Appu Rathinavelu

Biodegradable microparticles of influenza viral vaccine: comparison of the effects of routes of administration on the in vivo immune response in mice

The objective of this study was to investigate the comparative immune response following administration of biodegradable microparticles loaded with influenza viral vaccine using subcutaneous and oral routes. Influenza viral vaccine was entrapped in poly(d,l-lactide-co-glycolide) (PLG) and poly(isobutylcyanoacrylate) (PIBCA) microparticles. Stability and immunogenicity of entrapped antigen were retained, as evaluated by SDS-PAGE and immunoblot. Microparticles in the size range of <11 microm were evaluated for protein loading and in vitro antigen release. The mice were immunized with microparticle loaded antigen and IgG levels in blood and IgA levels in saliva and gastric secretions were monitored by ELISA method. When the mice were immunized with microparticle suspensions, IgG levels were higher if administered by subcutaneous primed by oral route compared to oral primed by subcutaneous route or subcutaneous or oral route. The IgA level in saliva and gastric secretions were also found to be higher when subcutaneous immunization was given followed by oral booster than oral priming followed by subcutaneous booster. The polymer types of the microparticles had effects on both IgG and IgA levels. This study provided insights into the design of microparticles of influenza vaccine for subcutaneous administration followed by an unlimited oral boosting, which will have high cost-effectiveness and patient compliance.

ANTICANCER ACTIVITIES OF GENISTEIN-TOPOTECAN COMBINATION IN PROSTATE CANCER CELLS

Prostate cancer is one of the leading causes of death in men aged 40 to 55. Genistein isoflavone (4′, 5′, 7‐trihydroxyisoflavone) is a dietary phytochemical with demonstrated anti‐tumour activities in a variety of cancers. Topotecan Hydrochloride (Hycamtin) is an FDA‐approved chemotherapy drug, primarily used for secondary treatment of ovarian, cervical and small cell lung cancers. This study was to demonstrate the potential anticancer efficacy of genistein‐topotecan combination in LNCaP prostate cancer cells and the mechanism of the combination treatment. The LNCaP cells were grown in complete RPMI medium, and cultured at 37°C, 5% CO2 for 24–48 hrs to achieve 70–90% confluency. The cells were treated with varying concentrations of genistein, topotecan and genistein‐topotecan combination and incubated for 24 hrs. The treated cells were assayed for (i) post‐treatment sensitivity using MTT assay and DNA fragmentation, (ii) treatment‐induced apoptosis using caspase‐3 and ‐9 binding assays and (iii) treatment‐induced ROS generation levels. The overall data indicated that (i) both genistein and topotecan induce cellular death in LNCaP cells, (ii) genistein‐topotecan combination was significantly more efficacious in reducing LNCaP cell viability compared with either genistein or topotecan alone, (iii) in all cases, cell death was primarily through apoptosis, via the activation of caspase‐3 and ‐9, which are involved in the intrinsic pathway, (iv) ROS generation levels increased significantly with the genistein‐topotecan combination treatment. Treatments involving genistein‐topotecan combination may prove to be an attractive alternative phytotherapy or adjuvant therapy for prostate cancer.

Phycocyanin Induces Apoptosis and Enhances the Effect of Topotecan on Prostate Cell Line LNCaP

C-phycocyanin (C-PC) from Spirulina has been previously shown to have anticancer properties. Here, we report on anticancer activity of C-PC that was isolated from the novel cyanobacterium Limnothrix sp. 37-2-1. C-PC from this organism exhibited anticancer properties in our in vitro systems; however, the required doses were well above the range of anticancer drugs normally used. Therefore, we conducted several experiments to test whether lower-than-usual doses of the anticancer drug topotecan (TPT) can offer the same level of cytotoxic effects as normal doses when combined with C-PC. For this purpose, cytotoxicities of C-PC and TPT were tested using the LNCaP (prostate cancer) cells. We found that when only 10% of a typical dose of TPT was combined with C-PC, the cancer cells were killed at a higher rate than when TPT was used alone at full dose. Similarly, we were also able to detect an increased level of radical oxygen species (ROS) generation as well as an increase in activities of caspase-9 and caspase-3 when these two compounds were used in combination. Taken together, our findings suggest that combining C-PC from Limnothrix sp. with the lower dose of TPT can induce apoptosis through generation of ROS and activation of caspases. In that respect, we suggest that C-PC can potentially improve the efficacy of the currently available anticancer drug, and therefore diminish its harsh side effects in the patient.

A novel regulation of VEGF expression by HIF-1α and STAT3 in HDM2 transfected prostate cancer cells.

On the basis of increasing roles for HDM2 oncoprotein in cancer growth and progression, we speculated that HDM2 might play a major role in hypoxia‐induced metastatic process. For verification of this hypothesis, wild‐type LNCaP prostate cancer cells and HDM2 transfected LNCaP‐MST (HDM2 stably transfected) cells were studied. The data obtained from our experiments revealed that the HDM2 transfected LNCaP‐MST cells possessed an ability to multiply rapidly and show distinct morphological features compared to non‐transfected LNCaP cells. During exposures to hypoxia HDM2 expression in the LNCaP and LNCaP‐MST cells was significantly higher compared to the normoxic levels. The LNCaP‐MST cells also expressed higher levels of HIF‐1α (hypoxia‐inducible factor‐1α) and p‐STAT3 even under the normoxic conditions compared to the non‐transfected cells. The HIF‐1α and p‐STAT3 expressions were increased several fold when the cells were subjected to hypoxic conditions. The HIF‐1α and p‐STAT3 protein expressions observed in HDM2 transfected LNCaP‐MST cells were 20 and 15 folds higher, respectively, compared to the non‐transfected wild‐type LNCaP cells. These results demonstrate that HDM2 may have an important regulatory role in mediating the HIF‐1α and p‐STAT3 protein expression during both normoxic and hypoxic conditions. Furthermore, the vascular endothelial growth factor (VEGF) expression that is typically regulated by HIF‐1α and p‐STAT3 was also increased significantly by 136% (P < 0.01) after HDM2 transfection. The overall results point towards a novel ability of HDM2 in regulating HIF‐1α and p‐STAT3 levels even in normoxic conditions that eventually lead to an up‐regulation of VEGF expression.

High-performance liquid chromatography using electrochemical detection for the determination of prazosin in biological samples

For the quantitation of prazosin a sensitive high-performance liquid chromatographic (HPLC) method was developed. This HPLC analysis method uses an electrochemical detection technique for the identification and quantitation of prazosin. In this assay the serum samples were deproteinized by using a simple acetonitrile precipitation technique that was followed by n-hexane extraction. Prazosin in the deproteinized serum sample was separated by an isocratic elution with an ODS Hypersil HPLC column (150 x 4.6 mm) using a mobile phase consisting of 0.05 M Na2HPO4-acetonitrile (60:40), pH 8.4. Prazosin that was eluted from the column was detected using a Coulochem II electrochemical detector. The precision of this assay method was assessed by performing inter- and intra-assay analyses by spiking prazosin free fetal bovine serum samples with 20 and 40 ng/ml concentrations of prazosin. In the intra-assay the recovery was 95.40 +/- 4.82% and 97.80 +/- 3.40%, respectively, for 20 and 40 ng/ml concentrations of prazosin that were used to spike the serum samples. This electrochemical detection HPLC assay method could be very useful in monitoring plasma levels of prazosin.

Basic Pharmacology : Understanding Drug Actions and Reactions

Contents PHYSICOCHEMICAL PROPERTIES OF DRUGS Basic Chemical Properties of Drugs Introduction The Periodic Table and Properties of Drugs The Periodic Table and Chemical Reactivity Chemical Bonds Magnitude of Ionic Charge Polyatomic Ions Covalent Bonds Coordinate Covalent Bonds Resonance Structures Shapes of Drug Molecules Electronegativity Polarity of Chemical Bonds Molecular Polarity Further Reading Acid-Base Properties of Drugs Hydrolysis of Salts Conjugate Acids and Conjugate Bases Strength of Acids and Bases Resonance and Inductive Effects Inductive Effects Resonance Effects The Henderson-Hasselbach Equation Further Reading Structural Determinants of Drug Action Structurally Nonspecific Drugs Volatile Anesthetics Structurally Specific Drugs Isosterism and Isosteres Structural Changes in Drug Molecules Further Reading Chemical Approaches to the Treatment of Cancer Normal vs. Malignant Cells Cell Cycle and Chemotherapy From Chemical Warfare to Chemotherapy Anticancer Drugs Further Reading PRINCIPLES OF BIOPHARMACEUTICS Administration and Absorption of Drugs Drug Administration Drug Absorption Physiological and Physicochemical Factors in Drug Absorption Drug Absorption, Bioavailability, and First-Pass Metabolism Further Reading Distribution and Excretion of Drugs Factors in Drug Distribution Patterns of Distribution Determination of the Volume of Distribution Clearance and Elimination Rate Clearance and the Maintenance Dose Rate Half-Life and the Steady State Bioavailability Drug Elimination Further Reading Metabolic Changes of Drugs Cytochromes P450 Redox Reactions and the CYP450 Enzyme Complex NAD + /NADH System FAD/FADH System The Cytochrome P450 Cycle Phase I and Phase II Reactions of Drug Metabolism Dose-Dependent Toxicity of Acetaminophen Further Reading PRINCIPLES OF DRUG-RECEPTOR AND DRUG-ENZYME INTERACTIONS Drug Receptors and Pharmacodynamics Mechanisms of Drug Action Chemical Signaling and Receptor Function Models of Drug-Receptor Interaction Affinity and Intrinsic Activity Agonists, Antagonists, and Partial Agonists Differential Effects of Agonists Cholinergic Neurotransmission Drug-Induced Enzyme Inhibition Drug Effects Mediated through Enzyme Inhibition Competitive, Uncompetitive, and Noncompetitive Inhibition Examples of Drug-Enzyme Interactions Pharmacological Effects Produced by Transpeptidase-Penicillinase Inhibition Suicide Inhibition of Enzymes References Drug-Receptor Dynamics and Theories Occupation Theory (Clark) Modified Occupancy Theory (Arien) Rate Theory (Paton) Relationship between Concentration and Response Concentration-Effect Curves Drug Antagonism Schilds Equation What Is the Importance of This Mathematical Relationship? Irreversible Antagonism Noncompetitive Antagonism Partial Agonists Various Factors That Can Regulate a Drugs Effect Efficacy and Potency Therapeutic Index Time-Action Curves Practice Problems Answers for the Problems References Receptor Regulation and Signaling Mechanisms Spare Receptors Overshoot Down Regulation Other Factors That Can Affect Drug Response Receptor Signaling and Second Messenger Systems Hormones Acting through Intracellular Receptors Receptor-Coupled (Membrane-Bound) Guanylate Cyclase (GC) Soluble Guanylate Cyclase Receptors Linked to G-Proteins and Second Messenger Production Activation of Adenylate Cyclase and G-Protein Function Downstream cAMP Second Messenger Pathway Ca++ /Phosphoinositide/PKC Signaling Pathway What Is the Purpose of G-Proteins or Any Other Second Messenger System? Ligand-Gated Ion Channels Signaling through Voltage-Dependent Ion Channels Ligand-Gated Sodium Channel Calcium Channels Location(s) of the voltage-sensitive Ca2+ channels (VSCC): How Does Calcium Contract the Skeletal Muscles and the Vascular Smooth Muscles? Chloride Channel References Index

Identification of HDM2 as a regulator of VEGF expression in cancer cells

Overexpression of vascular endothelial growth factor (VEGF) and the extent of neoangiogenesis are closely correlated with tumor development and cancer metastases. To assess whether VEGF mediated angiogenesis is regulated by HDM2, we treated the GI-101A and HL-60 cells with HDM2 gene specific antisense phosphorothioate oligodeoxynucleotide (AS5). The antisense treatment resulted in a significant reduction of both basal as well as phorbol 12,13-dibutyrate (PDB) and Diethylstilbestrol (DES) induced VEGF mRNA and protein expressions. Furthermore, when the Human Umbilical Vein Endothelial Cells (HUVECs) were exposed to medium obtained from AS5 transfected GI-101A and HL-60 cells, the angiogenesis was significantly reduced compared to the controls in the in vitro angiogenesis assay. On the contrary, the medium obtained from PDB treated cells that expressed HDM2 and VEGF at a higher level showed an increase in the tube formation by HUVEC. Thus, our present study suggests that modulation of HDM2 expression could play an important role in tumor angiogenesis and the metastatic process via transcriptional regulation of VEGF.

Effect of cisplatin on dopamine release from PC12 cells.

The effect of cisplatin, carboplatin and cyclophosphamide treatments on the neurotransmitter release process and the changes in intracellular Ca2+ level was determined by using PC12 cell experimental model. The neurotransmitter releasing ability of selected anticancer drugs was assessed by estimating the amount of dopamine release induced by them from PC12 cells. Among the three anticancer drugs tested in this study, cisplatin induced a maximum release of dopamine from PC12 cells. In addition, cisplatin pretreatment significantly increased the dopamine release stimulated by carbachol (0.5 mM) and KCl (55 mM). The additive effect exerted by cisplatin over KCl (55 mM) induced dopamine release was effectively blocked by verapamil (10 microM). The intracellular Ca2+ studies performed with cisplatin, carbachol and KCl (55 mM) treatments indicated that cisplatin does not induce any changes in the intracellular Ca2+ levels on its own, however it potentiates the effect of carbachol and KCl. Our study suggests that PC12 cells can be used as an experimental model to test the neurotransmitter releasing ability of emetic anticancer drugs.

Matrix Metalloproteinases: A challenging paradigm of cancer management

Matrix metalloproteinases (MMPs) are members of zinc-dependent endopeptidases implicated in a variety of physiological and pathological processes. Over the decades, MMPs have been studied for their role in cancer progression, migration, and metastasis. As a result, accumulated evidence of MMPs incriminating role has made them an attractive therapeutic target. Early generations of broad-spectrum MMP inhibitors exhibited potent inhibitory activities, which subsequently led to clinical trials. Unexpectedly, these trials failed to meet the desired goals, mainly due to the lack of efficacy, poor oral bioavailability, and toxicity. In this review, we discuss the regulatory role of MMPs in cancer progression, current strategies in targeting MMPs for cancer treatment including prodrug design and tumor imaging, and therapeutic value of MMPs as biomarkers in breast, lung, and prostate cancers.

Regulation of mdm2 mRNA expression in human breast tumor-derived GI-101A cells.

The MDM2 oncoprotein (p90) binds to p53 and inhibits its function. Here, the expression of mdm2 mRNA subsequent to phorbol 12,13-dibutyrate (PDB) or diethylstilbestrol (DES) treatment was analyzed in human breast tumor-derived GI-101A cell line. Expression of mdm2 mRNA was detected in rapidly growing GI-101A cells and that was similar to the expression seen in HL-60 cells. On the other hand PC12 (rat adrenal pheochromocytoma cells) did not show any mdm2 expression. GI-101A cells were treated with varying concentrations of DES or PDB, and mdm2 mRNA levels were determined by RT-PCR analysis. The RT-PCR results clearly showed that mdm2 mRNA expression was increased with increasing concentrations of PDB and DES treatments. To determine the specificity of the effects produced by DES and PDB the cells were treated with estrogen receptor antagonist tamoxifen and protein kinase C (PKC) specific inhibitor chelerythrine. Tamoxifen and chelerythrine co-treatments inhibited DES and PDB stimulated increases of mdm2 transcription respectively, in GI-101A cells. In an attempt to determine the upstream signaling pathway, the effects of PDB or DES on the mitogen activated protein kinase (MAPK) levels were determined by western blot analysis in the presence and absence of PD098059, a specific inhibitor of mitogen activated protein kinase kinase (MAPKK). The phospho-MAPK (p44/42) levels, an activated form of MAPK, increased in DES and PDB stimulated cells whereas PD098059 treatment inhibited this increase. Our data implicate MAPK as an upstream regulator of mdm2 expression and help to speculate on the intracellular regulation of mdm2 expression.