Thu Annelise Nguyen

Synthesis and Anti-Breast Cancer Activities of Substituted Quinolines

Promising anti-breast cancer agents derived from substituted quinolines were discovered. The quinolines were readily synthesized in a large scale from a sequence of reactions starting from 4-acetamidoanisole. The Michael addition product was isolated as the reaction intermediate in the ring closing reaction of 4-amino-5-nitro-2-(3-trifluoromethylphenyloxy)anisole with methyl vinyl ketone leading to 6-methoxy-4-methyl-8-nitro-5-(3-trifluoromethylphenyloxy)quinoline (14). The amino function of 8-amino-6-methoxy-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, prepared from 14, was connected to various side chains via alkylation with N-(3-iodopropyl)phthalimide, Michael addition with acrylonitrile, and reductive amination with various heterocycle carboxaldehydes, such as imidazole-4-carboxaldehyde, thiophene-2-carboxaldehyde, and 2-furaldehyde. Effects of the substituted quinolines on cell viability of T47D breast cancer cells using trypan blue exclusion assay were examined. The results showed that the IC(50) value of 6-methoxy-8-[(2-furanylmethyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline is 16+/-3nM, the lowest IC(50) out of all the quinolines tested. IC(50) values of three other quinolines are in the nanomolar range, a desirable range for pharmacological testing.

Peptide hydrogelation and cell encapsulation for 3D culture of MCF-7 breast cancer cells

Three-dimensional (3D) cell culture plays an invaluable role in tumor biology by providing in vivo like microenviroment and responses to therapeutic agents. Among many established 3D scaffolds, hydrogels demonstrate a distinct property as matrics for 3D cell culture. Most of the existing pre-gel solutions are limited under physiological conditions such as undesirable pH or temperature. Here, we report a peptide hydrogel that shows superior physiological properties as an in vitro matrix for 3D cell culture. The 3D matrix can be accomplished by mixing a self-assembling peptide directly with a cell culture medium without any pH or temperature adjustment. Results of dynamic rheological studies showed that this hydrogel can be delivered multiple times via pipetting without permanently destroying the hydrogel architecture, indicating the deformability and remodeling ability of the hydrogel. Human epithelial cancer cells, MCF-7, are encapsulated homogeneously in the hydrogel matrix during hydrogelation. Compared with two-dimensional (2D) monolayer culture, cells residing in the hydrogel matrix grow as tumor-like clusters in 3D formation. Relevant parameters related to cell morphology, survival, proliferation, and apoptosis were analyzed using MCF-7 cells in 3D hydrogels. Interestingly, treatment of cisplatin, an anti-cancer drug, can cause a significant decrease of cell viability of MCF-7 clusters in hydrogels. The responses to cisplatin were dose- and time-dependent, indicating the potential usage of hydrogels for drug testing. Results of confocal microscopy and Western blotting showed that cells isolated from hydrogels are suitable for downstream proteomic analysis. The results provided evidence that this peptide hydrogel is a promising 3D cell culture material for drug testing.

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.

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.

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 μmol/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 μmol/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 μmol/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.

Anti-breast cancer agents, quinolines, targeting gap junction.

Cancer cells exhibit many defects in cell communication that contribute to the loss of tissue homeostasis (excess cell proliferation, invasion, and metastasis). The process of cancer formation causes a disruption in cell homeostasis, affecting the ability to respond to extracellular signals, as well as triggering some intracellular events which alter gap junctional intercellular communication (GJIC). Previous research has shown that the first two generations of substituted quinolines have anti-cancer effects in human breast cancer cells. This report presents the synthesis and bioactivities of third generation substituted quinolines. Scrape load/dye transfer studies showed that 100 nM of PQ15, a third generation substituted quinoline, causes a 4.5-fold increase of gap junction activity in T47D breast cancer cells. Furthermore, a significant decrease of cell proliferation and viability was observed in the presence of 200 nM PQ15 compared to control. The expression of α-survivin was reduced to <40% in the treatment of 200 nM PQ15 compared to solvent alone. Alpha-survivin expression is upregulated in human cancers and associated with resistance to chemotherapy, suggesting that α-survivin prolongs the survival of cancer cells. Thus, it has been shown that substituted quinolines stimulate gap junction activity, decrease alpha survivin expression, and subsequently inhibit cancer cell growth. Our findings demonstrate that PQ15 has a promising role in exerting anti-cancer activity in human breast cancer cells.

Gap Junction Enhancer Increases Efficacy of Cisplatin to Attenuate Mammary Tumor Growth

Cisplatin treatment has an overall 19% response rate in animal models with malignant tumors. Increasing gap junction activity in tumor cells provides the targets to enhance antineoplastic therapies. Previously, a new class of substituted quinolines (PQs) acts as gap junction enhancer, ability to increase the gap junctional intercellular communication, in breast cancer cells. We examined the effect of combinational treatment of PQs and antineoplastic drugs in an animal model, showing an increase in efficacy of antineoplastic drugs via the enhancement of gap junctions. Mice were implanted with estradiol-17ß (1.7 mg/pellet) before the injection of 1×107 T47D breast cancer cells subcutaneously into the inguinal region of mammary fat pad. Animals were treated intraperitoneally with DMSO (control), cisplatin (3.5 mg/kg), PQ (25 mg/kg), or a combining treatment of cisplatin and PQ. Cisplatin alone decreased mammary tumor growth by 85% while combinational treatment of cisplatin and PQ1 or PQ7 showed an additional reduction of 77% and 22% of tumor growth after 7 treatments at every 2 days, respectively. Histological results showed a significant increase of gap junction proteins, Cx43 and Cx26, in PQ-treated tissues compared to control or cisplatin. Furthermore, evidence of highly stained caspase 3 in tumors of combinational treatment (PQ and cisplatin) was seen compared to cisplatin alone. We have showed for the first time an increase in the efficacy of antineoplastic drugs through a combinational treatment with PQs, a specific class of gap junction enhancers.

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.

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.