Rita Mulherkar

Identification of human papillomaviruses in tumors of the oral cavity in an Indian community

Oral cancers and other squamous cell cancers of the head and neck are common cancers in India, primarily due to tobacco chewing/smoking and alcohol consumption. Recent reports indicate involvement of human papillomavirus (HPV), HPV 16, in a subset of squamous cell carcinoma of head and neck (SCCHN) cases. To investigate the types of HPVs present in 83 oral cancers and 19 other head and neck tumors, degenerate primers directed to consensus regions in the HPV L1 open reading frame (ORF) were employed to amplify genomic DNA from tumor and when available, the adjacent normal mucosa. PCR‐amplified products were cloned and sequenced. Similar studies were done on exfoliated buccal cells of 102 individuals visiting a dental hospital for dental complaints. HPV was detected in 32 out of 102 patients (31%), in either the tumor or the adjacent normal mucosa, while 5% (5/102) of the comparative group were found to be HPV‐positive. Sequence analysis revealed a number of cutaneous HPVs, predominantly HPV types of the genus Beta‐Papillomavirus, in the oral cavity. Multiple HPV infections were also commonly observed in patients (14/102; 14%). HPV 16 and 18 were each detected in 6 patients (6/102; 6%). Neither high‐risk HPVs nor multiple infections were observed in the mouthwash samples of the comparative group. We report that the oral cavity harbors a variety of different HPVs. These viruses, in conjunction with the carcinogens present in tobacco could contribute to carcinogenesis.

HDAC inhibitor valproic acid enhances tumor cell kill in adenovirus-HSVtk mediated suicide gene therapy in HNSCC xenograft mouse model.

Safety, efficacy and enhanced transgene expression are the primary concerns while using any vector for gene therapy. One of the widely used vectors in clinical trials is adenovirus which provides a safe way to deliver the therapeutic gene. However, adenovirus has poor transduction efficiency in vivo since most tumor cells express low coxsackie and adenovirus receptors. Similarly transgene expression remains low, possibly because of the chromatization of adenoviral genome upon infection in eukaryotic cells, an effect mediated by histone deacetylases (HDACs). Using a recombinant adenovirus (Ad‐HSVtk) carrying the herpes simplex thymidine kinase (HSVtk) and GFP genes we demonstrate that HDAC inhibitor valproic acid can bring about an increase in CAR expression on host cells and thereby enhanced Ad‐HSVtk infectivity. It also resulted in an increase in transgene (HSVtk and GFP) expression. This, in turn, resulted in increased cell kill of HNSCC cells, following ganciclovir treatment in vitro as well as in vivo in a xenograft nude mouse model.

Lecithin-based novel cationic nanocarriers (LeciPlex) I: fabrication, characterization and evaluation.

AIMS In the present investigation, the feasibility of fabricating novel self-assembled cationic nanocarriers (LeciPlex) containing cetyltrimethylammonium bromide (CTAB) or didodecyldimethylammonium bromide (DDAB) and soybean lecithin using pharmaceutically acceptable biocompatible solvents such as 2-Pyrrolidone (Soluphor P) and diethyleneglycol monoethyl ether (Transcutol) was established. MATERIALS & METHODS The interaction between DDAB/CTAB and soybean lecithin in the nanocarriers was confirmed by differential scanning calorimetry and in vitro antimicrobial studies. The positive charge on the nanocarriers was confirmed by zeta potential analysis. RESULTS Transmission electron microscopy analysis could not reveal sufficient information regarding the internal structure of the nanocarriers, whereas cryotransmission electron microscopy studies indicated that these novel nanocarriers have unilamellar structure. Small-angle neutron scattering studies confirmed interaction of cationic surfactant (DDAB) and lecithin in the nanocarriers and confirmed the presence of unilamellar nanostructures. CONCLUSION Various hydrophobic drugs could be encapsulated in the CTAB/DDAB-based lecithin nanocarriers (CTAB-LeciPlex or DDAB-LeciPlex) irrespective of their difference in log p-values. In vitro antimicrobial studies on triclosan-loaded LeciPlex confirmed entrapment of triclosan in the nanocarriers. The ability of CTAB-LeciPlex and DDAB-LeciPlex to condense plasmid DNA was established using agarose gel electrophoresis. DDAB-LeciPlex could successfully transfect pDNA in HEK-293 cells indicating potential in gene delivery.

Apoptosis induction and anti-cancer activity of LeciPlex formulations.

PurposeCationic agents have been reported to possess anti-neoplastic properties against various cancer cell types. However, their complexes with lipids appear to interact differently with different cancer cells. The purpose of this study was to (i) design and generate novel cationic lecithin nanoparticles, (ii) assess and understand the mechanism underlying their putative cytotoxicity and (iii) test their effect on cell cycle progression in various cancer-derived cell lines. In addition, we aimed to evaluate the in vivo potential of these newly developed nanoparticles in oral anti-cancer delivery.MethodsCationic lecithin nanoparticles were generated using a single step nanoprecipitation method and they were characterized for particle size, zeta potential, stability and in vitro release. Their cytotoxic potential was assessed using a sulforhodamine B assay, and their effect on cell cycle progression was evaluated using flow cytometry. The nanoparticle systems were also tested in vivo for their anti-tumorigenic potential.ResultsIn contrast to cationic agents alone, the newly developed nanoformulations showed a specific toxicity against cancer cells. The mechanism of toxic cell death included apoptosis, S and G2/M cell cycle phase arrest, depending on the type of cationic agent and the cancer-derived cell line used. Both blank and drug-loaded systems exhibited significant anti-cancer activity, suggesting a synergistic anti-tumorigenic effect of the drug and its delivery system.ConclusionsBoth in vitro and in vivo data indicate that cationic agents themselves exhibit broad anti-neoplastic activities. Complex formation of the cationic agents with phospholipids was found to provide specificity to the anti-cancer activity. These formulations thus possess potential for the design of effective anti-cancer delivery systems.

Cholic acid-modified polyethylenimine: in vitro and in vivo studies

Low-molecular-weight polyethylenimine has lower cytotoxicity than high molecular weight polyethylenimine, but it is not an efficient transfection agent because of limitations of DNA delivery into the cytoplasm. Therefore, in the present study, the hydrophobic modification of low-molecular-weight polyethylenimine (PEI 2 kDa [PEI2]) by cholic acid (ChA) was performed to form PEI2-ChA, and in vitro and in vivo studies were performed. Results indicate that the nanoplexes of PEI2-ChA with gWIZ-GFP have greater transfection efficiency (27%) in NT8e cell lines as evaluated by flow cytometry and also observed by fluorescence imaging. The present study concluded that the transferrin-containing nanoplexes of PEI2-ChA conjugates with plasmid p53 warrant clinical trials in humans after exhaustive animal studies for use as a novel gene delivery system.