Rajagopalan Sridhar

Techniques for physicochemical characterization of nanomaterials

Advances in nanotechnology have opened up a new era of diagnosis, prevention and treatment of diseases and traumatic injuries. Nanomaterials, including those with potential for clinical applications, possess novel physicochemical properties that have an impact on their physiological interactions, from the molecular level to the systemic level. There is a lack of standardized methodologies or regulatory protocols for detection or characterization of nanomaterials. This review summarizes the techniques that are commonly used to study the size, shape, surface properties, composition, purity and stability of nanomaterials, along with their advantages and disadvantages. At present there are no FDA guidelines that have been developed specifically for nanomaterial based formulations for diagnostic or therapeutic use. There is an urgent need for standardized protocols and procedures for the characterization of nanoparticles, especially those that are intended for use as theranostics.

Chemoprevention of DMBA-induced UV-B promoted, NOR-1-induced TPA promoted skin carcinogenesis, and DEN-induced phenobarbital promoted liver tumors in mice by extract of beetroot.

Our previous studies identified the extract of Beta vulgaris (beetroot), commercially also known as betanin, as a potent cancer chemopreventive agent in both in vitro Epstein-Barr early antigen activation assay and in an in vivo two-stage mouse lung and skin carcinogenesis. To explore this issue further, we have now investigated its cancer chemopreventive potentials in three different chemical carcinogen initiation-promotion experimental tumor models in mice. Following tumor initiation with 390 nmol of 7,12-dimethylbenz(a)anthracene (DMBA) in 100 microl of acetone, the mouse skin tumor promotion with 3430 J/m(2) of ultraviolet light-B (UV-B) as well as splenomegaly was significantly inhibited by oral administration of 0.0025% betanin. At the same dose, betanin also afforded significant protection in the mouse skin cancer model following the topical application of 390 nmol of (+/-)-(E)-4-methyl-2-[(E)-hydroxyamino]-5-nitro-6-methoxy-3-hexanamide (NOR-1) in 100 microl of acetone and promoted by topical administration of 1.7 nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA). In the two-stage model of hepatocarcinogenesis in mice with N-nitrosodiethylamine (DEN, 30 mg/kg) as the initiator and phenobarbital as the promoter, oral administration of 0.0025% betanin also showed a very significant inhibition of both the incidence and multiplicity of the liver tumors. These findings along with our initial reports suggest that betanin which is a regularly consumed natural product colorant is an effective cancer chemopreventive agent in mice. The most interesting observation is that the cancer chemopreventive effect was exhibited at a very low dose used in the study and thus indicating that beetroot warrants more attention for possible human applications in the control of malignancy.

Protein kinases as therapeutic targets.

Protein kinases and phosphatases are likely targets for the development of therapeutic drugs since they are involved in specific signaling pathways which regulate cell functions such as metabolism, cell cycle progression, cell adhesion, vascular function and angiogenesis. Protein phosphorylation and dephosphorylation serve as molecular switches for modulating these processes and the level and duration of each is a highly regulated process in normal cells. Several compounds that inhibit the activity of tyrosine kinases are being evaluated as cancer therapeutic agents in clinical trials. Diabetes and complications of diabetes also involve deregulated levels of protein kinases. New approaches for regulating kinase gene expression include specific antisense oligonucleotides for inhibiting post-transcriptional processing of the messenger RNA, naturally occurring products and their chemical derivatives to inhibit kinase activity, and monoclonal antibodies to inhibit receptor linked kinases. Inhibition of phosphatases also serves to alter the duration of phosphorylation by kinases. Considerations for development of effective inhibitors include non-specific actions of compounds, cellular uptake, multiple intracellular targets that can dilute the effective cellular concentration of an agent, and tissue specificity. Kinase inhibitors may allow other therapeutic agents additional time to become effective and they may act synergistically with current treatments.

Vitamin E Succinate Induces Ceramide-Mediated Apoptosis in Head and Neck Squamous Cell Carcinoma In vitro and In vivo

Purpose: Vitamin E succinate (α-TOS) inhibits the growth of cancer cells without unacceptable side effects. Therefore, the mechanisms associated with the anticancer action of α-TOS, including ceramide-mediated apoptosis, were investigated using head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo. Experimental Design: Five different human HNSCC cell lines (JHU-011, JHU-013, JHU-019, JHU-022, and JHU-029) were treated with α-TOS, and its effects on cell proliferation, cell cycle progression, ceramide-mediated apoptosis, and ceramide metabolism were evaluated. The anticancer effect of α-TOS was also examined on JHU-022 solid tumor xenograft growth in immunodeficient mice. Results: α-TOS inhibited the growth of all the HNSCC cell lines in vitro in a dose- and time-dependent manner. Thus, JHU-013 and JHU-022 cell lines were more sensitive to α-TOS than the other cell lines. Cellular levels of ceramide, sphingomyelinase activity, caspase-3, and p53 were elevated with increasing time of exposure to α-TOS. The degradation of poly(ADP-ribose) polymerase protein in JHU-022 cells treated with α-TOS provided evidence for apoptosis. The amounts of nuclear factor κB, Bcl-2, and Bcl-XL proteins were reduced in the cells treated with α-TOS for 6 hours. The levels of caspase-9, murine double minute-2, and IκB-α proteins were unchanged after α-TOS treatment. I.p. administration of α-TOS slowed tumor growth in immunodeficient mice. Conclusions: α-TOS showed promising anticancer effects to inhibit HNSCC growth and viability in vivo and in vitro. The induction of enzymes involved in ceramide metabolism by α-TOS suggests that ceramide-mediated apoptosis may expand therapeutic strategies in the treatment of carcinoma.

Salvianolic acid B inhibits growth of head and neck squamous cell carcinoma in vitro and in vivo via cyclooxygenase-2 and apoptotic pathways

Overexpression of cyclooxygenase‐2 (COX‐2) in oral mucosa has been associated with increased risk of head and neck squamous cell carcinoma (HNSCC). Celecoxib is a nonsteroidal anti‐inflammatory drug, which inhibits COX‐2 but not COX‐1. This selective COX‐2 inhibitor holds promise as a cancer preventive agent. Concerns about cardiotoxicity of celecoxib, limits its use in long‐term chemoprevention and therapy. Salvianolic acid B (Sal‐B) is a leading bioactive component of Salvia miltiorrhiza Bge, which is used for treating neoplastic and chronic inflammatory diseases in China. The purpose of this study was to investigate the mechanisms by which Sal‐B inhibits HNSCC growth. Sal‐B was isolated from S. miltiorrhiza Bge by solvent extraction followed by 2 chromatographic steps. Pharmacological activity of Sal‐B was assessed in HNSCC and other cell lines by estimating COX‐2 expression, cell viability and caspase‐dependent apoptosis. Sal‐B inhibited growth of HNSCC JHU‐022 and JHU‐013 cells with IC50 of 18 and 50 μM, respectively. Nude mice with HNSCC solid tumor xenografts were treated with Sal‐B (80 mg/kg/day) or celecoxib (5 mg/kg/day) for 25 days to investigate in vivo effects of the COX‐2 inhibitors. Tumor volumes in Sal‐B treated group were significantly lower than those in celecoxib treated or untreated control groups (p < 0.05). Sal‐B inhibited COX‐2 expression in cultured HNSCC cells and in HNSCC cells isolated from tumor xenografts. Sal‐B also caused dose‐dependent inhibition of prostaglandin E2 synthesis, either with or without lipopolysaccharide stimulation. Taken together, Sal‐B shows promise as a COX‐2 targeted anticancer agent for HNSCC prevention and treatment.

Inactivation of lysozyme by sonication under conditions relevant to microencapsulation.

Controlled release dosage forms of proteins and other biomolecules can be prepared by microencapsulating them in polymeric microspheres. Proteins are subjected to potentially damaging effects of sonication and exposure to organic solvents during the microencapsulation process. The relatively stable enzyme lysozyme was dissolved in aqueous buffer and sonicated in the presence of methylene chloride to mimic the initial step of the microencapsulation process. The stability of lysozyme was evaluated by determining the enzyme activity before and after sonication, size-exclusion chromatography, native polyacrylamide gel electrophoresis, and by measuring the amount of precipitates formed. Following sonication, the total protein introduced was distributed between a soluble and an insoluble fraction. Sonication of lysozyme solutions in the presence of methylene chloride led to an increase in precipitates. The precipitates were enzymatically inactive, did not dissolve easily, and were held by non-covalent interactions. No fragments or aggregates of lysozyme were detectable in the soluble fraction. Sonicating aqueous lysozyme solutions with and without methylene chloride decreased the specific activity of the enzyme in the soluble fraction. Excipients such as dimethyl sulfoxide (DMSO), mannitol, sucrose, and tween 80 were included in the sonication mixtures containing lysozyme. With the exception of tween 80, the addition of the excipients to aqueous solutions of lysozyme led to a greater decrease in the specific activity of lysozyme when sonicated in the presence of methylene chloride. DMSO caused the greatest loss of enzyme activity following sonication. Sonication of lysozyme with water, methylene chloride, and DMSO yielded methyl radicals, which were trapped with alpha-phenyl N-tert-butylnitrone and detected by ESR spectroscopy.

Investigation of muscle lipid metabolism by localized one- and two-dimensional MRS techniques using a clinical 3T MRI/MRS scanner

To demonstrate the feasibility of estimating the relative intra‐ and extramyocellular lipid (IMCL and EMCL) pool magnitudes and calculating the degree of lipid unsaturation within soleus muscle using single‐voxel localized one‐ and two‐dimensional (1D and 2D) MR spectroscopy (MRS).

A shift from normal to high glucose levels stimulates cell proliferation in drug sensitive MCF-7 human breast cancer cells but not in multidrug resistant MCF-7/ADR cells which overproduce PKC-βII

Glucose concentration may be an important factor in breast cancer cell proliferation because the prevalence of breast cancer is high in diabetic patients. To determine the role of protein kinase C (PKC)‐βII in regulating MCF‐7 cell proliferation at different glucose concentrations, the effects of glucose and a PKC‐βII‐specific inhibitor (CGP53353) were examined in cultures of MCF‐7 human breast cancer cell line and its multidrug resistant variant (MCF‐7/ADR). Cell proliferation and DNA synthesis of MCF‐7 were increased when glucose concentration in the culture medium was increased from normal (5.5 mM) to high (25 mM) levels. However, MCF‐7/ADR cell proliferation and DNA synthesis were unaffected by the increase in glucose. PKC‐βII protein and the corresponding mRNA levels were 4‐ to 5‐fold higher in MCF‐7/ADR than in MCF‐7 cells. High glucose‐induced decreases of PKC‐βII protein and mRNA levels were observed during the DNA synthesis phase in MCF‐7 but not in MCF‐7/ADR cells. Decreases in PKC‐βII mRNA and protein levels below a critical threshold in response to high glucose levels may account for glucose‐stimulated proliferation of MCF‐7 cells. Cultures of multidrug resistant MCF‐7/ADR cells reach maximal cell density in medium containing normal (5.5 mM) glucose levels and are not induced to grow further in response to high (25 mM) glucose. Our results demonstrate a link between high glucose‐induced PKC‐βII isozyme down‐regulation with concomitant acceleration of cell cycle progression in MCF‐7 cells. Int. J. Cancer 83:98–106, 1999.

Cancer chemopreventive activity of synthetic colorants used in foods, pharmaceuticals and cosmetic preparations

In continuation with our studies to uncover cancer chemopreventive effects of non-toxic natural colorants and other products of biologic and synthetic origin, we tested several Food and Drug Administration-approved synthetic colorants for antitumor promoting potential by the in vitro Epstein-Barr virus early antigen activation in Raji cells in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Among 29 such colorants used in foods, pharmaceuticals and cosmetics and evaluated in vitro, six of the 10 most effective had an azo group. Three structurally unrelated colorants tested in this assay were also studied in vivo for chemoprevention of 7,12-dimethylbenz[a]anthracene (DMBA)-induced TPA-promoted mouse skin carcinogenesis. The results indicate that tartrazine, indigo carmine and erythrosine are potent inhibitors of skin tumor promotion in mice treated with DMBA and TPA.

Visualizing Head and Neck Tumors In Vivo Using Near- Infrared Fluorescent Transferrin Conjugate

Transferrin receptor (TfR) is overexpressed in human head and neck squamous cell carcinomas (HNSCCs). This study was carried out to investigate the feasibility of imaging HNSCC by targeting TfR using near-infrared fluorescent transferrin conjugate (TfNIR). Western blot analysis of four HNSCC cell lines revealed overexpression of TfR in all four lines compared with that in normal keratinocytes (OKFL). Immunocytochemistry further confirmed the expression of TfR and endocytosis of TfNIR in JHU-013 culture cells. Following intravenous administration of TfNIR (200 μL, 0.625 μg/μL), fluorescent signal was preferentially accumulated in JHU-013 tumor xenografts grown in the lower back (n = 14) and oral base tissues (n = 4) of nude mice. The signal in tumors was clearly detectable as early as 10 minutes and reached the maximum at 90 to 120 minutes postinjection. The background showed an increase, followed by a decrease at a much faster pace than tumor signal. A high fluorescent ratio of the tumor to muscle was obtained (from 1.42 to 4.15 among tumors), usually achieved within 6 hours, and correlated with the tumor size (r = .74, p = .002). Our results indicate that TfR is a promising target and that TfNIR-based optical imaging is potentially useful for noninvasive detection of early HNSCC in the clinic.