Dietary Polyphenols, Resveratrol and Pterostilbene Exhibit Antitumor Activity on an HPV E6-Positive Cervical Cancer Model: An in vitro and in vivo Analysis

Human papilloma virus (HPV)-induced cervical cancer is one of the most frequent cancers in women residing in underdeveloped countries. Natural compounds like polyphenols continue to be of scientific interest as non-toxic effective alternative treatments. Our previous work showed the efficacy of two polyphenols, resveratrol, and pterostilbene on human HeLa cells. Here we explored the in vitro anti-cancer activity and in vivo anti-tumor potential of these two structurally similar compounds on HPV oncogene E6 and E7 positive murine TC1 cells. In vitro analysis confirmed the cytotoxic potential of both resveratrol and pterostilbene compounds with each having a low IC50 value and each showing the ability to downregulate viral oncogene E6. Further in vivo studies on TC1 tumors developing in mice indicated that treatment with either resveratrol or pterostilbene can significantly inhibit tumor development, with both compounds capable of downregulating E6 and VEGF tumor protein levels. Interestingly, the decrease in tumor size in pterostilbene was associated with tumor cell apoptosis, as indicated by an upregulation of activated caspase-3 whereas in resveratrol-treated mice it was accompanied by arrest of cell cycle, as indicated by a downregulation of PCNA. Thus, resveratrol and pterostilbene can serve as potential antineoplastic agents against HPV E6+ tumors and may suppress tumor growth via two different mechanisms

Nongenomic Mechanisms of PTEN Regulation

A large amount of data supports the view that PTEN is a bona fide tumor suppressor gene. However, recent evidence suggests that derailment of cellular localization and expression levels of functional nonmutated PTEN is a determining force in inducing abnormal cellular and tissue outcomes. As the cellular mechanisms that regulate normal PTEN enzymatic activity resolve, it is evident that deregulation of these mechanisms can alter cellular processes and tissue architecture and ultimately lead to oncogenic transformation. Here we discuss PTEN ubiquitination, PTEN complex formation with components of the adherens junction, PTEN nuclear localization, and microRNA regulation of PTEN as essential regulatory mechanisms that determine PTEN function independent of gene mutations and epigenetic events

Dietary Polyphenols, Resveratrol and Pterostilbene Exhibit Antitumor Activity on an HPV E6-Positive Cervical Cancer Model: An in vitro and in vivo Analysis

Human papilloma virus (HPV)-induced cervical cancer is one of themost frequent cancers in women residing in underdeveloped countries. Natural compounds like polyphenols continue to be of scientific interest as non-toxic effective alternative treatments. Our previous work showed the efficacy of two polyphenols, resveratrol, and pterostilbene on human HeLa cells. Here we explored the in vitro anti-cancer activity and in vivo anti-tumor potential of these two structurally similar compounds on HPV oncogene E6 and E7 positive murine TC1 cells. In vitro analysis confirmed the cytotoxic potential of both resveratrol and pterostilbene compounds with each having a low IC50 value and each showing the ability to downregulate viral oncogene E6. Further in vivo studies on TC1 tumors developing in mice indicated that treatment with either resveratrol or pterostilbene can significantly inhibit tumor development, with both compounds capable of downregulating E6 and VEGF tumor protein levels. Interestingly, the decrease in tumor size in pterostilbene was associated with tumor cell apoptosis, as indicated by an upregulation of activated caspase-3 whereas in resveratrol-treated mice it was accompanied by arrest of cell cycle, as indicated by a downregulation of PCNA. Thus, resveratrol and pterostilbene can serve as potential antineoplastic agents against HPV E6+ tumors and may suppress tumor growth via two different mechanisms

The metastasis-promoting protein S100A4 regulates mammary branching morphogenesis

AbstractHigh levels of the S100 calcium binding protein S100A4 also called fibroblast specific protein 1 (FSP1) have been established as an inducer of metastasis and indicator of poor prognosis in breast cancer. The mechanism by which S100A4 leads to increased cancer aggressiveness has yet to be established; moreover, the function of this protein in normal mammary gland biology has not been investigated. To address the role of S100A4 in normal mammary gland, its spatial and temporal expression patterns and possible function in branching morphogenesis were investigated. We show that the protein is expressed mainly in cells of the stromal compartment of adult humans, and during active ductal development, in pregnancy and in involution of mouse mammary gland. In 3D culture models, topical addition of S100A4 induced a significant increase in the TGFα mediated branching phenotype and a concomitant increase in expression of a previously identified branching morphogen, metalloproteinase-3 (MMP-3). These events were found to be dependent on MEK activation. Downregulation of S100A4 using shRNA significantly reduced TGFα induced branching and altered E-cadherin localization. These findings provide evidence that S100A4 is developmentally regulated and that it plays a functional role in mammary gland development, in concert with TGFα by activating MMP-3, and increasing invasion into the fat pad during branching. We suggest that S100A4-mediated effects during branching morphogenesis provide a plausible mechanism for how it may function in breast cancer progression

Resveratrol and Pterostilbene Exhibit Anticancer Properties Involving the Downregulation of HPV Oncoprotein E6 in Cervical Cancer Cells

Cervical cancer is one of the most common cancers in women living in developing countries. Due to a lack of affordable effective therapy, research into alternative anticancer compounds with low toxicity such as dietary polyphenols has continued. Our aim is to determine whether two structurally similar plant polyphenols, resveratrol and pterostilbene, exhibit anticancer and anti-HPV (Human papillomavirus) activity against cervical cancer cells. To determine anticancer activity, extensive in vitro analyses were performed. Anti-HPV activity, through measuring E6 protein levels, subsequent downstream p53 effects, and caspase-3 activation, were studied to understand a possible mechanism of action. Both polyphenols are effective agents in targeting cervical cancer cells, having low IC50 values in the M range. They decrease clonogenic survival, reduce cell migration, arrest cells at the S-phase, and reduce the number of mitotic cells. These findings were significant, with pterostilbene often being more effective than resveratrol. Resveratrol and to a greater extent pterostilbene downregulates the HPV oncoprotein E6, induces caspase-3 activation, and upregulates p53 protein levels. Results point to a mechanism that may involve the downregulation of the HPV E6 oncoprotein, activation of apoptotic pathways, and re-establishment of functional p53 protein, with pterostilbene showing greater efficacy than resveratrol

Interaction of E-cadherin and PTEN regulates morphogenesis and growth arrest in human mammary epithelial cells

PTEN is a dual function phosphatase with tumor suppressor function compromised in a wide spectrum of cancers. Because tissue polarity and architecture are crucial modulators of normal and malignant behavior, we postulated that PTEN may play a role in maintenance of tissue integrity. We used two non-malignant human mammary epithelial cell lines (HMECs) that form polarized, growth-arrested structures (acini) when cultured in 3-dimensional laminin-rich extracellular matrix gels (3D lrECM). As acini begin to form, PTEN accumulates in both the cytoplasm, and at cell-cell contacts where it colocalizes with E-cadherin/{beta}-catenin complex. Reduction of PTEN levels by shRNA in lrECM prevents formation of organized breast acini and disrupts growth arrest. Importantly, disruption of acinar polarity and cell-cell contact by E-cadherin function-blocking antibodies reduces endogenous PTEN protein levels and inhibits its accumulation at cell-cell contacts. Conversely, in SKBR3 breast cancer cells lacking endogenous E-cadherin expression, exogenous introduction of E-cadherin gene causes induction of PTEN expression and its accumulation at sites of cell interactions. These studies provide evidence that E-cadherin regulates both the PTEN protein levels and its recruitment to cell-cell junctions in 3D lrECM indicating a dynamic reciprocity between architectural integrity and the levels and localization of PTEN. This interaction thus appears to be a critical integrator of proliferative and morphogenetic signaling in breast epithelial cells

A novel curcumin-based vaginal cream Vacurin selectively eliminates apposed human cervical cancer cells

Objective. Human papillomavirus (HPV) infections remain a leading cause of mortality worldwide. In the U.S. strategies via screening and vaccination prevent HPV-associated cervical neoplasms, but consume im- mense healthcare costs. The spice component curcumin has potent anticancer and antiviral properties, which have been difficult to harness as a treatment, due to its poor systemic bioavailability. This project tests the possibility of developing a curcumin-based therapy for cervical cancer. Methods. Using four HPV(+) cervical cancer cell lines and normal fibroblasts we first tested the selectivity and potency of curcumin in eliminating HPV(+) cells. Subsequently, we developed a curcumin-based cervical cream and tested its efficacy in eliminating apposed HPV(+) cells and also its possible side effects on the vaginal epithelium of healthy mice. Results. Curcumin selectively eliminates a variety of HPV(+) cervical cancer cells (HeLa, ME-180, SiHa, and SW756), suppresses the transforming antigen E6, dramatically inhibits the expression of the pro-cancer protein epidermal growth factor receptor (EGFR), and concomitantly induces p53. Additionally, Vacurin, a uniform colloidal solution of curcumin in a clinically used amphipathic vaginal cream, eliminates apposed HeLa cells while suppressing the expression of EGFR. In mice, daily intravaginal application of Vacurin for three weeks produced no change in body weight and when the mice were sacrificed, the vaginal tract epithelium showed no Vacurin-evoked adverse effects. Conclusion. We have developed a curcumin-based vaginal cream, which effectively eradicates HPV(+) cancer cells and does not affect non-cancerous tissue. Our preclinical data support a novel approach for the treatment of cervical HPV infection

Rapid bactericidal activity of an amphiphilic polyacrylate terpolymer system comprised of same-centered comonomers with 2-carbon and 6- carbon spacer arms and an uncharged repeat unit

The global health threat of antimicrobial resistance has created a pressing need to develop practical alternatives to conventional antibiotic agents. Peptide mimetic synthetic amphiphilic polymers are known to non-specifically disrupt the bacterial cell surface thus leading to highly hindered bacterial resistance development. We investigated the antibacterial activities of a terpolymer macromolecular architecture with a combination of 6- carbon and 2-carbon spacer arms (distance from polymer backbone to pendent cationic center) interspersed with counits with hydrophobic side groups. A random copolymer with a combination of 6-carbon spacer arm repeat units (60 mol%) and 2-carbon spacer arm (40 mol%) units is moderately active against bacteria and shows very low hemolytic activity. Incorporation of comonomer units with alkyl side groups, by replacing different levels of 2-carbon spacer arm counit, led to substantial increments in antibacterial activities without detrimental effects on hemolytic activities leading to highly selective (bacteria over red blood cells) antibacterial activity. Time-kill studies revealed rapid bactericidal activity of the terpolymer against both Staphylococcus aureus and Escherichia coli with 100% killing efficiency achieved within 1 h of polymer treatment, corresponding to a 5-log reduction of bacterial colony forming units. These results indicate the high potential of this amphiphilic terpolymer architecture in the development of alternatives to antibiotics

Diagnostic imaging of cervical intraepithelial neoplasia based on hematoxylin and eosin fluorescence

Background Pathological classification of cervical intraepithelial neoplasia (CIN) is problematic as it relies on subjective criteria. We developed an imaging method that uses spectroscopy to assess the fluorescent intensity of cervical biopsies derived directly from hematoxylin and eosin (H&E) stained tissues. Methods Archived H&E slides were identified containing normal cervical tissue, CIN I, and CIN III cases, from a Community Hospital and an Academic Medical Center. Cases were obtained by consensus review of at least 2 senior pathologists. Images from H&E slides were captured first with bright field illumination and then with fluorescent illumination. We used a Zeiss Axio Observer Z1 microscope and an AxioVision 4.6.3-AP1 camera at excitation wavelength of 450–490 nm with emission captured at 515–565 nm. The 32-bit grayscale fluorescence images were used for image analysis. Results We reviewed 108 slides: 46 normal, 33 CIN I and 29 CIN III. Fluorescent intensity increased progressively in normal epithelial tissue as cells matured and advanced from the basal to superficial regions of the epithelium. In CIN I cases this change was less prominent as compared to normal. In high grade CIN lesions, there was a slight or no increase in fluorescent intensity. All groups examined were statistically different. Conclusion Presently, there are no markers to help in classification of CIN I-III lesions. Our imaging method may complement standard H&E pathological review and provide objective criteria to support the CIN diagnosis