Subramanian Krishnakumar

Biochemical changes accompanying apoptotic cell death in retinoblastoma cancer cells treated with lipogenic enzyme inhibitors.

Retinoblastoma (RB) is a malignant intra-ocular neoplasm that affects children (usually below the age of 5years). In addition to conventional chemotherapy, novel therapeutic strategies that target metabolic pathways such as glycolysis and lipid metabolism are emerging. Fatty acid synthase (FASN), a lipogenic multi-enzyme complex, is over-expressed in retinoblastoma cancer. The present study evaluated the biochemical basis of FASN inhibition induced apoptosis in cultured Y79 RB cells. FASN inhibitors (cerulenin, triclosan and orlistat) significantly inhibited FASN enzyme activity (P<0.05) in Y79 RB cells. This was accompanied by a decrease in palmitate synthesis (end-product depletion), and increased malonyl CoA levels (substrate accumulation). Differential lipid profile was biochemically estimated in neoplastic (Y79 RB) and non-neoplastic (3T3) cells subjected to FASN inhibition. The relative proportion of phosphatidyl choline to neutral lipids (triglyceride+total cholesterol) in Y79 RB cancer cells was found to be higher than the non-neoplastic cells, indicative of altered lipid distribution and utilization in tumor cells. FASN inhibitor treated Y79 RB and fibroblast cells showed decrease in the cellular lipids (triglyceride, cholesterol and phosphatidyl choline) levels. Apoptotic DNA damage induced by FASN inhibitors was accompanied by enhanced lipid peroxidation.

Nanotechnology in ocular drug delivery.

Despite numerous scientific efforts, efficient ocular drug delivery remains a challenge for pharmaceutical scientists. Most ocular diseases are treated by topical drug application in the form of solutions, suspensions and ointment. These conventional dosage forms suffer from the problems of poor ocular bioavailability, because of various anatomical and pathophysiological barriers prevailing in the eye. This review provides an insight into the various constraints associated with ocular drug delivery, summarizes recent findings and applications of various nanoparticulate systems like microemulsions, nanosuspensions, nanoparticles, liposomes, niosomes, dendrimers and cyclodextrins in the field of ocular drug delivery and also depicts how the various upcoming of nanotechnology like nanodiagnostics, nanoimaging and nanomedicine can be utilized to explore the frontiers of ocular drug delivery and therapy.

Solitary fibrous tumor of the orbit: a clinicopathologic study of six cases with review of the literature☆

Solitary fibrous tumor of the orbit is a rare spindle cell neoplasm. There are 42 cases of solitary fibrous tumor of the orbit available in the literature. We present six more cases of orbital solitary fibrous tumors, which presented to our institute between 1999 and 2001. We highlight the need for clinical recognition of these tumors as a distinct entity and inclusion of this tumor in the etiological differential diagnosis of well-circumscribed orbital lesions presenting as unilateral proptosis in both children and in adults. The diagnosis may be suspected based on radiological features supported by histopathologic and immunohistochemical study. The strong CD34 immunoreactivity of this tumor supports its diagnosis. Complete surgical resection is the most important prognostic factor of this tumor.

Stem cell markers: ABCG2 and MCM2 expression in retinoblastoma

Backgound/aim: The authors studied the expression of cancer stem cell surface marker, ABCG2, and neural stem cell marker, MCM2, in retinoblastoma and correlated clinicopathologically. Methods: Among 39 retinoblastomas, 18 tumours were not subjected to preoperative/postoperative chemotherapy, 15 tumours underwent postoperative chemotherapy, and six tumours had preoperative chemotherapy. There were 20 tumours with no invasion and 19 tumours with invasion of choroid/optic nerve. ABCG2 and MCM2 expression was studied by immunohistochemistry. Results: ABCG2 was positive in six of six and MCM2 was positive in five of six tumours that had recurred in the orbit or metastasised. ABCG2 was positive in 15/19 tumours with invasion. MCM2 was positive in 16/19 tumours with invasion. Invasive tumours showed higher expression of ABCG2 (p<0.01) and MCM2 (p<0.01) proteins. There was no correlation with differentiation and laterality of the tumours. Non-neoplastic retina was positive for ABCG2 and MCM2. Conclusion: ABCG2 and MCM2 were expressed more in invasive tumours. Further studies are needed to understand the significance of ABCG2 and MCM2 expression in retinoblastoma.

Multidrug resistant proteins: P-glycoprotein and lung resistance protein expression in retinoblastoma

Background/aim: Retinoblastoma is the commonest primary intraocular tumour in children. Chemotherapy now plays a big part in the treatment of these tumours. There is not much information about the role of the multidrug resistance proteins (MDR)—P-glycoprotein (P-gp) and vault protein lung resistance protein (LRP)—in retinoblastoma. The authors investigated the expression of P-gp and LRP in retinoblastoma and correlated them clinicopathologically. Methods: Among 60 retinoblastomas, 40 tumours were not subjected to preoperative or postoperative chemotherapy and 20 tumours were subjected to postoperative chemotherapy. In this cohort 27 tumours had no invasion and 33 tumours had invasion of choroid, optic nerve, and orbit. P-gp and LRP expression were studied by immunohistochemistry. Immunoanalysis was done semiquantitatively. Results: Among the 60 tumours P-gp was expressed in 23 (38%) tumours and LRP was expressed in 35 (58%). P-gp was expressed in 11/27 (40%) tumours with no invasion and in 12/33 (36%) tumours with invasion. LRP was expressed in 15/27 (55%) tumours with no invasion and in 20/33 (60%) tumours with invasion. Both P-gp and LRP were negative in three tumours with invasion, which had later developed bone marrow metastasis. There was no correlation between P-gp and LRP expression with invasion, differentiation and laterality of the tumours and response to treatment. Conclusion: Retinoblastoma expresses P-gp and LRP intrinsically before chemotherapy and none of these proteins predicted the response to chemotherapy. Thus, further studies are needed to understand the significance of the expression of the P-gp and LRP proteins in retinoblastoma.

Expression of Epidermal Growth Factor Receptor, Ezrin, Hepatocyte Growth Factor, and c-Met in Uveal Melanoma: An Immunohistochemical Study

The immunoreactivity of epidermal growth factor receptor (EGFR) ezrin, hepatocyte growth factor receptor (HGF), and c-Met was studied in 60 uveal melanomas and was correlated with clinicopathologic parameters. Metastases were diagnosed in the patients with uveal melanoma between 5 years and 8 years (median, 6.5 years) after enucleation. Using Kaplan-Meier statistical analysis, we found a significant association between high c-Met expression and death due to uveal melanoma (p < 0.03). EGFR was expressed in 18 of 60 (30%) tumors; ezrin was expressed in 30 of 60 (50%) tumors. Tumors with liver metastasis (n = 6) showed higher expression of c-Met (p = 0.0009) compared with the tumors with no extension/extrascleral extension without liver metastasis (groups A-45 and B-9). HGF was negative in all the six tumors that had liver metastasis. Further studies are required to understand the possible mechanism of ligand-independent c-Met activation in patients with uveal melanoma.

Lysyl Oxidase Activity in the Ocular Tissues and the Role of LOX in Proliferative Diabetic Retinopathy and Rhegmatogenous Retinal Detachment

PURPOSE Lysyl oxidase (LOX) cross-links the side chain of collagen and elastin and thereby contributes to extracellular matrix (ECM) integrity. ECM remodeling is seen in various ocular diseases. Until now, there have been no reports on the LOX enzymes activity in ocular tissues. The purpose of this study was to estimate LOX activity and expression in human donor ocular tissues and to measure the specific activity of LOX in the vitreous of proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RRD). METHOD Human donor eyeballs obtained from an eye bank were used to study tissue distribution of LOX. Human vitreous specimens were obtained during vitreoretinal surgery from PDR (n = 16) and RRD (n = 10). LOX activity was estimated by N-acetyl-3,7-dihydroxyphenoxazine assay, immunohistochemistry, and real-time polymerase chain reaction (RT-PCR). Matrix metalloprotease (MMP)-2 and -9 were quantified in the vitreous by sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS The specific activity of LOX in ocular tissues was on the order of vitreous, iris ciliary body, lens, choroid RPE, and retina, which were comparable by mRNA expression and immunolocalization. The vitreous level of LOX activity decreased significantly in PDR and RRD, with an increase in total MMP-2 and -9 levels compared with normal donor vitreous. CONCLUSIONS LOX activity showed a statistically significant decrease in the vitreous of PDR and RRD relative to control specimens. This effect can contribute to the inadequate collagen cross-linking that causes the ECM changes that occur in these diseases.

Promises of Nanotechnology for Drug Delivery to Brain in Neurodegenerative Diseases

Brain is a delicate organ, isolated from general circulation and characterized by the presence of relatively impermeable endothelial cells with tight junctions, enzymatic activity and the presence of active efflux transporter mechanisms. These formidable obstacles often block drug delivery to the brain across the blood-brain barrier (BBB). Although several promising molecules have the potential in the in vitro settings but lack of in vivo response is probably because the molecule cannot reach the brain in a sufficient concentration. Drug delivery across the BBB is a major limitation in the treatment of central nervous system (CNS) disorders and CNS infections. This review deals with the role of nanobiotechnology in CNS drug delivery, in which three categories of carbon nanotubes, nanowires and nanoparticles (NPs) are explained. The small size of the NPs makes them an ideal choice to penetrate the BBB. Several mechanisms are involved in this process and various strategies are used. There are some concerns about the safety of NP entry in the brain that need to be resolved before human use. Although there is no approved nanotechnology-based CNS drug available the future for such neuro-nanobiotechnology based delivery system developments is promising.

Effect of Curcumin on miRNA Expression in Human Y79 Retinoblastoma Cells

Purpose: Retinoblastoma (RB) is the most common intraocular malignancy in children. Deregulation of several miRNAs has been identified in RB, suggesting a potential role in tumorigenesis. Recent evidence suggests that many dietary components like folate, retinoids and curcumin act as potential anticancer/antiproliferative agents by regulating the expression of miRNA. In this study, we investigated the effect of phenolic compound curcumin on miRNA expression in Y79 RB cells. Materials and Methods: We analyzed the expression profile of miRNA by microarray analysis and quantitative real-time polymerase chain reaction (qRT-PCR) in curcumin-treated Y79 RB cells. Transfection of miR-22 was performed using Lipofectamine 2000. Cell viability, in vitro scratch migration assay, prediction of miRNA targets and Western blot analysis were performed to determine the biological function of miR-22 in Y79 RB cells. Results: In Y79 RB cells treated with curcumin, 5 human miRNAs were upregulated and 16 were downregulated as detected with the miRNA microarray analysis. miR-22, a tumor-suppressor miRNA was one of the miRNA which was upregulated by curcumin. Transfected miR-22 Y79 cells inhibited the cell proliferation and reduced the migration, and erythoblastic leukemia viral oncogene homolog 3 (Erbb3) was confirmed to be the target gene of miR-22. Conclusion: These observations suggest that curcumin modulate the miRNA expression profile, thereby exerting its anticancer effects on RB cells.

Clinical aspects for survivin: a crucial molecule for targeting drug-resistant cancers

Drug resistance is frequently found in cancer patients who have prolonged chemotherapeutic treatments. Overcoming this phenomenon to make therapy available to these patients is one of the most important features in developing effective cancer therapeutic strategies. Identification of drug resistance causative molecules is one of the most focused areas of cancer research today. Many molecules have been identified in conferring cancer cells the property of drug resistance, and various small molecule inhibitors have been developed to target these molecules to restore the sensitivity of different traditional chemotherapeutic agents, which are frequently found to exhibit reduced potency during prolonged treatment, in cancer patients. Survivin, a member of the inhibitor of apoptosis proteins (IAP) family, has been identified as one of the most crucial biomarkers in the recognition of drug resistance. Survivin is overexpressed in tumor cells, helping in its proliferation and survival, and its overexpression is positively correlated with poor prognosis for cancer patients. Targeted therapeutic measures to inhibit survivin in cancers, particularly drug-resistant tumors, are the recent focus of research for cancer treatment.