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

Abstract

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.

Introduction

Gap junctional intercellular communication (GJIC) plays an important role in controlling cell growth, regulating cell differentiation, and maintaining homeostasis in normal cells and tissues [1,2]. Gap junction is a hydrophilic channel that is formed by transmembrane proteins, connexins [3]. Six connexins oligomerize into a hexameric structure known as connexon. Connexon at the plasma membrane may stand alone as a hemichannel or may dock with another connexon of an adjacent cell to form a gap junction [4]. The gap junction channel allows cells to exchange small molecules less than 1.2 kDa in size including small metabolites, electrical signals, and secondary messengers including cAMP, Ca²⁺, K⁺, etc. [5]. This maintenance of communication retains cells in homeostasis. The literature shows that mutations in connexin genes or deficiencies in GJIC are related to various human diseases, such as deafness, peripheral neuropathy, skin disorders, cataracts, and even cancers [6,7].

Diminished connexin expression and deficiency in GJIC are considered to be two characteristics of tumorigenesis [8,9]. Although the function of connexins in invasion, intravasation, extravasation, and metastasis is still controversial, it has been widely accepted that connexins are tumor suppressors because of both GJIC-dependent and GJIC-independent mechanisms [10–14]. Restoration or/and activation of GJIC in cancer cells are suggested to have the ability to reduce cancer cell proliferation and tumor growth [15,16]. In addition to a directly suppressive function, upregulation of GJIC in cancer cells is also important to increase the efficacy of anticancer drugs. Reestablishment of GJIC may aid in the delivery of a drug or a prodrug throughout a tumor, and subsequently mediates the bystander effect, a mechanism by which cytotoxic molecules are transported from a treated cell to a neighboring cell [13]. This mechanism has been shown to be an effective way to potentiate drug effect. The application of the bystander effect in gene therapy has shown that after enhancing connexin 43 (Cx43) and GJIC by 8-bromo-cyclic-AMP treatment, the effect of gene therapy was strengthened by the herpes simplex virus thymidine kinase/gancyclovir system [17]. Besides gene therapy, the bystander effect is also responsible for improving radiation therapy and chemotherapy [18,19]. Therefore, the development of novel agents or methods to enhance or restore GJIC in cancer cells is a new research strategy in cancer treatment.

PQ1 as shown in Fig. 1 is a quinoline derivative that has been reported to be a gap junction enhancer in T47D breast cancer cells. Previously, PQ1 increased GJIC in T47D cells, whereas it had no effect on GJIC in normal human mammary epithelial cells [20]. One μmol/l of PQ1 decreased cell viability to 50% in T47D cells and attenuated 70% of xenograft tumors in nude mice [20]. Combinational treatment of PQ1 and tamoxifen showed that PQ1 potentiated the effect of tamoxifen in T47D cells [21]. All these studies indicate the therapeutic potential of PQ1 in the treatment of breast cancer. However, data on PQ1 in normal tissues are needed before a preclinical trial of PQ1.

Fig. 1.

Chemical structure of PQ1 (C₂₁H₂₂F₃N₃O₂). Its molecular weight is 405.3744, the exact mass is 405.1431.

In this study, the effect of PQ1 was evaluated in healthy C57BL/6J mice. Drug distribution to vital organs was determined and the effect of PQ1 on apoptosis was determined. We also examined the expression of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that regulates the transcription and activity of several important drug-metabolizing enzymes. Further analysis using a histological observation of PQ1-treated tissues showed no alteration in structure. Our results showed that the distribution of PQ1 could be assessed through oral administration in mice, and low toxicity in vital organs was found.

Materials and methods

PQ1

A quinoline derivative, PQ1, was obtained as described by Shi et al. [22].

Animals

Female C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, Maine, USA). All mice were housed together in a temperature-controlled environment (72°F) with a 12-h light–dark cycle and unlimited access to standard mouse chow and water. Five-week-old mice, with an average weight of 24 g, were used. A measure of 25 mg/kg PQ1 was administered by an oral gavage to each animal. Animal care and used protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Kansas State University, following NIH guidelines.

Extraction of PQ1 from organs

Organs were cut into small pieces and diluted with 4ml of deionized water and 10ml of a solution of a 9 : 1 ratio of ethyl acetate and 1-propanol. The tissue mixture was sonicated for 40 min, and the organic layer was separated from a separatory funnel. The aqueous layer was extracted twice with 10ml of a 9 : 1 mixture of ethyl acetate and 1-propanol. The organic layers were combined, washed with 5 ml of brine, dried over anhydrous MgSO₄, and concentrated to dryness on a rotary evaporator. The residue was diluted with 1 ml of 1-propanol, filtered through a 0.2 μm filter disc (PTFE 0.2 μm; Fisherbrand, Pittsburgh, Pennsylvania, USA), and analyzed using high-performance liquid chromatography (HPLC) and mass spectrometry as described below.

Quantification of PQ1 in tissue extracts using high-performance liquid chromatography

HPLC analysis was carried out on a Varian Prostar 210 with a ultraviolet–visible detector and a reverse phase column (250×21.20mm, 10 micron; Phenomenex Inc., Torrance, California, USA). A flow rate of 4ml/min and a detection wavelength of 254nm were used. A gradient elution of solvent A, containing deionized water and 0.01% of trifluoroacetic acid; and solvent B, containing acetonitrile and 0.01% of trifluoroacetic acid, was applied for the analysis. 1,2,4,5-Benzenetetracarboxylic acid (BTA) was used as an internal standard to quantify the amount of PQ1 in the tissue extracts. Solutions of 100 μl of various mixtures of authentic PQ1 and BTA were injected into an HPLC instrument, the peak areas corresponding to PQ1 and BTA were integrated from the HPLC chromatogram, and the ratios of the peaks were obtained. The results of the ratios of HPLC peak areas and ratios from PQ1 and BTA concentrations were plotted, and a linear correlation line was obtained from the graph. Hence, using this correlation diagram, the ratio of HPLC peak areas of PQ1 and BTA from tissue extract, and the added known amount of BTA to the tissue extract, the amount of PQ1 in the tissue extract was determined. Moreover, the peak that had the same retention time as that of PQ1 from the injection of the tissue extract was collected, and its mass was determined using a mass spectrometer. The mass spectrum acquired from the collected peak of PQ1 from the tissue extract was identical to that of the authentic PQ1 mass spectrum. Hence, the molecular identity of PQ1 in the tissue extract was verified by mass spectrometry.

Mass spectroscopy

An Applied Biosystem API 2000 LS/MS/MS (Applied Bio-systems, Foster City, California, USA) mass spectrometer was used in the analysis. The eluent corresponding to the PQ1 peak from the HPLC was collected and injected into the mass spectrometer. A mass of 406 corresponding to M+1 of PQ1 was found in the mass spectra, and the fragmentation pattern of this M+1 mass was identical to that of authentic PQ1.

Western blot analysis

Organs from treated or untreated mice were collected and homogenized with lysis buffer (Cell Signaling Technology Inc., Danver, Massachusetts, USA) using a Vibra-Cell sonicator (Sonics & Materials Inc., Danbury, Connecticut, USA). The mixture was centrifuged at 13 000 rpm (15 700 g using an Eppendorf centrifuge 5415R with rotor F-45-24-11, Eppendorf North America, Hauppauge, New York, USA) for 30 min at 4°C, and the supernatant was collected. The total protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad Life Science Research, Hercules, California, USA). A measure of 40 μg of protein extract was separated by 4–20% sodium dodecylsulfate polyacrylamide gel electrophoresis for 35 min at 200 V and the proteins separated were transferred onto a nitrocellulose membrane. The membrane was immunoblotted against the protein of interest. The goat anti-survivin antibody and mouse anti-caspase-8 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, California, USA). The rabbit anticleaved caspase-3 and rabbit anti-Cx43 antibodies were obtained from Cell Signaling Technology. The rabbit anti-AhR and rabbit anti-actin antibodies were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Immunoreactions using chemiluminescence were visualized by a FluoChem E Imaging Instrument (Cell Biosciences Inc., Santa Clara, California, USA). The intensities of the bands were digitized using Un-Scan-It software (Silk Scientific Inc., Orem, Utah, USA).

Hematoxylin and eosin staining

Hematoxylin and eosin (H&E) staining was performed on paraffin-embedded tissues by following the standard protocol. Sections of 5 μm were dewaxed and rehydrated in xylene and decreasing ethanol concentrations to water. Sections were stained with H&E and mounted for microscopic imaging.

Statistical analysis

Pixel intensities of protein bands were normalized to pixel intensities of loading control protein, actin, or GAPDH. All protein expression data presented were expressed as mean ± SD of at least three independent experiments from different animals. Significant differences were analyzed by comparing the data of treated animals and control (untreated) animals. Significance was considered at P value less than 0.05 using Student's t-test.

Results

Distribution of PQ1

Examination of distribution is important for the development of PQ1 as an anticancer drug. A measure of 25 mg/kg of PQ1 was administered to 5-week-old female C57BL/6J mice by an oral gavage. The total amount of PQ1 administered to each animal was defined as 100%. One hour after treatment, the majority of PQ1 was detected in the liver and the brain at levels of 10 and 5% of the total amount administered, respectively. PQ1 was at a low detectable level in the heart with 1%, the lung with 1.5%, the kidney with 1%, and the uterus with 2.5% (Fig. 2a). Interestingly, the PQ1 distribution changed after 12 h of administration. The percentage of PQ1 in the liver decreased from 10 to 5% and the percentage of PQ1 in brain decreased from 5 to 2%. In contrast, PQ1 in the kidney increased from 1 to 3%, indicating that a shift in PQ1 from the liver to the kidney had occurred. The amounts of PQ1 in the heart, lung, and uterus remained consistent at 12 h of administration (Fig. 2b). After a 24-h treatment, no PQ1 was found in the brain and the heart. The percentage of PQ1 decreased to 3% in the liver and 1% in the kidney. The average percentage of PQ1 in the uterus remained at 3%. PQ1 in the lung a increased slightly from 1.5 to 2.6% at the 24-h time point (Fig. 2c).

Fig. 2.

PQ1 distribution in mice. Mice, treated with 25 mg/kg of PQ1, were killed at 1 h (a), 12 h (b), and 24 h (c). The total amount of PQ1 administered to each animal was defined as 100%. Percentages of PQ1, normalized to the total amounts of PQ1 in the brain, heart, lung, liver, kidney, and uterus, are presented. Data of each experiment were obtained from four mice. Data points represent the percentage of PQ1 in an organ of each mouse, and the dashed lines show the average of PQ1 in four mice.

Effect of PQ1 on apoptosis in normal tissues

Apoptosis is a programmed cell death, an important event in homeostasis of healthy organs [23,24]. Drugs that can affect apoptosis in healthy organs may induce apoptosis-related side effects [25]. Cell proliferation or cell death often depends on the balance of proapoptotic and antiapoptotic factors. Thus, expressions of survivin, an antiapoptotic factor, and caspases, proapoptotic proteins, were evaluated. Two specific members of the caspase family were examined in the presence of PQ1: cleaved caspase-3 is the checkpoint protein of both intrinsic and extrinsic apoptotic pathways and caspase-8 is the key reporter of the extrinsic apoptotic pathway [26].

The results showed that the level of survivin increased in PQ1-treated organs, whereas both cleaved caspase-3 and caspase-8 decreased in these organs (Fig. 3a, b, c). The level of survivin increased by 14% in the liver, 28% in the heart, and 44% in the lung at 1 h after the administration of PQ1 compared with the controls. These effects are consistent with the level of PQ1 detected. Interestingly, the level of survivin in these organs was reduced to the same level as the controls at the 24-h time point. In the brain and kidney, there were no detectable changes in survivin expression at any time point. The uterus was the only organ in which survivin decreased more than 25% after PQ1 treatment (Fig. 3a). As for caspase-8 expression, the brain, heart, lung, liver, and uterus of the treated animals showed a slight decrease in expression ranging from 12 to 37% compared with the untreated animals; however, there was no significant change in the kidney (Fig. 3b). Cleaved caspase-3 was only detected in the uterus, liver, and lung of untreated animals; thus, the change in cleaved caspase-3 upon PQ1 treatment was measured in these three organs. A significant decrease in cleaved caspase-3 ranging from 37 to 45% was observed at 12 h after treatment compared with the control (Fig. 3c). The results of caspases and survivin suggest that PQ1 inhibits proapoptotic factors and promotes antiapoptotic proteins in different normal organs.

Fig. 3.

Effect of PQ1 on apoptosis in normal tissues. Vital organs from PQ1-treated and untreated animals were subjected to western blot analysis, examining the effect of 1 h, 12 h, and 24 h of treatments of PQ1 on the levels of survivin (a), caspase-8 (b), and cleaved caspase-3 (c). Immunblotting images and graphical data are presented. C, control animals without treatment; T, PQ1-treated animals. Two fragments of activated caspase-3 were detected in the uterus and the liver. The upper band is a 19 kDa fragment and the lower band is a 17 kDa fragment. In the bar graph, the pixel intensities of protein bands were normalized to the pixel intensities of loading control protein, actin, and the results of treated animals are normalized to the results of the control animals. Graphical presentations of three experiments are shown with ± SD and statistical significance (*P<0.05).

Effect of PQ1 on aryl hydrocarbon receptor levels in normal tissues

AhR is a transcriptional factor involved in the metabolic pathway of aromatic hydrocarbon compounds [27]. The main adaptive response of AhR is the binding of AhR and hydrocarbon compounds, inducing metabolizing enzymes that are involved in its metabolic pathway [27]. Aromatic hydrocarbon compounds have been shown to trigger the AhR-mediated pathway for its metabolism; thus, the effect of PQ1, an aromatic hydrocarbon compound, on AhR was examined.

The results showed that the level of AhR in the brain, heart, and liver increased significantly by 161, 167, and 124% at 12 h after PQ1 treatment compared with the controls, respectively; however, there was a delay in detecting AhR in the kidney. A 114% AhR was detected in the kidney at the 24-h time point (Fig. 4a). From the drug/tissue distribution data, the amounts of PQ1 peaked at 1 h in the brain, heart, and liver, but peaked at the 12-h time point in the kidney (Fig. 2a and b). These suggest that there is a time-delay response in AhR in these organs. Interestingly, the level of AhR fluctuated from 117% at 1 h of dosing to 63% at 12 h of dosing. Furthermore, only 57, 61, and 54% of AhR were detected in the treated uterus at 1-, 12-, and 24-h time points, respectively, compared with the controls (Fig. 4a). An early onset of AhR downregulation after the administration of PQ1 implies that PQ1 might be involved in a different mode of action in the uterus. At 1 h of PQ1 administration, the level of AhR changed proportionally along with the amount of PQ1 in the liver, indicating a direct dependent function of AhR to PQ1 in the liver (Fig. 4b). The data showed that PQ1 can trigger a change in the expression of AhR in the brain, heart, liver, and kidney.

Fig. 4.

Effect of PQ1 on aryl hydrocarbon receptor (AhR) levels in normal tissues. (a) Western blot analysis, examining the effect of 1 h, 12 h, and 24 h of treatments of PQ1 on the level of AhR, was performed. Mice without PQ1 treatment were used as a control. Immunoblotting images and graphical data are presented. C, control animals without treatment; T, PQ1-treated animals. In the bar graph, the pixel intensities of protein bands were normalized to the pixel intensities of loading control protein, actin. Graphical presentations of three experiments are shown with ± SD and statistical significance, *P<0.05. (b) The level of AhR changes proportionally with the amounts of PQ1 in the liver after a 1-h treatment. Immunoblotting images are also shown above the graph. A line indicates the percentage of PQ1 normalized to the amount of PQ1 in the liver of a corresponding animal. The AhR levels normalized to the control group are shown by the bar graph. All the data as well as the body weight of each mouse have been normalized.

Effect of PQ1 on connexin in normal tissues

As PQ1 has been shown to enhance GJIC [20] and increase Cx43 expressions (data not shown) in breast cancer cells, the expressions of Cx43 in PQ1-treated and PQ1-untreated organs was determined. Cx43 was detected in the heart, brain, and lung in the absence of PQ1 treatment; however, the level of Cx43 decreased in all PQ1-treated organs. A statistically significant decrease of 31% compared with the control was found at the 24-h time point in the heart. A constant level of Cx43 in the lung was observed at all time points. Interestingly, the level of Cx43 in the brain declined gradually over time (Fig. 5). These results suggest that the function of PQ1 in normal cells may involve a different mode of action as compared with that observed previously in cancer cells.

Fig. 5.

Effect of PQ1 on connexin 43 (Cx43) expression in normal tissues. The brain, heart, and lung from treated and untreated mice were subjected to western blot analysis, examining the effect of 1 h, 12 h, and 24 h of treatments of PQ1 on the level of Cx43. Mice without PQ1 treatment were used as a control. Both immunoblotting images and graphical data are presented. Both the phosphorylated Cx43 and the unphosphorylated Cx43 were detected in the heart. The upper band indicates the phosphorylated Cx43 and the lower band indicates the unphosphorylated Cx43. The pixel intensities of protein bands were normalized to the pixel intensities of loading control protein, GAPDH, in the bar graph. Graphical presentations of three experiments are shown with ± SD and statistical significance, *P<0.05.

Histological analysis of normal tissues

The liver is an important organ in drug metabolism. H&E staining of PQ1-treated organs was performed. All 24 mice were assessed grossly or microscopically for histological changes. Histological results showed that the PQ1-treated liver remained unchanged compared with the control, which indicated no observable toxicity of PQ1 to the liver at the treated dosage and time (Fig. 6a). Other tissues including the heart, adrenal gland, kidney, and reproductive tract were also examined and no histological change was observed (Fig. 6b). Twenty-one of the histologically PQ1-treated mice showed no evidence of hemorrhage or inflammatory cells. These mice showed no histological evidence of lesions compared with control mice that did not receive PQ1 treatment at any time point.

Fig. 6.

A hematoxylin and eosin (H&E) staining of whole organs. PQ1-treated tissues were examined by H&E staining. (a) Liver sections from untreated animals (A) and PQ1-treated animals at 1 h (B), 12 h (C), and 24 h (D) are presented. The toxicity of PQ1-treated liver was examined by H&E staining using ×40 magnification. Histological results indicated that PQ1-treated livers showed no change compared with the control. (b) Histology of PQ1-treated animals for the heart (A), adrenal gland (B), and reproductive tract (D) was observed under ×4 magnification, and that of the kidney (C) was observed under ×10 magnification. The results showed no histological alterations in the treated animals compared with the control.

Discussion

Cancer is a complicated disease, with multiple deregulation pathways, necessitating cancer treatment with multiple and combinational approaches [28]. The deficiency of GJIC in cancer cells adds to the complexity of cancer treatment in which the lack of drug transfer to the surrounding area creates challenges to cancer therapy [14]. Some anticancer drugs are reported to inhibit GJIC and reduce connexin expression [29,30]. Hence, restoration of GJIC in cancer cells is a focal point in combinational treatment by potentiating the effect of anticancer drugs. In addition to combinational treatment, overexpression of connexin and activation of GJIC also play a suppressive role in tumors [13]. Therefore, the development of molecules and agents that increase the connexin expression and GJIC can be a useful therapeutic strategy in cancer therapy.

Quinolines are known for their anticancer effects by targeting tumor hypoxia and modulating multidrug resistance [31,32]. Previous reports have shown that PQ1, a quinoline derivative, enhances GJIC, inhibits cell and tumor growth, and increases the potential of the combinational treatment with tamoxifen in T47D breast cancer cells [20,21]. Therefore, the current study presents data on drug/tissue distribution and examines the key factors of apoptotic pathways in normal mice.

Oral gavage, a desirable and safe route of administration, was used in this study. The uptake of any drug depends on the rate of blood flow; thus, the level of PQ1 was evaluated in five vital organs (brain, heart, lung, kidney, and liver) that have a high rate of blood flow. PQ1 was measured in each vital organ after oral administration. The effective dosage of PQ1 falls in the nmol/l range in cells and xenograft tumors [20]. To examine the toxicity in normal organs, a higher concentration of PQ1 was administered at 25 mg/kg body weight, which is equivalent to 47.7 μmol/l. The concentrations of PQ1 in the organs examined were more than 20-fold higher than the effective dosage. PQ1 was detected in all tested organs after a 1-h treatment and reduced at 24 h after treatment, suggesting that PQ1 can be eliminated or excreted after 24 h (Fig. 2). The highest concentrations of PQ1 were found in the liver and kidney at different time points (Fig. 2a and b). A high percentage of PQ1 was detected in the brain at 1 h. This detectable level may be because of the processing of tissue in which PQ1 in the blood vessels could not be excluded during the whole-tissue extraction (Fig. 2a). Our results show that PQ1 can be absorbed, distributed to vital organs, and metabolized in C57B/6J mice.

A major side effect of therapeutic drugs is the potential activation of the apoptosis pathway in normal cells. For example, diarrhea, a common side effect of chemotherapy, is partly caused by induced apoptosis in normal cells of the small intestinal epithelium [25]. It has also been reported that both chemotherapeutic drugs and irradiation can induce apoptosis in normal thymocytes [33,34]. In this report, the presence of PQ1 through an oral gavage led to a decrease in cleaved caspase-3 and an increase in survivin of normal tissues, indicating the inactivation of apoptosis (Fig. 3a and c). Further study of the extrinsic apoptotic pathway showed a decrease in caspase-8 after treatment with PQ1, which further shows that PQ1 cannot activate the extrinsic pathway of apoptosis in normal tissues (Fig. 3b). The effect on apoptosis in normal organs indicates a minor, apoptosis-related side effect caused by PQ1. Interestingly, PQ1 increased caspase-3 cleavage [20] and the level of caspase-8 protein in T47D cells. The opposing aspect of PQ1 on apoptosis in cancer cells compared with normal tissues implies that PQ1 may have a different mechanism in cancer cells. The difference between cancer and normal cells is also shown by the function of PQ1 on connexin expression. PQ1 enhances GJIC [20] and increases connexin expression in T47D breast cancer cells; however, it decreases the expression of Cx43 in a normal heart, brain, and lung (Fig. 5). The PQ1 mechanism of opposing effects in normal and cancer cells is not clear. Further studies are needed to clarify the causes of antitumor effects.

AhR, a ligand-dependent transcription factor involved in the transcription of many important drug-metabolizing enzymes [35], is widely expressed in rodent and human tissues [36]. An increase in the AhR protein level in PQ1-treated mice was observed in vital organs, indicating the possible involvement of PQ1 in the activation of ligand-dependent transcription of the AhR pathway (Fig. 4a). The proportional relation between AhR expression and the level of PQ1 detected in the liver at 1 h indicated a direct impact of PQ1 on AhR expression. However, AhR was decreased by PQ1 treatment in the lung compared with the control. A previous report has shown that an increase in AhR was found in the early stage of lung adenocarcinoma [37], suggesting that the low level of AhR in PQ1-treated lung is because of tissue specificity. Furthermore, an increase in AhR in PQ1-treated organs implies that PQ1 is involved in the AhR-mediated pathway. Further analysis of gene regulation and enzyme activities in AhR-mediated pathways is needed to elucidate the metabolism of PQ1.

Gap junction has been studied for more than 40 years. Until recently, the involvement of gap junction in cancer has been reported and discussed widely. Although several molecules have been developed to modulate different levels of gap junctional proteins and GJIC [13], none of these molecules has been examined in clinical trials for the treatment of cancer. Our present findings support the notion that PQ1 is a promising anti-breast cancer candidate and may serve as a lead compound for drug development.