Anti-tumor effect of novel cationic biomaterials in prostate cancer.

BACKGROUND: Tumor cells expressing excessive anionic-charged sialic acid can be potentially targeted by cationic polymers which may inhibit tumor growth. In the present study, three new families of cationic polymers were synthesized to assess their effects on prostate cancer cells. MATERIALS AND METHODS: Cationic polymers effects on PC3 prostate cancer cells and normal prostate epithelial cell (RWPE-1) were assessed using cell viability, DNA fragmentation, apoptosis assays and confocal microscopy. RESULTS: The dextran-based polymer (Dex-PA-3X) (40 µg/ml) and the vinyl-based PolyAETA (5 µg/ml) induced a significant reduction in cell viability in PC3 cells (85% and 50%, respectively; p<0.05) in comparison to RWPE-1 cells. Furthermore, Dex-PA-3X induced a 50%, and PolyAETA induced a 35% increase in cell death in PC3 cells compared to RWPE-1 cells measured by DNA fragmentation assay. Lower concentrations of both polymers induced apoptosis while higher concentrations induced both apoptosis and necrosis by immunostaining. Confocal microscopy indicated the localization of Dex-PA in the cytoplasm of PC3 but not RWPE-1 cells, while PolyAETA was seen in both PC3 and RWPE-1 cells, but at lower intensity in RWPE-1 cells. CONCLUSION: The newly-synthesized cationic polymers Dex-PA-3X and PolyAETA selectively bind to, reduce viability and induce cell apoptosis in prostate cancer cells, suggesting that targeting negatively-charged tumor cells could be a novel strategy to treat prostate cancer.

In vitro method to observe E-selectin-mediated interactions between prostate circulating tumor cells derived from patients and human endothelial cells.

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche. The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines. Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions. Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs. Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions. However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly. CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs. Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques. In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system. This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

Circulating tumor cells from prostate cancer patients interact with E-selectin under physiologic blood flow.

Hematogenous metastasis accounts for the majority of cancer-related deaths, yet the mechanism remains unclear. Circulating tumor cells (CTCs) in blood may employ different pathways to cross blood endothelial barrier and establish a metastatic niche. Several studies provide evidence that prostate cancer (PCa) cell tethering and rolling on microvascular endothelium via E-selectin/E-selectin ligand interactions under shear flow theoretically promote extravasation and contribute to the development of metastases. However, it is unknown if CTCs from PCa patients interact with E-selectin expressed on endothelium, initiating a route for tumor metastases. Here we report that CTCs derived from PCa patients showed interactions with E-selectin and E-selectin expressing endothelial cells. To examine E-selectin-mediated interactions of PCa cell lines and CTCs derived from metastatic PCa patients, we used fluorescently-labeled anti-prostate specific membrane antigen (PSMA) monoclonal antibody J591-488 which is internalized following cell-surface binding. We employed a microscale flow device consisting of E-selectin-coated microtubes and human umbilical vein endothelial cells (HUVECs) on parallel-plate flow chamber simulating vascular endothelium. We observed that J591-488 did not significantly alter the rolling behavior in PCa cells at shear stresses below 3 dyn/cm(2). CTCs obtained from 31 PCa patient samples showed that CTCs tether and stably interact with E-selectin and E-selectin expressing HUVECs at physiological shear stress. Interestingly, samples collected during disease progression demonstrated significantly more CTC/E-selectin interactions than samples during times of therapeutic response (p=0.016). Analysis of the expression of sialyl Lewis X (sLe(x)) in patient samples showed that a small subset comprising 1.9-18.8% of CTCs possess high sLe(x) expression. Furthermore, E-selectin-mediated interactions between prostate CTCs and HUVECs were diminished in the presence of anti-E-selectin neutralizing antibody. CTC-Endothelial interactions provide a novel insight into potential adhesive mechanisms of prostate CTCs as a means to initiate metastasis.

Functional characterization of circulating tumor cells with a prostate-cancer-specific microfluidic device.

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45- cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2-400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents.

Second-generation substituted quinolines as anticancer drugs for breast cancer.

Cancer cells have reduced capacity for gap junctional inter-cellular communication (GJIC). One feasible approach to reduce growth of cancer cells is to enhance GJIC. This report shows that a second-generation substituted quinoline, PQ7, has anti-tumor effect. Scrape load/dye transfer and colony growth assays were performed to measure GJIC and tumor formation of T47D breast cancer cells. PQ7 at 500 nM induced a 16-fold increase in the GJIC in T47D cells. In addition to an increase in GJIC, a 50% decrease of colony growth was observed with 100 nM of PQ7. PQ7-treated nu/nu mice showed a 100% regression of xenograft tumor growth of T47D cells. The results show that PQ7 has a promising role in exerting anti-tumor activity in human breast cancer cells.

Bioactivity and molecular targets of novel substituted quinolines in murine and human tumor cell lines in vitro.

Substituted quinolines (PQ code number), which reduce colony formation and increase gap junctional intercellular communication, were tested for their ability to interact with various molecular targets in murine and human tumor cell lines in vitro. Various markers of tumor cell metabolism, DNA fragmentation, mitotic disruption, apoptosis induction and growth factor receptor signaling pathways were assayed in vitro to evaluate drug cytotoxicity. Based on its ability to inhibit the metabolic activity of suspension cultures of leukemic L1210 cells at days 2 and 4 in vitro, PQ1 succinic acid salt is the most effective antiproliferative agent among the synthetic quinoline analogs tested. Moreover, antiproliferative PQ1 is effective across a spectrum of monolayer cultures of pancreatic Pan02, epidermoid A-431 and mammary SK-BR-3 and BT-474 tumor cells. PQ1 also blocks Ki-67 expression, a marker of tumor cell proliferation. A 1.5- to 3-h treatment with PQ1 is sufficient to inhibit the incorporations of [3H]-thymidine into DNA, [3H]-uridine into RNA and [3H]-leucine into protein used to assess the rates of macromolecule syntheses over a 0.5- or 1-h period of pulse-labeling in L1210 tumor cells. A 15-min pretreatment with PQ1 inhibits the cellular transport of both purine and pyrimidine nucleosides over a 30-sec period in vitro, suggesting that PQ1 may prevent the incorporation of [3H]-adenosine and [3H]-thymidine into DNA because it rapidly blocks the uptake of these nucleosides by the tumor cells. Since PQ1 does not reduce the fluorescence of the ethidium bromide-DNA complex, it does not directly bind to or destabilize double-stranded DNA. Over a 6- to -48-h period, PQ1 has very little effect on the mitotic index of L1210 cells but stimulates the formation of many binucleated cells and a few micronuclei, suggesting that this compound might increase mitotic abnormality, induce chromosomal damage or missegregation, and block cytokinesis. The fact that PQ1 induces initiator caspase-2 and effector caspase-3 activities and poly(ADP-ribose) polymerase-1 cleavage within 1-4 h and internucleosomal DNA fragmentation within 24 h in L1210 cells suggests that this antitumor drug can trigger the early and late events required for cells to undergo apotosis. Whole-cell immunodetection and Western blot analysis indicate that, in contrast to 17-(allylamino)-17-demethoxygeldanamycin and radicicol, PQ1 fails to down-regulate the protein level at 24 h and autophosphorylation at 3 h of membrane-anchored HER1 in A-431 cells and HER2 in SK-BR-3 cells, suggesting that this antitumor compound is unlikely to interact with and inhibit Hsp90 and the epidermal growth factor (EGF) receptor signaling pathways. In conclusion, antiproliferative PQ1 is effective against a spectrum of tumor cells and might interact with various membrane and nuclear targets to enhance gap junctions, inhibit nucleoside transport and block cytokinesis but does not appear to disrupt the EGF receptor-mediated signaling pathways to induce growth arrest and apoptosis.

A novel role of gap junction connexin46 protein to protect breast tumors from hypoxia.

Connexin proteins are the principle structural components of the gap junctions. Colocalization and tissue-specific expression of diverse connexin molecules are reported to occur in a variety of organs. Impairment of gap junctional intercellular communication, caused by mutations, gain of function or loss of function of connexins, is involved in a number of diseases including the development of cancer. Here we show that human breast cancer cells, MCF-7 and breast tumor tissues express a novel gap junction protein, connexin46 (Cx46) and it plays a critical role in hypoxia. Previous studies have shown that connexin46 is predominantly expressed in lens and our studies find that Cx46 protects human lens epithelial cells from hypoxia induced death. Interestingly, we find that Cx46 is upregulated in MCF-7 breast cancer cells and human breast cancer tumors. Downregulation of Cx46 by siRNA promotes 40% MCF-7 cell death at 24 hr under hypoxic conditions. Furthermore, direct injection of anti-Cx46 siRNA into xenograft tumors prevents tumor growth in nude mice. This finding will provide an exciting new direction for drug development for breast cancer treatment and suggests that both normal hypoxic tissue (lens) and adaptive hypoxic tissue (breast tumor) utilize the same protein, Cx46, as a protective strategy from hypoxia.

Combinational treatment of gap junctional activator and tamoxifen in breast cancer cells.

Tamoxifen is a drug of choice for endocrine-responsive breast tumor patients. However, tamoxifen resistance has become a major concern for the treatment of breast cancer. Combinational therapies of tamoxifen and different drugs are being frequently studied. In this study, we tested the efficacy of substituted quinolines (code name=PQ1; a gap junctional activator) in combination with tamoxifen in T47D cells. Colony growth assay was performed using soft agar to measure the colony growth, whereas cell proliferation was measured by the MTT assay in T47D cells. The level of Ki67, survivin, and BAX was measured using confocal microscopy along with western blot analysis. Apoptosis-bromodeoxyuridine triphosphate labeling was also examined in the induced treatment of T47D cells. We observed a 55% decrease in the colony growth in the presence of combination of PQ1 and tamoxifen, whereas tamoxifen alone had little effect. A combination of 10 micromol/l tamoxifen and 200 or 500 nmol/l PQ1 resulted in only 16% cell viability compared with controls at 48 h in T47D cells by the MTT assay. We found a significant increase in BAX protein at 1 h in the presence of 500 nmol/l PQ1 alone, 10 micromol/l tamoxifen alone, and the combination of PQ1 and tamoxifen. A two-fold increase was observed in active caspase 3 in the presence of combinational treatment of 10 micromol/l tamoxifen and 200 or 500 nmol/l PQ1. In addition, flow cytometric analysis showed a 50% increase in the number of apoptotic cells in the presence of the combination of tamoxifen and PQ1 compared with the control. Furthermore, the results show that combinational treatment of tamoxifen and PQ1 significantly reduces the expression of survivin in T47D cells. Gap junction inhibitor studies with carbenexolone were also performed confirming the role of gap junctions in cell proliferation and cell death. The combinational treatment of PQ1 and tamoxifen has a significant increase in BAX expression, caspase 3 activation, and DNA fragmentation. Tamoxifen alone and in combination with PQ1 showed a decrease in the expression of survivin, whereas PQ1 alone was shown to be independent of the survivin-mediated pathway. This suggests that an increase in gap junction activity can potentiate the effect of tamoxifen. The combinational treatment of tamoxifen and PQ1 also showed a significant decrease in cell viability compared with tamoxifen treatment alone. The gap junction inhibitor carbenexolone was shown to increase cell proliferation by increased cyclin D1 expression, MTT assay, and Ki67 expression. It further decreased cell death. This study shows for the first time that combinational treatment of tamoxifen and PQ1 (a gap junctional activator) can be used to potentiate apoptosis of T47D human breast cancer cells. Thus, a gap junctional activator, PQ1, could potentially alter either the length or dose of tamoxifen clinically used for breast cancer patients.

Regulation of gap junctional intercellular communication by TCDD in HMEC and MCF-7 breast cancer cells.

Previous studies suggest that many neoplastic tissues exhibit a decrease in gap junctional intercellular communication (GJIC). Many hydrocarbons and organochlorine compounds are environmental pollutants known to be carcinogenic. The effect of an organochlorine compound, TCDD, on GJIC in human breast cell lines has not been established. In the present study, we showed that TCDD causes an inhibition in the gap junctional activity in MCF-7 (breast cancer cells). In MCF-7 cells, an increase in the phosphorylated form of gap junctional protein, connexin 43 (Cx43), and PKC alpha was seen in the presence of TCDD. Gap junctional plaque formation was significantly decreased in MCF-7 cells in the presence of TCDD. Immunoprecipitation studies of PKC alpha showed that TCDD caused a significant 40% increase in the phosphorylated Cx43 in MCF-7 cells. TCDD also modulated the translocation of PKC alpha from the cytosol to the membrane and caused a 2-fold increase in the PKC alpha activity at 50 nM TCDD in MCF-7 cells. Calphostin C, an inhibitor of PKC alpha, showed a significant inhibition of PKC alpha activity in the presence of TCDD. Furthermore, TCDD also caused a decrease in the gap junctional activity and Cx43 protein in human mammary epithelial cells (HMEC). However, we observed a shift in the Cx43 plaques towards the perinuclear membrane in the presence of TCDD by confocal microscopy and Western blot. Overall, these results conclude that TCDD decreases GJIC by phosphorylating Cx43 via PKC alpha signaling pathway in MCF-7 cells; however, TCDD decreases the GJIC by affecting the localization of Cx43 in HMEC. These new findings elucidate the differential mode of effect of TCDD in the downregulation of GJIC in HMEC and MCF-7 cells.