The antitumor activity of a lactosaminated albumin conjugate of doxorubicin in a chemically induced hepatocellular carcinoma rat model compared to sorafenib

Accepted Manuscript Title: The antitumor activity of a lactosaminated albumin conjugate of doxorubicin in a chemically induced hepatocellular carcinoma rat model compared to sorafenib Author: Bakheet Elsadek Ahmed Mansour Tahia Saleem Andr´e Warnecke Felix Kratz PII: DOI: Reference:

S1590-8658(16)30749-6 http://dx.doi.org/doi:10.1016/j.dld.2016.10.003 YDLD 3274

To appear in:

Digestive and Liver Disease

Received date: Revised date: Accepted date:

15-2-2016 22-9-2016 3-10-2016

Please cite this article as: Elsadek Bakheet, Mansour Ahmed, Saleem Tahia, Warnecke Andr´e, Kratz Felix.The antitumor activity of a lactosaminated albumin conjugate of doxorubicin in a chemically induced hepatocellular carcinoma rat model compared to sorafenib.Digestive and Liver Disease http://dx.doi.org/10.1016/j.dld.2016.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Revised version of DLD-16-128R1

The antitumor activity of a lactosaminated albumin conjugate of doxorubicin in a chemically induced hepatocellular carcinoma rat model compared to sorafenib

Bakheet Elsadek (PhD)1,*, Ahmed Mansour (Associate Professor) 2, Tahia Saleem (Professor)3, André Warnecke (PhD)4, Felix Kratz (PhD)4

1

Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, Al-

Azhar University, Assiut Branch, P. O. Box 71524, Assiut, Egypt; 2Department of Pharmacology and Toxicology, Faculty of Pharmacy, Al-Azhar University, Nasr City, P. O. Box 11651, Cairo, Egypt; 3Department of Biochemistry, Faculty of Medicine, Assiut University, P. O. Box 71526, Assiut, Egypt; 4

Tumor Biology Center, Division of Macromolecular Prodrugs, Breisacher

Strasse 117, 79106 Freiburg, Germany**

*

Corresponding author: To whom correspondence should be addressed:

Dr. Bakheet Elsadek, Lecturer of Biochemistry and Molecular Biology, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, P. O. Box 71524, Assiut, Egypt. E-mail: [email protected]; Tel.: +201110596270; Fax: +20934801212.

**

New address: CytRx Corporation, Drug Discovery Branch, Engesserstrasse

4, D-79108 Freiburg, Germany.

1

Financial Disclosure: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Author's contributions: Bakheet Elsadek carried out the biochemical assays, molecular and hematological analyses, wrote the first draft of manuscript and shared in animal model design, statistical analyses and interpretation of results. Ahmed Mansour carried out the animal works including model design, dosage forms administration, survival study and shared in hematological analyses. Tahia Saleem shared in research design and interpretation of results. André Warnecke shared in research design, synthesized and characterized L-HSADOXO, estimated the MTD of L-HSA-DOXO and shared in statistical analyses. Felix Kratz design the study concept, shared in synthesis and characterization of L-HSA-DOXO and shared in writing the first draft of manuscript and interpretation of results. All authors revised and edited the manuscript and approved the submitted version.

2

Abstract Background: Worldwide, consistent survival benefit for chemotherapy in hepatocellular carcinoma (HCC) is a golden goal for concerned researchers. Nexavar® (sorafenib) is the only approved agent that achieved touchable successes in this regard. Thus, there is a pressing medical need for new promising drugs to improve HCC therapy. Aims: our designed lactosaminated albumin

conjugate

of

doxorubicin

(L-HSA-DOXO)

that

rapidly

and

preferentially accumulates in the liver is compared, for the first time at its MTD, with doxorubicin and sorafenib, not only for antitumor efficacy but also for overall survival. Methods: HCC was induced in male Wistar rats with Nnitrosodiethylamine added to drinking water (100 mg/L) for 8 weeks. Endpoints were antitumor efficacy, tolerability and overall survival. Results: LHSA-DOXO proved to be superior at least over doxorubicin in the majority of assessed endpoints. Circulating AFP-L3% was diminished in L-HSA-DOXO (14.5%) and sorafenib (18.4%) groups compared to DENA (31.1%) and doxorubicin (29.5%) groups. This superiority was further confirmed by Western blot analyses of some novel HCC biomarkers. Survival study reinforced

consistent

benefits

of

both

L-HSA-DOXO

and

sorafenib.

Conclusions: L-HSA-DOXO shows at least comparable activity to sorafenib which clinically achieves only ~3 months overall survival benefit. Combination of these two agents could act beneficially or synergistically via two different modes of action to fight HCC.

Keywords: Drug targeting; Human serum albumin; Prodrug.

3

1. Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide that represents the third most common cause of cancer mortality [1]. It is associated closely with cirrhosis, and its prognosis is very poor because the diagnosis is generally made at an advanced stage of the disease [2]. HCC is the most frequent liver tumor and its development is preceded, both in humans and rodents, by the appearance of foci of altered hepatocytes (FAHs) and dysplastic nodules in the liver. These lesions are referred to as premalignant since they consist of phenotypically altered cells exhibiting a higher risk of malignant evolution than normal cells [3]. Various morphologic, biochemical, and molecular features of these lesions show substantial overlaps between species suggesting that the basic mechanisms

of

HCC

development

are

similar.

Animal

models

of

hepatocarcinogenesis have been developed in order to clarify the phenotypic, biochemical, and biological events occurring during the transformation of normal hepatocytes to cancer cells. The analysis of the different steps preceding full malignancy is essential for the knowledge of these events and the evaluation of risk factors for liver cancer as well as the discovery of diagnostic and prognostic biomarkers and targets for chemopreventive and therapeutic approaches [3]. The prognosis of HCC is poor because only 10–20% of HCCs can be removed completely using surgery. If the cancer cannot be completely removed, the overall survival without treatment is in the range of 3–8 months. Furthermore, with the exception of the receptor tyrosine kinase inhibitor

4

sorafenib (Nexavar®), no consistent survival benefits for anticancer agents in HCC have been recorded during the past 30 years in approximately 100 randomized studies including systemic and intra-arterial chemotherapy (predominantly doxorubicin-based or platinum-based), various hormonal therapies (tamoxifen and anti-androgens), and immunotherapy (usually interferon alpha) [4-9]. Sorafenib may be used in patients with advanced HCC increasing the overall benefit in survival by nearly 3 months as shown in a phase III trial compared to a placebo group [10]. Thus, there is an unmet medical need to improve the therapy of HCC. Due to fact that the asialoglycoprotein receptor (ASGP-R) is overexpressed on proliferating liver carcinoma cells [11] and ASGP-R exhibits a high binding selectivity for sugar-bearing ligands and that their localization is restricted to a single cell type, these low-molecular weight ligands can be exploited to deliver ligand-bound drugs to the liver. This inherent disadvantage was circumvented by using lactosaminated human serum albumin (L-HSA) as a hepatotropic drug carrier [12, 13]. When 20–30 lactose residues are attached to one albumin molecule, the neoglycoprotein L-HSA is selectively taken up by parenchymal liver cells [14]. Based on these results, LHSA was conjugated to the anticancer drug doxorubicin (DOXO). Coupling of doxorubicin

to

L-HSA

was

carried

out

using

the

(6-

maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH) (Fig. 1). [Fig. 1] The objective of the current study was two-fold: To compare, for the first time, L-HSA-DOXO at its maximum tolerated dose (MTD) with the

5

conventional doxorubicin and the approved tyrosine kinase inhibitor sorafenib (Nexavar®), not only for antitumor efficacy but also for overall survival benefits. Secondly, the study aimed to evaluate and compare, for the first time, the following surrogate parameters in all groups; glypican-3 (GPC-3), golgi protein 73 (GP73), heat shock protein 70 (HSP70) in addition to serum alpha L-fucosidase (AFU) activity, alpha-fetoprotein (AFP) and the Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3). The chemically induced rat model of HCC using N-nitrosodiethylamine (DENA) has been proven to be a feasible model for these evaluations. In general, hepatic chemical carcinogenesis is a multi-step process in experimental animals. Carcinogens initiate the process, which is followed by regeneration, growth and clonal proliferation, eventually leading to cancer [15]. DENA is a representative alkylating agent of a family of carcinogenic Nnitroso compounds and its administration to animals has been shown to cause cancer in liver and also at low incidence in other organs. Initiation during or after DENA exposure is thought to be a rapid metabolism of DENA to reactive metabolites that interact with DNA forming various DNA adducts that can lead to mutations. Hence, the O4-ethyldeoxythymidine adduct accumulates in hepatocyte DNA following DENA administration which is believed to be important in tumor initiation [15]. 2. Materials and methods 2.1. In vivo animal experiments Animal experiments were performed after approval by the Institutional Animal Care and Use Committee of the Faculty of Medicine, Assiut University, Egypt (IRB00008718). All experiments were performed using male Wistar rats

6

weighing 150–160g (El-Neil Pharmaceutical Company, Cairo, Egypt) which were allowed to acclimatize in the experimental laboratory for 2 weeks. Rats were housed (4 per cage) and were kept in a regulated environment (temperature, 20–22 °C; humidity, 50±5%; night/day cycle, 12 hours) with free access to standard diet pellets and sterile tab water ad libitum. Animal weights were recorded regularly two times per week and animal behaviors were monitored daily. After the 2 weeks acclimatization period, animals were randomized into 2 groups of 16 and 64 rats, respectively. The first group served as a negative control and the animals of the large group received DENA (Sigma-Aldrich GmbH, Munich, Germany), given in their drinking water (100 mg/L) for 8 weeks [16]. The DENA solution was administered in dark bottles and was prepared as a fresh solution every other day. One month after the end of DENA administration, the 64 rats of the second group were allocated to four subgroups of 16 animals each. The first subgroup (DENA group) received 5% glucose solution (i.v., weekly for 4 consecutive weeks). The second subgroup (DOXO group) received doxorubicin (Yick-Vic Chemicals & Pharmaceuticals, HK., China), 2.5 mg/kg (i.v., weekly for 4 consecutive weeks). The third subgroup (L-HSA-DOXO group) received a lactosaminated human serum albumin conjugate of doxorubicin (L-HSA-DOXO; Fig. 1) that was prepared and characterized according to the literature [17], in the dose of 5.0 mg/kg doxorubicin equivalents (i.v., weekly for 4 consecutive weeks). The fourth subgroup (sorafenib group) received sorafenib (Selleck Chemicals, China), 60 mg/kg, daily, per os.

7

One week after the last treatment, blood samples were collected from 50% of the animals per group (i.e. 8 animals) via retro-orbital vein plexus for hematological and clinical chemistry investigations. Those animals were then sacrificed by cervical decapitation under isoflurane anesthesia. Subsequent to autopsy, the livers were excised, purified from adhering fat and connective tissues, washed in ice-cold isotonic saline and then divided into two parts; the first part was stored in 10% neutral buffered formalin solution and subjected for histopathological examinations. The second part was homogenized in icecold Tris-HCl lysis buffer, pH 7.4 containing 1% protease inhibitor cocktail (Cell Signaling Technology, Inc., MA, USA) using Potter-Elvehjem rotor-stator homogenizer, fitted with a Teflon pestle (Omni International, Kennesaw, GA, USA). Samples were subsequently aliquoted and stored at -80 °C until use in Western blot assays. The remaining 50% of the animals, i.e., 8 rats in each group were monitored for overall survival. 2.2. Biochemical and hematology assays Total proteins levels (g/dL), serum albumin levels (g/dL), total serum bilirubin levels (mg/dL), serum ALT activity (U/L), serum AST activity (U/L), serum GGT activity (U/L) and serum ALP activity (U/L) were estimated using commercially available colorimetric and kinetic assay kits together with the photometer 5010 V5+ (ROBERT RIELE GmbH & Co KG - Berlin - Germany). Serum AFU activity (nmol ml-1 h-1) was estimated using colorimetric procedures [18]. Serum AFP and AFP-L3 were assayed using commercially available rat specific ELISA kits (Elabscience Biotechnology Co., Ltd, Wuhan, China) on a Stat Fax microplate ELISA reader (FL, USA) and results are expressed as the ratio of AFP-L3 to total AFP (%). Complete blood pictures

8

were performed for each rat using an ABX Micros 60 hematology analyzer (ABX Diagnostics - Horiba group – France). 2.3. Western blot assessments Proteins in the liver tissue homogenates were denatured at 95 °C for 5 minutes in 2× Laemmli buffer followed by addition of 5% 2-mercaptoethanol. SDS–PAGE electrophoresis was achieved by loading 50 µg protein per lane at 75 V through 12% resolving gel followed by 125 V during approximately 2 hours and transferred to a PVDF membrane using T-77 ECL semidry transfer unit (AmershamBioSciences UK Ltd) for 2 hours. Immunoblotting was performed by incubating the PVDF membrane in TBS buffer containing 0.1% Tween and 5% non fat milk for one hour at 4°C, followed by overnight incubation at 4°C with rabbit polyclonal antibodies of GPC-3 (Novus Biologicals, LLC, Littleton, CO, USA), GP73 (Novus Biologicals, LLC, Littleton, CO, USA), and HSP70 (Bioss Inc., Woburn, Massachusetts, USA) at a dilution of 1:1500. After being washed three times with TBST buffer, each membrane was incubated for 1 hour at room temperature with an alkaline phosphatase-conjugated

goat

anti-rabbit

secondary

antibody

(Novus

Biologicals, LLC, Littleton, CO, USA) at a dilution of 1:5000. Subsequently, each membrane was washed four times in TBST and the bound antibodies were detected with a commercially available BCIP/NBT substrate detection Kit (Genemed Biotechnologies, Inc., CA, USA). Equivalent protein loading for each lane was confirmed by stripping and re-blotting each membrane at 4°C against rabbit polyclonal anti β-actin antibody (Novus Biologicals, LLC, Littleton, CO, USA) at a dilution of 1:5000. The analysis was repeated to

9

assure reproducibility of results. Quantification of each corresponding analysis was further performed using Image J software and expressed as a β-actin%.

2.4. Histopathological examinations Histopathological examinations of the obtained sections from samples of all liver lobes were carried out according to standard protocol [19]. 2.5. Nuclear morphometric analyses Morphometric analyses of the obtained histological images were performed at the Pathology Department, National Research Center, Egypt using Leica Qwin 500 Image Analyzer

(LEICA Imaging Systems Ltd, Cambridge,

England) which consists of Leica DM-LB microscope with JVC color video camera attached to a computer system Leica Q 500IW. On routine haematoxylin and eosin stained slides from each group, 100 clearly defined cells with predominant staining were selected manually in a random fashion from different fields, and in order to avoid measuring and counting the same cells again, the microscope stage was moved from left to right, and then down and across in a step-wise manner. The nuclear morphometric parameters studied were nuclear area and nuclear length. Nuclear area is the area enclosed inside the contour and the length is the longest orthogonal projection. All measurements were made under 200X magnification and expressed in micrometers. 2.6. Survival study The remaining 50% of the animals per group (i.e. 8 animals) were monitored for overall survival in order to establish Kaplan-Meier plots. Hence, animals were inspected daily, and mortality was monitored and recorded.

10

2.7. Statistical analyses Statistical analyses of the data were carried out using GraphPad prism 5.0. Data comparisons were performed using analysis of variance (ANOVA) followed by Tukey’s t-test. The levels of significance were accepted with p<0.05 and all relevant results were graphically displayed as mean ± SEM. Overall survival was depicted as a Kaplan-Meier plot. 3. Results 3.1. Chemical synthesis of L-HSA-DOXO: L-HSA-DOXO with a drug loading ratio of 6-8 drug molecules per protein molecule was synthesized by coupling of doxorubicin to L-HSA using the (6maleimidocaproyl) hydrazone derivative of the drug (DOXO-EMCH) as previously described in literatures [17]. 3.2. Changes in the animal body weights In comparison to the healthy control group, all DENA drinking animals lost weight during the course of the experiment before treatment. This induced decrease in body weight gain was apparently attenuated following treatment with L-HSA-DOXO or sorafenib, especially when compared to the group treated with doxorubicin – see Fig. 2. [Fig. 2] 3.3. Morphological changes in the liver features Livers of animals in the DENA group showed abnormal morphological features with faint red irregular rough surface incorporating a number of HCC nodules. Some livers showed loose consistency with diffuse massive heterogeneous lesions beside dark area of necrotic mass as appear in Fig. 3B1. Conversely, livers in L-HSA-DOXO group (Fig. 3E) showed normal

11

morphological aspects with healthy reddish brown color, normal firm consistency and smooth surface, and borders in a similar manner to that of animals in the control group (Fig. 3A). On the other hand, livers from the doxorubicin (Fig. 3C) and sorafenib (Fig. 3D) treated groups showed relatively altered morphological features with irregular rough pale surface incorporating some scattered micronodules (arrows) of different sizes throughout the liver. [Fig. 3] 3.4. Liver function tests In comparison to the healthy control group, animals in the DENA and doxorubicin groups showed signs of impaired liver functions including significant hypoalbuminemia (p<0.01 and p<0.001, respectively), as well as significant elevations in the serum ALT (p<0.01 and p<0.01, respectively), AST (p<0.001 and p<0.001, respectively), GGT (p<0.001 and p<0.001, respectively), total bilirubin (p<0.001 and p<0.05, respectively), and ALP (p<0.001 and p<0.01, respectively). These impaired liver functions were tolerated to a high extent in the groups treated with L-HSA-DOXO or sorafenib as illustrated in Table 1. [Table 1] 3.5. HCC-related tumor markers Circulating AFP-L3% was apparently diminished in L-HSA-DOXO (14.5%) and sorafenib (18.4%) treated groups in comparison to the DENA (31.1%) and doxorubicin (29.5%) groups – see Fig. 4A. Additionally, in comparison to the DENA and doxorubicin groups, serum AFU activity was significantly lower in L-HSA-DOXO (p<0.01 and p<0.01, respectively), and sorafenib (p<0.01 and p<0.001, respectively) – see Fig. 4B. Moreover, L-HSA-DOXO treatment

12

markedly attenuated the DENA-induced over-expressions of GPC-3, GP73 and HSP70 in liver tissue homogenates. Conversely, treatment with sorafenib seemed to have little effects while treatment with doxorubicin had no effects on the expression of these markers as depicted in Fig. 4 (C-E), [Fig. 4] 3.6. Histopathological examinations Livers of rats from healthy control group revealed normal histological structure of hepatic lobules (Fig. 5A). Contrariwise, liver sections from animals in DENA group showed severe histopathological alterations revealing micronodular lesions of HCC characterized by carcinoma cells with large vesicular nuclei, more than one nucleoli, mitotic figure and intracytoplasmic eosinoplilic hyaline bodies in addition to fibroblastic cells proliferation and inflammatory cells infiltration (Fig. 5B). Meanwhile, liver sections from rats treated with free doxorubicin showed numerous foci of altered hepatocytes confined as HCC with the normal trabecular structure of the liver is distorted by carcinoma cells, proliferation of oval cells and mitotic figures as well as marked atypia (Fig. 5C). However, liver sections of animals in sorafenib group showed improvement in the histopathological structure with steatosis of hepatocytes, vacuolation of hepatocytes, dilatation and congestion of hepatic sinusoids besides ballooning degeneration in focal manner at the hepatic parenchyma (Fig.

5D).

Preferentially,

L-HSA-DOXO

therapy

resulted

in

marked

improvement in the hepatic tissue histological architecture as the examined sections revealed only small focal clear hepatocytes with no evidence of fibrosis, and dysplasia or noticed malignancy (Fig. 5E). [Fig. 5]

13

3.7. Nuclear morphometry The nuclear morphometric results revealed that, in comparison to the control group, both the nuclear area and nuclear length were significantly higher in the DENA (p<0.001) and free doxorubicin (p<0.05) groups. Neither the nuclear area nor the nuclear length were significantly affected in the groups treated with sorafenib or L-HSA-DOXO which exhibited relatively equivalent corresponding values to that of the control group. Although, there were no significant differences in the nuclear area and nuclear length between the LHSA-DOXO group and the sorafenib group, the utopia in the nuclear morphometric features was more distinctive in the former group – see Table S4 and Fig. S2 in the supporting information. 3.8. Hematology study Hemoglobin concentration was significantly diminished in doxorubicin group in comparison to the healthy control (p<0.001), DENA (p<0.001), sorafenib (p<0.001) and L-HSA-DOXO (p<0.001) groups. Also, therapy with doxorubicin induced significant alterations in some other hematologic parameters causing leucopenia,

erythrocytopenia

and

thrombocytosis,

which

were

less

pronounced or absent in the groups treated with L-HSA-DOXO or sorafenib as observed in Table 1. 3.9. Survival study As shown in the Kaplan-Meier analysis (Fig. 5F), free doxorubicin was unable to improve the overall survival of animals with a median survival of approximately 20 days that was comparable to the untreated DENA group

14

(approximately 25 days). On the contrary, both L-HSA-DOXO and sorafenib prolonged the overall median survival of rats in each corresponding group to approximately 100 and 105 days, respectively, from the date of therapy cessation. 4. Discussion Globally, HCC resembles the fifth most common cancer and the third-leading cause of cancer-related deaths with a steadily increasing incidence of about 625,000 new cases per year accounting for approximately 600,000 worldwide deaths annually [20, 21]. Less than 30% of patients diagnosed with HCC are eligible for curative treatment and during the course of the natural evolution of HCC a significant proportion of patients are candidates for systemic therapies. In recent years, by virtue of the considerable progresses that have been made in furthering the knowledge of molecular pathogeneses of HCC, tangible advances have been achieved in the development of some targeted therapies for fighting this disease [20-23]. Nevertheless, with only three months of survival gain compared to placebo, only sorafenib, a multikinase inhibitor, remains till date the sole approved drug in advanced HCC [24]. Despite this, many practitioners and country health authorities consider the cost-efficacy ratio of sorafenib somewhat insufficient [25, 26]. Additionally, some newly published data and clinical practices highlighted a great inter-individual and even intra-individual variation regarding clinical benefit and toxicity of sorafenib. Otherwise, the drug is not even approved for patients with advanced HCC in some emerging countries [27-29]. Thus, there is an urgent unmet medical need to develop novel targeted therapies for more efficient attack of HCC resulting in more satisfactory outcomes.

15

Several years ago, it was reported that ASGP-R, a glycoprotein receptor present only on the surface of hepatocytes, mediates uptake and lysosomal degradation of galactosyl terminating peptides [30]. This receptor has also been histochemically detected on surface of well and poorly differentiated cells of HCC [11, 31]. This result supported the attempts to develop selective HCC chemotherapy through the ASGP-R as a vector for selectively delivering the drugs to parenchymal liver cells. In this case, the chemotherapeutic index of the drugs which display the main side effects on extrahepatic tissues could be increased by coupling them to macromolecules that are taken up by this receptor. In

the

light

of

this

knowledge

besides

our

experience

in

macromolecular prodrugs design, doxorubicin was coupled to lactosaminated human serum albumin (L-HSA) using the (6-maleimidocaproyl) hydrazone derivative of the drug (DOXO-EMCH). For a drug-protein conjugate to be pharmacologically active the bond between the drug and the carrier must be sufficiently stable in the bloodstream but should release of the drug at its site of action, i.e. the tumor cells. After cellular uptake, L-HSA-DOXO is transported to the acidic pH value of endosomal-lysosomal compartments where doxorubicin is rapidly released to reach levels 7–20 times higher than those raised in extra-hepatic tissues [32, 33]. DOXO-EMCH bound to L-HSA fulfills these requirements. This was demonstrated in rats bearing chemically induced HCC by intravenous injection of L-HSA-DOXO at a dose of 1 mg/kg. Two to three hours after administration of the conjugate, the intracellular release of doxorubicin from the carrier was essentially complete [34].

16

Biodistribution studies in healthy rats have demonstrated that L-HSADOXO produced a very rapid and selective uptake in the liver with very low levels in healthy organs, whereas in rats injected with free doxorubicin comparable levels of doxorubicin were measured in the liver, heart, intestine, kidney and spleen [35]. Further in vivo experiments were performed in rats bearing DENA induced HCCs in order to evaluate the doxorubicin levels in tumor nodules after administration of the prodrug [11, 36]. In these experiments it was unexpectedly observed that administration of L-HSADOXO can increase doxorubicin concentrations also in poorly differentiated HCCs, which lack the ASGP-R. The mechanism by which L-HSA-DOXO is actively taken up by neoplastic hepatocytes, which do not express ASGP-R, is not precisely known but preliminary data suggest that uptake of L-HSA-DOXO by these cells is mediated by adsorptive endocytosis [37]. In order to predict the therapeutic efficacy of the L-HSA-DOXO conjugate, it was found that L-HSA-DOXO in a relatively low dose (1 mg/kg) significantly reduced the number of HCCs developed from neoplastic lesion without causing significant body weight loss compared with an equivalent dose of free doxorubicin, which was completely ineffective and therapy produced a body weight loss of ~10% [31]. Furthermore, in order to examine the potential of the L-HSA-DOXO in reducing the growth of already formed tumors, another study was performed by monitoring tumor nodules in a DENA induced HCC rat model using high frequency ultrasound imaging [16]. It was observed that L-HSA-DOXO, in a dose of 1 mg/kg, hindered the development of new tumors from pre-neoplastic lesions as well as significantly inhibiting the growth of established HCCs. However, equivalent free doxorubicin dose

17

showed an effect on tumor growth only in the first period of administration [16]. Accordingly, it was summarized that L-HSA-DOXO is a promising liver targeting doxorubicin conjugate to treat HCC. In the current study, we aimed to investigate, for the first time, not only the antitumor efficacy but also the overall survival benefits of L-HSA-DOXO at its MTD (four cycles of 5.0 mg/kg doxorubicin equivalents) in comparison to sorafenib and free doxorubicin on a chemically induced HCC rat model. We have observed that treatment with L-HSA-DOXO or sorafenib was superior over free doxorubicin with respect to systemic toxicity as indicated by body weight changes. All DENA drinking animals lost weight during the course of the experiment before treatment, suggesting primary tumor burden or the metastatic spread of the tumor as a possible cause. A similar observation was also reported using the same animal model [38]. As shown in Fig. 2, the induced decrease in body weight gain was apparently attenuated following treatment with L-HSA-DOXO or sorafenib, especially when compared to the group treated with doxorubicin which might serve as an indicator for a better tolerability of the first two drugs. It was also noticed macroscopically that the livers from the DENA group showed abnormal morphological features. Comparable morphological changes have also previously been reported in several DENA-induced HCC rat models [31, 34, 36]. Conversely, it was observed that the livers in both the control and L-HSA-DOXO groups showed normal morphological aspects with healthy appearance, whilst the livers from the doxorubicin and sorafenib treated group showed relatively altered morphological features as seen in Fig. 3.

18

Additionally, in comparison to the healthy control group, it was found that animals in the DENA and doxorubicin groups showed signs of impaired liver functions including significant hypoalbuminemia, as well as significant elevations in the serum ALT, AST, GGT, total bilirubin, and ALP. These observed DENA-forced alterations in blood parameters of liver functions could be secondary events following lipid peroxidation of hepatocyte membranes with the consequent increase in the leakage of these indices from damaged liver tissues as has been previously reported in many models of DENAinduced hepatocellular degeneration [31, 39, 40]. Noteworthy, these observations of impaired liver function were tolerated to a high extent in the groups treated with L-HSA-DOXO or sorafenib to match its corresponding levels in the healthy control group with minor superiority of L-HSA-DOXO over sorafenib as illustrated in Table 1. Additional evidence for supporting the tumor repressing nature of LHSA-DOXO was found in the levels of new generation tumor markers of HCC including AFP-L3%, AFU, GPC-3, GP73 and HSP70. These markers have been recently used by some investigators for monitoring treatment response and recurrence as well as surrogate markers of clinicopathological variables of HCC and as prognostic factors for the overall survival [41-48]. As depicted in Fig. 4, circulating AFP-L3% and AFU activity were apparently diminished in L-HSA-DOXO and sorafenib groups compared to the DENA and doxorubicin groups. Moreover, Western blot assessments of GPC3, GP73 and HSP70 as HCC-related molecular markers provide further evidence for superiority of L-HSA-DOXO not only over doxorubicin but also over sorafenib. As shown in Fig. 4, DENA administration induced strong

19

expressions of GPC-3, GP73 and HSP70 in hepatic tissues, proving the occurrence of premalignant liver changes in this group. Especially for the LHSA-DOXO treated group, the observed expression pattern of these markers was attenuated to relatively notable levels. Conversely, treatment with sorafenib seemed to have little effects while doxorubicin had no effects on the expression of these markers as depicted in Fig. 4. Likewise, histolopathological examinations of liver tissues with the subsequent quantitative analyses of the histological images that attempts to enhance the objectivity and reproducibility of the diagnostic pathology presented another strong testimony for L-HSA-DOXO advantageous effects in fighting DENA-induced HCC. In accordance with histological findings of other previous publications, DENA administration induced severe histopathological alterations in the hepatic cells [49, 50] with significant increases in both the nuclear area and nuclear length, possibly due to the presence of tumor cells with gigantic nuclei and multinucleate cells that expresses abnormal divisions and genetic anomalies associated with the malignant changes. Obviously, both L-HSA-DOXO and sorafenib therapies resulted in marked attenuation in the DENA-induced alterations in the liver histological architecture and significantly decreased the nuclear area and nuclear length with a relative superiority to L-HSA-DOXO over sorafenib. Contrariwise, the residual alterations that were observed in histopathological pictures obtained from liver sections of animals after free doxorubicin (Fig. 5C) administration reflect its minor efficacy and firmly suggest the distinctive tumor suppressing nature of L-HSA-DOXO in DENA-induced HCC.

20

Meanwhile, hematology data provided an additional proof for the enhanced tolerability of L-HSA-DOXO over free doxorubicin. As observed in Table 1, hemoglobin concentration was significantly diminished in doxorubicin group in comparison to the control, DENA, sorafenib and L-HSA-DOXO groups. It should be noted that, in comparison to the healthy group, neither LHSA-DOXO nor sorafenib induced significant alteration in the hemoglobin concentration. At the same time, therapy with doxorubicin induced significant alterations in some other hematologic parameters causing leucopenia, erythrocytopenia and thrombocytosis, which were less pronounced or absent in the groups treated with L-HSA-DOXO or sorafenib. Over and above, the obtained survival data suggested that both LHSA-DOXO and sorafenib have significant potential to increase the overall survival rate in rats bearing HCC. As shown in the Kaplan-Meier analysis (Fig. 5F), both L-HSA-DOXO and sorafenib prolonged the overall median survival of rats in each corresponding group to approximately 100 and 105 days, respectively, from the date of therapy cessation. Analogous reports concluded that sorafenib has the potential to reduce mortality rate and prolong the overall survival of HCC bearing subjects, perhaps because the recurrent tumors that developed after sorafenib treatment progressed more slowly as a result of the direct antitumoral effects or indirectly through the anti-angiogenic activity of sorafenib [51, 52]. On the contrary, free doxorubicin failed in improving overall survival with a median survival of approximately 20 days that was comparable to the untreated DENA group (approximately 25 days). Since the majority of HCCs in western countries arise in cirrhotic livers, there is concern that high local doxorubicin concentration in the liver that

21

result from treatment with L-HSA-DOXO could lead to hepatic damage in a clinical use of the conjugate. This potential issue was previously addressed by studying the effect of the conjugate applied in the efficacy study, on serum parameters of liver function and viability in normal rats, in rats with regenerating liver after partial hepatectomy and in rats with fibrosis/cirrhosis induced by CCl4 or DENA. In normal rats, L-HSA-DOXO did not modify any clinically relevant serum liver parameter [53, 54]. Administered to partially hepatectomised rats, it neither impaired the viability of regenerating hepatocytes nor produced changes in their ultra-structure and caused only a small delay of hepatic DNA recovery [53]. In rats with fibrosis/cirrhosis induced by CCl4, it only caused moderate increases in AST and ALT serum levels, not statistically different from those produced by the free drug [54]. In these

animals,

free

doxorubicin

produced

a

decrease

in

albumin

concentration which was probably due to kidney damage and proteinuria caused by the drug in rats. The only effect observed in conjugate treated rats was an increase in ALP activity. The excellent tolerability of L-HSA-DOXO was also confirmed in HCCs bearing rats. In these animals the free drug caused a marked decrease in body weight, but systemic toxicity was not observed in rats administered the conjugate [16, 53]. These reduced side effects can be easily explained by the lower drug concentrations caused by the conjugate in the extra-hepatic tissues [31]. In conclusion, we confirm that L-HSA-DOXO, at its MTD, has interesting anticancer activity and acceptable tolerability against DENAinduced HCC in rats with a trend towards an enhanced overall survival. These promising results are on the whole comparable to sorafenib whereas, L-HSA-

22

DOXO might have an advantage due a different mode of action and that as an outlook a next step is to evaluate the potential synergistic effect of a combination of the kinase inhibitor sorafenib and the liver tumor targeting conjugate L-HSA-DOXO. This may shed new light on a novel therapeutic strategy for patients with HCC. Conflict of interest: None declared. Acknowledgements The authors are grateful to both Dr. Adel B. Kholoussy, Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Egypt and Dr. Marwa Shabana, Department of Pathology, National Research Center, Egypt for carrying out the histopathological examinations and the morphometric analyses.

23

5. References (1) Yim SH, Chung YJ. An Overview of Biomarkers and Molecular Signatures in HCC. Cancers. 2010;2:809-23. (2) el-Houseini ME, Mohammed MS, Elshemey WM, et al. Enhanced detection of hepatocellular carcinoma. Cancer control : journal of the Moffitt Cancer Center. 2005;12:248-53. (3) Feo F, Pascale R, Calvisi D. Models for Liver Cancer: John Wiley & Sons, Ltd.; 2007. (4) Bruix J, Sherman M, Llovet JM, et al. Clinical management of hepatocellular

carcinoma.

Conclusions

of

the

Barcelona-2000

EASL

conference. European Association for the Study of the Liver. Journal of hepatology. 2001;35:421-30. (5) Bruix J, Sherman M, Practice Guidelines Committee AAftSoLD. Management of hepatocellular carcinoma. Hepatology. 2005;42:1208-36. (6) Llovet JM, Bruix J. Systematic review of randomized trials for unresectable hepatocellular carcinoma: Chemoembolization improves survival. Hepatology. 2003;37:429-42. (7) Llovet JM, Burroughs A, Bruix J. Hepatocellular carcinoma. Lancet. 2003;362:1907-17. (8) Lopez PM, Villanueva A, Llovet JM. Systematic review: evidence-based management of hepatocellular carcinoma--an updated analysis of randomized controlled trials. Alimentary pharmacology & therapeutics. 2006;23:1535-47. (9) Yeo W, Mok TS, Zee B, et al. A randomized phase III study of doxorubicin versus

cisplatin/interferon

alpha-2b/doxorubicin/fluorouracil

(PIAF)

24

combination chemotherapy for unresectable hepatocellular carcinoma. Journal of the National Cancer Institute. 2005;97:1532-8. (10) Llovet JM, Ricci S, Mazzaferro V, et al. Sorafenib in advanced hepatocellular

carcinoma.

The

New

England

journal

of

medicine.

2008;359:378-90. (11) Di Stefano G, Fiume L, Bolondi L, et al. Enhanced uptake of lactosaminated human albumin by rat hepatocarcinomas: implications for an improved chemotherapy of primary liver tumors. Liver international : official journal of the International Association for the Study of the Liver. 2005;25:85460. (12) Fiume L, Busi C, Mattioli A, et al. Hepatocyte targeting of adenine-9-betaD-arabinofuranoside

5'

-monophosphate

(ara-AMP)

coupled

to

lactosaminated albumin. FEBS letters. 1981;129:261-4. (13) Fiume L, Cerenzia MR, Bonino F, et al. Inhibition of hepatitis B virus replication by vidarabine monophosphate conjugated with lactosaminated serum albumin. Lancet. 1988;2:13-5. (14) Zarski JP, Barange K, Souvignet C, et al. Efficacy and safety of lactosaminated human serum albumin-adenine arabinoside monophosphate in chronic hepatitis B patients non-responders to interferon therapy: a randomised clinical trial. Journal of hepatology. 2001;34:486-8. (15) Sivalokanathan S, Ilayaraja M, Balasubramanian MP. Antioxidant activity of

Terminalia

arjuna

bark

extract

on

N-nitrosodiethylamine

induced

hepatocellular carcinoma in rats. Molecular and cellular biochemistry. 2006;281:87-93.

25

(16) Di Stefano G, Fiume L, Baglioni M, et al. Efficacy of doxorubicin coupled to lactosaminated albumin on rat hepatocellular carcinomas evaluated by ultrasound imaging. Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver. 2008;40:278-84. (17) Di Stefano G, Lanza M, Kratz F, et al. A novel method for coupling doxorubicin to lactosaminated human albumin by an acid sensitive hydrazone bond: synthesis, characterization and preliminary biological properties of the conjugate. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences. 2004;23:393-7. (18) Bukofzer S, Stass PM, Kew MC, et al. Alpha-L-fucosidase as a serum marker of hepatocellular carcinoma in southern African blacks. British journal of cancer. 1989;59:417-20. (19) Banchroft JD, Stevens A, Turner DR. Theory and practice of histoloicl techniques, fourth ed. New york, London, San Francisco, Tokyo: Churchil Livingstone1996. (20) Bouattour M, Payance A, Wassermann J. Evaluation of antiangiogenic efficacy in advanced hepatocellular carcinoma: Biomarkers and functional imaging. World journal of hepatology. 2015;7:2245-63. (21) Faivre S, Bouattour M, Raymond E. Novel molecular therapies in hepatocellular carcinoma. Liver international : official journal of the International Association for the Study of the Liver. 2011;31 Suppl 1:151-60. (22) Kudo M. Signaling pathway/molecular targets and new targeted agents under

development

in

hepatocellular

carcinoma.

World

journal

of

gastroenterology. 2012;18:6005-17.

26

(23) Psyrri A, Arkadopoulos N, Vassilakopoulou M, et al. Pathways and targets in hepatocellular carcinoma. Expert review of anticancer therapy. 2012;12:1347-57. (24) Cheng AL, Kang YK, Chen Z, et al. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. The Lancet Oncology. 2009;10:25-34. (25) Camma C, Cabibbo G, Petta S, et al. Cost-effectiveness of sorafenib treatment in field practice for patients with hepatocellular carcinoma. Hepatology. 2013;57:1046-54. (26) Palmer DH, Hussain SA, Smith AJ, et al. Sorafenib for advanced hepatocellular carcinoma (HCC): impact of rationing in the United Kingdom. British journal of cancer. 2013;109:888-90. (27) Arrondeau J, Mir O, Boudou-Rouquette P, et al. Sorafenib exposure decreases over time in patients with hepatocellular carcinoma. Investigational new drugs. 2012;30:2046-9. (28) Iavarone M, Cabibbo G, Piscaglia F, et al. Field-practice study of sorafenib therapy for hepatocellular carcinoma: a prospective multicenter study in Italy. Hepatology. 2011;54:2055-63. (29) Mir O, Coriat R, Blanchet B, et al. Sarcopenia predicts early dose-limiting toxicities and pharmacokinetics of sorafenib in patients with hepatocellular carcinoma. PloS one. 2012;7:e37563. (30) Trere D, Fiume L, De Giorgi LB, et al. The asialoglycoprotein receptor in human hepatocellular carcinomas: its expression on proliferating cells. British journal of cancer. 1999;81:404-8.

27

(31) Fiume L, Bolondi L, Busi C, et al. Doxorubicin coupled to lactosaminated albumin inhibits the growth of hepatocellular carcinomas induced in rats by diethylnitrosamine. Journal of hepatology. 2005;43:645-52. (32) Di Stefano G, Kratz F, Lanza M, et al. Doxorubicin coupled to lactosaminated human albumin remains confined within mouse liver cells after the intracellular release from the carrier. Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver. 2003;35:428-33. (33) Greenfield RS, Kaneko T, Daues A, et al. Evaluation in vitro of adriamycin immunoconjugates synthesized using an acid-sensitive hydrazone linker. Cancer research. 1990;50:6600-7. (34) Di Stefano G, Fiume L, Baglioni M, et al. Doxorubicin coupled to lactosaminated albumin: effect of heterogeneity in drug load on conjugate disposition and hepatocellular carcinoma uptake in rats. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences. 2008;33:191-8. (35) Fiume L, Di Stefano G. Lactosaminated human albumin, a hepatotropic carrier of drugs. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences. 2010;40:253-62. (36) Di Stefano G, Fiume L, Baglioni M, et al. A conjugate of doxorubicin with lactosaminated albumin enhances the drug concentrations in all the forms of rat hepatocellular carcinomas independently of their differentiation grade. Liver international : official journal of the International Association for the Study of the Liver. 2006;26:726-33.

28

(37) Goormaghtigh E, Chatelain P, Caspers J, et al. Evidence of a specific complex

between

adriamycin

and

negatively-charged

phospholipids.

Biochimica et biophysica acta. 1980;597:1-14. (38) Bansal AK, Bansal M, Soni G, et al. Protective role of Vitamin E pretreatment on N-nitrosodiethylamine induced oxidative stress in rat liver. Chemico-biological interactions. 2005;156:101-11. (39) Sayed-Ahmed MM, Aleisa AM, Al-Rejaie SS, et al. Thymoquinone attenuates diethylnitrosamine induction of hepatic carcinogenesis through antioxidant signaling. Oxidative medicine and cellular longevity. 2010;3:25461. (40) Sivaramakrishnan V, Shilpa PN, Praveen Kumar VR, et al. Attenuation of N-nitrosodiethylamine-induced hepatocellular carcinogenesis by a novel flavonol-Morin. Chemico-biological interactions. 2008;171:79-88. (41) Behne T, Copur MS. Biomarkers for hepatocellular carcinoma. International journal of hepatology. 2012;2012:859076. (42) Cheng J, Wang W, Zhang Y, et al. Prognostic role of pre-treatment serum AFP-L3% in hepatocellular carcinoma: systematic review and metaanalysis. PLoS One. 2014;9:e87011. (43) Gan Y, Liang Q, Song X. Diagnostic value of alpha-L-fucosidase for hepatocellular carcinoma: a meta-analysis. Tumour biology : the journal of the International

Society

for

Oncodevelopmental

Biology

and

Medicine.

2014;35:3953-60. (44) Jain D. Tissue diagnosis of hepatocellular carcinoma. Journal of clinical and experimental hepatology. 2014;4:S67-73.

29

(45) Lagana SM, Salomao M, Bao F, et al. Utility of an immunohistochemical panel consisting of glypican-3, heat-shock protein-70, and glutamine synthetase in the distinction of low-grade hepatocellular carcinoma from hepatocellular

adenoma.

Applied

immunohistochemistry

&

molecular

morphology : AIMM / official publication of the Society for Applied Immunohistochemistry. 2013;21:170-6. (46) Shi M, Huang Z, Yang Y, et al. [Diagnostic value of serum Golgi protein73 (GP73) combined with AFP-L3% in hepatocellular carcinoma: a metaanalysis]. Zhonghua Gan Zang Bing Za Zhi. 2015;23:189-93. (47) Vora SR, Zheng H, Stadler ZK, et al. Serum alpha-fetoprotein response as a surrogate for clinical outcome in patients receiving systemic therapy for advanced hepatocellular carcinoma. The oncologist. 2009;14:717-25. (48) Wang NY, Wang C, Li W, et al. Prognostic value of serum AFP, AFP-L3, and GP73 in monitoring short-term treatment response and recurrence of hepatocellular carcinoma after radiofrequency ablation. Asian Pacific journal of cancer prevention : APJCP. 2014;15:1539-44. (49) Afzal M, Kazmi I, Gupta G, et al. Preventive effect of Metformin against N-nitrosodiethylamine-initiated

hepatocellular

carcinoma

in

rats.

Saudi

pharmaceutical journal : SPJ : the official publication of the Saudi Pharmaceutical Society. 2012;20:365-70. (50) Golla K, Bhaskar C, Ahmed F, et al. A target-specific oral formulation of Doxorubicin-protein nanoparticles: efficacy and safety in hepatocellular cancer. Journal of Cancer. 2013;4:644-52. (51) Liu L, Cao Y, Chen C, et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in

30

hepatocellular

carcinoma

model

PLC/PRF/5.

Cancer

research.

2006;66:11851-8. (52) Zhang W, Zhao G, Wei K, et al. Adjuvant sorafenib reduced mortality and prolonged overall survival and post-recurrence survival in hepatocellular carcinoma patients after curative resection: a single-center experience. Bioscience trends. 2014;8:333-8. (53) Di Stefano G, Derenzini M, Kratz F, et al. Liver-targeted doxorubicin: effects on rat regenerating hepatocytes. Liver international : official journal of the International Association for the Study of the Liver. 2004;24:246-52. (54) Di Stefano G, Fiume L, Domenicali M, et al. Doxorubicin coupled to lactosaminated albumin: Effects on rats with liver fibrosis and cirrhosis. Digestive and liver disease : official journal of the Italian Society of Gastroenterology and the Italian Association for the Study of the Liver. 2006;38:404-8.

31

Table 1: Circulating levels of clinically relevant liver function indices, tumor markers and hematological parameters in different groups. Control

ALT (U/L)

AST (U/L)

GGT (U/L)

T.

31.50

73.00

5.69**

3.90$

± 140.40 ± 140.50

± 95.00

6.16***

4.77***

± 1.35 0.20*** 23.74***

14.45

(g/dL)

0.12

Proteins 7.14

± 2.72

0.96 ± 0.22*

± 2.45

± 6.99

AFP (ng/mL)

3.87

± 74.82

± 20.53

(ng/mL)

0.015

2.63***

3.85***

14.09

± 12.43

Hb (g/dL)

0.44***

±

0.30†† 26.23$$, ††† ± 35.68 8.51$ ± 6.56

± 13.04

0.84

0.65

RBCs

7.73

± 6.88

(106/mm3)

0.46

0.29 NS

0.23***, $$$

PLT

694.80±

651.40

1636.00

(103/mm3)

60.16

±44.09NS

89.07***, $$$

± 3.08

0.36

0.10$$$, ††† ±

0.28††† ± 423.10

†††

± 7.32 0.18††† ± 623.10 49.67†††

±

34.30$$, †† ± 31.57

±

6.48$,† ± 4.57

1.70$$$,†† , $$$

±

± 6.32

± 413.80

± 7.01

NS

‡‡

22.67$

13.40***

± 23.26

0.17***

± 6.24

± 69.61

0.073

± 0.69 ± 0.10

19.66$$

34.61***

AFP-L3

1.51

± 223.40

0.20***, $$$

10.44***

1.35

± $$$,

†††

$$$, †††

± 4.74

35.51***

20.93

3.75

***,

± 3.69 ± 0.15 3.70

(nmol 382.10 ± 596.80 ± 610.30

ml-1 h-1)

± 59.38 $$$,

± 190.40

0.16***

0.19NS

0.24

4.31

±

8.12***

†††

29.82**

0.13**

± 141.60

57.00 ***,

±

3.64NS

7.07*** ±

154.90 ± 325.00 ± 277.60

3.55

(g/dL)

±

DOXO ± 40.50

± 142.00

13.68***

± 92.50

2.75

Albumin

AFU

5.14**

0.04

ALP (U/L)

T.

± 40.25

10.01***

L-HSA-

Sorafenib

± 54.00

5.77 23.63

DOXO

± 57.88

2.32

Bilirubin 0.26

(mg/dL)

DENA

±

1.35$$$,††† ± 11.86 0.42

±

†††

± 6.16

±

0.14**,††† ± 783.30

±

45.39†††

32

Total 3

WBCs 8.21 3

(10 /mm )

0.55

± 12.38 1.51

NS

± 14.16 1.85*

± 8.03 0.78

± †

8.55 ± 1.15†

Data are presented as mean ± SEM (n = 8). *, $, †, and ‡ indicate significant change from control, DENA, DOXO and sorafenib respectively, where NS indicates no significant difference. *, $, †, and ‡ indicate significant change at p<0.05; **, $$, ††, and ‡‡ indicate significant change at p<0.01; ***, $$$, †††, and ‡‡‡ indicate significant change at p<0.001. AFP, alpha-fetoprotein; AFPL3, Lens culinaris agglutinin-reactive fraction of AFP; AFU, alpha Lfucosidase; ALP, Alkaline phosphatase; ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; DENA, N-nitrosodiethylamine; DOXO, Doxorubicin; GGT, Gamma-glutamyl transferase; Hb, Hemoglobin; L-HSA, Lactosaminated human serum albumin; PLT, Platelet ; RBCs, Red blood cells; T. Bilirubin, Total Bilirubin; T. Proteins, Total Proteins; WBCs, White Blood Cells.

33

Figure Captions

Fig. 1. Structures of doxorubicin, the (6-maleimidocaproyl)hydrazone derivative of doxorubicin (DOXO-EMCH) and its acid-sensitive conjugate with L-HSA (L-HSA-DOXO). DOXO, Doxorubicin; L-HSA, Lactosaminated human serum albumin. Fig. 2. Development of animal body weight in different groups. Data are presented as mean ± SEM (n = 16). DENA, N-nitrosodiethylamine; DOXO, Doxorubicin; L-HSA, Lactosaminated human serum albumin. Fig. 3. Effect of tested compounds on the morphological aspect of the livers. Representative photographs of livers from the control (A 1-4) and LHSA-DOXO (E 1-4) groups showed normal morphological aspects. Liver from the DENA group (B 1-4) showed faint red irregular rough surface and mostly loose consistency. Liver from the DOXO group (C 1-4) showed irregular rough pale yellow surface incorporating central non-diffuse macronodules (arrow). Liver from the sorafenib group (D 1-4) showed firm consistency with irregular rough surface incorporating some scattered micronodules (arrows) of different sizes throughout the liver. Fig. 4. Serum AFP-L3% (A); AFU activity (B) as well as representative Western blot analysis of GPC-3 (C), GP73 (D) and HSP70 (E) expressions in liver tissues of different groups. In (A) bar chart shows percentage of AFP-L3 (red) to total AFP (green). In (B) data are presented as mean ± SEM (n = 8). *, $ and † indicate significant change from control group, DENA group and DOXO group respectively. *, $ and † indicate significant change at

34

p<0.05; **, $$ and †† indicate significant change at p<0.01; ***, $$$ and ††† indicate significant change at p<0.001. AFP, Alpha-fetoprotein; AFP-L3, Lens culinaris agglutinin-reactive fraction of AFP; AFU, Alpha L-fucosidase; DENA, N-nitrosodiethylamine; DOXO, Doxorubicin; L-HSA, Lactosaminated human serum albumin; GP73, Golgi protein 73;

GPC-3, Glypican-3; HSP70, Heat

shock protein. Fig. 5. (A-E) Representative photomicrographs of liver sections from different groups (H&E X400); (F) Overall of survival rate of animals in each group as estimated by Kaplan-Meier analysis. [A] Hepatic tissues in the control group showed normal histological structure; [B] Hepatic tissues in DENA group revealed HCC, carcinoma cells with large vesicular nuclei with more than one nucleoli, mitotic figure and intracytoplasmic eosinoplilic hyaline bodies; [C] Livers in DOXO group showed severe histopathological alterations confined as HCC. The normal trabecular structure of the liver is distorted by carcinoma cells, proliferation of oval cells and mitotic figures; [D] Livers of rats from sorafenib group showed improvement in the histopathological structure with steatosis of hepatocytes, vacuolation of hepatocytes, dilatation and congestion of hepatic sinusoids; [E] Livers of rats from L-HSA-DOXO group showed marked improvement in the histopathological picture as the examined sections revealed only small focal clear hepatocytes; [F] Overall survival rate in animals treated with both L-HSA-DOXO (blue line) and sorafenib (yellow line) was significantly higher than that in animals treated with DOXO (red line) and the untreated DENA group (brown line). DENA, N-nitrosodiethylamine; DOXO, Doxorubicin; L-HSA, Lactosaminated human serum albumin.

35