Maryam Zahin

Punicalagin and Ellagic Acid Demonstrate Antimutagenic Activity and Inhibition of Benzo[a]pyrene Induced DNA Adducts

Punicalagin (PC) is an ellagitannin found in the fruit peel of Punica granatum. We have demonstrated antioxidant and antigenotoxic properties of Punica granatum and showed that PC and ellagic acid (EA) are its major constituents. In this study, we demonstrate the antimutagenic potential, inhibition of BP-induced DNA damage, and antiproliferative activity of PC and EA. Incubation of BP with rat liver microsomes, appropriate cofactors, and DNA in the presence of vehicle or PC and EA showed significant inhibition of the resultant DNA adducts, with essentially complete inhibition (97%) at 40 μM by PC and 77% inhibition by EA. Antimutagenicity was tested by Ames test. PC and EA dose-dependently and markedly antagonized the effect of tested mutagens, sodium azide, methyl methanesulfonate, benzo[a]pyrene, and 2-aminoflourine, with maximum inhibition of mutagenicity up to 90 percent. Almost all the doses tested (50–500 μM) exhibited significant antimutagenicity. A profound antiproliferative effect on human lung cancer cells was also shown with PC and EA. Together, our data show that PC and EA are pomegranate bioactives responsible for inhibition of BP-induced DNA adducts and strong antimutagenic, antiproliferative activities. However, these compounds are to be evaluated in suitable animal model to assess their therapeutic efficacy against cancer.

Scalable Production of HPV16 L1 Protein and VLPs from Tobacco Leaves.

Cervical cancer is the most common malignancy among women particularly in developing countries, with human papillomavirus (HPV) 16 causing 50% of invasive cervical cancers. A plant-based HPV vaccine is an alternative to the currently available virus-like particle (VLP) vaccines, and would be much less expensive. We optimized methods to express HPV16 L1 protein and purify VLPs from tobacco (Nicotiana benthamiana) leaves transfected with the magnICON deconstructed viral vector expression system. L1 proteins were extracted from agro-infiltrated leaves using a series of pH and salt mediated buffers. Expression levels of L1 proteins and VLPs were verified by immunoblot and ELISA, which confirmed the presence of sequential and conformational epitopes, respectively. Among three constructs tested (16L1d22, TPL1d22, and TPL1F), TPL1F, containing a full-length L1 and chloroplast transit peptide, was best. Extraction of HPV16 L1 from leaf tissue was most efficient (> 2.5% of total soluble protein) with a low-salt phosphate buffer. VLPs were purified using both cesium chloride (CsCl) density gradient and size exclusion chromatography. Electron microscopy studies confirmed the presence of assembled forms of HPV16 L1 VLPs. Collectively; our results indicated that chloroplast-targeted transient expression in tobacco plants is promising for the production of a cheap, efficacious HPV16 L1 VLP vaccine. Studies are underway to develop plant VLPs for the production of a cervical cancer vaccine.

Human papillomavirus E7 serology and association with p16 immunohistochemistry in squamous cell carcinoma of the head and neck

BACKGROUND Human papillomavirus (HPV)-positive oropharyngeal cancer is associated with improved survival and treatment response as compared to HPV-negative cancers. P16 overexpression is widely accepted as a surrogate marker for HPV positivity. METHODS A total of 92 serum samples from 75 head and neck squamous cell carcinoma (HNSCC) patients were examined for HPV16 and 18 E7 antibodies by ELISA. Available tissue was tested for HPV-DNA by PCR, and p16 immunohistochemistry was obtained from a deidentified database. RESULTS Of 75 HNSCC patients, 25 were HPV E7 seropositive. Seropositivity was strongly associated with cancers of the oropharynx, and correlated with positive p16 immunohistochemistry (IHC) and HPV-DNA. Post-treatment serum was available in a limited subset of patients, revealing a decrease in antibody titers following response to treatment. CONCLUSIONS HPV E7 seropositivity correlated with positive tumor HPV-DNA and p16 expression, and was strongly associated with cancers of the oropharynx. E7 serology warrants further study as a potential biomarker in HPV-positive HNSCC.

Targeting synthetic Human Papillomavirus (HPV) L2 disulfide-induced N-terminus conformational epitopes for pan-HPV vaccine development

BACKGROUND Current vaccines against Human Papillomavirus (HPV) are highly effective and based on recombinant virus-like particles (VLPs) of the major capsid protein L1. Since these vaccines are HPV type-specific and expensive for global implementation, an alternative, broader-spectrum immunogen would be the N-terminus of the minor capsid protein L2 that induces low titered broadly cross-neutralizing antibodies. Here we analyzed the reactivity of different synthetic L2 peptides containing N-terminus amino acids 17-36 in order to test their antigenicity. METHODS Different synthetic peptides were designed to target the 17-36 amino acid sequences, present in highly antigenic amino-terminus of L2 protein. Six different peptides including Cys22-Cys28 disulfide bonded cyclized L2 peptide were examined for their antigenicity against mouse monoclonal antibody RG-1 and rabbit polyclonal antisera to HPV L2 by enzyme-linked immunosorbent assay (ELISA). RESULTS Here we report that the cyclized form of synthetic L2 peptide, which is formed through Cys22-Cys28 disulfide bridges, has the highest reactivity to antibodies than other synthetic L2 peptides. CONCLUSION A cyclized L2 peptide has potential to be an excellent candidate to formulate a low-cost, broadly protective pan-oncogenic HPV vaccine.

Identification of G-quadruplex forming sequences in three manatee papillomaviruses

The Florida manatee (Trichechus manatus latirotris) is a threatened aquatic mammal in United States coastal waters. Over the past decade, the appearance of papillomavirus-induced lesions and viral papillomatosis in manatees has been a concern for those involved in the management and rehabilitation of this species. To date, three manatee papillomaviruses (TmPVs) have been identified in Florida manatees, one forming cutaneous lesions (TmPV1) and two forming genital lesions (TmPV3 and TmPV4). We identified DNA sequences with the potential to form G-quadruplex structures (G4) across the three genomes. G4 were located on both DNA strands and across coding and non-coding regions on all TmPVs, offering multiple targets for viral control. Although G4 have been identified in several viral genomes, including human PVs, most research has focused on canonical structures comprised of three G-tetrads. In contrast, the vast majority of sequences we identified would allow the formation of non-canonical structures with only two G-tetrads. Our biophysical analysis confirmed the formation of G4 with parallel topology in three such sequences from the E2 region. Two of the structures appear comprised of multiple stacked two G-tetrad structures, perhaps serving to increase structural stability. Computational analysis demonstrated enrichment of G4 sequences on all TmPVs on the reverse strand in the E2/E4 region and on both strands in the L2 region. Several G4 sequences occurred at similar regional locations on all PVs, most notably on the reverse strand in the E2 region. In other cases, G4 were identified at similar regional locations only on PVs forming genital lesions. On all TmPVs, G4 sequences were located in the non-coding region near putative E2 binding sites. Together, these findings suggest that G4 are possible regulatory elements in TmPVs.

Medicinal Plants and Phytocompounds: A Potential Source of Novel Antibiofilm Agents

Medicinal plants and plant-derived bioactive compounds are well known for their contribution to primary health care as a source novel drug discovery for various ailments. Emergence and spread of microbial drug resistance due to various mechanisms has impacted the efficacy of almost all old and new antibacterial drugs. The biofilm mode of microbial growth has significantly increased the survival strategies and resistance levels of microbes to drugs, making the treatment of infections more difficult. Currently, efforts are going on to develop novel strategies including targeting biofilms to treat infections. Various natural products are known to inhibit biofilm formation or preformed biofilms. In recent years medicinal plants and phytocompounds were reported with promising antibiofilm activity in vitro from different parts of the world. In this chapter we have reviewed the current literature on antibiofilm agents derived from medicinal plants and/or plant-derived compounds. Plant extracts and phytocompounds of various classes have been found effective against bacterial or fungal biofilms, with some compounds showing activity against both. Such compounds are expected to be effective against mixed biofilms. Interestingly, certain quorum-sensing inhibiting plant extracts or compounds can also inhibit biofilms made by bacteria such as Pseudomonas aeruginosa. The majority of the antibiofilm phytocompounds identified so far have been tested in vitro; however, only a few compounds have been reported effective under in vivo condition. This could be due to the lack of access of the investigators to suitable animal models for different diseases to assess the therapeutic efficacy of these antibiofilm agents. The results of this chapter indicated that the phytocompounds may be effective alone or in combination with antibiotics, as in the treatment of systemic infection. However, further investigation on their mode of action and in vivo efficacy are prerequisites to obtain broad-spectrum antifungal agents of clinical value.

Viral DNA integration and methylation of human papillomavirus type 16 in high-grade oral epithelial dysplasia and head and neck squamous cell carcinoma

This study evaluated the integration and methlyation of human papillomavirus type 16 (HPV16) in head and neck squamous cell carcinoma (HNSCC) and its oral precursor, high-grade oral epithelial dysplasia (hgOED). Archival samples of HPV16-positive hgOED (N = 19) and HNSCC (N = 15) were evaluated, along with three HNSCC (UMSCC-1, -47 and -104) and two cervical cancer (SiHa and CaSki) cell lines. HgOED cases were stratified into three groups with increasing degrees of cytologic changes (mitosis, karyorrhexis and apoptosis). The viral load was higher and the E2/E6 ratio lower (indicating a greater tendency toward viral integration) in group 3 than in groups 1 or 2 (p = 0.002, 0.03). Methylation was not observed in hgOED cases and occurred variably in only three HNSCC cases (26.67%, 60.0% and 93.3%). In HNSCC cell lines, lower E7 expression correlated with higher levels of methylation. HgOED with increased cytologic change, now termed HPV-associated oral epithelial dysplasia (HPV-OED), exhibited an increased viral load and a tendency toward DNA integration, suggesting a potentially increased risk for malignant transformation. More detailed characterization and clinical follow-up of HPV-OED patients is needed to determine whether HPV-OED is a true precursor to HPV-associated HNSCC and to clarify the involvement of HPV in HNSCC carcinogenesis.

T cell-mediated antitumor immune response eliminates skin tumors induced by mouse papillomavirus, MmuPV1

Previous studies of naturally occurring mouse papillomavirus (PV) MmuPV1-induced tumors in B6.Cg-Foxn1nu/nu mice suggest that T cell deficiency is necessary and sufficient for the development of such tumors. To confirm this, MmuPV1-induced tumors were transplanted from T cell-deficient mice into immunocompetent congenic mice. Consequently, the tumors regressed and eventually disappeared. The elimination of MmuPV1-infected skin/tumors in immunocompetent mice was consistent with the induction of antitumor T cell immunity. This was confirmed by adoptive cell experiments using hyperimmune splenocytes collected from graft-recipient mice. In the present study, such splenocytes were injected into T cell-deficient mice infected with MmuPV1, and they eliminated both early-stage and fully formed tumors. We clearly show that anti-tumor T cell immunity activated during tumor regression in immunocompetent mice effectively eliminates tumors developing in T cell-deficient congenic mice. The results corroborate the notion that PV-induced tumors are strongly linked to the immune status of the host, and that PV antigens are major anti-tumor antigens. Successful anti-PV T cell responses should, therefore, lead to effective anti-tumor immune therapy in human PV-infected patients.

Broad Spectrum Antioxidant Properties of 20 Indian Medicinal Plants

ABSTRACT Antioxidant properties of methanol extracts of 20 plants were studied by four different assays over a range of concentrations (12.5–400 µg mL−1), and compared with standard antioxidants’ ascorbic acid and butylated hydroxytoluene. Ascorbic acid–equivalent antioxidant activity of extracts by the phosphomolybdenum method ranged from 392.8 to 2942.7 µmoles g−1 at 400 µg mL−1, whereas DPPH radical scavenging was between 30.9% and 95.2%; extracts also showed strong activity by FRAP and CUPRAC assays. The extracts demonstrated dose-dependent activity and strong correlation between antioxidant activities and total phenolics. Total phenolic content was the highest in Syzygium aromaticum (279 mg GAE g−1) and the lowest in Sesame indicum (28.16 mg GAE g−1).