehydrogenase and alkaline phosphatase) were normalized with a decrease in serum urea and serum creatinine. Concurrent treatment reduced changes in the architecture of renal tissue and also decreased the size of crystals thereby helping in quick expulsion of the crystals. The present results indicated that Achyranthes aspera had an ability to maintain renal functioning and reduced renal injury

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

Protective effect of >3 kDa fraction on oxalate injured renal cells (MTT assay).

<p>(A) Assessment of viability of oxalate injured NRK-52E cells in the presence of >3 kDa fraction of <i>T</i>. <i>arjuna</i> by MTT assay. Data are mean ± S.D of three independent observations. ns: not significant. All treatment groups were simultaneously compared via one-way ANOVA using Dunnett’s multiple comparisons test. ‘*’ represents P values versus untreated cells (control) and ‘#’ represents P values versus oxalate treated cells, where * p< 0.05 vs control, ** p < 0.005 vs control, *** p < 0.001 vs control, # p < 0.05 vs oxalate, ## p < 0.01 vs oxalate. (B) Assessment of viability of oxalate injured MDCK cells in the presence of >3 kDa fraction of <i>T</i>. <i>arjuna</i> by MTT assay. Data are mean ± S.D of three independent observations. ns: not significant. All treatment groups were simultaneously compared via one-way ANOVA using Dunnett’s multiple comparisons test. ‘*’ represents P values versus untreated cells (control) and ‘#’ represents P values versus oxalate injured cells, where * p< 0.05 vs control, ** p < 0.005 vs control, *** p < 0.001 vs control, # p < 0.05 vs oxalate, ## p < 0.005 vs oxalate.</p