Combinational treatment of gap junction enhancers and paclitaxel attenuates mammary tumor growth.

A class of substituted quinolines (PQs) acts as a gap junction enhancer with the ability to increase the gap junctional intercellular communication in breast cancer cells. This study examined the effect of a combinational treatment of PQs and the antineoplastic drug paclitaxel in a xenograft animal model. Mice were implanted with estradiol-17ß (1.7 mg/pellet) before the injection of 1 × 10 T47D breast cancer cells subcutaneously into the inguinal region of mammary fat pad. Animals were treated intraperitoneally with DMSO (control), paclitaxel (10 mg/kg), PQ (25 mg/kg), or a combinational treatment of paclitaxel and PQ. There was no significant difference between the paclitaxel-treated animals and the control group after seven injections of treatment for 2 weeks. All mice treated with PQ had a significant decrease in mammary tumor growth. The combinational treatment of paclitaxel and PQ1 or PQ7 showed a reduction in tumor growth by 2.3- and 2.2-fold, respectively, compared to paclitaxel alone after seven treatments every 2 days. Molecular analysis indicated a significant increase of gap junction proteins and caspase signaling in PQ and paclitaxel-treated tissues compared to control. Furthermore, there is evidence of an upregulation of Cyclin D1 expression in paclitaxel-treated tumors compared to control, suggesting that the neoplastic cells were highly proliferative and nonresponsive to the paclitaxel alone. We have showed for the first time an increase in the efficacy of antineoplastic drugs via the enhancement of gap junctions with PQs, a specific class of gap junction enhancers.

The anticancer effect of PQ1 in the MMTV-PyVT mouse model.

Animal models are commonly used to analyze the mechanism of carcinogenesis as well as the development and screening of potent drugs. Here the transgenic strain FVB/N-Tg(MMTV-PyVT)634Mul/J (also known as PyVT) was used as a model system for measuring tumor burden, drug sensitivity, and metastasis of mammary carcinomas. Loss of gap junctional intercellular communication and the down regulation of connexin expression are characteristic of neoplastic cells. The substituted quinoline, 6-methoxy-8-[(3-aminopropyl)amino]-4-methyl-5-(3-trifluoromethyl-phenyloxy)quinolone (PQ1), has been shown to restore GJIC and increase connexin expression in breast cancer cell lines while not affecting normal mammary cells, suggesting that it may provide effective anticancer treatment with less detrimental effects. The PyVT spontaneous mammary tumor mouse model was used to determine the biological and histological effects of PQ1 on tumorigenesis and metastasis at three stages of development: Pretumor, early tumor and late tumor formation. Treatment with PQ1 at all three stages of development significantly reduced tumor growth. PQ1 treatment further increased Cx43 expression during pre- and early-tumor formation, while it prevented an increase in Cx46 expression during late stage tumor formation. This study shows that Cx43 expression and neoplastic cellular growth are inversely related, but that PQ1 can alter tumor growth through targeting gap junction proteins to prove clinical efficacy in the treatment of spontaneous mammary tumors.

PQ1, a quinoline derivative, induces apoptosis in T47D breast cancer cells through activation of caspase-8 and caspase-9.

Apoptosis, a programmed cell death, is an important control mechanism of cell homeostasis. Deficiency in apoptosis is one of the key features of cancer cells, allowing cells to escape from death. Activation of apoptotic signaling pathway has been a target of anti-cancer drugs in an induction of cytotoxicity. PQ1, 6-methoxy-8-[(3-aminopropyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, has been reported to decrease the viability of cancer cells and attenuate xenograft tumor growth. However, the mechanism of the anti-cancer effect is still unclear. To evaluate whether the cytotoxicity of PQ1 is related to induction of apoptosis, the effect of PQ1 on apoptotic pathways was investigated in T47D breast cancer cells. PQ1-treated cells had an elevation of cleaved caspase-3 compared to controls. Studies of intrinsic apoptotic pathway showed that PQ1 can activate the intrinsic checkpoint protein caspase-9, enhance the level of pro-apoptotic protein Bax, and release cytochrome c from mitochondria to cytosol; however, PQ1 has no effect on the level of anti-apoptotic protein Bcl-2. Further studies also demonstrated that PQ1 can activate the key extrinsic player, caspase-8. Pre-treatment of T47D cells with caspase-8 or caspase-9 inhibitor suppressed the cell death induced by PQ1, while pre-treatment with caspase-3 inhibitor completely counteracted the effect of PQ1 on cell viability. This report provides evidence that PQ1 induces cytotoxicity via activation of both caspase-8 and caspase-9 in T47D breast cancer cells.

The effect of the PQ1 anti-breast cancer agent on normal tissues.

Gap junctions are intercellular channels connecting adjacent cells, allowing cells to transport small molecules. The loss of gap junctional intercellular communication (GJIC) is one of the important hallmarks of cancer. Restoration of GJIC is related to the reduction of tumorigenesis and increase in drug sensitivity. Previous reports have shown that PQ1, a quinoline derivative, increases GJIC in T47D breast cancer cells, and subsequently attenuates xenograft breast tumor growth. Combinational treatment of PQ1 and tamoxifen can lower the effective dose of tamoxifen in cancer cells. In this study, the effects of PQ1 were examined in normal C57BL/6J mice, evaluating the distribution, toxicity, and adverse effects. The distribution of PQ1 was quantified by high-performance liquid chromatography and mass spectrometry. The expressions of survivin, caspase-8, cleaved caspase-3, aryl hydrocarbon receptor (AhR), and gap junction protein, connexin 43 (Cx43), were assessed using western blot analysis. Our results showed that PQ1 was absorbed and distributed to vital organs within 1 h and the level of PQ1 decreased after 24 h. Furthermore, PQ1 increased the expression of survivin, but decreased the expression of caspase-8 and caspase-3 activity. Interestingly, the expression of AhR increased in the presence of PQ1, suggesting that PQ1 may be involved in the AhR-mediated response. Previously, PQ1 caused an increase in Cx43 expression in breast cancer cells; however, PQ1 induced a decrease in Cx43 in normal tissues. Hemotoxylin and eosin staining of the tissues showed no histological change between the treated and the untreated organs. Our studies indicate that the administration of PQ1 by an oral gavage can be achieved with low toxicity to normal vital organs.

Feline sarcomatoid renal cell carcinoma with peritoneal carcinomatosis and effusion.

A 9-y-old, castrated male, domestic medium-hair cat diagnosed previously with chronic kidney disease developed anorexia and vomiting. Ultrasonography revealed abdominal effusion and a left renal perihilar mass. Cytologic evaluation of the peritoneal fluid and mass identified atypical epithelioid cells suspected to be of renal epithelial or possible mesothelial origin. Immunohistochemical (IHC) evaluation of a formalin-fixed, paraffin-embedded peritoneal fluid cell block indicated both pancytokeratin and vimentin expression in the atypical epithelioid cell population. With scanning electron microscopic evaluation, similar epithelioid cells lacked the cell-surface microvilli expected of mesothelium, supporting an antemortem diagnosis of probable carcinoma. On postmortem examination, the left kidney was effaced by an infiltrative neoplasm with myriad similar nodules throughout the peritoneum. The neoplasm was composed primarily of polygonal-to-spindle-shaped cells with strong vimentin and weak pancytokeratin cytoplasmic immunolabeling. Further IHC characterization with PAX8, CK18, KIT, napsin A, SMA, desmin, CD18, and claudin 5 was performed. Histologic and IHC findings supported a diagnosis of sarcomatoid renal cell carcinoma with peritoneal carcinomatosis. An in vitro cell culture line of neoplastic cells harvested from the primary tumor was successfully established for future research endeavors.

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.

Connexin 26 and Connexin 43 in Canine Mammary Carcinoma.

Incidence of canine mammary carcinoma is two times higher than the rate of human breast cancer. Mammary tumors are the most common type of cancer in intact female dogs and account for about half of all neoplasms in these dogs. Well-established models of breast cancer have shown that neoplastic cells often have a loss of intercellular communication, particularly gap junction proteins. Thus, the objective of this study is to explore the aspect of gap junction intercellular communication in canine mammary carcinoma, non-cancerous (CMEC) and cancerous (CMT12, CMT27, and CF41.Mg) cells, and patient-derived tumors. Both non-cancerous and cancerous mammary cells express connexins 26 and 43 using immunofluorescence; however, the level of expression is significantly different in quantitative analysis using western blot in which connexin 43 in both CMT12 and CMT27 is significantly decreased compared to CMEC. Furthermore, a decrease of gap junction capacity in CMT12 and CMT27 was observed compared to CMEC. Immunostaining of CMT27-xenograft tumors revealed positive Cx26 and negative Cx43 expression. Similarly, immunostaining of spontaneous canine mammary tumors revealed that Cx26 is present in all tumors while Cx43 is present in 25% of tumors. Overall, the study provides for the first time that a differential pattern of connexin expression exists between non-cancerous and cancerous mammary cells in dogs. This study will pave the path for further in vitro work of connexins in comparative canine models and possibly allow for novel therapeutics to be developed.

Bioavailability and efficacy of a gap junction enhancer (PQ7) in a mouse mammary tumor model.

The loss of gap junctional intercellular communication is characteristic of neoplastic cells, suggesting that the restoration with a gap junction enhancer may be a new therapeutic treatment option with less detrimental effects than traditional antineoplastic drugs. A gap junction enhancer, 6-methoxy-8-[(2-furanylmethyl) amino]-4-methyl-5-(3-trifluoromethylphenyloxy) quinoline (PQ7), on the normal tissue was evaluated in healthy C57BL/6J mice in a systemic drug distribution study. Immunoblot analysis of the vital organs indicates a reduction in Cx43 expression in PQ7-treated animals with no observable change in morphology. Next the transgenic strain FVB/N-Tg(MMTV-PyVT) 634Mul/J (also known as PyVT) was used as a spontaneous mammary tumor mouse model to determine the biological and histological effects of PQ7 on tumorigenesis and metastasis at three stages of development: Pre tumor, Early tumor, and Late tumor formation. PQ7 was assessed to have a low toxicity through intraperitoneal administration, with the majority of the compound being detected in the heart, liver, and lungs six hours post injection. The treatment of tumor bearing animals with PQ7 had a 98% reduction in tumor growth, while also decreasing the total tumor burden compared to control mice during the Pre stage of development. PQ7 treatment increased Cx43 expression in the neoplastic tissue during Pre-tumor formation; however, this effect was not observed in Late stage tumor formation. This study shows that the gap junction enhancer, PQ7, has low toxicity to normal tissue in healthy C57BL/6J mice, while having clinical efficacy in the treatment of spontaneous mammary tumors of PyVT mice. Additionally, gap junctional intercellular communication and neoplastic cellular growth are shown to be inversely related, while treatment with PQ7 inhibits tumor growth through targeting gap junction expression.

Gap Junction Enhancer Potentiates Cytotoxicity of Cisplatin in Breast Cancer Cells.

Cisplatin is one of the most widely used anti-cancer drugs due to its ability to damage DNA and induce apoptosis. However, increasing reports of side effects and drug resistance indicate the limitation of cisplatin in cancer therapeutics. Recent studies showed that inhibition of gap junctions diminishes the cytotoxic effect and contributes to drug resistance. Therefore, identification of molecules that counteract gap junctional inhibition without decreasing the anti-cancer effect of cisplatin could be used in combinational treatment, potentiating cisplatin efficacy and preventing resistance. This study investigates the effects of combinational treatment of cisplatin and PQ1, a gap junction enhancer, in T47D breast cancer cells. Our results showed that combinational treatment of PQ1 and cisplatin increased gap junctional intercellular communication (GJIC) as well as expressions of connexins (Cx26, Cx32 and Cx43), and subsequently decreased cell viability. Ki67, a proliferation marker, was decreased by 75% with combinational treatment. Expressions of pro-apoptotic factors (cleaved caspase-3/-8/-9 and bax) were increased by the combinational treatment with PQ1 and cisplatin; whereas, the pro-survival factor, bcl-2, was decreased by the combinational treatment. Our study demonstrates for the first time that the combinational treatment with gap junction enhancers can counteract cisplatin induced inhibition of gap junctional intercellular communication and reduction of connexin expression, thereby increasing the efficacy of cisplatin in 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.

Inhibition of gap junction activity through the release of the C1B domain of protein kinase Cgamma (PKCgamma) from 14-3-3: identification of PKCgamma-binding sites.

We have shown previously that insulin-like growth factor-I or lens epithelium-derived growth factor increases the translocation of protein kinase Cgamma (PKCgamma)to the membrane and the phosphorylation of Cx43 by PKCgamma and causes a subsequent decrease of gap junction activity (Nguyen, T. A., Boyle, D. L., Wagner, L. M., Shinohara, T., and Takemoto, D. J. (2003) Exp. Eye Res. 76, 565-572; Lin, D., Boyle, D. L., and Takemoto, D. J. (2003) Investig. Ophthalmol. Vis. Sci. 44, 1160-1168). Gap junction activity in lens epithelial cells is regulated by PKCgamma-mediated phosphorylation of Cx43. PKCgamma activity is stimulated by growth factor-regulated increases in the synthesis of diacylglycerol but is inhibited by cytosolic docking proteins such as 14-3-3. Here we have identified two sites on the PKCgamma-C1B domain that are responsible for its interaction with 14-3-3epsilon. Two sites, C1B1 (residues 101-112) and C1B5 (residues 141-151), are located within the C1 domain of PKCgamma. C1B1 and/or C1B5 synthetic peptides can directly compete for the binding of 14-3-3epsilon, resulting in the release of endogenous cellular PKCgamma from 14-3-3epsilon, in vivo or in vitro, in activation of PKCgamma enzyme activity, phosphorylation of PKCgamma, in the subsequent translocation of PKCgamma to the membrane, and in inhibition of gap junction activity. Gap junction activity was decreased by at least 5-fold in cells treated with C1B1 or C1B5 peptides when compared with a control. 100 microM of C1B1 or C1B5 peptides also caused a 10- or 4-fold decrease of Cx43 plaque formation compared with control cells. The uptake of these synthetic peptides into cells was verified by using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry. We have demonstrated that the activity and localization of PKCgamma are regulated by its binding to 14-3-3epsilon at the C1B domain of PKCgamma. Synthetic peptides corresponding to these regions of PKCgamma successfully competed for the binding of 14-3-3epsilon to endogenous PKCgamma, resulting in inhibition of gap junction activity. This demonstrates that synthetic peptides can be used to exogenously regulate gap junctions.

LEDGF activation of PKC gamma and gap junction disassembly in lens epithelial cells.

Lens epithelium-derived growth factor (LEDGF) has been shown to enhance survival of lens epithelial cells (LECs) against stress. The objectives of these studies are to determine how LEDGF controls PKC gamma activity in normal LECs: how this control of PKC gamma regulates the phosphorylation of Connexin 43, the inhibition of gap junction activity, and the prevention of assembly of gap junctions in LECs. A rabbit LEC line, N/N1003A, was grown in the absence or presence of LEDGF. PKC gamma protein was translocated from the cytosolic fractions to the membrane fractions upon addition of LEDGF at 10 ng ml(-1). In whole cell extracts of N/N1003A cells, co-immunoprecipitation assays showed a protein-protein interaction between PKC gamma and Connexin 43. In the presence of LEDGF the activation of PKC gamma enhanced the phosphorylation of Connexin 43 by four-fold compared to the absence of LEDGF. The addition of LEDGF for 30 min resulted in a 65% decrease in gap junction Connexin 43 at the cell surface and a 70% decrease in gap junction activity. These results suggest that the activation of PKC gamma by LEDGF plays a major role in gap junction assembly/disassembly, which may enhance survival of LECs against osmolarity-stress induced by high sugar concentration.