LQFM030 reduced Ehrlich ascites tumor cell proliferation and VEGF levels

Accepted Manuscript LQFM030 reduced Ehrlich ascites tumor cell proliferation and VEGF levels

Mariana Flavia da Mota, Flávio Silva de Carvalho, Renato Ivan de Ávila, Paulo Henrique Marcelino de Ávila, Alane Pereira Cortez, Ricardo Menegatti, José Ricardo Sabino, Thais Rosa Marques dos Santos, Sandro Antônio Gomes, Luiz Carlos da Cunha, Marize Campos Valadares PII: DOI: Reference:

S0024-3205(17)30676-8 https://doi.org/10.1016/j.lfs.2017.12.029 LFS 15491

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

1 June 2017 15 December 2017 22 December 2017

Please cite this article as: Mariana Flavia da Mota, Flávio Silva de Carvalho, Renato Ivan de Ávila, Paulo Henrique Marcelino de Ávila, Alane Pereira Cortez, Ricardo Menegatti, José Ricardo Sabino, Thais Rosa Marques dos Santos, Sandro Antônio Gomes, Luiz Carlos da Cunha, Marize Campos Valadares , LQFM030 reduced Ehrlich ascites tumor cell proliferation and VEGF levels. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), https://doi.org/ 10.1016/j.lfs.2017.12.029

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ACCEPTED MANUSCRIPT LQFM030 reduced Ehrlich ascites tumor cell proliferation and VEGF levels

Mariana Flavia da Motaa,#, Flávio Silva de Carvalhob,#, Renato Ivan de Ávilaa, Paulo Henrique Marcelino de Ávilaa, Alane Pereira Corteza, Ricardo Menegattib, José Ricardo Sabinoc, Thais Rosa Marques dos Santosa,

a

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Sandro Antônio Gomesd, Luiz Carlos da Cunhad and Marize Campos Valadaresa,*

Laboratório de Farmacologia e Toxicologia Celular, Faculdade de Farmácia, Universidade Federal de Goiás,

Goiânia, GO, Brazil. b

Laboratório de Química Farmacêutica Medicinal (LQFM), Faculdade de Farmácia, Universidade Federal de

Goiás, Goiânia, GO, Brazil.

d

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Laboratório de Cristalografia, Instituto de Física, Universidade Federal de Goiás, Goiânia, GO, Brazil. Núcleo de Estudos e Pesquisas Tóxico-Farmacológicas (NEPET), Faculdade de Farmácia, Universidade

Federal de Goiás, Goiânia, GO, Brazil.

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c

These authors equally contributed for this study.

*

Corresponding author. Address: Faculdade de Farmácia – Universidade Federal de Goiás, Rua 240 esquina

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#

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com 5ª Avenida, s/n, Setor Universitário, Goiânia, GO, Brazil. CEP: 74605.170. E-mail address: [email protected] / [email protected]

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Phone / fax: +55 62 3209-6039 ext. 202.

ACCEPTED MANUSCRIPT Abstract

Aims: This study reports the biological properties of LQFM030 in vivo, a molecular simplification of the compound nutlin-1.

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Main methods:

Ehrlich ascites tumor (EAT)-bearing mice were treated intraperitoneally with LQFM030 (50, 75 or 150 mg/kg) for 10 days to determine changes in ascites tumor volume, body weight, cytotoxicity and angiogenesis. Moreover, flow cytometric expression of p53 and p21 proteins and caspase-3/7, -8 and

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-9 activation were investigated in EAT cells from mice treated. Acute oral systemic toxicity potential of LQFM030 in mice was also investigated using an alternative method.

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Key findings:

Treatment of EAT-bearing mice with LQFM030 resulted in a marked decline in tumor cell

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proliferation and the vascular endothelial growth factor (VEGF) levels along with enhanced survival of the mice. Apoptotic tumor cell death was detected through p53 and p21 modulation and increase

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of caspase-3/7, -8 and -9 activity. LQFM030 also showed orally well tolerated, being classified in the

Significance:

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UN GHS category 5 (LD50 > 2000-5000 mg/Kg).

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LQFM030 seems to be a promising antitumor candidate for combinatory therapy with typical cytotoxic compounds, reducing the toxicity burden while allowing a superior anticancer activity. Moreover, these data also open new perspectives for LQFM030 as an antiangiogenic agent for treatment of diseases involving VEGF overexpression.

Keywords: Ehrlich ascites tumor; nutlins; p53; p21; VEGF; MDM2 inhibitors.

ACCEPTED MANUSCRIPT Introduction

The tumor suppressor p53 coordinates a signal transduction network designed to combat oncogenic stress.[1-3] Among the pathways coordinated by this multifunctional transcription factor, it is known that p53 modulates its transcriptional target p21, which plays an important role in

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anticancer pharmacology treatment since it can trigger tumor cell cycle arrest associated with cell death by apoptotic mechanisms.[4] In this sense, anticancer drug discovery field has focused in the development of novel compounds that can activate or restore the p53 pathway[5,6], such as small molecules as nutlins, RG7112, an optimization of nutlin-3a, RO5503781, MI 773, DS 3032b and

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PRIMA 1MET, which are in phase I clinical trial.[6,7]

Among the small molecules developed until now, nutlins have showed promising antitumor

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effects. These cis-imidazoline analogues showed to be non-genotoxic murine double minute 2 (MDM2) protein antagonists binding MDM2 in the p53-binding pocket, preventing p53

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degradation.[8] This class of compounds also promotes cytotoxic and apoptotic events in leukemic cells, in contrast to normal CD34+ hematopoietic progenitor cells that showed low cytotoxicity when

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exposed to this compound.[9] In addition, it was demonstrated that nutlins trigger cell cycle arrest

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and/or apoptosis independent of the p53 status in leukemic cells (wild type or mutant).[10] In addition, it has been showed that treatment of hematological malignancies with nutlin

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promotes an increase of p53 levels as well as of its transcriptional targets, including MDM2 and p21 proteins, triggering cell cycle arrest and/or apoptotic cell death[9,11-15]. Nutlins also showed effects on drug resistance in chronic leukemia[13], synergize with genotoxic drugs[16,17] and anti-angiogenic activity.[18] Nutlin-3a showed antitumor and antiangiogenic effects via targeting the vascular endothelial growth factor (VEGF) and p53 pathways using an in vivo neuroblastoma model.[19] Moreover, in vitro VEGF suppression and in vivo VEGF-mediated antiangiogenic effects have been

ACCEPTED MANUSCRIPT triggered by MDM2 inhibitors, antisense oligonucleotides (ASO) and nutlin-3 in various human breast cancer models with different p53 status.[20] Recently, we reported the synthesis and several in vitro biological properties of the compound LQFM030 [1-(4-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl) piperazin-1-yl) ethanone] (Figure 1), obtained through molecular simplification of the lead compound nutlin-1. This

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heterocyclic compound induced DNA fragmentation, phosphatidylserine externalization and activation of initiators and effector caspases. Furthermore, it was demonstrated that LQFM030 did not affect p53 gene transcription; although MDM2 mRNA showed increased and MDM2 protein was decreased.[21]

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Considering the important findings obtained with nutlins and their analogues, represented here by the compound LQFM030, this study evaluated the possible antitumor effects triggered by

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LQFM030 in an in vivo Ehrlich ascites tumor (EAT) model as well as its tumor cell death mechanisms involved. Furthermore, acute oral systemic toxicity hazard potential of LQFM030 in

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mice was also investigated using the alternative approach focused on reducing animals use proposed

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by OECD[24].

ACCEPTED MANUSCRIPT Materials and Methods

Animals

Considering that animal experimentation in cancer research remains essential to

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understand the fundamental mechanisms of antitumor drugs as well as to meet regulatory requirements, the in vivo analyses conducted in this study were focused on animal welfare. The experimental design considered the minimum number of animals without reducing the scientific integrity of data generated. Also, procedures as reduced volume of injections, anesthesia and

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analgesia were carried out.

Male and female Swiss albino mice (25-30 g) were obtained from Indústria Química do

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Estado de Goiás (IQUEGO). The animals were kept under constant ambient conditions, including 12 h/12 h light-dark cycle and controlled temperature (23 ± 2ºC). Standard granulated chow (Presence

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Nutrição Animal, São Paulo, SP, Brazil) and drinking water were provided ad libitum. All experimental procedures and protocols realized in this study were reviewed and approved by the

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Research Ethics Committee of the Federal University of Goiás (CEUA - PRPPG/UFG Nº. 018/12).

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At the end of the experiments, mice were previously anesthetized using xylazine (10 mg/kg) (Syntec, Cotia, SP, Brazil) and ketamine hydrochloride (100 mg/kg) (König, Santana de Parnaíba, SP, Brazil)

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administered intraperitoneally, and then euthanized by cervical dislocation [22].

LQFM030 treatment LQFM030 was synthesized and characterized as previously described

[21]

. A LQFM030 (5

mg/mL)-containing emulsion was then prepared as follows: to 5 mg of LQFM030 was added 100 µL of sunflower oil (Caramuru Alimentos, Itumbiara, GO, Brazil), 100 µg of soy phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL, USA) and 50 µL of ethanol (Sigma-Aldrich, St. Louis, MO,

ACCEPTED MANUSCRIPT USA). The final volume was completed with ultrapure water. In addition, a blank emulsion (without LQFM030) was also prepared. Mouse EAT model was maintained in mice through serial intraperitoneal transplantation as previously described.[23] For the assay, EAT cells (1 x 107 cells/mL) were injected intraperitoneally into each mouse. The animals were then randomly weighted and divided into four groups

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(n=6/group). After 24 h of the tumor transplantation, mice were treated intraperitoneally with doses (0.1 mL/animal) of LQFM030 (50, 75 or 150 mg/kg) or its vehicle (EAT control) for 10 days. A fifth group containing healthy mice (non-EAT-bearing animals, n=6), named control group, received only

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phosphate buffered saline (PBS) (control).

In vivo antitumor activity evaluation

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The antitumor activity of LQFM030 was determined by analysis of ascites tumor volume, body weight, viable tumor cell count and angiogenesis. To determine whether the compound

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inhibited tumor growth and angiogenesis, the tumor volume was obtained by the difference in body weight between tumor implantation and the last day of treatment. Ascites fluids were removed, the

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cells were harvested and resuspended in PBS followed incubation for 2 h at 37ºC in a humidified

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atmosphere of 5% CO2 in air. The non-adherent population, excluding macrophage, was aspirated

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out and viable cells were counted using trypan blue dye exclusion method.

VEGF level measurement VEGF levels secreted by EAT cells into the peritoneal ascites was measured using mouse VEGF quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions. In brief, 50 µL assay diluent and 50 µL ascites from control or treated mice were added to a 96-well microplate pre-coated with a polyclonal antibody specific for mouse VEGF. Recombinant mouse VEGF was used to set up the standard curve. After incubation for 2 h at room

ACCEPTED MANUSCRIPT temperature, the plate was washed and polyclonal anti-mouse VEGF antibodies conjugated to horseradish peroxidase were added followed additional incubation of 2 h. After that, the wells were washed again and substrate added in each well followed incubation of 30 min. Absorbance was then measured at 450 nm using a microplate reader (Awareness Technology, Germany).

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Hematological and biochemical analyses

Anesthetized animals had blood samples collected and separated into two vials, one containing EDTA anticoagulant (Newprov, Pinhais, PR, Brazil) to perform a complete blood count and another one to separate the serum for biochemical analysis. The hematological test was

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performed using an ABX Micros 60 apparatus (Horiba, São Paulo, SP, Brazil). Biochemical analyses

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were performed using LabmaxPlenno apparatus (Labtest, Belo Horizonte, MG, Brazil).

Flow cytometric analysis of p53 and p21 proteins

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EAT cells (1 x 106 cells/mL) from treated and non-treated animals were washed with PBS, suspended in 250 µL of BD Cytofix/Cytoperm™ solution (BD Biosciences, San Jose, CA, USA)

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followed incubation of 20 min at 4°C. After that, cells were washed twice in PBS-Tween 20 and

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incubated, for 15 min at room temperature, with mouse monoclonal anti-p53 (pAb 122, sc-56182 FITC) or anti-p21 (F-5, sc-6246 FITC) antibodies, both purchased from Santa Cruz Biotechnology

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(Dallas, TX, USA). Afterward, cells were washed in PBS-Tween 20, suspended in PBS and analyzed using BD FACSCanto II flow cytometer (Becton Dickinson, San Jose, CA, USA) by acquisition of a total of 10,000 events.

Flow cytometric analysis of the activities of caspases-3/7, -8 and -9 The activities of caspases in EAT cells (1 x 106 cells/mL) from treated and non-treated animals were carried out using a CaspaTagTM Caspase-3/7, -8 and -9 In Situ Assay kit (MilliporeTM,

ACCEPTED MANUSCRIPT Temecula, CA, USA), following the manufacturer’s instructions. A total of 10,000 events was acquired and analyzed using BD FACSCanto II flow cytometer.

Acute oral systemic toxicity assessment The evaluation of in vivo acute oral toxicity of LQFM030 was performed using an

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alternative method focused on reducing animals use, the OECD test guideline Nº 423 - Acute Oral Toxicity Class.[24] First, the initial dose selected for LQFM030 (300 mg/kg) was chosen based on the data obtained from an in vitro alternative test of the incorporation of neutral red into 3T3 basal cells (data not shown). A dose of 2000 mg/kg was also investigated. As recommended by OECD test

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guideline Nº 423, the compound was prepared in sunflower oil and a single dose of LQFM030 or its vehicle was administered orally (gavage) to female Swiss mice (0.2 mL/animal, n=3). After

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treatment, the animals were observed at 5-30 min and every hour up to the twelfth hour of the first day. Hippocratic screening method was employed to evaluate the effects of the LQFM030 on the

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consciousness, disposal, activity and coordination of the motor system and muscle tone, activity of the autonomic and central nervous system of the animal. In the end of experiment, LD50 (dose that

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causes death in 50% of animals) was obtained and used to classify LQFM030 in accordance with

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United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS). In addition, after euthanasia of the animals, abdominal cavity of each mouse was opened to

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check possible macroscopic changes. Organs (liver, kidney, spleen, heart, lung and stomach) were also collected for histological evaluation using routine procedures for inclusion of tissue sections on paraffin, sectioning and hematoxylin-eosin staining. A histopathologist performed a complete examination of the tissues examined.

Statistical analysis

ACCEPTED MANUSCRIPT Data expressed as means ± SD are representative of at least two different experiments. The difference between groups was evaluated by t test or one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test or Tukey’s post-test, using GraphPad Prism version 5.01 software for Windows (San Diego, CA, USA). Statistical significance was established

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as p<0.05.

ACCEPTED MANUSCRIPT Results

Effects of LQFM030 treatment in EAT-bearing mice The effects of LQFM030 on the viability of tumor cells from EAT-bearing mice were evaluated after 10 days of treatment using the trypan blue exclusion method. Treatment of EAT-

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bearing mice with 50, 75 or 150 mg/kg of LQFM030 resulted in a marked dose-dependent decline in EAT cell proliferation (p<0.05) (Figure 2a). The 50, 75 and 150 mg/kg doses decreased cell viability by 39.4, 42.1 and 55.4%, respectively. The administration of LQFM030 doses also resulted in a dose-dependent decrease of the body weight of animals (p<0.05) (Figure 2b) associated with

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reduction of the volumes of ascites, in special at 150 mg/kg/day dose (p<0.05) (Figure 2c), when compared to the untreated group. Treatment with the 150 mg/kg/day dose promoted a 54.67%

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reduction in intraperitoneal tumor cell burden. In additional, hematological alterations induced by the tumors in these animals were partially reversed by the treatment (Table 1). Similar results were also

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observed for the serum biochemical parameters in EAT-bearing mice treated with LQFM030 (Table 2).

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Regarding the antiangiogenic activity of LQFM030, analysis of the vascular pattern of the

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peritoneal wall showed morphological changes (Figure 3a). Untreated EAT-bearing mice showed tortuous dilated and congested vessels in comparison to healthy mice (Figure 3a). After treatment

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with 150 mg/kg/day of LQFM030, the capillaries were markedly reduced in diameter and appeared similar to those in the healthy group. Moreover, the treatment with 150mg/kg resulted in a biological tendency towards a reduction in VEGF levels in the peritoneal washing supernatant; however, no statistical significance was detected (Figure 3b). In addition, untreated EAT-bearing mice died within 20 days. On the other hand, treatment with 150 mg/kg/day of LQFM030 significantly enhanced the survival rate to 25 days (p<0.05) (data not shown).

ACCEPTED MANUSCRIPT Apoptosis in LQFM030-treated EAT-bearing mice The effects of LQFM030 on p53 and p21 expressions were investigated in EAT cells from mice treated with 150 mg/kg of the compound for 10 days. The treatment promoted a 3.5-fold increase in p53 protein levels (p<0.05) (Figures 4a, b and c) associated with an increase of 2.0-fold in p21 protein levels (p<0.05) (Figures 4d, e and f).

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Since caspases are important in the apoptotic process modulated by p53, the roles of caspase-3/7, caspase-8 and caspase-9 in LQFM030-induced EAT cell death were evaluated. EAT cells from mice treated with LQFM030 (150 mg/kg) for 10 days showed a significant increase in the activities of all caspases examined when compared to control (p<0.01) (Figures 5a-i). These findings

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resulted in 1.8, 1.3 and 1.7-fold increases in caspase-3/7, -8 and -9 activities, respectively (p<0.01).

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In vivo oral acute toxicity study

In view of the positive effect of LQFM030 in preventing cancer progression in vivo, we

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evaluated the acute oral systemic toxicity hazard of this compound using the OECD test guideline Nº 423. The initial doses selected for LQFM030 were 300 mg/kg followed by 2000 mg/kg based on

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previous results from our group. There was no signal of toxicity or death in animals exposed to a

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single LQFM030 dose at 300 mg/kg. In relation to the single LQFM030 dose at 2000 mg/kg, it promoted piloerection in the first two hours after exposure. In the next 14 days, no other signs of

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abnormality or death were observed. The necropsy showed no macroscopic changes in the animals. Thus, LQFM030 showed low acute oral systemic toxicity, since LD50 obtained classified the compound as UN GHS category 5 (LD50 > 2000-5000 mg/Kg). Moreover, histological analysis of liver, kidney, spleen, heart and lung showed normal architecture in LQFM030-treated animals in comparison to control (Figure 6). However, the stomachs of treated animals showed atypical architectures, with signs of hyperplasia (Figure 7). Furthermore, there were no differences in body weight of LQFM030-treated animals when compared to untreated group (data not shown).

ACCEPTED MANUSCRIPT Discussion

In this study, the potential antitumor effects promoted by the treatment of EAT-bearing mice with LQFM030 doses (50, 75 or 100 mg/kg) for 10 days were investigated. In parallel, oral acute toxicity assessment of LQFM030 was also carried out. The main findings are highlighted as

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follows: (1) LQFM030, especially at dose of 150 mg/kg, inhibited EAT cell proliferation and, consequently, reduced tumor growth; (2) LQFM030 partially reversed EAT-induced hematological and biochemical alterations; (3) LQFM030 changed the vascular pattern of the peritoneal wall of EAT-bearing mice associated with tendency towards a reduction in VEGF levels, suggesting that this

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compound has an antiangiogenic activity; (4) LQFM030 triggered apoptosis in EAT cells from mice treated by modulation of p53 and p21 levels and caspase activation; (5) LQFM030 increased the

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survival rate of EAT-bearing mice; (6) LQFM030 was not able to promote severe acute oral toxicity hazard, being classified in the UN GHS category 5 (LD50 > 2000-5000 mg/Kg).

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Antiangiogenic agents reduce tumor progression by decreasing tumor blood supply; in addition, they have the advantage of not inducing tumor resistance. Under conditions of MDM2

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overexpression, the expression of VEGF mRNA and protein is enhanced suggesting that MDM2 is a

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key event in the development of tumor and metastasis.[25,26] Secchiero et al. [18] reported that nutlin-3 inhibits endothelial cell migration, induces of cell cycle arrest and increases apoptosis in endothelial

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cells. Patterson et al.[19] investigated whether inhibition of both MDM2 and VEGF signaling increased antitumor effects and showed that treatment with bevacizumab plus nutlin-3a promoted reduction of tumor growth associated with angiogenesis inhibition in vivo, with marked changes on tumor vascularity. These results suggest that concomitantly targeting the VEGF and p53 pathways enhances tumor growth suppression. Chavala et al.[25] showed that nutlin triggered retinal angiogenesis inhibition with advantages over available cytokine-targeted antiangiogenic therapies. Through in vitro and in vivo breast cancer models, Xiong et al.[20] investigated the role of MDM2 in

ACCEPTED MANUSCRIPT VEGF-mediated tumor angiogenesis and its action as an antiangiogenic target for oncology treatment by inhibiting MDM2 triggered by antisense oligonucleotides (ASO) or nutlin-3. The authors concluded that the ASO construct targeting MDM2 suppressed VEGF expression in vitro and VEGFmediated tumor angiogenesis in vivo. Moreover, VEGF suppression followed by MDM2 inhibition in p53 mutant cells implies that MDM2 regulates VEGF expression in a p53-independent manner.

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Apoptosis triggered through p53 pathway can increase caspases activities.[27,28] Corroborating this, LQFM030 also mediated apoptosis through modulation of p53 and p21 expression and caspase-3/7, -8, -9 activities. Thus, these data suggests that both extrinsic and intrinsic apoptotic pathways are triggered by LFQM030. Although the exact mechanism by which

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LQFM030 inhibits tumor progression is not totally clear, we can suggest that this compound increases p53 probably through MDM2 inhibition[21], resulting in an increase in p21 levels, cell cycle

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arrest, apoptosis and regulatory effects on VEGF expression and, in consequence, antiangiogenic effects. Similarly, it has been showed that the oral treatment of medulloblastoma tumor-bearing nude

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mice with nutlin-3 increased survival rate of animals and also inhibited tumor development through caspase-3 activation, re-activating p53 function as well as an increase of p21 levels.[29]

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Considering that LQFM030 showed potential as an anticancer agent, we evaluated the acute

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oral toxicity of this compound using an alternative method focused on reducing animals use, the OECD test guideline Nº 423.[24] The treatment of animals with a single dose of LQFM030 (300 or

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2000 mg/kg) was well tolerated by mice, with no death or changes observed on liver, kidney, spleen, heart or lungs. Thus it was categorized as UN GHS category 5, which is characterized by chemicals with probable low acute oral toxicity risk.[24] However, as it is common for cytotoxic compounds, the stomachs of animals orally exposed to LQFM030 showed atypical architecture with signs of hyperplasia. Künkele et al.[29] showed that treatment of medulloblastoma tumor-bearing nude mice with nutlin-3 dose (200 mg/kg), orally administered twice daily for approximately 20 days or treated

ACCEPTED MANUSCRIPT with three doses of nutlin-3 in 24 h, was also well tolerated and did not promote any clinical signs of toxicity.

Conclusions

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Taken together, our results demonstrate that LQFM030, a molecular simplification of nutlin 1, inhibits EAT progression via p53 and p21 level modulation, resulting in apoptosis and antiangiogenic effects. Furthermore, LQFM030 showed to be orally well tolerated as observed by oral acute toxicity assessment, being classified in the UN GHS category 5. Thus, LQFM030 seems to

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be a promising antitumor compound for combinatory therapy with typical cytotoxic agents, reducing the toxicity burden while allowing a superior anticancer action. Moreover, these data open new

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perspectives for LQFM030 as an antiangiogenic agent for treatment of diseases involving VEGF

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overexpression.

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Conflict of interest

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Acknowledgements

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The authors declare that there are no conflicts of interest.

The authors are grateful to Fundação de Apoio à Pesquisa (FUNAPE) of Federal University of Goiás, Fundação de Apoio à Pesquisa do Estado de Goiás (FAPEG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support.

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and antiangiogenic activity of Synadenium umbellatum Pax. J Ethnopharmacol 2008; 120(3):

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474–478.

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toxic class method. Guideline for the Testing of Chemicals, n. 423, 2001. 25. Chavala SH, Kim Y, Tudisco L, Cicatiello V, Milde T, Kerur N, Claros N, Yanni S, Guaiquil VH, Hauswirth WW, Penn JS, Rafii S, De Falco S, Lee TC, Ambati J. Retinal angiogenesis suppression through small molecule activation of p53. J Clin Invest 2013; 123(10): 4170– 4181. 26. Zhou S, Gu L, He J, Zhang H, Zhou M. MDM2 regulates vascular endothelial growth factor mRNA stabilization in hypoxia. Mol Cell Biol 2011; 31(24): 4928–4937.

ACCEPTED MANUSCRIPT 27. Saha MN, Jiang H, Mukai A, Chang H. RITA inhibits multiple myeloma cell growth through induction of p53-mediated caspase-dependent apoptosis and synergistically enhances nutlininduced cytotoxic responses. Mol Cancer Ther 2010; 9(11): 3041–3051. 28. Ray RM, Bhattacharya S, Johnson LR. Mdm2 inhibition induces apoptosis in p53deficient human colon cancer cells by activating p73- and E2F1-mediated expression of PUMA and

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Neuro Oncol 2012; 14(7): 859–869.

ACCEPTED MANUSCRIPT Table 1. Hematological parameters of Ehrlich ascites tumor (EAT)-bearing mice before and after LQFM030 treatment. Healthy

Group of animals

mice

EAT control

LQFM030 50 mg/kg

75 mg/kg

150 mg/kg

4.8 ± 0.6

13.8 ± 2.6*

10.6 ± 3.2*

11.7 ± 4.0*

11.1 ± 4.4*

Erythrocytes (106/mm3)

8.2 ± 0.08

7.4 ± 0.7

7.1 ± 1.2

6.4 ± 1.6

5.0 ± 2.5*#a

Hemoglobin (g/dL)

11.2 ± 0.6

9.6 ± 1.2

9.4 ± 1.8

9.4 ± 1.6

8.2 ± 3.1

Hematocrit (%)

20.1 ± 1.4

17.6 ± 1.8

714.2 ± 34.2

1140.0 ±

Platelets (103/mm3)

84.5* Packed cell volume

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Leukocytes (103/mm3)

18.2 ± 3.2

17.5 ± 2.7

19.0 ± 2.6

816.6 ±

748.5 ±

739.1 ±

23.4#

68.5#

83.5#

99.1 ± 5.5

97.7 ± 5.0

98.0 ± 7.2

37.6 ± 8.5*

45.1 ± 11.4*

47.2 ± 12.0*

98.6 ± 2.4

96.5 ± 3.9

Lymphocyte (%)

68.7 ± 6.9

44.6 ± 8.0*

Monocyte (%)

14.0 ± 1.8

32.7 ± 6.4*

29.4 ± 3.0*

29.5 ± 7.1*

33.0 ± 5.7*

Granulocyte (%)

16.6 ± 1.5

28.2 ± 2.7*

33.6 ± 1.5*#

26.5 ± 2.0*a

23.5 ± 3.6*a

Lymphocyte (103/mm3)

3.1 ± 0.1

5.5 ± 1.0*

4.3 ± 0.3

3.5 ± 0.6#

6.4 ± 1.7*ab

Monocyte (103/mm3)

0.6 ± 0.1

2.5 ± 0.3*#

4.3 ± 0.4*

1.6 ± 0.3*a

4.0 ± 1.3*#b

Granulocyte (103/mm3)

0.5 ± 0.2

3.3 ± 0.4*

2.6 ± 0.5*

2.3 ± 0.5*

2.5 ± 0.1*

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(µm3)

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p values were generated by ANOVA and Newman-Keuls Multiple Comparison Test a posteriori. *p<0.05 vs. healthy mice; #p<0.05 vs. EAT control; ap<0.05 vs. 50 mg/kg; bp<0.05 vs. 75 mg/kg.

ACCEPTED MANUSCRIPT Table 2. Effect of LQFM030 on serum biochemical parameters of mice. Group of animals

Healthy mice

EAT control

ALT/TGP (U/L)

35.4 ± 8.1

AST/TGO (U/L)

136.7 ± 5.8

CPK (U/L)

50 mg/kg

75 mg/kg

150 mg/kg

33.5 ± 8.4

32.1 ± 8.9

24.1 ± 2.3

31.0 ± 3.6

506 ± 25.6*

446.3 ± 25.8*

233.6 ±

302.3 ±

25.5*#a

76.0*#ab

2010.0 ±

4478.0 ±

1515.0 ±

1628.0 ±

163.3

334.5

172.0 ± 21.9

342.3 ± 36.7*

4505.0 ±

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LDH (U/L)

LQFM030

483.5*#

555.7*a

62.2*#b

283.0 ±

175.3 ±

351.5 ±

21.2*#

10.6#a

12.0*ab

0.37 ± 0.05

0.39 ± 0.03

0.38 ± 0.06

0.38 ± 0.01

0.34 ± 0.05

Urea (mg/dL)

48.6 ± 5.0

49.0 ± 5.7

46.2 ± 7.9

40.7 ± 4.0

41.2 ± 5.8

Glucose (mg/dL)

192.0 ± 11.8

179.3 ±

139.0 ± 14.8*

153.5 ± 20.5

183.7 ± 29.1

4.4 ± 0.2*

4.4 ± 0.1*

4.3 ± 0.2*

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Creatinine (mg/dL)

5.4 ± 0.3

4.7 ± 0.1*

Albumin (g/dL)

2.1 ± 0.3

1.7 ± 0.1*

1.6 ± 0.1*

1.6 ± 0.1*

1.7 ± 0.2*

Direct bilirubin

0.06 ± 0.02

0.06 ± 0.02

0.05 ± 0.01

0.04 ± 0.0

0.06 ± 0.01

0.17 ± 0.06

0.15 ± 0.05

0.09 ± 0.0

0.2 ± 0.02*ab

158.3 ±

132.5 ±

146.0 ± 4.2*

122.5 ± 17.6#

13.7*

113.0#

Total bilirubin

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(mg/dL)

0.12 ± 0.01

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(mg/dL) Total cholesterol

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Total protein (g/dL)

111.3 ± 3.8

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(mg/dL)

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p values were generated by ANOVA and Newman-Keuls Multiple Comparison Test a posteriori. *p<0.05 vs. healthy mice; #p<0.05 vs. EAT control; ap<0.05 vs. 50 mg/kg; bp<0.05 vs. 75 mg/kg.

Figure 1. Chemical structure of LQFM030 [1-(4-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl) piperazin-1-yl) ethanone].

Figure 2. Effects of LQFM030 in tumor growth, cell viability and ascitic fluid of Ehrlich ascites tumor (EAT)-bearing mice. Animals (n=6/group) were treated intraperitoneally with LQFM030 (50,

ACCEPTED MANUSCRIPT 75 or 150 mg/kg) or its vehicle (EAT control) for 10 days to determine changes in (a) tumor cell viability, (b) body weight and (c) ascites tumor volume. (*p< 0.05 vs. control).

Figure 3. Effects of LQFM030 in tumor angiogenesis of Ehrlich ascites tumor (EAT)-bearing mice. Animals (n=6/group) were treated intraperitoneally with LQFM030 (50, 75 or 150 mg/kg) or its

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vehicle (EAT control) for 10 days to determine changes in (a) peritoneal angiogenesis and (b) VEGF production.

Figure 4. Effects of LQFM030 in p53 and p21 expression of Ehrlich ascites tumor (EAT)-bearing

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mice. Animals (n=6/group) were treated intraperitoneally with LQFM030 (150 mg/kg) or its vehicle (EAT control) for 10 days and EAT cells were collected to evaluate p53 and p21 expression by flow

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cytometer. Representative histograms of EAT cell control (a and d) and EAT cells from LQFM030treated animals (b and e). Figures (c) and (f) represent flow cytometer analysis of p53 and p21 levels,

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respectively. (*p< 0.05 vs. control).

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Figure 5. Effects of LQFM030 in caspase activity of Ehrlich ascites tumor (EAT)-bearing mice.

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Animals (n=6/group) were treated intraperitoneally with LQFM030 (150 mg/kg) or its vehicle (EAT control) for 10 days and EAT cells were collected to evaluate caspases-3/7, -8 and -9 activities by

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flow cytometer. Representative graphics of EAT cell control (a, d and g) and EAT cells from LQFM030-treated animals (b, e and h). Figures (c), (f) and (h) represent flow cytometer analysis of caspases-3/-7, -8 and -9 activities, respectively (*p< 0.05 vs. control).

Figure 6. Representative photomicrographs of different organs obtained from healthy mice treated without or with single doses of LQFM030 (300 or 2000 mg/kg): (a) liver, (b) kidney, (c) spleen, (d) lung and (e) heart. Hematoxylin and eosin (H&E), 20x magnification.

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Figure 7. Representative photomicrographs of stomach obtained from healthy mice treated without or with single doses of LQFM030 (300 or 2000 mg/kg): (a) 100x magnification, (b) control (40x

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magnification), 300 and 2000 mg/kg LQFM030 (20x magnification). Hematoxylin and eosin (H&E).

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Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7