Biochimie 94 (2012) 2467e2474

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Biochimie
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Review

A novel pro-apoptotic effector lactaptin inhibits tumor growth in mice models

Olga A. Koval a, *, Alexandr S. Fomin a, Vasily I. Kaledin b, Dmitry V. Semenov a, Miraslava O. Potapenko a, Elena V. Kuligina a, Valery P. Nikolin b, Eugeny V. Nikitenko c, Vladimir A. Richter a
a

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentiev av. 8, Novosibirsk 630090, Russia Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, Lavrentiev av. 10, Novosibirsk 630090, Russia c The Perinatal Centre Maternity Hospital, Lezhena 32, Novosibirsk 630089, Russia
b

a r t i c l e i n f o
Article history: Received 3 July 2012 Accepted 20 August 2012 Available online 30 August 2012 Keywords: Lactaptin Antitumor agents Human milk peptides Apoptosis

a b s t r a c t
Lactaptin, a human milk-derived protein, induces apoptosis in cultured tumor cells. We designed a recombinant analog of lactaptin (RL2) and tested its antitumor activity. The sensitivity of hepatocarcinoma A-1 (HA-1), Lewis lung carcinoma, and Ehrlich carcinoma to RL2 were tested to determine the most reliable in vitro animal model. HA-1 cells, which had the highest sensitivity to RL2, were transplanted into A/Sn mice to investigate RL2 antitumor activity in vivo. Investigation of the molecular effects of RL2 shows that RL2 induces apoptotic transformation of HA-1 cells in vitro: phosphatidylserine translocation from inner side of the lipid bilayer to the outer one and dissipation of the mitochondrial membrane potential. Repetitive injections of RL2 (5e50 mg/kg) for 3e5 days effectively inhibited ascites and solid tumor transplant growth when administered intravenously or intraperitoneally, without obvious side effects. The solid tumor inhibitory effect of RL2 (5 i.v. injections, cumulative dose 125 mg/kg) was comparable with that of cyclophosphamide at a therapeutic dose (5 i.v. injections, cumulative dose 150 mg/kg). In combination therapy with cyclophosphamide, RL2 had an additive antitumor effect for ascites-producing tumors. Histomorphometric analysis indicated a three-fold reduction of spontaneous metastases in the liver of RL2-treated mice with solid tumor transplants in comparison with control animals. Repeated RL2 treatment substantially prolonged the lifespan of mice with intravenously injected tumor cells. Recombinant analog of lactaptin effectively induced apoptosis of tumor cells in vitro and suppressed the growth of sensitive tumors and metastases in vivo. 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction Surgery, radiation therapy, and chemotherapy are currently the most effective clinical strategies for cancer treatment [1,2]. The inability of conventional chemotherapeutic agents to discriminate tumor cells from normal rapidly-dividing cells, however, is the primary cause of the high general toxicity of chemotherapeutics. This worldwide problem has motivated investigators to design novel nontoxic pharmaceuticals using rationale design based on the molecular and cellular biology characteristics of tumor tissues. Much effort has been directed toward nding new efcacious and specic antitumor agents [3,4]. Among them, pro-apoptotic molecules are of special interest for the development of novel antitumor and antimetastatic drugs [5]. A number of naturally occurring proteins that selectively induce apoptosis of cancer cells were recently used to develop novel

* Corresponding author. Tel.: 7 383 3635190; fax: 7 383 3635153. E-mail address: o_koval@ngs.ru (O.A. Koval). 0300-9084/$ e see front matter 2012 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biochi.2012.08.017

anticancer drug substances: TRAIL, apoptin and HAMLET [6e8]. Human milk is a source of multiple bioactive peptides, some of which are activated by proteolysis [9e11]. Lactaptin is an 8.6-kDa proteolytic fragment of kappa-casein isolated from human milk. Lactaptin decreases the viability of cultured human adenocarcinoma MCF-7 cells by inducing apoptosis [12]. A recombinant analog of lactaptin (RL2) containing lactaptin as a subsequence and a C-terminal His6-tag was recently designed and produced in Escherichia coli [13]. RL2 has been extensively studied in vitro in various transformed cell lines and induces apoptosis of cultured human cancer cells: MCF-7, A549, and Hep-2, without inducing cytotoxic effects on normal mesenchymal stem cells. RL2 induces apoptosis of MCF-7 cells by phosphatidylserine externalization; activation of caspases 7, 8, and 9; and by the loss of mitochondria membrane potential but the cellular target responsible for RL2dependent cancer cell apoptosis are not yet fully elucidated [14]. In the present study, the therapeutic effects of RL2 were tested using a murine cancer model. The study focused on the development of efcient RL2 treatment protocols, including the type of administration, effective dose, and course of treatment.

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An RL2-sensitive in vivo murine tumor model was selected based on in vitro responses. RL2 inhibited tumor growth in a dose- and course-dependent manner and prolonged the lifespan of tumorbearing animals. Moreover, RL2 therapy noticeably decreased HA-1 spontaneous metastases progression. 2. Materials and methods 2.1. Recombinant analog of lactaptin The recombinant analog of lactaptin RL2 was expressed in E. coli BL21 strain (DE3) (Stratagene, La Jolla, CA) and puried from the host E. coli cells in two stages by afnity chromatography on the His-afnity Gel (Zymo Research, Orange, CA) and by ion-exchange chromatography on DEAE-cellulose [13]. The purity of the protein (98%) was conrmed by reversed phase-high performance liquid chromatography on a C5 RP-column (Discovery BIO Wide Pore C5, SigmaeAldrich Chemical Co., St. Louis, MO). 2.2. Tumors A strain-specic transplantable HA-1 cell line established and cryopreserved by Kaledin et al. [15] was used in the in vivo experiments. Thawed tumor cells were injected into the abdominal cavity of A/Sn mice. The ascites tumor, which developed within 1 week after tumor inoculation, was transplanted into the experimental animals. For in vitro experiments, HA-1 hepatoma, Lewis lung carcinoma (LLC), and Ehrlich ascites adenocarcinoma were used to prepare cell cultures grown in Iscoves Modied Dulbeccos Media (Sigma) with 10% FBS (Gibco BRL Co., Gaithersburg, MD). 2.3. MTT assay Cell survival in response to RL2 treatment was determined by MTT assay (MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide), SigmaeAldrich). Aliquots (100 mL) containing 1 103 tumor cells were plated onto 96-well plates and incubated overnight. The next day, 100 mL of experimental media containing different concentrations of RL2 was added to each well. The cells were incubated for 48 h; the media were replaced with 200 mL of RPMI 1640 medium containing 0.25 mg/mL MTT-solution, and the plates were incubated at 37  C for 4 h. The medium was removed, followed by the addition of 0.15 mL of DMSO to each well. The plates were read at 570 and 620 nm using an Apollo LB912 plate reader (Berthold Technologies, Oak Ridge, TN). Cell viability was determined as the absorbance at A570 with reference to A620 and expressed as a percentage of control SD for triplicate independent experiments. 2.4. Apoptosis detection HA-1 tumor cells (1 106) in 6-well plates were treated with 60 mg/mL RL2 for 18 h. The cells were detached with 0.025% trypsin, washed twice with Iscoves Modied Dulbeccos Media containing 10% FBS, and treated using uorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit I (BD Pharmingen, San Jose, CA) or with 5 mg/mL JC1 (50 60 ,60 -tetrachloro-1,10,3,30 -tetraethylbenzimidazolcarbocyanin iodide) (SigmaeAldrich) for 20 min at 37  C. Cells without RL2 treatment were used as a control. The stained cells were scanned using a FACSCanto II ow cytometer (BD Biosciences, Franklin Lakes, NJ) and the data were analyzed by FACSDiva Software (BD Biosciences). For microscopic analysis of the mitochondrial membrane changes HA-1 cells were seeded (1 104) onto 4-well CultureSlides (BD Falcon, Bedford, MA), incubated with 60 mg/mL RL2 for 18 h and

then 5 mg/mL JC1 were added for 20 min. Stained (nonxed) cells were visualized with uorescence microscope Axioscop 2 PLUS (Carl Zeiss, GmbH). 2.5. Mice and treatment Female A/Sn mice between 10 and 12 weeks of age (colony of the Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia) weighing 20e25 g were used for the in vivo experiments. The mice were maintained under controlled lighting conditions at 65% humidity, 25  C, and allowed food and water ad libitum. All procedures involving animals were performed in compliance with the protocols and recommendations for proper use and care of laboratory animals (ECC Directive 86/609/EEC). Ascites HA-1 tumor cells were washed and suspended in saline and 0.1 mL of the suspension containing 2 105 cells was inoculated subcutaneously (s.c.) on the dorsal side or intramuscularly (i.m.) in the right thigh of the mice. To obtain articial metastases, 1.5 105 tumor cells in 0.2 mL saline were injected into the mice intravenously (i.v.) in the lateral tail vein. After tumor transplantation, the mice were randomly divided into experimental and control groups and treated with RL2 (or saline in control) injections (i.p. or i.v.). To treat articially disseminated tumor cells and experimental metastases, RL2 in 0.4 mL saline was administered intravenously at 24 and 48 h after tumor cells inoculation. To treat solid tumor transplants, RL2 was administered intraperitoneally and intravenously when the tumor nodules had grown to 30e50 mm3 in size. The dosing, course duration, and scheme of treatment are described in the Results section and in the gure legends. Mice were observed and weighed daily, and the tumor growth rate was determined by measuring the nodes using calipers every 3 days. The median tumor volume (V) was calculated as V(p/ 6 a2 b), where a was the smaller of the two perpendicular tumor diameters [16]. At the end of the experiment, the animals were killed by cervical dislocation and the tumors were removed and weighed. 2.6. Histomorphometric analysis The excised livers were xed in 4% paraformaldehyde and transverse 6e7-mm sections were prepared at ve different levels to cover the entire liver. The sections were stained with hematoxylin-eosin and picro-fuchsin (by van Giesons), the volume fraction of liver occupied by metastases was measured by the previously described morphometric procedure using an Axioplan 2 Imaging (Carl Zeiss, GmbH) [17,18]. 2.7. Statistical analysis For all mouse experiments, data are expressed as mean SE. The ManneWhitney U-test was used for comparison between the two groups. A p value of less than 0.05 was considered statistically signicant. 3. Results 3.1. RL2 induced apoptosis and inhibited viability of mouse tumor cells in vitro The cytotoxic effects of RL2 were studied in a variety of murine tumor cell lines: LLC, Ehrlich adenocarcinoma and HA-1 hepatoma. As indicated by the MTT assay, HA-1 hepatoma had the highest sensitivity to RL2 (IC50 0.01 mg/mL, exposure time 48 h). LLC and Ehrlich adenocarcinoma tumor cell lines appeared less sensitive or totally resistant to RL2 treatment (Fig. 1A). During incubation with RL2, the HA-1 cells underwent obvious morphologic alterations: cell shrinkage, nuclear condensation, and the appearance of

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Fig. 1. In vitro effects of RL2 on viability and apoptosis of murine tumor cells. A. Antiproliferative effect of RL2. Cells were treated with 0.06 mg/mL RL2 for 48 h and viability was analyzed by MTT assays. Data are presented as the mean of at least three separate experiments for each cell line (*p < 0.05); B. RL2 induces phosphatidylserine exposure. HA-1 cells were treated with 0.06 mg/mL RL2 for 18 h and analyzed by ow cytometry after annexin V/propidium iodide (PI) staining. Cell populations with the FITC/propidium iodide (PI) phenotype were designated as viable cells, FITC/PI e as apoptotic cells, and FITC/PI e as secondary apoptotic cells. *-signicantly different from control, p < 0.001.

apoptotic bodies (data are not shown). To determine whether the RL2 effect on cell viability was due to apoptosis, the RL2-treated HA-1 cells were analyzed by ow cytometry using annexin V/PI staining. In RL2-treated cells, the annexin V/PI subpopulation increased substantially compared with that in untreated cells (Fig. 1B). For the HA-1 cells, the apoptotic population increased to about 15% after 18 h of incubation with RL2. Transition of the mitochondrial transmembrane potential which occurs during RL2-dependent death of HA-1 cells was identied by uorescence microscopy and measured by ow cytometry. We used membrane-permeable lipophilic cationic uorochrome JC1 to

detect its potential-dependent accumulation in mitochondria reected by a uorescence emission shift. JC1 monomers display green cytoplasmic uorescence (w525 nm) while its aggregates accumulated in functional mitochondria display orange uorescence (w590 nm) [19]. Changing in JC1 mitochondrial binding is reected by a decrease in the red/ green uorescence ratio. FACS analysis of the JC1 uorescence intensity distribution allows us to identify the decrease of red uorescence and increase of green uorescence in RL2-treating cells in comparison with control cells (Fig. 2A). Fluorescence microscopy of JC1 stained cells

Fig. 2. Effect of RL2 on mitochondria membrane potential of HA-1 tumor cells. HA-1 tumor cells were incubated with RL2 (60 mg/mL) for 18 h and stained by mitochondrial dye JC1. A. Flow cytometry of the transition of the mitochondrial membrane potential of HA-1 cells. B. Fluorescence microscopy of JC-1 distribution into HA-1 cells. Bar corresponds to 37 mm.

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also showed the disruption of mitochondrial membrane potential after RL2 treatment (Fig. 2B). Therefore RL2 could activate the mitochondrial pathway of programmed cell death in mouse HA-1 cells. 3.2. Direct effect of RL2 on HA-1 tumor cells in vivo Eighteen mice were each inoculated intraperitoneally with 2 106 of HA-1 tumor cells and the animals were then randomly divided into experimental (8 animals) and control (9 animals) groups. The next day, the experimental mice were injected with 0.25 mL (0.25 mg, w12.5 mg/kg) of RL2 solution and the controls with 0.25 mL saline, and the injections were repeated 3 days later. The experiment was complete when all the mice were dead. The mean lifespan of the RL2-treated mice was 18.3 0.53 days compared with 12.2 0.43 days (p < 0.001) for the control mice (Fig. 3A). 3.3. Distant tumor transplants and RL2 injection site We then evaluated the route of tumor cell inoculation and RL2 injections were changed: HA-1 tumor cells were inoculated subcutaneously, and RL2 was administrated either intraperitoneally or intravenously. Treatment was started when the tumor nodes had grown to 3 mm in diameter. Three RL2 injections (every second day) were performed in a single dose of 25 mg/kg. Control mice received equal volume injections of saline. As shown in Fig. 3B at the end of the experiment (32 days after tumor transplantation), mean tumor weight in control mice was higher then in intraperitoneally RL2-treated mice, and in intravenously RL2-treated mice. Thus, systemic administration of RL2 inhibited the growth of local tumor transplants. 3.4. RL2 inhibited tumor growth in a dose-dependent manner HA-1 tumor cells were transplanted into the thigh muscles of the mice at day 0, and RL2 was administered intravenously ve times (every second day) at doses of 5, 10, 25, and 50 mg/kg, starting from day 10. In the experiments, the RL2 doses for intravenous injections were restricted by the limited solubility of the peptide at a neutral pH. Moreover, as required by the guidelines for the welfare and use of animals in cancer research [20], the maximum injection volume in the experiments was 20 mL/kg.

Conforming to these limitations, the maximum single dose of intravenous RL2 treatment was 50 mg/kg (0.5 mL, 4 mg/mL). The tumor transplants were repeatedly measured and their volume calculated. On day 25, the mice were killed by cervical dislocation, and the tumors were excised and weighed. The two highest doses of RL2, 25 mg/kg and 50 mg/kg, had the greatest tumor growth inhibitory effects (Fig. 4A). Weight loss was monitored as an indicator of RL2 toxicity. Animal weights were recorded at the time of tumor volume measurement [21]. No weight loss was observed in RL2-treated mice as compared with the control group. Hence, the RL2 dose of 25 mg/kg effectively delayed tumor growth without visible toxic effects. To optimize the treatment protocols, the 25-mg/kg dose was chosen for subsequent experiments. 3.5. RL2 inhibited tumor growth in a course-dependent manner Courses with two and four 25-mg/kg RL2 injections were compared to nd a more effective scheme for inhibiting tumor growth (Fig. 4B). Tumor cells were inoculated to mice subcutaneously and RL2 was administered intravenously two or four times (additional details are given in the gure legend). As few as two RL2 doses were sufcient to delay tumor growth, while four injections had a more substantial and prolonged inhibitory effect on tumor growth. 3.6. Comparison with cyclophosphamide When the dose- and course-dependent experiments were nished, we performed experiments aimed to compare the therapeutic effects of RL2 with those of cyclophosphamide, a widely used chemotherapeutic agent [22]. As the rst step, HA-1 cells were inoculated into mice subcutaneously and 5 days later the tumor transplants became palpable. The animals were divided into three groups and therapeutic treatment was started. A single dose of RL2 (25 mg/kg) or cyclophosphamide (30 mg/kg) was intravenously administered to the mice every third day for 5 cycles. The experiment was terminated 20 days after tumor transplantation, when one mouse receiving cyclophosphamide appeared moribund. In RL2-treated mice, tumor size (Table 1) was comparable to that in cyclophosphamide-treated mice. In the control group, tumor size was approximately 2e2.5 times greater than that in cyclophosphamide- and RL2-treated mice.

Fig. 3. Direct and distant effects of RL2. A. Viability of HA-1 tumor-bearing mice (i.p.) after RL2 i.p. Injections (12.5 mg/kg). B. RL2 suppression effect on solid HA-1 tumor growth. RL2 was injected intravenously or intraperitoneally at a dose of 25 mg/kg three times.

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Fig. 4. The dose and course-dependent antitumor activity of RL2. A. RL2 was administered every second day at the indicated doses (mg/kg) starting on day 5 after tumor transplantation. Statistical differences in mean tumor weight between control and experimental groups are indicated by * for p < 0.01; ** for p < 0.05 (statistically signicant differences between groups). B. HA-1 tumor cells were subcutaneously implanted in A/Sn mice and RL2 was intravenously injected at a dose of 25 mg/kg two or four times, starting on day 8. Arrows indicate the days of RL2 injections. Statistical differences in mean tumor volume between control and experimental groups are indicated by * for p < 0.01; ** for p < 0.05 (statistically signicant differences between RL2-treated groups).

Based on the signicant suppressive effect of RL2 on local tumor growth, we next examined the antimetastatic activity. Hepatoma HA-1 is a rapidly growing tumor that metastasizes specically to the liver. To estimate the effect of RL2 on spontaneous metastasis, morphometric examinations of the livers of control and RL2treated animals were performed (Fig. 5) and metastases were estimated as the percent of the total liver volume. The morphometric analysis revealed that nearly 3 times fewer metastases developed in the liver of RL2-treated mice than in control mice (6.7 0.7% versus 2.3 0.4%, p < 0.05). Therefore, RL2 provided greater protection against spontaneous metastasis. Although either RL2 or cyclophosphamide alone slowed HA-1 tumor growth, a combined treatment modality was not included in this experiment. The effect of cyclophosphamide on liver metastasis progression was also not studied due to the labor intensity of the morphometric analyses. Alternatively, a model experiment was performed in which the effect of the tested drugs on HA-1 tumors developing in the abdominal cavity in ascites form was evaluated based on the prolongation of the lifespan of tumorbearing animals. After tumor cell transplantation (1 106 cells/ animal), the mice were divided into 6 groups; then 2 of the groups (receiving 2 or 5 saline injections) were clustered as a control group. Two RL2 injections increased the lifespan by 43% and ve RL2 injections increased the lifespan by 53% compared with

Table 1 Therapeutic effect of RL2 vs. cyclophosphamide in mice bearing subcutaneous HA-1 tumor transplants and liver metastases. Group 1 (Control) Number of animals Initial body weight (g) Tumor weight, g (%) Tumor growth retardation (%) Final body weight, g (% of initial) Volume density of liver metastases 6 20.0 1.10 0.22 (100) e 19.3 0.6 (96.5) 6.7 0.7 Group 2 (cyclophosphamide) 6 19.3 0.53 0.08 (48.2) 51.8 17.7 0.6 (91.7) N.D. Group 3 (RL2) 7 20.6 0.47 0.05 (42.7) 57.3 20.2 0.5 (98.1) 2.3 0.4a Fig. 5. Metastasis suppression effect of RL2. Photographs of sections prepared from the livers of HA-1 tumor-bearing mice. HA-1 cells were subcutaneously inoculated into mice and 5 days later experimental animals were intravenously injected with RL2 (25 mg/kg/dose) every third day for 5 cycles. Typical patterns of spontaneous liver metastasis are shown for mice in control and experimental groups. Arrows indicate visible metastases. Bar corresponds to 25 mm.

N.D. e not determined. a Signicantly different from control, p < 0.001.

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controls (Table 2). At the same time, a 2-fold increase in the lifespan was observed in cyclophosphamide-treated mice and a 240.6% increase (2.5-fold) was observed in mice receiving a combination of cyclophosphamide and RL2. The ndings indicated that RL2 treatment at the tested doses moderately retarded tumor growth, and the effect was only slightly enhanced by longer exposure. In combination therapy, cyclophosphamide at near-optimal doses exerted a strong antitumor effect and RL2 had an additive antitumor effect. These observations suggest that RL2 is a potential candidate drug for use in combination with other chemotherapeutic agents or other modalities of adjuvant therapy, rather than for the rst-line tumor treatment. 4. Discussion Tumors usually arise as small local formations with obvious borders separating them from the surrounding tissues. Neoplasms acquire the signs of malignancy (insusceptibility to regulatory body activity, invasiveness, and metastatic ability) in the process of growth and development, known as tumor progression. Tumors acquire particular signs of malignancy, e.g., metastatic ability, independently throughout their lifespan and, in some cases, do not have time to develop some of these signs [23,24]. This dictates the strategy to radically eliminate the tumors as early as possible. With regard to surface tumors that are easily accessible for therapy, surgical removal and destruction by radiation are thought to be the best methods. More advanced neoplasms require chemotherapy. A major breakthrough in cancer treatment, particularly breast cancer, was systemic adjuvant (postsurgical) chemotherapy targeting of disseminated and distant metastatic tumor cells [25]. Indeed, drug treatment for cancer was initially based on the ability of a chemotherapeutic drug to freely disseminate in the body and to reach tumor nodes and individual tumor cells regardless of their number or location. Conventional chemotherapeutic drugs (such as cyclophosphamide, methotrexate, 5-uorouracil) target the DNA of dividing cells. Due to their insufcient selectivity, however, these drugs also attack rapidly dividing cells of normal tissues (i.e., hematopoietic or intestine) [26]. This fact motivated researchers to search for new classes of anticancer drugs that act on specic receptors present in cancer cells that are absent from or of little importance for normal cells. The monoclonal antibody drug trastuzumab (Herceptin, Genentech, South San Francisco, CA, and Roche, Basel, Switzerland) targeting HER-2 receptors (HER-2 neu or ErbB2) and involved in signal transduction in cancer cells, the epidermal growth factor receptor tyrosine kinase inhibitors erlotinib and getinib (Terceva, OSI Pharmaceuticals, Farmingdale, NY; OSI-774) are recent examples of novel drugs [27e29]. The lack of specicity of the actions of these drugs on cancer cells in a number of cases, however, causes severe side effects [30].
Table 2 Therapeutic effect of RL2 and cyclophosphamide with separate and combined administration to mice with intraperitoneal growing HA-1 ascites tumors. Groups Number Mean lifespan, M m of animals (Days) (%) 12.8 0.36 18.3 0.53a 19.6 1.1a 25.6 0.6
a

1. Control (saline) 15 2. RL2 12.5 mg/kg twice (2 and 8 5 days after tumor transplantation) 3. RL2 12.5 mg/kg 5 times (every 5 3d day) after tumor transplantation 4. Cyclophosphamide 30 mg/kg 5 5 times (every 3d day) after tumor transplantation 5. RL2 as in group 3 cyclophosphamide 5 as in group 4
a

100 143 153.1 200

30.8 1.1a

240.6

Signicantly different from control, p < 0.001.

Recently, a number of tumor-selective naturally occurring proteins that induce apoptosis in cancer cells, i.e., TRAIL, apoptin, mda-7/IL-24, and HAMLET, have been used for anticancer therapeutic investigations [6e9,31]. A valuable additional option to this line of cancer treatment could be apoptosis-inducing peptides, such as RL2. This peptide specically attacks the cells of susceptible tumors and ignores other tumors or normal cells [13]. Selection of a novel compounds for further clinical development relies predominantly on whether the compound shows specic cytotoxic activity in certain in vitro tumor cell lines. At the same time, there is no universal tumor model for screening novel antitumor agents for specic action due to the different apoptotic sensitivities and expression of different drug resistance transporters in different cancer cells, which could inuence therapeutic outcomes [32]. Moreover, current anticancer treatments usually do not eradicate the clones of cancer stem cells, and instead facilitate expansion of the cancer stem cell pool or the selection of resistant clones, or both, which eventually leads to treatment failure [33]. Standard testing procedures are based on screening of several tumor cell lines (tumor panel). We previously reported that RL2 decreases the viability of cultured tumor cells in vitro with different efciencies [34]. In this study, susceptibility to the cytotoxic actions of the recombinant analog of lactaptin was tested using several mouse cell lines developed from tumors originating from different tissues e mammary (Ehrlich ascites carcinoma), liver (HA-1 hepatoma), and lungs (LLC). Hepatoma A-1 was highly sensitive to RL2 treatment in vitro and in vivo. Here we demonstrated that recombinant analog of lactaptin RL2 induces apoptotic changes in HA-1 cells with specic alteration of plasma membrane of the cells. We display activation of mitochondrial signal pathway in HA-1 cells undergoing apoptosis upon RL2 treatment. Therefore the RL2 activity towards HA-1 hepatocellular tumors used in our experiments suggests that the agents action is not conned to MCF-7 breast tumor. Transition of the mitochondrial transmembrane potential (Djm) is an early and universal event in apoptosis. Formation of permeability transition pores in the outer membrane leaet of mitochondria allows mitochondrial proteins to ux into the cytosol [35]. We demonstrated that RL2 induced dissipation of Djm of RL2treated HA-1 cancer cells. Chronic Djm dissipation is associated with cell death. Currently, several compounds e peptide derivatives, small molecules and cationic lipophilic agents with mitochondriotoxic activity to cancer cells are used in clinical trials for cancer therapy [36]. Thus, it is possible recombinant analog of lactaptin mediated mitochondrial damage and then apoptotic death pathway. We have found RL2 could easy penetrate into the cytoplasm of cancer and normal cells (data not shown) and interacts with cellular targets. Dissipation of mitochondrial transmembrane potential realizes through the opening of the mitochondrial permeability transition pore (MPTP). For today, the composition of this multiprotein complex is still unknown and we assumed RL2 could directly interact with components of the MPTP of HA-1 cells. Predominant glycolytic metabolism of cancer cells makes their mitochondrial transmembrane potential higher and therefore cancer cells could be more sensitive to compounds inducing Djm disruption [36,37]. This could be one of the possible explanations of tumor-selective RL2 activity and which have to be investigated more carefully. One obstacle in studies of potential antitumor drugs during the transition from in vitro experiments to in vivo experiments is the method of the test drug administration. For example, for the HAMLET complex, effective suppression of tumor growth was demonstrated following prolonged direct contact of the agent with tumor cells: skin papillomas were treated by topical drug

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application while bladder cancers and glioblastomas were treated by intravesical and intracranial HAMLET instillation, respectively [38e40]. Technologic solutions for drug delivery directly to tumor sites, however, are not available for all tumors. In our study, RL2 effectively suppressed tumor growth both in direct contact with tumor cells (direct effect) and at a location of tumor transplants distant from the RL2 injection site. Because RL2 shows high cytotoxicity to HA-1 cells in vitro, we suppose that its antitumor activity is specied by direct intercommunication with tumor cells in mice. Though, it may be speculated that mice immune system is stimulated by RL2 potentiating direct antitumor effects. Therefore, RL2 can be used to treat sensitive tumors in different locations. Moreover, our ndings showed no toxicity of RL2 at a dose of 50 mg/kg. Thus, RL2 has a modest therapeutic index, is easily administered, and has promising potential as an anticancer agent. In addition, a comparison of actual antitumor approaches suggests that it is unlikely that we will nd a single compound that satises both the criteria of selectivity and efcacy. Moreover, a combination of drugs exerting their effect through different therapeutic targets minimizes the risk of developing resistance [3,41]. The additive antitumor effect of RL2 and cyclophosphamide provides solid grounds for the recombinant analog of lactaptin to be considered as a potential candidate for combination anticancer therapy. In combination therapies, the additive actions of the drugs allow for a reduction of the cyclophosphamide dose without compromising efcacy while lowering risk of serious side effects. 5. Conclusions RL2 effectively induced apoptosis in vitro and suppressed the growth of sensitive tumors and metastases in vivo. The results of this pilot study indicate that RL2 is a promising candidate for the development of adjuvant therapy for specic metastasizing experimental and possibly some clinical tumors in humans. Conict of interest We conrm that there is no conict of interest in this manuscript. Acknowledgment The study was supported by grants from the Russian Foundation for Basic Research (grants nos. 10-04-01109-a and 11-04-12100), grants from Russian Federal Target Programs (nos. 02.740.11.0715 and 16.N08.12.1009). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biochi.2012.08.017. References
[1] C.L. Creutzberg, R.A. Nout, The role of radiotherapy in endometrial cancer: current evidence and trends, Curr. Oncol. Rep. 13 (2011) 472e478. [2] J. Alsina, M.A. Choti, Liver-directed therapies in colorectal cancer, Semin. Oncol. 38 (2011) 561e567. [3] V. Pavet, M.M. Portal, J.C. Moulin, R. Herbrecht, H. Gronemeyer, Towards novel paradigms for cancer therapy, Oncogene 30 (2011) 1e20. [4] M. Avadisian, S. Fletcher, B. Liu, W. Zhao, P. Yue, D. Badali, W. Xu, A.D. Schimmer, J. Turkson, C.C. Gradinaru, P.T. Gunning, Articially induced protein-membrane anchorage with cholesterol-based recognition agents as a new therapeutic concept, Angew. Chem. Int. Ed. Engl. 50 (2011) 6248e6253. [5] H.B. Kim, M.J. Kim, D.Y. Kim, J.W. Lee, J.H. Bae, D.W. Kim, S.D. Kang, S.H. Kim, High susceptibility of metastatic cells derived from human prostate and colon cancer cells to TRAIL and sensitization of TRAIL-insensitive primary cells to TRAIL by 4,5-dimethoxy-2-nitrobenzaldehyde, Mol. Cancer 10 (2011) 46.

[6] P. Holoch, T.S. Grifth, TNF-related apoptosis-inducing ligand (TRAIL): a new path to anti-cancer therapies, Eur. J. Pharmacol. 625 (2009) 63e72. [7] R.A. Schoop, D.E. Baatenburg, R.J. Jong, M.H. Noteborn, Apoptin induces apoptosis in an oral cancer mouse model, Cancer Biol. Ther. 7 (2008) 1368e 1373. [8] A.-K. Mosseberg, Y. Hou, M. Svensson, B. Holmqvist, C. Svanborg, HAMLET treatment delays bladder cancer development, Invest. Urol. 183 (2010) 1590e 1597. [9] I. Lpez-Expsito, I. Recio, Protective effect of milk peptides: antibacterial and antitumor properties, Adv. Exp. Med. Biol. 606 (2008) 271e293. [10] H. Meisel, W. Bockelmann, Bioactive peptides encrypted in milk proteins: proteolytic activation and thropho-functional properties, Antonie Van Leeuwenhoek 1e4 (1999) 207e215. [11] J.R. Kanwar, R.K. Kanwar, X. Sun, V. Punj, H. Matta, S.M. Morley, A. Parratt, M. Puri, R. Sehgal, Molecular and biotechnological advances in milk proteins in relation to human health, Curr. Protein Pept. Sci. 10 (2009) 308e338. [12] V.V. Nekipelaya, D.V. Semenov, M.O. Potapenko, E.V. Kuligina, Y.Y. Kit, I.V. Romanova, V.A. Richter, Lactaptin is a human milk protein inducing apoptosis of MCF-7 adenocarcinoma cells, Dokl. Biochem. Biophys. 419 (2008) 58e61. [13] D.V. Semenov, A.S. Fomin, E.V. Kuligina, O.A. Koval, V.A. Matveeva, I.N. Babkina, N.V. Tikunova, V.A. Richter, Recombinant analogs of a novel milk pro-apoptotic peptide, lactaptin, and their effect on cultured human cells, Protein J. 3 (2010) 174e180. [14] A.S. Fomin, O.A. Koval, D.V. Semenov, M.O. Potapenko, E.V. Kuligina, Y.Y. Kit, V.A. Richter, Analysis of biochemical markers of MCF-7 cell apoptosis induced by a recombinant analogue of lactaptin, Bioorg. Chem. 38 (2012) 1e7. [15] V.I. Kaledin, N.A. Matienko, V.P. Nikolin, Y.V. Gruntenko, V.G. Budker, Intralymphatic administration of liposome-encapsulated drugs to mice: possibility for suppression of the growth of tumor metastases in the lymph nodes, J. Natl. Cancer Inst. 66 (1981) 881e887. [16] A.J. Minn, G.P. Gupta, P.M. Siegel, P.D. Bos, W. Shu, D.D. Giri, A. Viale, A.B. Olshen, W.L. Gerald, J. Massagu, Genes that mediate breast cancer metastasis to lung, Nature 436 (2005) 518e524. [17] G.G. Avtandilov, Medical Morphometry, Medicine, Moscow, 1990. [18] F. Vidal-Vanaclocha, C. Amzaga, A. Asumendi, G. Kaplanski, C.A. Dinarello, Interleukin-1 receptor blockade reduces the number and size of murine B16 melanoma hepatic metastases, Cancer Res. 54 (1994) 2667e2672. [19] S.T. Smiley, M. Reers, C. Mottola-Hartshorn, M. Lin, A. Chen, T.W. Smith, G.D. Steele, L.B. Chen, Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 3671e3675. [20] P. Workman, E.O. Aboagye, F. Balkwill, A. Balmain, G. Bruder, D.J. Chaplin, J.A. Double, J. Everitt, D.A. Farningham, M.J. Glennie, L.R. Kelland, V. Robinson, I.J. Stratford, G.M. Tozer, S. Watson, S.R. Wedge, S.A. Eccles, Guidelines for the welfare and use of animals in cancer research, Br. J. Cancer 102 (2010) 1555e 1577. [21] R.J. Bernacki, R.J. Bergeron, C.W. Porter, Antitumor activity of N, N-bis(ethyl) spermine homologues against human MALME-3 melanoma xenografts, Cancer Res. 52 (1992) 2424e2430. [22] A. Emadi, R.J. Jones, R.A. Brodsky, Cyclophosphamide and cancer: golden anniversary, Nat. Rev. Clin. Oncol. 6 (2009) 638e647. [23] D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (2000) 57e70. [24] D. Dingli, F. Michor, T. Antal, J.M. Pacheco, The emergence of tumor metastases, Cancer Biol. Ther. 6 (2007) 383e390. [25] E. Rivera, Liposomal anthracyclines in metastatic breast cancer: clinical update, Oncologist 8 (2003) 3e9. [26] R.R. Misra, S.E. Bloom, Roles of dosage, pharmacokinetics, and cellular sensitivity to damage in the selective toxicity of cyclophosphamide towards B and T cells in development, Toxicology 66 (1991) 239e256. [27] T. Mukohara, Role of HER2-targeted agents in adjuvant treatment for breast cancer, Chemother. Res. Pract. (2011) 730360. [28] K. Nakatomi, Y. Nakamura, I. Tetsuya, S. Kohno, Treatment with getinib after erlotinib-induced liver injury: a case report, J. Med. Case Rep. 5 (2011) 593. [29] N. Zhukov, S.A. Tjulandin, Targeted therapy in the treatment of solid tumors: practice contradicts theory, Biochemistry 73 (2008) 751e768. [30] J. Arribas, J. Baselga, K. Pedersen, J.L. Parra-Palau, p95HER2 and breast cancer, Cancer Res. 71 (2011) 1515e1519. [31] M.H. Noteborn, Proteins selectively killing tumor cells, Eur. J. Pharmacol. 625 (2009) 165e173. [32] M.J. Adams, A.W. Harris, C.A. Pinkert, L.M. Corcoran, W.S. Alexander, S. Cory, R.D. Palmiter, R.L. Brinster, The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice, Nature 318 (1985) 533e538. [33] B.B. Zhou, H. Zhang, M. Damelin, K.G. Geles, J.C. Grindley, P.B. Dirks, Tumorinitiating cells: challenges and opportunities for anticancer drug discovery, Nat. Rev. Drug Discov. 8 (2009) 806e823. [34] A.S. Fomin, D.V. Semenov, E.V. Kuligina, O.A. Koval, I.N. Babkina, N.V. Tikunova, V.A. Richter, Genetically engineered analogs of potentially antitumor peptide e lactaptin. Vestnik Novosibirsk State Univers. Biol. Clinic. Med. 8 (2010) 17e25 [35] I. Vermes, C. Haanen, C. Reutelingsperger, Flow cytometry of apoptotic cell death, J. Immunol. Methods 243 (2000) 167e190. [36] G. Kroemer, L. Galluzzi, C. Brenner, Mitochondrial membrane permealization in cell death, Physiol. Rev. 87 (2007) 99e163.

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O.A. Koval et al. / Biochimie 94 (2012) 2467e2474 Glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival, Cancer Res. 64 (2004) 2105e2112. [40] A.-K. Mossberg, B. Wullt, L. Gustafsson, W. Mansson, E. Ljunggren, C. Svanborg, Bladder cancer respond to intravesical instillation of HAMLET (human a-lactalbumin made lethal to tumor cells), Int. J. Cancer 121 (2007) 1352e1359. [41] M.D. Pegram, G.E. Konecny, C. OCallaghan, M. Beryt, R. Pietras, D.J. Slamon, Rational combinations of trastuzumab with chemotherapeutic drugs used in the treatment of breast cancer, J. Natl. Cancer Inst. 96 (2004) 739e749.

[37] A. Lena, M. Rechichi, A. Salvetti, B. Bartoli, D. Vecchio, V. Scarcelli, R. Amoroso, L. Benvenuti, R. Gagliardi, V. Gremigni, L. Rossi, Drug targeting the mitochondrial pore act as cititoxic and cytostatic agents in temozolomide-resistant glioma cells, J. Transl. Med. 7 (2009) 13. [38] L. Gustafsson, I. Leijonhufvud, A. Aronsson, A.-K. Mossberg, C. Svanborg, Treatment of skin papillomas with topical a-lactalbumin-oleic acid, N. Engl. J. Med. 350 (2004) 2663e2672. [39] W. Fischer, L. Gustafsson, A.-K. Mossberg, J. Gronli, S. Mork, R. Bjerkvig, Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human