Deeksha Pal

Alkaline Phosphatase: An Overview

Alkaline phosphatase (ALP; E.C.3.I.3.1.) is an ubiquitous membrane-bound glycoprotein that catalyzes the hydrolysis of phosphate monoesters at basic pH values. Alkaline phosphatase is divided into four isozymes depending upon the site of tissue expression that are Intestinal ALP, Placental ALP, Germ cell ALP and tissue nonspecific alkaline phosphatase or liver/bone/kidney (L/B/K) ALP. The intestinal and placental ALP loci are located near the end of long arm of chromosome 2 and L/B/K ALP is located near the end of the short arm of chromosome 1. Although ALPs are present in many mammalian tissues and have been studied for the last several years still little is known about them. The bone isoenzyme may be involved in mammalian bone calcification and the intestinal isoenzyme is thought to play a role in the transport of phosphate into epithelial cells of the intestine. In this review, we tried to provide an overview about the various forms, structure and functions of alkaline phosphatase with special focus on liver/bone/kidney alkaline phosphatase.

Over-Expression of Telomere Binding Factors (TRF1 & TRF2) in Renal Cell Carcinoma and Their Inhibition by Using SiRNA Induce Apoptosis, Reduce Cell Proliferation and Migration Invitro

Telomere binding factors viz. TRF1 and TRF2 are a part of sheltrin complex that are present exclusively at the ends of chromosomes. These factors play an important role in maintaining chromosomal integrity at the ends. However, their status and role are not clear in renal cell carcinoma (RCC). Therefore, the present study was conducted to evaluate TRF1 and TRF2 expressions in RCC tissues. Further, the role of these factors involved in tumorigenesis was elucidated by gene silencing using siRNA in RCC cell line (A498). The present study documented a significant over-expression of TRF1 (P = 0.005) and TRF2 (P = 0.0048) mRNAs by real time PCR in RCC tissues as compared with adjacent normal kidney tissues. Immunohistochemistry studies also revealed higher expression of TRF1 and TRF2 proteins in RCC. Moreover, TRF1 or TRF2 gene silencing using siRNA showed marked reduction in proliferation of RCC cells (P = 0.000). Further, significantly induced cell cycle arrest (P = 0.000) and apoptosis of RCC cells (P = 0.000) was documented upon TRF1 or TRF2 gene silencing. Henceforth, the results deduce that TRF1 or TRF2 inhibitions play an important role in the induction of apoptosis in A498 cells, which may serve as a potential therapeutic target in RCC.

Implication of BBM lipid composition and fluidity in mitigated alkaline phosphatase activity in renal cell carcinoma

Previous study has documented reduced alkaline phosphatase (ALP) activity in brush border membrane (BBM) isolated from renal cell carcinoma (RCC). Diminished activity of ALP is associated with alteration in both increased Km as well as decreased Vmax of enzyme suggests that there may be a change in the conformation of enzyme as well as decreased number of ALP active molecules. The present study was conducted to find out any role of BBM lipid composition and its fluidity in diminished activity of alkaline phosphatase in renal cell carcinoma. Total phospholipids and glycolipids were significantly augmented in BBM from RCC as compared to control. Fractional analysis of total phospholipids revealed significantly increased phosphatidylethanolamine. Decreased fractions of sphingomyelin and phosphatidylinositol were observed. Cholesterol-to-total phospholipid molar ratios in tumor BBM was a significantly lower in tumor BBM. A significant reduction in polarization and microviscosity was found in BBM from RCC. Therefore, we conclude that alteration in membrane lipid composition and fluidity may play a substantial role in reduced activity of ALP in RCC.

Association between ZIP10 gene expression and tumor aggressiveness in renal cell carcinoma

Zinc is an indispensable trace element which is vital for the functioning of numerous cellular processes like cell replication and growth. Cellular zinc homeostasis is tightly regulated by zinc transporters involved in zinc influx and efflux processes. Notwithstanding, the association of zinc transporters with the aggressiveness of cancer, especially renal cell carcinoma (RCC), is unknown. In view of the fact, the present study was initiated to ascertain whether ZIP10 transporter expression is modulated during RCC progression. A total of 57 samples of RCC and corresponding normal renal tissue were analyzed for ZIP10 gene expression by real time PCR. We observed significantly higher expression of ZIP10 mRNA (P=0.002) in high grade clear cell RCC tissue (Grades III & IV) as compared to low grade clear cell RCC tissue (Grades I & II). A significant difference was also observed in the ZIP10 expression in different types of RCC (P=0.001). This is the first study which shows a significant correlation between ZIP10 mRNA expressions with aggressiveness of RCC. Therefore, ZIP10 mRNA expression could be used as a possible biomarker for the aggressive behavior of RCC and a promising target of novel treatment strategies.

Augmented telomerase activity, reduced telomere length and the presence of alternative lengthening of telomere in renal cell carcinoma: plausible predictive and diagnostic markers.

In this study, we analyzed 100 cases of renal cell carcinoma (RCC) for telomerase activity, telomere length and alternative lengthening of telomeres (ALT) using the TRAP assay, TeloTTAGGG assay kit and immunohistochemical analysis of ALT associated promyelocytic leukemia (PML) bodies respectively. A significantly higher (P=0.000) telomerase activity was observed in 81 cases of RCC which was correlated with clinicopathological features of tumor for instance, stage (P=0.008) and grades (P=0.000) but not with the subtypes of RCC (P = 0.355). Notwithstanding, no correlation was found between telomerase activity and subtypes of RCC. Strikingly, the telomere length was found to be significantly shorter in RCC (P=0.000) to that of corresponding normal renal tissues and it is well correlated with grades (P=0.016) but not with stages (P=0.202) and subtypes (P=0.669) of RCC. In this study, telomere length was also negatively correlated with the age of patients (r(2)=0.528; P=0.000) which supports the notion that it could be used as a marker for biological aging. ALT associated PML bodies containing PML protein was found in telomerase negative cases of RCC. It suggests the presence of an ALT pathway mechanism to maintain the telomere length in telomerase negative RCC tissues which was associated with high stages of RCC, suggesting a prevalent mechanism for telomere maintenance in high stages. In conclusion, the telomerase activity and telomere length can be used as a diagnostic as well as a predictive marker in RCC. The prevalence of ALT mechanism in high stages of RCC is warranted for the development of anti-ALT inhibitors along with telomerase inhibitor against RCC as a therapeutic approach.

Inhibition of hTERT and telomerase activity via hampering MAPK kinase signaling in renal cell carcinoma cell lines

C that has a reputation of a deadly disease all around the world is a broad group of diseases that involves uncontrolled cell growth and proliferation. The best method for its prevention and control is its early diagnosis and treatment. Before molecular diagnostics, scientists relied on the results obtained from biopsies and microscopic examination of affected tissue samples but nowadays molecular diagnosis is emerging as a new and eye opening approach that merges genomics and proteomics for early detection and diagnosis of cancer. Genomics and proteomics tools are used to detect different molecular signatures like genetic and epigenetic signatures, changes in gene expressions, protein biomarker profiles and other metabolite profile changes, which in turn allows to identify the combinations of biomarkers which may detect best the presence or risk of cancer or monitor cancer therapies. Molecular diagnosis uncovers the different changes that occur during the transformation of a normal cell to a tumour cell and capture this information as expression patterns. Robotically printed microarrays, Real time PCR and mi RNAs are widely used techniques for measuring these expression pattern and help researchers to differentiate between a normal and a cancer cell. Molecular diagnosis through proteomics make use of surface enhanced laser desorption/ ionization time of flight mass spectrometry and peptide receptors in mapping of protein patterns that are involved in malignant growth. Nanotechnology is an evolving science that can be successfully used for cancer diagnosis in future. Nanoscale devices quantum dots and carbon nano-tubes can be promising nano-tools for effective measurement of malignancy. All these techniques offer great promise for revolutionizing the diagnosis of cancer. This article recaps some scientific considerations about different relevant molecular diagnostics of cancer and consideration about future challenges.Results: The BTSCs could be obtained from human GBM tissues, and formed neuroshpheres after cultured 7d in vitro, and were CD133positive and nestin-positive. Theses BTSCs could regenerate a GBM tumor in vivo when implanted intraventricularly. Cell migration assay showed that BTSCs have the capacity to actively attract hNSCs. The fate of hNSCs in vivo can be visualized under confocal fluorescence microscope. hNSCs exhibit extensive tropism toward the site of the tumor and infiltrate tumor foci.T lecture will address a variety of topics related to the use of middle gastrectomy with regional lymph node resection as a new therapeutics in early gastric cancer in the middle one-third of the stomach. And will include discussion of: the therapeutics for gastric cancer in recent years; compare the advantage and disadvantage of these therapeutics; compare the difference of surgery time, lymph node resection, aerating time, hospitalization time postoperation, feed after 3 months and Visick graded index between group A (middle gastrectomy with regional lymph node resection), B (total gastrectomy with D1 lymph node resection) and C (endoscopic mucosal resection). We found that middle gastrectomy with regional lymph node resection in early gastric cancer will not increase the risk and time of surgery, and it can reduce the risk of lymph node migrating, complication related to surgery, and improve the quality of life.Introduction: In recent years HSP90 has been studied intensively as a therapeutic target. Hsp90 is an abundant protein in mammalian cells which is responsible for the consolidation of a many of cellular pathways and processes. Human amniotic membrane (hAM) has recently been considered as an upcoming anti-cancer therapy. The aim of this study was investigation the effect of hAMP on the expression levels of HSP60 gene in HepG-2 Hepatocarcinoma cell line.A an orange–pink β-carotenoid, is a potent anti-oxidant found in nature. It crosses blood-brain and brain-eye barriers, hence is important in treating many disorders and diseases such as cancer. The pink colored appearance to the flesh and carapace of most of the sea animals such as lobsters, crab, shrimps etc. is imparted by astaxanthinCattle and humans do not synthesize astaxanthin pigment naturally. Therefore commercially produced pigment is added into food products such as animal feed, krill oil, etc. In this study, shrimp waste was collected from local hotels of Nashik and Trimbakeshwar. Bacteria were isolated from the body parts of the shrimpsusing de ManRogosa – Sharpe and Mac Conkey’s media. Overall 28 isolates of Lactobacillus from various shrimp body parts viz., head, stomach, tail etc.were obtained and identified using morphobiochemical characteristics. The microbial Diversity index was determined for body parts of the shrimp on SPC agar plates. Diversity index of stomach and head ranged between 0.99 to 1 indicating high microbial diversity of shrimps. Astaxanthin extracted from dried shrimp waste using hexane/acetone gave 4% recovery (~0.4 g/10 g of shrimp). Microbial fermentation using Lactobacillus isolates showed enhanced production up to 8% recovery (~0.8 g/10 g of shrimp). The detection of the extracted pigment was done using thin layer chromatography (TLC). The concentrate was subjected to TLC using silica gel G250 sheet and run with solvent system of hexane:acetone (2:3). Thin layer chromatographic separation of carotenoid extract from shrimp waste produced a distinct band at Rf ∼0.3 corresponding to free Astaxanthin. It was further characterized using spectrophotometry with absorption maxima at 473 nm and using High Performance Liquid Chromatography (HPLC). Microbial fermentation was employed for astaxanthin production and fermentation medium was optimised by fractional Factorial Design (FFD) utilizing waste in the form of molasses (1-5%), coconut milk from rotten coconuts (10-30%), whey (1-3%) and soyabean extract (20-60%). By microbial fermentation, the yield of Astaxanthin (g/10 gm of shrimp) was 1.74, 1.46 and 1.14 using various substrates such as 20% soyabean water, 1% molasses and 20% coconut milk respectively. Subsequently, extraction of astaxanthin was also made cost-effective with waste oils viz., sesame, soyabean and mustard oil, collected from Shani temples and local eatery in the town. Molar extinction was estimated and co-related with the final yield of pigment. Astaxanthin yield was made prolific by optimizing both, Production and extraction, production of pigment using microbial fermentation in the presence of eco-substrates and extraction utilizing the waste oils, which being the thrust area of the present work.T lecture will address a topics related to the use of rodent models for pre-clinical evaluation of colon therapeutics for treating colon and its related disease. Use of therapeutic micro particles and associated options. In addition to these subjects, attendees of this lecture will be familiarized with the need for rigorous and stringent testing of candidate carrier system, so that only the most promising therapies are advanced to clinical trial evaluation of efficacy when treating second brain (colon) cancer.Sulfur is an important element has many practical applications in present as nanoparticles. Nanosize sulfur particles also have many important applications like in pharmaceuticals, medicine, synthesis of nanocomposites for lithium batteries, modification of carbon nano tubes [1]. Different methods were used for nano-sized particle synthesis; among those, chemical precipitation, electrochemical method, micro emulsion technique, composing of oil, surfactant, co-surfactant, aqueous phases with the specific compositions and ultrasonic treatment of sulfur-cystine solution [2]. In this work Sulfur nanoparticles (S NPs) were prepared by a quick precipitation method with and without using a surfactant to stabilize the formed S NPs.Result: Monodisperse particles, high colloidal stability and superparamagneticproperties were observed, by using the simple method with the certain molar concentration of Co2+ in 3M NaOH at 100°C. The design of NP surface was done by grafting an appropriate amount of PEG onto the TMC backbone based on structure and physiology of tumor cells. In vivo, thetherapeutic success of magnetic NPs relies on thehydrodynamic sizes of NPs between 10 and 100 nm and maximize blood half-life of NPs. The hydrodynamic diameters of synthesized PEG-g-TMC-CoFe2O4 NPs were between 50-80 nm by DLS. Also, the negative and positive charge of copolymer coating improves the uptake of cationic nanoparticles in cancer cells and increases the distribution of anionic nanoparticles throughout the tumor environment. The properties of CoFe2O4 and PEG-g-TMCCoFe2O4 NPs were analyzed by TEM, DLS, XRD, IR and VSM. Conclusion: Results showed that,CoFe2O4with 12 nm were synthesized by appropriate anisotropy and coercivity instead of iron-oxide NPs with low coercivity in the same size. Also, the amino and hydroxyl groups of copolymer can be utilized as a linker for the protein and drug conjugationby covalent bonds or by weak interactions. So, this polymer nanocomposites canbe apply for selective and targeted delivery of drugs or a targeted antibody (nanobody) that recognize tumor antigens on the cancer cells in cancer and radio therapy.Data Synthesis: Surgery evolved rapidly from the improvement in anesthetic techniques, antibiotics, blood replacement, new reconstruction procedures, management strategies of neck metastasis and laryngeal cancer. Recognized as an effective staging and therapeutic procedure, elective neck dissection diminished functional and aesthetic sequelae. Partial laryngectomies and endoscopic transoral laser resection could keep the function of speech and swallowing without definitive tracheostomy. Advances in creating algorithms calculation and distribution of more accurate dose enabled the development of dosimetry and quality control in radiotherapy,providing a more conservative approach. The radiotherapy with intensity modulation has high precision with better protection of organs at risk. Altered fractionation schemes can reduce the late toxicity with survival benefit. Tomotherapy, volumetric modulated archoterapy, stereotactic radiotherapy and FDG-PET CT are recent approaches. Induction and sequential chemotherapy is a key component in the treatment of locally advanced head and neck cancer. The receiver of Epidermal Growth Factor IgG1 monoclonal antibody (EGFR) showed significant clinical benefits in the treatment of locally advanced, recurrent and/or metastatic cancer.

Therapeutic Anticancer Approaches Targeting Telomerase and Telomeres

Telomeres and telomerase are attractive targets for anticancer therapy. This is evidenced with the facts that majority of human cancers express the enzyme telomerase which is utmost important to maintain the telomere length, thereby to ensure indefinite cell proliferation – a hallmark of cancer. In human cells, a structure referred to as telomere has been identified to cap the terminal regions of chromosomes which can protect the ends of DNA strands from degradation and fusion, whereas telomerase plays a pivotal role in cellular immortality and tumorigenesis. Henceforth, strategies have been made to induce telomerase inhibition target virtually all of the major components of ribonucleoprotein holoenzyme and related cell signal pathways that regulates its activity which includes telomerase reverse transcriptase (hTERT) catalytic subunit, the telomere RNA component (hTERC), and associated proteins. It is noteworthy here that most of the cancers have an alternative lengthening of telomere termed as ALT even in the absence of telomerase activity. In these cases, there is an urgent need to understand the cell signaling pathways for ALT mechanism which can be used as therapeutic targets. Other strategies have been developed to target the protein associated with telomerase at the telomeric ends of chromosomes such as tankyrase. Increasing evidences suggest that directly targeting telomeric DNA using agents directed against the shelterin complex may also have anticancer activity. The limitations of strategies remain to be resolved to facilitate the clinical applications. In this chapter, recent development of strategies against these targets shall be discussed.

Metallothionein gene expression in renal cell carcinoma.

Introduction: Metallothioneins (MTs) are a group of low-molecular weight, cysteine-rich proteins. In general, MT is known to modulate three fundamental processes: (1) the release of gaseous mediators such as hydroxyl radical or nitric oxide, (2) apoptosis and (3) the binding and exchange of heavy metals such as zinc, cadmium or copper. Previous studies have shown a positive correlation between the expression of MT with invasion, metastasis and poor prognosis in various cancers. Most of the previous studies primarily used immunohistochemistry to analyze localization of MT in renal cell carcinoma (RCC). No information is available on the gene expression of MT2A isoform in different types and grades of RCC. Materials and Methods: In the present study, total RNA was isolated from 38 histopathologically confirmed cases of RCC of different types and grades. Corresponding adjacent normal renal parenchyma was taken as control. Real-time polymerase chain reaction (RT PCR) analysis was done for the MT2A gene expression using β-actin as an internal control. All statistical calculations were performed using SPSS software. Results: The MT2A gene expression was found to be significantly increased (P < 0.01) in clear cell RCC in comparison with the adjacent normal renal parenchyma. The expression of MT2A was two to three-fold higher in sarcomatoid RCC, whereas there was no change in papillary and collecting duct RCC. MT2A gene expression was significantly higher in lower grade (grades I and II, P < 0.05), while no change was observed in high-grade tumor (grade III and IV) in comparison to adjacent normal renal tissue. Conclusion: The first report of the expression of MT2A in different types and grades of RCC and also these data further support the role of MT2A in tumorigenesis.