Balakrishna L. Lokeshwar

GAP-JUNCTIONAL COMMUNICATION IN NORMAL AND NEOPLASTIC PROSTATE EPITHELIAL CELLS AND ITS REGULATION BY CAMP

Gap‐junctional communication and expression of gap junction‐forming proteins were investigated in normal human prostate epithelial cells and in several malignant prostate cell lines. In comparison with normal cells, gap‐junctional communication in malignant cells, as assayed by the transfer of 443‐Da fluorescent tracer Lucifer yellow, was either reduced or not detected. Malignant cells expressed mRNA transcripts for connexin (Cx) 43, whereas normal cells expressed mRNA transcripts for Cx32 and Cx40. In both normal and malignant cells, gap‐junctional communication was enhanced twofold to fivefold by treatment with forskolin, an agent known to increase intracellular levels of cAMP. Immunocytochemical staining with a Cx43‐specific antibody revealed that in malignant cells this enhancement correlated with the number of gap junctions and occurred without any qualitative or quantitative alteration in Cx43 mRNA or protein. Moreover, western blot analyses showed that both control and forskolin‐treated malignant cells expressed only one form of Cx43. Our data suggest that gap‐junctional communication in both normal and malignant prostate cells may be regulated by hormones that work via a cAMP‐dependent signal transduction pathway. Thus, both normal and malignant cells offer a new experimental model system in which interactions between a hormonal form of cellular communication and intercellular communication mediated via gap junctions can be studied.

Potential application of a chemically modified non-antimicrobial tetracycline (CMT-3) against metastatic prostate cancer.

The matrix metalloproteinases (MMPs) are known to play a significant role in tumor cell invasion and metastasis (Liotta and Stetler-Stevenson, 1990; Stetler-Stevenson et al., 1993; Chambers et al, 1995; Lawrence and Steeg, 1996). Typically, aggressive tumor cells secrete a large excess of MMPs and low levels of endogenous inhibitors (e.g., TIMPs) (Liotta et al, 1991). This imbalance is known to be a causative factor in invasion and metastasis (Liotta et al., 1991; Lokeshwar et al, 1993). Recently, several synthetic inhibitors of MMPs have been tested for their potential to prevent or reduce tumor metastases (Davies et al, 1993; Brown, 1995; Sledge et al, 1995; Wojtowicz-Praga et al, 1997). Several of these inhibitors have limited effectiveness as therapeutic agents due to poor solubility (Davies et al, 1993), lack of oral availability (Brown, 1995), or toxicity (Sledge et al, 1995). An orally administrable, non-toxic inhibitor which neutralizes the MMPs would be a prime therapeutic candidate. Cytotoxicity to proliferating cells, with little systemic toxicity, would be additional virtues of such a drug. Tetracyclines such as doxycycline (DC) and several chemically modified non-antimicrobial tetracyclines (CMTs)

Experimentally induced dry eye produces ocular surface inflammation and epithelial disease.

Dry eye results from decreased production, increased evaporation or decreased clearance of tears.1,2 It causes ocular irritation and ocular surface disease, termed keratoconjunctivitis sicca (KCS), that causes blurred and fluctuating vision and increases the risk of sight-threatening corneal infection and ulceration.3 The histological features of KCS are abnormal proliferation and differentiation of the ocular surface epithelium with decreased density of conjunctival goblet cells and decreased and abnormal production of mucus by the ocular surface epithelium.4 The most severe KCS develops in conditions where the ability to tear responsively to neural stimulation is lost.5–7

Neuroendocrine peptides stimulate adenyl cyclase in normal and malignant prostate cells.

Elevations of intracellular cAMP in human prostate cancer cells have been shown to increase invasiveness and to promote neuronal differentiation. Since neuroendocrine peptides capable of activating adenyl cyclase are present in prostatic nerves and epithelial neuroendocrine cells, we investigated normal and malignant human prostate cells for changes in intracellular cAMP in response to the prostatic peptides vasoactive intestinal peptide (VIP), calcitonin (CT), and calcitonin gene-related peptide (CGRP). Normal prostate epithelial cells and LNCaP prostate cancer cells exhibited, respectively, 6- and 30-fold increases in intracellular cAMP in response to VIP. ALVA-31 and PPC-1 prostate cancer cells demonstrated 20- to 200-fold increases in cAMP in response to CGRP, while normal epithelial cells and LNCaP cells exhibited smaller (2- to 6-fold) responses. Only DU-145 cells increased cAMP substantially in response to CT. VIP receptor mRNA was identified by Northern blot analysis only in those cells that responded to VIP. CT receptor mRNA was identified only in DU-145 cells by polymerase chain reaction and Southern blot analysis. These results suggest that VIP and possibly CGRP receptors are likely to be present in both normal and malignant prostate cells. VIP or CGRP may regulate secretion of proteases by normal or prostate cancer cells and may influence epithelial cell differentiation.

Anti-proliferative and anti-cancer properties of Achyranthes aspera: specific inhibitory activity against pancreatic cancer cells.

AIMS OF THE STUDY Achyranthes aspera (Family: Amaranthacea) is a medicinal plant used as an anti-cancer agent in ayurveda, a traditional system of medicine practiced in subcontinental India. The aim of the study was to systematically investigate the anti-proliferative properties of Achyranthes aspera leaves extracted in methanol (LE) on human cancer cells in vitro. MATERIALS AND METHODS We tested time, dose dependent and specific anti-proliferative activity of LE by clonogenic cell survival assay on human cancer and normal epithelial cell lines in vitro. We further investigated its effect on the expression of metastatic and angiogenic genes by real time polymerase chain reaction. On silica gel column, we carried out initial fractionation analysis. RESULTS LE exhibited time and dose dependent cytotoxicity on several tumor cells. Compared to cancer cells of colon, breast, lung and prostate origin, pancreatic cancer cells were significantly more sensitive to LE. Preliminary mechanistic studies suggested that LE selectively suppressed the transcription of metalloproteases (MMP-1 and -2), inhibitors of MMPs (TIMP-2) and angiogenic factors (VEGF-A and VEGF-B). Fractionation of LE on methanol equilibrated silica gel column resolved into three fractions of which fraction (F 3) was found to be enriched with anti-proliferative activity. CONCLUSION Methanolic extract of Achyranthes aspera contains potent anti-proliferative compound with specific activity against pancreatic cancer. Further studies are needed to confirm the in vivo anti-tumorigenicity and subsequent chemical characterization of the active molecule(s).

In Vitro Global Gene Expression Analyses Support the Ethnopharmacological Use of Achyranthes aspera

Achyranthes aspera (family Amaranthaceae) is known for its anticancer properties. We have systematically validated the in vitro and in vivo anticancer properties of this plant. However, we do not know its mode of action. Global gene expression analyses may help decipher its mode of action. In the absence of identified active molecules, we believe this is the best approach to discover the mode of action of natural products with known medicinal properties. We exposed human pancreatic cancer cell line MiaPaCa-2 (CRL-1420) to 34 μg/mL of LE for 24, 48, and 72 hours. Gene expression analyses were performed using whole human genome microarrays (Agilent Technologies, USA). In our analyses, 82 (54/28) genes passed the quality control parameter, set at FDR ≤ 0.01 and FC of ≥±2. LE predominantly affected pathways of immune response, metabolism, development, gene expression regulation, cell adhesion, cystic fibrosis transmembrane conductance regulation (CFTR), and chemotaxis (MetaCore tool (Thomson Reuters, NY)). Disease biomarker enrichment analysis identified LE regulated genes involved in Vasculitis—inflammation of blood vessels. Arthritis and pancreatitis are two of many etiologies for vasculitis. The outcome of disease network analysis supports the medicinal use of A. aspera, viz, to stop bleeding, as a cure for pancreatic cancer, as an antiarthritic medication, and so forth.

Abstract P3-03-03: Phenolic compounds from pimenta dioica berries (allspice) inhibit breast cancer cell proliferation by autophagy induction

Background and methods:Recent studies have shown that plant-derived dietary compounds have potential as chemopreventive agents to prolong survival and reduce breast cancer deaths. Lack of specific mechanism of action is the leading concern for adopting these edible compounds as chemopreventive or adjuvant therapeutics against breast and other cancers. The unripe berries of Pimenta dioica, also called Allspice, are widely used in cuisines worldwide, contain a cornucopia of bioactive compounds with potential therapeutic properties. Limited studies on the potential anticancer compounds present in Allspice prompted us to hypothesize and test potential antiproliferative agents in an aqueous extract of Allspice (AAE).An aqueous extract of Certified-organic Allspice (AAE) was prepared and tested on several BrCa cell lines for potential antitumor activities by techniques as western blotting, clonogenic survival, ELISA, flow cytometry, quantitative-RT- PCR, promoter reporter assay and microscopy. Results: AAE reduced the viability and clonogenic growth of BrCa cells (IC50∼100μg/ml) with significantly reduced cytotoxicity against non-tumorigenic quiescent cells (IC50 >200μg/ml). AAE induced antiproliferative activity was inconsistent with cell cycle arrest, apoptosis or necrosis, but was strongly associated with characteristics of autophagy, including high vacuolation, increase in levels of GFP-LC3-positive puncta, and LC3-II protein levels. Silencing the ATG gene expression in both ER+ and ER- cells by siRNA prevented AAE-induced cell death. In ERα+ cell line MCF-7, ATG7 silencing abrogated AAE cytotoxicity by 80% and in ERα- MDA-MB231 cells, 50% cell death was rescued. Inhibiting ATG5 gene yielded similar yet less pronouced results. Autophagy was induced by Endoplasmic reticulum (ER) stress, confirmed by increase in ER stress marker GRP78, but no change in ATP levels. Further, AAE caused inhibition of mTOR signaling and showed enhanced cytotoxicity when combined with an mTOR inhibitor, rapamycin. AAE reduced ERα protein as well ERα-mRNA levels in MCF-7 and T47D cells. Incubation with AAE (100μg/ml) caused 50% decrease in ERα protein level while its mRNA decreased by 25%. Furthermore, AAE induced down regulation of ERα was partially rescued by co-incubation with the 26S-proteasome inhibitor MG132. AAE also exhibited anti-metastatic activities against MDA-MB231 cells where it decreased invasive potential and caused decreases in mRNA levels of EMT markers N-cadherin, vimentin, slug and increase in Twist. Activity based purification combined with chemical identification strategies resulted in identification of several novel candidate compounds with characteristics of polyphenols. In conclusion, we determined the anti-cancer properties of an aqueous extract of Allspice on BrCa cells, which principally affects cell survival by inducing cell death by autophagy and ERα down regulation. It reduced EMT in metastatic BrCa cells thus enhancing the clinical potential of AAE as an adjuvant for BrCa treatment or dietary intervention. [Grant Support: Sheila and David Fuente Cancer Biology program of Sylvester Cancer Center (LZ) and PHS Grant No. R01 CA156776 (BLL)]. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-03.