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Anti-Cancer Drugs:
doi: 10.1097/CAD.0b013e328359affd
Preclinical Reports

Antitumor effects and preliminary systemic toxicity of ANISpm in vivo and in vitro

Li, Minga; Li, Qianb; Zhang, Ya-hongb; Tian, Zhi-yonga; Ma, Hong-xiab; Zhao, Jinb; Xie, Song-qianga; Wang, Chao-jieb

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Author Information

aInstitute of Chemical Biology

bThe Key Laboratory of Natural Medicine and Immuno-Engineering, Henan University, Kaifeng, China

Correspondence to Song-qiang Xie, Institute of Chemical Biology, Henan University, 475004 Kaifeng, China Tel: +86 378 2864665; fax: +86 378 3880680; e-mail: kfxsq@yahoo.com.cn and Correspondence to Chao-jie Wang, The Key Laboratory of Natural Medicine and Immuno-Engineering, Henan University, Kaifeng 475004, China Tel/fax: +86 378 3880680; e-mail: wcjsxq@henu.edu.cn

Received April 11, 2012

Accepted August 24, 2012

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Abstract

Polyamines as a vector to ferry toxic agents have attracted attention, and naphthalimide–polyamine conjugates show potent activity and tumor cell selectivity. The present study was carried out to evaluate the antitumor effects and preliminary systemic toxicity of ANISpm, a novel 3-amino-naphthalimide–spermine conjugate. The polyamine transport system recognition of ANISpm, supported by α-difluoromethylornithine (DFMO)/spermidine (Spd) experiments, is in accordance with its potent cell selectivity between human hepatoma HepG2 cells and normal QSG7701 hepatocyte. The antiproliferative effect is because of ANISpm-induced cell apoptosis, a common characteristic of both naphthalimide and polyamine analogs. Various apoptotic assessment assays have shown that ANISpm can induce apoptosis through the PI3K/Akt signal pathway. The apoptotic signaling cascade involves Akt inactivation, which results in a series of cellular events. The downstream pathway includes Bad dephosphorylation, dissociation of 14-3-3 and Bad, and binding to Bcl-xL, which triggers the disruption of the mitochondrial membrane, release of cytochrome c, and caspases’ cascade activation. Furthermore, the Akt/mTOR signal pathway is also involved in ANISpm-mediated cell-cycle arrest. Additive DFMO or Spd, which only enhances or attenuates ANISpm-mediated cell apoptosis, respectively, does not alter the signal pathway. In addition, preliminary toxicology evaluation showed that ANISpm had no obvious system toxicity at a dose of 2.5 mg/kg, which exerted potent antitumor activity in vivo, especially hematotoxicity. Thus, ANISpm merits further investigation as a potential chemotherapeutic agent against hepatocellular carcinoma.

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Introduction

Hepatocellular carcinoma (HCC) is one of the most prevalent life-threatening human cancers and is increasing in terms of incidence worldwide 1. Therefore, an understanding of the molecular mechanisms that underlie imbalanced proliferation/apoptosis processes during the course of hepatocarcinogenesis is required. Several pieces of evidence support the predominant role of phosphatidylinositol-3-kinase (PI3K) signaling in hepatocyte proliferation and suggest that upregulation of Akt signaling may contribute toward the progression of preneoplastic lesions as HCC 2. The PI3K/Akt pathway provides a link between extracellular survival signals and apoptosis. Activation of a receptor tyrosine kinase by extracellular stimuli results in the phosphorylation of PI3K. This activates Akt and results in the phosphorylation of key survival proteins, such as Bcl-2, Bad, mTOR, caspase-9, and caspase-3 by Akt 3–5. Phosphorylation of these proteins decreases the susceptibility of tumor cells to apoptotic stress 6.

The PI3K/Akt pathway is involved in the regulation of multiple cellular processes, such as cell survival, proliferation, growth, angiogenesis, migration, and transformation 5. Numerous reports have shown that constitutive activation of the PI3K/Akt signal pathway is closely associated with tumor development, aggressive progression, increased metastasis, and resistance to cancer therapies 7,8. Consequently, efforts are currently under way to develop drugs that target this signal pathway 9,10.

The natural polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm), play an essential role in cellular growth and differentiation 11. The requirement of tumor cells for polyamines, leading to increased polyamine biosynthesis and/or external uptake, makes the polyamine pathway attractive for tumor-targeted chemotherapy 12. Polyamines are also potential carriers for drug delivery because the polyamine transport system (PTS) in tumor cells seems to have a wide structural tolerance for a wide variety of substrates. In this respect, the antineoplastic drugs are conjugated to polyamines to facilitate their entrance into cells with active PTS. Several related drug–polyamine conjugates have been designed to increase their cytotoxicity to tumor cells and to reduce the side effects on normal cells 13–15.

Polyamine derivatives have the potential to inhibit HCC. For example, DENSpm, a symmetrically disubstituted polyamine derivative, is being studied in clinical trials against hepatoma 16. In a recent report, we found that some naphthalimide–polyamine conjugates exert enhanced cytotoxicity to hepatoma BEL 7402 cells over their normal cell counterparts (QSG7701) in vitro, and may induce apoptosis on hepatoma cell lines 17. However, knowledge of the upstream target of naphthalimide–polyamine conjugates-induced apoptosis is rudimentary and remains to be expanded. Therefore, a novel compound, 3-amino-naphthalimide–spermine conjugate (ANISpm), was synthesized in our laboratory (structure shown in Fig. 1a) and evaluated for both apoptotic activity as well as Akt-inhibitory activity in human HepG2 cells.

Fig. 1
Fig. 1
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Materials and methods

Materials

RNase A, rhodamine123 (Rh123), rapamycin, wortmannin (Wor), α-difluoromethylornithine (DFMO), propidium iodide (PI), and 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma (St Louis, Missouri, USA). RPMI1640, F-12, and fetal calf serum (FCS) were purchased from Gibco (Grand Island, New York, USA). The Annexin V-FITC apoptosis detection kit was purchased from Cell Signaling Technology (Beverly, Massachusetts, USA). Proteins were detected by western blotting using the following primary antibodies: Akt, mTOR phospho-Akt (Thr308), phospho-Akt (Ser473), phospho-p70S6 kinase (Thr389), phospho-PTEN (Ser380/Thr382/383), PTEN, and phospho-mTOR, which were purchased from Cell Signaling Technology. Bcl-xL, Bad, phospho-Bad (Ser136), 14-3-3, caspase-9, caspase-3, Bcl-2 and cytochrome c, as well as horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, California, USA). All other chemicals used in the experiments were commercial products of reagent grade. ANISpm (purity: 99.2%) was synthesized in our laboratory and the structural formula is shown in Fig. 1a.

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Synthesis of ANISpm

A mixture of 4-amino-1,8-naphthalic anhydride and polyamine (in a 1 : 1 molar ratio) in EtOH was heated at the refluxing temperature, and the reaction process was monitored by TLC. After completion, the reaction mixture was cooled to room temperature and concentrated under vacuum to yield an oily residue, which was further purified to obtain the Boc-protected intermediates 2. The respective N-Boc-protected intermediates 2 (1.2 mmol) was dissolved in EtOH (20 ml) and stirred at 0°C for 10 min. A volume of 4 mol/l HCl was added dropwise at 0°C. The reaction mixture was stirred at room temperature overnight. The solution typically yielded a white solid as a precipitate. The solid was filtered, washed several times with absolute ethanol, and dried under vacuum to yield the pure target compounds 3:

2-{3-[4-(3-Butoxycarbonyamino-aminopropyl-butoxycarbonylamino)butyl-butoxycarbonyamino]-propyl}-5-amino-1H-benz-[de]isoquinoline-1,3(2H)-dione(2): yield 86%, pale yellow oil, 1H NMR (400 MHz, CDCl3) δ: 7.62∼7.66 (m, 2H, Ar-H); 7.40 (d, 1H, J=7.6 Hz, Ar-H);7.19 (t, J=8.0 Hz, 1H, Ar-H); 7.11∼7.14 (m, J=8.0 Hz, 1H, Ar-H); 4.18(t, J=7.2 Hz, 2H, 1×N-CH2); 3.13∼3.15 (m, 10 H, 5×N-CH2); 1.62∼1.73 (m, 6H, 1×CH2); 1.41∼1.46 (m, 6H, 3×CH2); 1.41∼1.46 (m, 33H, 3×CH2+9×CH3). ESI-MI m/z: 722.4[M+Na]+;

2-{3-[4-(3-aminopropylamino)butylamino]-propyl}-5-amino-1H-benz-[de]isoquinoline-1,3(2H)-dione tetrahydrochloride(3): yield 82%, white solid, 1H NMR (400 MHz, D2O) δ: 7.63∼7.67(m, 2H, Ar-H); 7.41(d, 1H, J=7.6 Hz, Ar-H); 7.20 (t, J=8.0 Hz, 1H, Ar-H); 7.13∼7.16 (m, J=8.0 Hz, 1H, Ar-H); 3.48 (t, J=2.8 Hz, 2H, 1×N-CH2); 2.95∼3.08 (m, 8H, 4×N-CH2); 1.73∼1.76 (m, 8H, 4×CH2); 1.62∼1.63 (m, 2H, 1×CH2); 1.23∼1.29 (m, 2H, 1×CH2). ESI-MI m/z: 400.3[M+H–3HCl]+, Anal. calculated for C22H37Cl4N5O2·0.5H2O: C 47.66%, H 6.91%, N 12.63%; found: C 47.70%, H 7.01%, N 12.60% (Scheme 1).

Scheme 1
Scheme 1
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Study in vitro
Cell culture and treatment

The HepG2 cell line and the QSG7701 cell line, purchased from Shanghai Institute for Biological Science, Chinese Academy of Sciences (Shanghai, China), were supplemented with 10% (v/v, 20% for QSG7701 cells) FCS and 1 mmol/l glutamine. The Chinese hamster ovary cell line with green fluorescence protein EGFP–Akt1 stable expression (CHO-Akt1) was purchased from Thermo Scientific Company and was cultured in Ham’s F-12 nutrient mixture supplemented with 10% FCS. For the in-vitro activation of Akt and mTOR kinases, cells were seeded overnight and then serum starved for 24 h, followed by treatment with 100 ng/ml insulin-like growth factor-I (IGF-1) (Calbiochem, San Diego, California, USA) for 10 min. Aminoguanidine (1 mmol/l) was added as an inhibitor of amine oxidase derived from FCS and had no effect on the various parameters of the cells measured in this study 18.

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Cytotoxicity assay

The cytotoxicity of ANISpm, in the presence or absence of 100 μmol/l DFMO or 500 μmol/l Spd, was evaluated in HepG2 cells and QSG7701 cells by the conversion of MTT into a purple formazan precipitate as described previously 18. The concentration of DFMO (100 μmol/l) and Spd (500 μmol/l) was selected for no obvious cytotoxicity on HepG2 cells and QSG7701 cells and the inhibition ratio was 2.76, 0.34% and 0.74, 0.18%, respectively. Cells were seeded into 96-well plates at 5×103 cells/well. Various concentrations of ANISpm with or without 100 μmol/l DFMO or 500 μmol/l Spd were subsequently added and incubated for 48 and 72 h. The inhibited rate was calculated from the plotted results using untreated cells as 100%.

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Intracellular fluorescence intensity assay

The intracellular fluorescence intensity of ANISpm was detected in HepG2 and QSG7701 cells by high content screening (HCS) (Thermo Scientific Cellomics ArrayScan Vti; Cellomics Inc., Pittsburgh, Pennsylvania, USA) analysis, which is a new quantitative cytometric technique. Briefly, HepG2 or QSG7701 cells were seeded into 96-well plates at 5×103 cells/well. After 24 h, the cells were stained with acridine orange (AO) (50 μg/ml) for 15 min and then washed three times with PBS. ANISpm (10, 30, or 50 μmol/l) was added into a 96-well plate alone or in combination with DFMO (100 μmol/l) and Spd (500 μmol/l), and then the intracellular fluorescence intensity of ANISpm for different time intervals (15, 30, and 60 min) was determined using Thermo Scientific Cellomics ArrayScan Vti (Cellomics Inc.). Cell numbers were determined by AO staining, and the intracellular fluorescence intensity of ANISpm was then determined at an excitation of 350 nm and an emission of 460 nm.

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Cell apoptosis evaluation

Cell apoptosis was evaluated using three methods. First, it was determined by morphologic observation. After treatment with ANISpm and/or DFMO or Spd, cells were harvested and stained with AO and ethylene dibromide (EB) and assessed by fluorescence microscopy (Leica, Wetzlar, Germany). Briefly, 1 μl of a stock solution (100 μg/ml AO and EB) was added to 25 μl of cell suspension. EB-negative cells with nuclear shrinkage, blebbing, or apoptotic bodies were counted as apoptotic cells. Second, after exposure to ANISpm (1 μmol/l) in the presence or absence of 100 μmol/l DFMO or 500 μmol/l Spd for 48 h, cells were fixed with ice-cold 70% ethanol and then stained with PI (100 μg/ml) after removing the RNA in the cells by RNase A treatment (50 μg/ml). The analysis was carried out using a FACScan (Becton Dickinson, Sparks, Maryland, USA) emitting an excitation laser light at 488 nm. Data acquisition and analysis were controlled by Modifit software (Becton Dickinson, San Jose, California, USA). Finally, apoptotic evaluation was carried out using the Annexin V-FITC apoptosis detection kit (Cell Signaling Technology). Cells were seeded in six-well plates and exposed to ANISpm in the absence or presence of 100 μmol/l DFMO or 500 μmol/l Spd for 48 h, and then harvested and stained according to the manufacturer’s instruction. The analysis was carried out using a FACScan (Becton Dickinson, San Jose, California, USA) emitting an excitation laser light at 488 nm. Fluorescence signals were detected at 518 and 620 nm for FITC and PI detection, respectively. Data acquisition and analysis were controlled using Cellquest software 18.

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Akt activity analysis by high content screening assay

HCS analysis was used to measure Akt activity on HepG2 and CHO-Akt1 cells using Thermo Scientific Cellomics ArrayScan Vti (Cellomics Inc.). Briefly, CHO-Akt1 cells were seeded at a density of 10 000 cells/well in black 96-well Packard Viewplates (Packard, Boston, Massachusetts, USA) coated with fibronectin and incubated overnight. Cells were then washed three times in cell wash buffer (Ham’s F-12 nutrient mixture containing glutamax-1 supplemented with 0.1% FCS, 0.1% BSA, and 5 mmol/l HEPES). The assay was initiated by adding 1 μmol/l ANISpm and/or 100 μmol/l DFMO or 500 μmol/l Spd for 48 h, which was prepared in assay buffer (Ham’s F-12 nutrient mixture containing glutamax-1 supplemented with 0.1% BSA and 5 mmol/l HEPES). Cells were fixed with 4% formalin buffer, washed three times with PBS and stained with 1 μmol/l Hoechst 33342, and read on Thermo Scientific Cellomics ArrayScan Vti (Cellomics Inc.) and Morphology Explorer BioApplication Software Module (Thermo Fisher Scientific–Cellomics, Pittsburgh, Pennsylvania, USA).

For the HepG2 cell, Akt1 translocation evaluation was carried out using the phospho-Akt activation kit (No. 8404102; Cellomics Inc.). Cells were seeded in 96-well plates, exposed to ANISpm in the absence or presence of 100 μmol/l DFMO or 500 μmol/l Spd for 48 h, and then stained according to the manufacturer’s instruction and read on Thermo Scientific Cellomics ArrayScan Vti (Cellomics Inc.) and Morphology Explorer BioApplication Software Module. IGF-I was used as a reference agonist, and compounds were assayed for their ability to inhibit IGF-I-stimulated membrane translocation of Akt1. The PI3K inhibitor Wor (200 nmol/l) was used as a reference antagonist.

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Measurement of mitochondrial membrane potential

Mitochondrial membrane potential (MMP) was determined by the retention of Rh123, a membrane-permeable fluorescent cationic dye. The uptake of Rh123 by mitochondria is proportional to the MMP 18. Briefly, cells (1×106) were incubated with 0.1 μg/ml Rh123 in the dark for 20 min at room temperature. After washing with PBS, the cells were analyzed by FACScan (Becton Dickinson, San Jose, California, USA) with an excitation and emission wavelength of 495 and 535 nm, respectively.

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Western blots

HepG2 cells, treated with ANISpm and/or DFMO and Spd for 48 h, were harvested and washed with PBS. Cytosolic and mitochondrial fractions were prepared as described in our previous study 18. Total cellular protein was isolated using the protein extraction buffer (containing 150 mmol/l NaCl, 10 mmol/l Tris (pH 7.2), 5 mmol/l EDTA, 0.1% Triton X-100, 5% glycerol, and 2% SDS). Protein concentrations were determined using the protein assay kit. Equal amounts of proteins (50 μg/lane) were fractionated using 8 or 12% SDS-PAGE and transferred to PVDF membranes. The membranes were incubated with primary antibodies. After washing with PBS, the membranes were incubated with peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibody, followed by enhanced chemiluminescence staining through the enhanced chemiluminescence system. β-Actin (45 kDa) was used to normalize for protein loading 18.

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Study in vivo
Evaluation of antitumor effects

Kunming male mice (6–8 weeks old) were purchased from the Laboratorial Animal Center of Henan (Zhengzhou, China). All animal procedures were performed following the protocol approved by the Institutional Animal Care and Use Committee at the Henan University. For the solid tumor model, the mice were injected subcutaneously in the right flank with 0.2 ml of a cell suspension containing 5×107 of H22 cells. Tumors were allowed to grow for ∼7 days before drug treatment. Tumor-bearing mice were randomly assigned to one of the following five treatment groups (n=10 mice/group): control (physiologic saline), ANISpm (2.5 mg/kg), DFMO+ANISpm, Spd+ANISpm, and amonafide (5 mg/kg). For the ascites tumor model, mice were injected in the abdominal cavity with 0.2 ml of a cell suspension containing 5×107 of H22 cells. The next day, the tumor-bearing mice were randomly assigned to one of the following five treatment groups (n=10 mice/group): control (physiologic saline), ANISpm (2.5 mg/kg), DFMO+ANISpm, Spd+ANISpm, and amonafide (5 mg/kg). ANISpm, physiologic saline, or amonafide was administered through a tail vein injection for 7 consecutive days. DFMO (0.5%) or Spd (0.1%), at doses that had no effect on tumor growth and metastasis, was administered ad libitum in tap water for 7 consecutive days. For the solid tumor study, the mice were killed by ether anesthesia after 24 h of drug withdrawal. Solid tumors were removed and weighed. The inhibitory rate was calculated as follows: inhibitory rate (%)=[(weightcontrol−weightdrug)/weightcontrol]×100. For each treatment group in the ascites model, mice were used for mean survival time (MST) analysis. The extended lifespan rate was calculated as follows: extend rate (%)=(MSTdrug/MSTcontrol)×100 19. For the tumor passive metastasis model, H22 cells (5×106 cells/mouse) for Kunming mice were injected intravenously through the tail vein. To ensure that all mice bore actively growing lung tumors before the drug treatment, pulmonary metastasis was allowed to develop for 10 days. On day 11, mice were injected with intravenous drug for 7 consecutive days. At day 18, mice were killed by ether anesthesia, the lungs were removed and weighed, and then fixed in Bouin’s fluid. After fixing, lung metastases nodus was numbered.

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Systemic toxicity evaluation

To study the toxic effects of ANISpm, as indicated by loss of body weight, food consumption, hematology, organ weights, and macroscopic evaluations, 30 mice without tumors were randomly assigned to one of three treatment groups (n=10 mice/group): control (physiologic saline), ANISpm (2.5 mg/kg), and amonafide (5 mg/kg). The weight of mice was recorded daily before the tail vein injection. The experiment was terminated on day 8. Blood and bone marrow were collected, and hematological profiling was carried out by determining the platelet, red blood cell, white blood cell, and bone marrow cell counts using a Coulter Counter T-890 (Coulter Electronics, Hialeah, Florida, USA). The organ index was investigated for the evaluation of systemic toxicity. Organ index (%)=(organ weight/body weight)×100.

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Data analysis

All data were presented as mean±SD and analyzed using Student’s t-test or analysis of variance, followed by the q test.

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Results

ANISpm triggers antiproliferation and cell death in HepG2 cells but not in QSG7701 cells

Hepatoma cells (HepG2) and normal hepatic cells (QSG7701) were stimulated with the indicated concentrations of ANISpm for 48 and 72 h, and the cell viability was assessed to determine whether ANISpm inhibits proliferation and has selectivity. As shown in Fig. 1b, ANISpm decreased the cell viability of HepG2 tumor cells in a dose-dependent and time-dependent manner. Cotreatment with ANISpm and DFMO, a specific inhibitor of ornithine decarboxylase activity, further decreased cell survival; however, Spd attenuated this effect. The IC50 value was decreased or increased after combination with DFMO or Spd, respectively. In contrast, nontumor QSG7701 cells were less growth inhibited with ANISpm treatment, even when cotreated with DFMO (Table 1). Thus, these data show that ANISpm decreases cell viability and inhibits cell proliferation in a tumor cell-specific manner that is synergistically enhanced with DFMO and reversed with Spd.

Table 1
Table 1
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More ANISpm uptake in HepG2 tumor cells than normal QSG7701 cells

To assay the uptake of ANISpm in HepG2 cells and QSG7701 cells, the intracellular fluorescence of ANISpm was measured using HCS following the addition of ANISpm to the culture medium. The intracellular fluorescence intensity of ANISpm increased in a dose-dependent and time-dependent manner in HepG2 cells (Fig. 1b). Furthermore, DFMO enhanced and Spd attenuated this fluorescence intensity, respectively (Fig. 1c). However, the uptake of ANISpm was considerably lower in QSG7701 cells compared with that in HepG2 cells, even when cotreated with DFMO (Fig. 1d and e), indicating that ANISpm uptake is mediated by the PTS.

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ANISpm induces HepG2 cell apoptosis

To determine whether apoptosis contributes toward the decreased cell survival following ANISpm treatment, we assessed cell apoptosis using fluorescence microscopy and flow cytometry. As shown in Fig. 2a, the fluorescence of control cells was diffuse and uniform, whereas the typical apoptotic morphology was discovered after treatment with ANISpm, including chromatin condensation, dense nucleoli, and apoptotic bodies. Furthermore, DFMO enhanced and Spd attenuated these apoptotic effects, respectively. In addition to morphological changes, we detected cell apoptosis using Annexin V and PI staining. The percentage of apoptotic cells increased significantly after treatment with ANISpm alone or cotreatment with DFMO, whereas Spd antagonized ANISpm-induced apoptosis (Fig. 2b). Taken together, the above data show that the antiproliferative effect of ANISpm is, at least in part, because of cell apoptosis.

Fig. 2
Fig. 2
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ANISpm inhibits the activity and translocation of the Akt

Using CHO cells transfected with GFP-tagged Akt1 protein, IGF-1 treatment stimulates the translocation of Akt1 from the cytoplasm to the plasma membrane. ANISpm alone or cotreated with DFMO inhibited this translocation, whereas Spd attenuated the inhibitory effect of ANISpm (Fig. 2c). Furthermore, the ANISpm inhibition of Akt1 translocation was also observed in HepG2 cells (Fig. 2d). Western blot analysis showed that the level of phosphorylated Akt kinase decreased significantly during incubation with ANISpm. Meanwhile, the level of total Akt protein was constant, indicating that the observed changes might reflect a decrease in kinase activity rather than a decrease in the cellular protein content. The expression of PTEN, an upstream-suppressor of Akt, was also constant (Fig. 2e).

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The molecular mechanism of ANISpm induces cell-cycle arrest

ANISpm significantly induced HepG2 cell-cycle arrest in the G0/G1 phase and this effect was enhanced or attenuated by DFMO and Spd, respectively (Fig. 3a). To determine the molecular mechanisms responsible for ANISpm-induced cell-cycle arrest, we examined the expression of proteins involved in cell-cycle regulation during the G1 phase, such as Cdk4 and p27Kip1. In comparison with untreated control cells, the expression of Cdk4 and p27Kip1 was downregulated or upregulated by treatment with ANISpm alone or with additive DFMO, respectively (Fig. 3a). The mTOR/p70S6K-signaling pathway is related to G1 phase arrest, and is therefore possibly modulated by ANISpm in HepG2 cells. Treatment of HepG2 cells with ANISpm resulted in downregulated phospho-mTOR and phospho-p70S6K, which are downstream effectors of the mTOR-signaling pathway after 48 h, whereas the total levels of mTOR and p70S6K remained unchanged at the same time points. These data indicated that ANISpm suppresses the mTOR-signaling pathway in HepG2 cells, probably because of inhibition of Akt activity. Furthermore, these effects were enhanced by DFMO and attenuated by Spd (Fig. 3a).

Fig. 3
Fig. 3
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The molecular mechanism of ANISpm induces cell apoptosis

Akt deactivation-mediated cell apoptosis is often correlated with the mitochondrial signal pathway; thus, we initially assessed the change in MMP after ANISpm treatment by flow cytometry using the fluorescent probe Rh123. As shown in Fig. 3b, compared with the control, ANISpm significantly decreased the MMP of HepG2 cells and DFMO augmented ANISpm-induced MMP loss. In contrast, Spd addition inhibited the ANISpm-induced MMP loss. Furthermore, western blot analysis also indicated that ANISpm-induced cytochrome c release from the mitochondria to the cytoplasm (Fig. 3b). Previous reports have shown that Bcl-2 family numbers are often involved in mitochondria-induced cell apoptosis. Western blot analysis showed that the treatment of HepG2 cells with 1 μmol/l ANISpm alone or cotreated with DFMO or Spd for 48 h only slightly alters the protein levels of Bad and Bcl-xL. However, ANISpm induced Bad Ser136 dephosphorylation accompanied by less expression of Bad–14-3-3, which normally complexes with phospho-Bad (Fig. 3b). In addition, ANISpm alone or cotreated with DFMO also obviously induced caspase-9 and caspase-3 activation, whereas Spd attenuated ANISpm-mediated caspase-9 and caspase-3 activation.

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Antitumor effect and systemic toxicity of ANISpm in vivo

ANISpm significantly suppressed tumor growth in comparison with the control mice in the solid tumor model (Fig. 4a). The tumor inhibitory ratio was 40.2% (1.37±0.43 g) for amonafide and 53.7% (1.06±0.47 g) for ANISpm, as compared with control mice (2.29±0.68 g), respectively. Furthermore, DFMO enhanced ANISpm-induced tumor growth inhibitory effect (67.2%, 0.75±0.44 g). In contrast, Spd attenuated this effect (14.8%, 1.95±0.56 g). The MST of the control mice was 13.3±3.9 days in the ascites model. After treatment with ANISpm, ANISpm+DFMO, ANISpm+Spd, or amonafide, the lifespan was increased by 2.3-fold (30.4±6.1 days) and 2.6-fold (34.4±5.8 days), 1.6-fold (21.3±4.6 days), and 1.5-fold (19.6±3.8 days), respectively (Fig. 4b). In the lung metastasis model, mice treated with ANISpm showed few visible metastases nodus, DFMO and Spd enhanced or attenuated this effect, respectively, whereas all mice treated with physiologic saline had an extensive tumor burden in the lungs, and amonafide only moderately decreased lung metastases nodus numbers (Fig. 4c).

Fig. 4
Fig. 4
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In systemic toxicity evaluation, we found no weight loss in the ANISpm-treated mice, whereas treatment with 5 mg/kg amonafide was associated with weight loss, indicating the dose toxicity to some extent. Moreover, we found that the food consumption of tested mice was decreased after treatment with amonafide, whereas ANISpm treatment had no obvious effect on the food consumption of mice in contrast with the control (data not shown). At the same time, no significant difference in the organic factors and pathological change of the heart, liver, kidney, brain, and lung was observed for both tested drugs and the control (Fig. 5a and d). In addition, amonafide downregulated spleen and thymus indexes, whereas ANISpm had no influence on immune function (Fig. 5b). Finally, ANISpm had no obvious influence on bone marrow cell numbers, indicating that ANISpm had no evident hematotoxicity in this test. In addition, amonafide resulted in a significant decrease in platelet, red blood cell, and white blood cell numbers compared with the control, whereas ANISpm did not (Fig. 5c).

Fig. 5
Fig. 5
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Discussion

The synthetic homospermidine as a vector exerts favorable PTS recognition in an anthracene–polyamine system, and may be potentially useful for the rational design of novel drug–polyamine conjugates 14,15. However, the conjugate with the native Spm incorporated into naphthalimide also showed potent antitumor activity and cell selectivity 17. Similar conjugates need to be investigated in detail to explore its potential as an efficient vector. In this report, 3-amino-naphthalimide, the key component of amonafide in clinical trials, was conjugated with Spm to form ANISpm.

Extensive evidence points to the fact that diverse polyamine conjugates could harness PTS for entrance into targeted cells 13–15. There are two well-accepted indirect methods to evaluate the transport of polyamine conjugates through PTS. Compared with the CHO/CHO–MG method, the DFMO/Spd approach has the advantage that it is suitable for the assessment of most cell lines 20,21. DFMO, an irreversible inhibitor of ornithine decarboxylase, is the rate-limiting enzyme in the biosynthesis of polyamines. The presence of added DFMO inhibits the cellular polyamine biosynthesis, which in turn facilitates the import of exogenous polyamine through PTS. Polyamine conjugates with some native polyamine properties will have more chances to enter the cells, and decreased IC50 values are often observed. In contrast, Spd, a natural PTS substrate, outcompetes the PAT-targeted polyamine conjugates for cellular entry and thus ‘rescues the cell’ from these cytotoxic drugs. Indeed, the additive Spd is an antagonist for drug uptake and exerts a significant ‘import-inhibition’ effect. Therefore, increased IC50 values are often observed in the presence of Spd. In our experiments, both the decreased IC50 values with DFMO and the increased IC50 values with Spd indicated that ANISpm can be transported through PTS.

In considering new targets and pathways for therapy, it is also important to be aware of the toxicity to normal cells. ANISpm significantly inhibited human hepatoma HepG2 cell proliferation, with little growth inhibition in ANISpm-treated normal liver QSG7701 cells. This indicates that ANISpm shows a potent antigrowth effect and excellent tumor selectivity. This selectivity may be because of the increased need for polyamines in the more rapidly proliferating tumor cells compared with normal ones 16. The rationale for the design of polyamine conjugates is that their PTS recognition could reduce the adverse effects on normal cells. The differential uptake of ANISpm and growth inhibition effects on normal and tumor cells support this hypothesis.

Tumor cells remain dependent on one or a few key genes for the maintenance of their malignant nature and survival. As the PI3K/Akt pathway is deregulated in a wide range of tumor types, inhibitors developed against this pathway may have broad therapeutic utility. In addition, the PI3K/Akt pathway includes many components that are kinases with gain-of-function mutations. These mutated kinases are attractive targets for the development of small-molecule inhibitors that inhibit these aberrant proteins 22. There are many examples in the literature showing that the activation of Akt in tumor cells is critical for mediating cell-survival signaling and protecting cells from apoptosis 23. We therefore investigated whether ANISpm-induced cell-cycle arrest and apoptosis were related to the Akt signal pathway. The results obtained showed that ANISpm significantly inhibited Akt1 phosphorylation and translocation from the cytoplasm to the plasma membrane. This effect was enhanced by DFMO and attenuated by Spd. However, ANISpm had no influence on PTEN and total Akt1, even in combination with DFMO or Spd. PTEN is a lipid phosphatase that dephosphorylates PI3K products, and thus prevents their phosphorylation and downstream activation of the serine/threonine kinase Akt 24. The effect of ANISpm was different from that of some vitamin analogs, which can upregulate the expression of the tumor suppressor PTEN in tumor cells 25. Our results show that ANISpm only inhibits Akt phosphorylation, which is critical for the activation of the Akt signal pathway, and DFMO enhances or Spd attenuates this effect but does not change the target of ANISpm.

p27Kip1 acts as an inhibitor of CDKs, and thereby inhibits progression through the G1 phase of the cell cycle. Previous work has shown that p27Kip1 is directly phosphorylated by Akt 26. Our data show that ANISpm induces HepG2 cell-cycle arrest in the G0/G1 phase, downregulates Cdk4, and upregulates p27Kip1. Previous research has shown that the downstream-signal molecules of the Akt signal pathway, such as mTOR kinase and p70S6 kinase, are associated with cell growth and cell-cycle progress, with mTOR regulating protein synthesis through activation of the ribosomal protein p70S6 kinase 27. Our results show that ANISpm alone or in combination with DFMO inhibits Akt/mTOR signaling. Furthermore, Spd could weaken ANISpm-induced effects but did not reverse them.

Mitochondrial dysfunction has been implicated as being a key mechanism in apoptosis in various cell death profiles 28. Two major events have been observed in apoptosis involving mitochondrial dysfunction. One event is the change in the membrane permeability and subsequent loss of membrane potential. The other is the release of apoptotic proteins, such as cytochrome c, from the intermembrane space of mitochondria into the cytosol 29. In agreement with these observations, we found that the ANISpm-induced loss of MMP was accompanied by the release of cytochrome c. Thus, it is plausible that mitochondrial dysfunction may be involved in ANISpm-induced HepG2 cell apoptosis.

Previous studies have shown that Bcl-2 family numbers are usually involved in the polyamine conjugate-mediated mitochondria apoptotic pathway 30. Accordingly, we focused on the proapoptotic Bcl-2 family member Bad, a key downstream effector of Akt, which can be phosphorylated at Ser136 by Akt 31. It has been found that in the absence of activated Akt, Bad forms a heterodimer with Bcl-xL, which is an antiapoptotic protein that prevents the release of cytochrome c from the mitochondria. This complex formation abrogates the antiapoptotic function of Bcl-xL, thereby facilitating cell apoptosis through the mitochondrial pathway. Conversely, when Akt is activated, Bad is phosphorylated and translocated into the cytoplasm by binding with the phosphoserine-binding protein 14-3-3. The sequestration of Bad from the mitochondria frees Bcl-xL to facilitate antiapoptotic signaling. As a consequence, the dynamic interaction between Bcl-xL and Bad represents a critical determinant of cell fate downstream of the PI3K/Akt cascade 32. In the present study, we found that ANISpm induces Akt inactivation and Bad dephosphorylation. Our results indicate that Bad dephosphorylation, followed by the dissociation of Bad from 14-3-3 and the subsequent association of Bad with Bcl-xL may be involved in ANISpm-induced mitochondrial dysfunction and apoptosis. In addition, ANISpm also induces Bcl-2 downregulation through the Akt/mTOR signal pathway. These results indicated that Akt inactivation, followed by Bcl-2 downregulation and Bad activation, the dissociation of Bad from 14-3-3, and the subsequent association of Bad and Bcl-xL, are involved in ANISpm-induced mitochondrial dysfunction, cytochrome c release, activation of caspases, and consequent cell apoptosis.

Furthermore, naphthalimide derivatives have been shown to be DNA intercalators. Amonafide, which, as indicated above, has completed several phase I and II clinical trials, acts as a topo II poison 33. We also found that polyamine–naphthalimide conjugates could affect DNA 17.

ANISpm also exerted more potent antitumor activity than amonafide in vivo. The growth of mouse hepatoma H22 cells was inhibited significantly in a subcutaneous xenograft model. ANISpm markedly extended the MST of H22 ascites-burden mice than control and amonafide. The emergence of disseminated metastases remains the major cause of mortality, being responsible for 90% of all cancer deaths; thus, the inhibition of tumor cells metastases is a vital factor for tumor therapeutics. Our results showed that ANISpm clearly prevented the lung metastases of H22 cells. Furthermore, DFMO and Spd enhanced or attenuated these against tumor effects in vivo, indicating that ANISpm possessed potent antitumor activity in vivo and in vitro. However, early naphthalimides, for example, amonafide, although effective in its phase 2 clinical trials when administered either alone or in combination with other antitumor drugs, had dose-limiting bone marrow toxicity, leading to thrombocytopenia, anemia, and leukopenia. To eliminate this unpredictable and unacceptable toxicity, persistent efforts have been made toward the elimination of the site on naphthalimides. Preliminary toxicology evaluation showed that ANISpm had no obvious system toxicity at a dose of 2.5 mg/kg, which exerted potent antitumor activity in vivo, especially hematotoxicity. Encouragingly, ANISpm could also induce weight gain in mice, whereas the weight decreased after treatment with amonafide.

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Conclusion

These results provide an ANISpm-mediated death-signaling pathway that preferentially targets the HepG2 tumor cells and not normal cells. The Akt inactivation is followed by a series of cellular events. The downstream pathway includes Bad dephosphorylation, dissociation of 14-3-3 and Bad, and binding to Bcl-xL, which leads to disruption of the mitochondrial membrane, release of cytochrome c, and caspase cascade activation. In addition, the Akt/mTOR-signaling pathway is also correlated with cell arrest and apoptosis. Thus, ANISpm merits further investigation as a potential chemotherapeutic agent against HCC.

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Acknowledgements

This project was supported by the National Natural Science Foundation of China (No. 20872027), the Henan Natural Science Foundation (No. 0821022700, 072102330028, 102300410095), and the China Postdoctoral Science Foundation Funded Project (No. 20090450092, 201003395).

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Conflicts of interest

There are no conflicts of interest.

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Keywords:

Akt signal; apoptosis; cell cycle; mTOR; polyamine conjugate

© 2013 Lippincott Williams & Wilkins, Inc.

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