Ability of Group IVB metallocene polyethers containing dienestrol to arrest the growth of selected cancer cell lines.

BACKGROUND: Monomeric Group IVB (Ti, Zr and Hf) metallocenes represent a new class of antitumor compounds. There is literature on the general biological activities of some organotin compounds. Unfortunately, there is little information with respect to the molecular level activity of these organotin compounds. We recently started focusing on the anti-cancer activity of organotin polymers that we had made for other purposes and as part of our platinum anti-cancer effort. METHODS: For this study, we synthesized a new series of metallocene-containing compounds coupling the metallocene unit with dienestrol, a synthetic, nonsteroidal estrogen. This is part of our effort to couple known moieties that offer antitumor activity with biologically active units hoping to increase the biological activity of the combination. The materials were confirmed to be polymeric using light scattering photometry and the structural repeat unit was verified employing matrix assisted laser desorption ionization mass spectrometry and infrared spectroscopy results. RESULTS: The polymers demonstrated the ability to suppress the growth of a series of tumor cell lines originating from breast, colon, prostrate, and lung cancers at concentrations generally lower than those required for inhibition of cell growth by the commonly used antitumor drug cisplatin. CONCLUSION: These drugs show great promise in vitro against a number of cancer cell lines and due to their polymeric nature will most likely be less toxic than currently used metal-containing drugs such as cisplatin. These drugs also offer several addition positive aspects. First, the reactants are commercially available so that additional synthetic steps are not needed. Second, synthesis of the polymer is rapid, occurring within about 15 seconds. Third, the interfacial synthetic system is already industrially employed in the synthesis of aromatic nylons and polycarbonates. Thus, the ability to synthesize large amounts of the drugs is straight forward.

The use of monoreassortants and reverse genetics to map reovirus lysis of a ras-transformed cell line.

Reovirus has been shown to lyse most transformed cells while establishing a persistent or abortive infection in non-transformed cells. Developing methods to identify the reovirus genes associated with oncolysis is an important step toward understanding the mechanisms involved. This report is the first to develop and apply the use of monoreassortants and reverse genetics to identify an individual reovirus gene associated with reovirus oncolysis. Infection with reovirus serotypes 1/Lang, 2/Jones or 3/Dearing of cells transformed with a normal copy of c-Ha-RAS (N1 cells) or with a normal copy of c-Myc (Myc-3 cells), produces large amounts of progeny virus of all three serotypes and results in lysis of both these cell lines. Infection of cells transformed with a mutant c-Ha-RAS gene (T1 cells) with either serotype 1/Lang and 2/Jones results in the production of large amounts of virus and lysis of the cells. In sharp contrast, serotype 3/Dearing virus infection of these cells produced small amounts of virus and resulted in limited lysis of these cells. Using monoreassortants and reverse genetics we exploited this phenotypic difference between the three serotypes to identify a single reovirus gene linked to the preferential lysis of the T1 cells, the S4 gene.

Tuning the Gold Nanoparticle Colorimetric Assay by Nanoparticle Size, Concentration, and Size Combinations for Oligonucleotide Detection.

Gold nanoparticle (GNP)-based aggregation assay is simple, fast, and employs a colorimetric detection method. Although previous studies have reported using GNP-based colorimetric assay to detect biological and chemical targets, a mechanistic and quantitative understanding of the assay and effects of GNP parameters on the assay performance is lacking. In this work, we investigated this important aspect of the GNP aggregation assay including effects of GNP concentration and size on the assay performance to detect malarial DNA. Our findings lead us to propose three major competing factors that determine the final assay performance including the nanoparticle aggregation rate, plasmonic coupling strength, and background signal. First, increasing nanoparticle size reduces the Brownian motion and thus aggregation rate, but significantly increases plasmonic coupling strength. We found that larger GNP leads to stronger signal and improved limit of detection (LOD), suggesting a dominating effect of plasmonic coupling strength. Second, higher nanoparticle concentration increases the probability of nanoparticle interactions and thus aggregation rate, but also increases the background extinction signal. We observed that higher GNP concentration leads to stronger signal at high target concentrations due to higher aggregation rate. However, the fact the optimal LOD was found at intermediate GNP concentrations suggests a balance of two competing mechanisms between aggregation rate and signal/background ratio. In summary, our work provides new guidelines to design GNP aggregation-based POC devices to meet the signal and sensitivity needs for infectious disease diagnosis and other applications.

The 3' sequences required for incorporation of an engineered ssRNA into the Reovirus genome.

BACKGROUND: Understanding how an organism replicates and assembles a multi-segmented genome with fidelity previously measured at 100% presents a model system for exploring questions involving genome assortment and RNA/protein interactions in general. The virus family Reoviridae, containing nine genera and more than 200 members, are unique in that they possess a segmented double-stranded (ds) RNA genome. Using reovirus as a model member of this family, we have developed the only functional reverse genetics system for a member of this family with ten or more genome segments. Using this system, we have previously identified the flanking 5' sequences required by an engineered s2 ssRNA for efficient incorporation into the genome of reovirus. The minimum 5' sequence retains 96 nucleotides and contains a predicted sequence/structure element. Within these 96 nucleotides, we have identified three nucleotides A-U-U at positions 79-81 that are essential for the incorporation of in vitro generated ssRNAs into new reovirus progeny viral particles. The work presented here builds on these findings and presents the results of an analysis of the required 3' flanking sequences of the s2 ssRNA. RESULTS: The minimum 3' sequence we localized retains 98 nucleotides of the wild type s2 ssRNA. These sequences do not interact with the 5' sequences and modifications of the 5' sequences does not result in a change in the sequences required at the 3' end of the engineered s2 ssRNA. Within the 3' sequence we discovered three regions that when mutated prevent the ssRNA from being replicated to dsRNA and subsequently incorporated into progeny virions. Using a series of substitutions we were able to obtain additional information about the sequences in these regions. We demonstrate that the individual nucleotides from, 98 to 84, 68 to 59, and 28 to 1, are required in addition to the total length of 98 nucleotides to direct an engineered reovirus ssRNA to be replicated to dsRNA and incorporated into a progeny virion. Extensive analysis using a number of RNA structure-predication software programs revealed three possible structures predicted to occur in all 10 reovirus ssRNAs but not predicted to contain conserved individual nucleotides that we could probe further by using individual nucleotide substitutions. The presence of a conserved structure would permit all ten ssRNAs to be identified and selected as a set, while unique nucleotides within the structure would direct the set to contain 10 unique members. CONCLUSION: This study completes the characterization and mapping of the 5' and 3' sequences required for an engineered reovirus s2 ssRNA to be incorporated into an infectious progeny virus and establishes a firm foundation for additional investigations into the assortment and encapsidation mechanism of all 10 ssRNAs into the dsRNA genome of reovirus. As researchers build on this work and apply this system to additional reovirus genes and additional dsRNA viruses, a complete model for genome assortment and replication for these viruses will emerge.

Characterization of in vivo anti-rotavirus activities of saponin extracts from Quillaja saponaria Molina.

Rotavirus is the leading cause of severe diarrhea disease in newborns and young children worldwide with approximately 300,000 pre-adolescent deaths each year. Quillaja saponins are a natural aqueous extract obtained from the Chilean soapbark tree. The extract is approved for use in humans by the FDA for use in beverages as a food addictive. We have demonstrated that Quillaja extracts have strong antiviral activities in vitro against six different viruses. In this study, we evaluated the in vivo antiviral activity of these extracts against rhesus rotavirus (RRV) using a mouse model. We established that at a dosage of 0.015 mg/mouse of saponin extract, RRV induced diarrhea can be significantly reduced from 79% to 11% when mice are exposed to 500 plaque-forming-units (PFU) for each of five consecutive days. Additionally, while a reduction of RRV induced diarrhea depended both on the concentration of virus introduced and on the amount of Quillaja extract given to each mouse, the severity and interval of diarrhea under a variety of conditions tested, in all the treated mice were greatly reduced when compared to those that did not receive the Quillaja extracts. Mechanistically, there is strong evidence that the Quillaja extracts are able to "block" rotavirus infection by inhibiting virus-host attachment through disruption of cellular membrane proteins and/or virus receptors. We believe that Quillaja extracts have promise as antivirals to reduce rotavirus infection and the severity of the disease in humans.

Effect of Electrical Conductivity Through the Bulk Doping of the Product of Titanocene Dichloride and 2-Nitro-1,4-phenylenediamine.

The condensation polymer derived from reaction between titanocene dichloride and 2-nitro-1,4-phenylenediamine was doped by mixing the polymer with different amounts of iodine. This bulk doping of the titanocene polyamine resulted in an increase in bulk conductivity from 10 to over 1,000 fold. Conductivity increased to a doping level of about 10 to 15% iodine. Conductivity decreased as the sample discs were heated returning to pre-doped levels after the samples were heated for eight minutes. It is believed that this decrease in conductivity is due to the surface evaporation of iodine as the samples were heated. MALDI MS and IR results are consistent with the formation of C-I compounds for doped materials.

Structural Consideration in Designing Organotin Polyethers to Arrest the Growth of Breast Cancer Cells In Vitro.

The ability to inhibit cancer is inherent in organotin materials yet the structural relationships that regulate/direct this activity remains unknown. We measured antitumor activity using a matched pair of cell lines MDA-MB-231 cells that are estrogen-independent, estrogen receptor negative and MCF-7 cells, a cell line that is estrogen receptor (ER) positive. Those polyethers that contained a O-phenyl unit were able to significantly inhibit the non-estrogen sensitive cell line but were much less effective against the estrogen sensitive cell line; that is, the human breast cancer cell line MDA-MB-231 showed better test results for polymers derived from diols containing the O-phenyl moiety than the breast cancer cell line MCF-7, a well-characterized estrogen receptor positive control cell line. Those polyethers that did not contain the O-phenyl unit inhibited both cell lines approximately the same. The differential activity of the O-phenyl-containing polyethers is likely due to the estrogen-sensitive cells combining with some of the organotin polyethers minimizing their ability to inhibit cell growth.

Antiviral Activity of Metal-Containing Polymers-Organotin and Cisplatin-Like Polymers.

Polymers containing platinum and to a lesser extent tin, have repeatedly demonstrated antitumor activity in vitro and in vivo against a variety of cell and tumor types. The mechanisms responsible for the antitumor activity include inducing a delay in cell proliferation and sister chromatid exchanges blocking tumor growth. As most DNA and some RNA viruses require, and even induce, infected cells to initiate DNA replication and subsequent cell division, compounds with antitumor activity will very likely also possess antiviral activity. This article examines the use of metal-containing polymers as a novel class of antivirals.

Prevention of rotavirus infections in vitro with aqueous extracts of Quillaja Saponaria Molina.

BACKGROUND: Rotavirus is the leading cause of severe diarrhea disease in newborns and young children worldwide, estimated to be responsible for over 300,000 childhood deaths every year, mostly in developing countries. Rotavirus-related deaths represent approximately 5% of all deaths in children younger than 5 years of age worldwide. Saponins are readily soluble in water and are approved by the US FDA for inclusion in beverages intended for human consumption. The addition of saponins to existing water supplies offers a new form of intervention into the cycle of rotavirus infection. We believe that saponins will 'coat' the epithelium of the host's small intestine and prevent attachment of rotavirus. DISCUSSION: This experiment provides in vitro data for the possibility of including saponin in drinking water to prevent infections of rotavirus. We demonstrate that microgram amounts of extract, while exhibiting no cell cytotoxicity or direct virucidal activity, prevent rotavirus from infecting its host cells. In addition, the presence of residual amounts of extract continue to block viral infection and render cells resistant to infection for at least 16 h after the removal of the extract from the cell culture media. CONCLUSION: We demonstrate that two Quillaja extracts possess strong antiviral activity at concentrations more than 1000-fold lower than concentrations exhibiting cell cytotoxicity. Extract concentrations as high as 1000 µg/ml are not cytotoxic, but concentrations as low as 1.0 µg/ml are able to block rotavirus and reovirus attachment and infection.

Antiviral and Anticancer Activity of Cisplatin Derivatives of Tilorone.

Cisplatin derivatives containing tilorone and a tilorone derivative were synthesized and characterized employing IR analysis, mass spectrometry, elemental analysis, and chain length. The polymeric cisplatin derivatives prevent growth of four transformed cell lines, L929, 143, Vero, and BS-C-1 and effectively inhibit four viruses including the DNA viruses; herpes simplex-1, vaccinia, and Varicella zoster, and the RNA virus, reovirus in the micrograms/mL range. The tilorone polymers display activity against both transformed cells and DNA and RNA viruses at biologically important concentrations.

Features of the mammalian orthoreovirus 3 Dearing l1 single-stranded RNA that direct packaging and serotype restriction.

A series of recombinant mammalian orthoreoviruses (mammalian orthoreovirus 3 Dearing, MRV-3De) were generated that express an MRV-3De lambda3-CAT fusion protein. Individual viruses contain L1CAT double-stranded (ds) RNAs that range in length from a minimum of 1020 bp to 4616 bp. The engineered dsRNAs were generated from in vitro-transcribed single-stranded (ss) RNAs and incorporated into infectious virus particles by using reverse genetics. In addition to defining the sequences required for these ssRNAs to be 'identified' as l1 ssRNAs, the individual nucleotides in these regions that 'mark' each ssRNA as originating from mammalian orthoreovirus 1 Lang (MRV-1La), mammalian orthoreovirus 2 D5/Jones (MRV-2Jo) or MRV-3De have been identified. A C at position 81 in the MRV-1La 5' 129 nt sequence was able to be replaced with a U, as normally present in MRV-3De; this toggled the activity of the MRV-1La ssRNA to that of an MRV-3De 5' l1. RNA secondary-structure predictions for the 5' 129 nt of both the biologically active MRV-3De l1 ssRNA and the U81-MRV-3De-restored MRV-1La 5' ssRNA predicted a common structure.

Localizing the reovirus packaging signals using an engineered m1 and s2 ssRNA.

Using in vitro engineered and transcribed reovirus m1 and s2 ssRNAs, we demonstrate that the nucleotides used to identify these ssRNAs are localized to the 5' and not the 3' termini. To demonstrate this, we used our previously reported S2-CAT reovirus and we report the creation of an engineered M1-CAT reovirus. The M1 gene of this virus retains 124 nucleotides from the wild type M1 gene preceding the CAT gene and 172 nucleotides from the wild type gene following the CAT gene. The engineered M1-CAT ssRNA is 1048 nucleotides in length, much shorter than the wild type M1 at 2304 nucleotides. We have used a set of chimeric s2.m1 ssRNAs to localize the packaging signals within these RNAs. By packaging signals we mean that the presence of these signals in engineered ssRNAs results in these ssRNAs being replicated to dsRNA and packaged into progeny virus. An engineered ssRNA with a 5' sequence identical with the wild type s2 ssRNA, supported by a 3' sequence from either the m1 or s2 ssRNA, is incorporated into a virus as an S2 dsRNA. Likewise, an ssRNA with an m1 5' end is incorporated as an M1 dsRNA.

Antiviral activity obtained from aqueous extracts of the Chilean soapbark tree (Quillaja saponaria Molina).

Natural, aqueous extracts of Quillaja saponaria, the Chilean soapbark tree, contain several physiologically active triterpenoid saponins that display strong adjuvant activity when used in either human or animal vaccines. In this paper, we describe studies that demonstrate a novel antiviral activity of Quillaja extracts against six viruses: vaccinia virus, herpes simplex virus type 1, varicella zoster virus, human immunodeficiency viruses 1 and 2 (HIV-1, HIV-2) and reovirus. We demonstrate that microgram amounts of extract, while exhibiting no cell cytotoxicity or direct virucidal activity, prevent each of the six viruses tested from infecting their host cells. In addition, the presence of residual amounts of extract continue to block virus infection and render cells resistant to infection for at least 16 h after the removal of the extract from the cell culture medium. We demonstrate that a Quillaja extract possesses strong antiviral activity at concentrations more than 100-fold lower than concentrations that exhibit cell cytotoxicity. Extract concentrations as high as 100 microg ml(-1) are not cytotoxic, but concentrations as low as 0.1 microg ml(-1) are able to block HIV-1 and HIV-2 virus attachment and infection.

Synthesis of Organotin Polyamine Ethers Containing Acyclovir and their Preliminary Anticancer and Antiviral Activity.

Organotin polyamine ethers containing acyclovir in their backbone were synthesized in moderate to high yield employing the aqueous interfacial polycondensation system. The products are high molecular weight polymers. Infrared spectroscopy of the products shows new bands characteristic of the formation of Sn-N and Sn-O bonds consistent with the proposed structure. MALDI-TOF MS below 2000 Da shows the presence of organotin and acyclovir units containing these two moieties. The products show moderate inhibition of a number of cancer cell lines and exhibit the ability to inhibit a number of viruses, particularly the herpes simplex virus-1 and varicella zoster virus that are responsible for herpes, chicken pox and shingles.

Identification of the 5' sequences required for incorporation of an engineered ssRNA into the Reovirus genome.

Using a reovirus reverse genetics system, we have identified the 5' sequences required of an engineered s2 ssRNA for efficient incorporation into the dsRNA genome of Reovirus. Employing an engineered, functionally active reovirus S2/CAT gene retaining the first 198 5' terminal nucleotides and the last 284 3' terminal nucleotides of the wild-type S2 segment, we have determined the 5' sequence required by a ssRNA to be recognized, replicated to dsRNA, and stably incorporated into an infectious reovirus. The 5' sequence retains 96 nucleotides of the wild-type s2 ssRNA and a predicted sequence-structure element. Within these 96 nucleotides, we have identified three nucleotides A-U-U at positions 79-81 that are essential for the incorporation of in vitro-generated ssRNAs into new reovirus progeny viral particles. This study establishes a firm foundation for additional investigation into the assortment and encapsidation mechanism of all 10 ssRNAs into the dsRNA genome of reovirus.