Inhibition of Tumor Growth by Recombinant ... - Springer Link

Arch Pharm Res Vol 34, No 6, 987-995, 2011 DOI 10.1007/s12272-011-0616-z

Inhibition of Tumor Growth by Recombinant Adenovirus Containing Human Lactoferrin through Inducing Tumor Cell Apoptosis in Mice Bearing EMT6 Breast Cancer Jianjie Wang1,3, Qingwang Li1,2, Yetao Ou1,3, Zengsheng Han1, Kun Li1, Peijun Wang1,3, and Shaobo Zhou4 1

Department of Biological Engineering, College of Environment and Chemical Engineering, Yanshan University, No.438 Hebei Street, Qinhuangdao 066004, China, 2School of Animal Science, Northwest A & F University, No.22 Xinong Street, Yangling 712100, China, 3College of Basic Medicine, Jiamusi University, No.148 Xuefu Street, Jiamusi 154007, China, and 4 LIRANS, Institute of Research in the Applied Natural Sciences, University of Bedfordshire, Luton, UK, LU2 8DL (Received June 18, 2010/Revised October 1, 2010/Accepted October 12, 2010)

Human lactoferrin (hLTF), an 80-kDa iron-binding glycoprotein, has antitumor activity. In this study, a recombinant adenovirus containing the human lactoferrin cDNA (ad-rhLTF) was constructed and its effect on tumor growth was investigated in mice bearing EMT6 breast cancer. Ad-rhLTF was injected seven times within 14 days into the tumor site at two concentrations (108 and 5 × 108 pfu/mL) in mice bearing EMT6 breast cancer. Injected ad-rhLTF had considerable cytotoxicity on mice breast cancer, and significantly reducing the weight of tumor produced and increasing the tumor inhibition rate up to 52.64%. The presence of apoptotic cells was confirmed using TUNEL staining and flow cytometry assays. At the same time, RTPCR and Western blot analyses demonstrated that ad-rhLTF also decreased expression of Bcl2 and increased Bax and caspase 3 expressions. Therefore, we conclude that ad-rhLTF inhibits tumor growth by inducing tumor cell apoptosis in mice with breast cancer by triggering the mitochondrial-dependent pathway and activation of caspase 3. The results indicate that adrhLTF might be a promising drug for breast cancer gene therapy. Key words: Lactoferrin, Adenovirus vector, Gene therapy, Breast cancer, Apoptosis, Pathway

Selected by Editors INTRODUCTION Lactoferrin (LTF), an iron-binding glycoprotein, is mainly present in mammalian milk colostrum. It is also found in exocrine secretions of mammals and is released from neutrophil granules during inflammation (Artym et al., 2003). LTF contains 703 amino acids and has a molecular weight of 80 kilodaltons (GonzálezChávez et al., 2009). The primary functions of LTF are to improve immunological responses (Zimecki et al., Correspondence to: Qingwang Li, Department of Biological Engineering, College of Environment and Chemical Engineering, Yanshan University, No.438 Hebei Street, Qinhuangdao 066004, China Tel: 86-335-8074662, Fax: 86-335-8074662 E-mail: [email protected]

2007), iron transport, storage and chelation (Ward and Conneely, 2004). LTF also exhibits many useful biologic functional activities that have been used in antibacterial, antivirus, antioxidant and immunoregulatory roles (Shimazaki, 2000; Conneely, 2001; Mulder et al., 2008). LTF also has antitumor effects and inhibits the proliferation of different tumor cells including esophageal carcinoma, oral cancer, lung cancer, liver cancer, colon carcinoma and blander cancer (Ward et al., 2002; Tsuda et al., 2002; Mader et al., 2005). However, the pharmacologic mechanisms of LTF on breast cancer are unknown. Commercial LTF is mainly bovine LTF that is extracted from whey proteins in bovine milk. The prohibitive cost has restricted therapeutic development efforts utilizing bovine LTF. The production of recombinant human LTF (rhLTF) provides a method for production of large volumes at low cost (Cerven et al., 2008; Tutykhina et al., 2009). Previously, we

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reported on the construction of adenoviral vectors containing human LTF cDNA (ad-rhLTF) and a green fluorescent protein (GFP)-reported gene fusion, and described the abundant expression of human LTF in the milk of goats and rabbits (Han et al., 2007, 2008). In this study, we repeatedly injected purified and titrated ad-rhLTF into the tumor sites of EMT6 bearing mice to explore the therapeutic potential of ad-rhLTF for breast cancer, unravel the possible mechanisms responsible for the antitumor activity of ad-rhLTF and to provide scientific experimental evidence for tumor gene therapy of ad-rhLTF.

Academy of Military Medical Sciences. Animal experiments were conducted in accordance with the NIH Guide for the care and use of laboratory animals (NIH Publication No. 80-23; revised 1978 and the number approved by Administrated-Committee of Laboratory Animals was 062310). The mice were randomly divided into five groups (n = 10 per group). One group was used for the preparation of EMT6 tumor cells. The other four groups were used for different administrations of drugs (see below). Animals were housed in plastic cages with free access to food and water and maintained on a regulated environment (20 ± 2°C).

MATERIALS AND METHODS

Prepared EMT6 tumor cells The EMT6 breast cancer cell line (5 × 106/mL EMT6 breast cancer cells) was injected into the right forelimb in one group of mice (0.2 mL/mouse). When each tumor had grown to 1 cm in size, it was removed and suspended in normal saline to a concentration of 5 × 106/mL under aseptic conditions.

Drugs and chemicals Human embryonic kidney (HEK) 293 and mouse breast cancer EMT6 cell lines were obtained from the Cancer Institute of the Chinese Academy of Medical Sciences. Prodium iodide, ribonuclease (RNase) and rabbit anti mouse LTF (L3262) monoclonal antibody were obtained from Sigma-Aldrich. The terminal deoxyribonucleotide transferase-mediated nick-end labeling assays (TUNEL) kit was purchased from KeyGEN Bio. Trizol was purchased from Gibco. Reverse transcriptase cDNA synthesis kit was purchased from Takara Bio. Bcl-2 (C21), Bax (B-9) and caspase 3(E-8) monoclonal antibodies were obtained from Santa Cruz Biotechnology. Horseradish peroxidase-labeled rabbit antigoat IgG antibody and actin polyclonal antibody were purchased from Biosynthesis Bio. The enhanced chemiluminescence kit was purchased from Amersham Pharmacia Biotech. All other chemicals used were of analytical reagent grade. Preparation of viral stock HEK 293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium containing 10% fetal bovine serum (FBS) and incubated at 37oC in an atmosphere of 5% CO2. One hundred microliters of adrhLTF suspension was added to HEK 293 cells grown to 80% confluence (Wang et al., 2010). After 48 h, the titer of the adenovirus stock was determined using GFP on semi-confluent 293 cells. The 293 cells were collected and the virus was by four cycles of freezing in liquid nitrogen and melting at 37oC, diluting the virus stock concentration based on the plaque-forming units (pfu)/mL with normal saline to the desired concentration, and storing at −70oC until use. Animals Fifty female 6-week-old Kunming mice were purchased from the Laboratory Animal Center of the

Animal model and drug treatment Four groups of mice were all injected with 0.2 mL/ mouse of 5 × 106/mL EMT6 breast cancer cells in the skin under the right forelimb. The select drug was administered when each tumor had attained a size of 0.3-0.5 cm. One group was injected with 100 µL of normal saline in the tumor site as the control group. One group was given the standard antitumor reference drug cyclophosphamide (CTX; 25 mg/kg body weight, i.p. daily for 14 days); this group was the positive control group (CTX group). The other two groups were injected with 100 µL ad-rhLTF in tumor sites at a dosage of 108 pfu/mL or 5 × 108 pfu/mL (ad-rhLTF groups). All of the injections were given once every two days for a total of seven times in 14 days. After 14 days, all mice were weighed and killed, and each tumor was removed and weighed. According to the mean weight of tumor, the rate of tumor inhibition was calculated as follows: rate of inhibition (%) = [(mean tumor weight of control group − mean tumor weight of treated group) ÷ mean tumor weight of control group] × 100. Morphologic analysis of tumor tissues Tumor specimens collected from the control group, CTX group and the 5 × 108 pfu/mL ad-rhLTF group were fixed in 10% (v/v) neutral formalin solution, dehydrated through a graded ethanol series and embedded in paraffin. Tissue serial sections 4 µm in thickness were stained with hematoxylin and eosin and then examined under the light microscope.

Effect of Recombination Human Lactoferrin

TUNEL assay Apoptotic cells in sections of mouse tumor tissue from the four groups were detected by using an in situ apoptosis detection kit according to the manufacturer’s instructions. The distinctly brown staining of cells was indicative of apoptosis and blue staining of cells was indicative of non-apoptotic cells when examined using a light microscope. Images were acquired using Leica Application suite software (Leica Microsystems), and the average number of positive cells was counted by using Leica QWin software (Leica Microsystems) in five randomly selected optical fields (200 tumor cells/ per field). The apoptotic index (%) was calculated as: (number of apoptotic cells ÷ total number of cells) × 100. Cell cycle and apoptosis assessments by flow cytometry Tumors collected from the control group, CTX group and ad-rhLTF group (5 × 108 pfu/mL) were minced, a single cell suspension was prepared with 200 mesh filtering, centrifuged at 956 g for 5 min, washed three times, the cell concentration was adjusted to 106/mL, cells were fixed with 70% ethanol for 30 min at 4oC, and DNA content and cell cycle were analyzed by flow cytometry after treatment with RNase and propidium iodide staining for 30 min. The proportion of cells in each cell cycle and apoptosis number were calculated. The data were analyzed with CellQuest safeware (Becton Dickinson). RNA extraction and reverse transcriptionpolymerase chain reaction (RT-PCR) Total RNA in tumor tissues was extracted using Trizol. RT-PCR was performed by using reverse transcriptase cDNA synthesis kit according to the manufacturer’s protocol. One microgram of total RNA was reverse transcribed into cDNA and then followed by PCR amplification using specific primers: Bcl-2, forward: (5'-CCTGGCACCTGGCGGATAGC-3') and reverse: (5'-CGACTGAAGAGTGAGCCCAGCAGAAC3'); Bax, forward: (5'-GCTCTGA ACAGATCATGAAGACAG-3') and reverse: (5'-CAATCCAAAGTGGACCTGAGG-3'); caspase-3, forward: (5'-TTTGTTTGTGTGCTTCTGAGCC-3') and reverse: (5'-GATGTTCTGGAGAGCCCCG-3'); β-actin, forward (5'-AATGGGTCAGAAGGACTCCTATGTGG-3') and reverse: (5′-CGCCTA GAAGCACTTGCGGTG-3'). Bcl-2 or Bax was amplified by 30 cycles at 94°C for 30 sec, 58°C for 30 sec, and 72°C for 40 sec in order. Caspase-3 was amplified by 30 cycles at 94°C for 45 sec, 52°C for 45 sec, and 72°C 1 min. All procedures were followed by a 7 min extension at 72°C. PCR products were electro-

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phoresed on a 1.0% agarose gel containing ethidium bromide and visualized by ultraviolet-induced fluorescence.

Western blot analysis Tumor tissues from the four groups were minced and lysed in 500 µL cell lysis buffer for 30 min, and centrifuged at 12,000 g for 15 min at 4°C. The supernatant was collected and protein concentrations were determined according to the Bradford method. Samples were subjected to 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) after they were boiled for 5 min, and the resolved proteins were electrophoretically transferred to polyvinylidene difluoride membranes by a semi-dry transfer method. The membranes were blocked with 5% non-fat dried milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature, washed three times with TBST, and incubated with TBST containing 5% of dried skim milk and primary antibody (LTF, Bcl-2, Bax or caspase 3) for 2 h at room temperature. After washing three times with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Proteins were visualized by using an enhanced chemiluminescence kit and exposed to X-ray film. At the same time, actin was used as an internal control for all Western blots. The intensity of protein bands was quantified by using LabWork 3.0 UVP software. Statistical analysis All values are expressed as mean ± S.D. One-way analysis of variance and Duncan’s multiple range tests were used for determining differences between groups, and p < 0.05 was regarded as statistically significant.

RESULTS Adenovirus-mediated hLTF cDNA expression in 293 cells and tumor tissues The intensity of GFP fluorescence indicated the high-expression level of ad-rhLTF in 293 cells under fluorescence microscopy (Fig. 1A). This result indicated that the ad-rhLTF could be used effectively in further experiments. The expression of ad-rhLTF in tumor tissues from the EMT6 bearing mice was analyzed by Western blotting. The level of hLTF increased substantially after administration with ad-rhLTF in a dose-dependent manner, with 5 × 108 pfu/mL being the better effective concentration for ad-rhLTF expression in vivo. Human LTF was not detected in either control or CTX groups not treated with ad-

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al., 2008), produced an inhibition rate of 58.68% (Table I).

Fig. 1. Expression of ad-rhLTF in 293 cells and tumor tissues. (A) Fluorescent micrograph of 293 cells transfected ad-rhLTF (×200). The intensity of GFP fluorescence indicates the high-expression level of ad-rhLTF in 293 cells. (B) Typical result of Western blot analysis performed to detect ad-rhLTF expression in tumor tissues from EMT6 bearing mice.

hLTF (Fig. 1B).

Cytotoxicity of ad-rhLTF on mice breast cancer Tumor-bearing mice were treated with ad-rhLTF (108 pfu/mL or 5 × 108 pfu/mL) seven times, or with CTX. There was no significant effect on the body weight of mice in either the ad-rhLTF group or CTX group after administration. However, compared with the control group, ad-rhLTF administration of 108 pfu/ mL and 5 × 108 pfu/mL significantly decreased the tumor weight, and the tumor inhibition rate was 42.80% and 52.64%, respectively. CTX, which is the standard chemotherapeutic (Li et al., 2007; Zhang et

Morphological changes of cell apoptosis Tumor cells in the control group were arranged closely in different sizes and shapes. The cells displayed a small cytoblastema and bigger nucleus with a thickly staining and obvious heteromorphism, and hyperplasia as well. As shown in Fig. 2, the number of tumor cells in the ad-rhLTF treatment groups decreased markedly, and tumor cell chromatin accumulated at the side of the nucleic membranes. The nucleic shape was irregular and the surface of the nucleic membrane was rough. The nucleus was broken but was encapsulated by intact membrane, containing intact organelles and apoptotic bodies. Light microscopy comparison with the control group revealed more apoptotic cells in the ad-rhLTF group. TUNEL staining results showed that the apoptotic index reached 21.24% in the ad-rhLTF group (5 × 108 pfu/mL) (Fig. 3), demonstrating that ad-rhLTF treatment produced markedly more apoptotic cells. Effect of ad-rhLTF on tumor cell cycle After administration of CTX and ad-rhLTF (5 × 108 pfu/mL), the percentage of tumor cells in G0/G1 phase was increased significantly from 48.3% in the control group to 64.7% in CTX group and 68.7% in ad-rhLTF group (p < 0.05). Correspondingly, the percentage of sub-G1 cells as apoptotic cells was also significantly increased from 4.2% in the control group to 8.9% in CTX group and 19.6% in ad-rhLTF group (p < 0.05). However, CTX and ad-rhLTF reduced the proportion of tumor cells in the S and G2/M phases, compared to the control group. These data indicated that ad-rhLTF arrested the cell cycle in G0/G1 phase and induced tumor cell apoptosis (Fig. 4). Expression of apoptosis related genes in tumor tissues To determine the mechanisms of apoptosis induced

Table I. The inhibitory effect of ad-rhLTF on EMT6 solid tumors Body weight (g) Groups

n

Start

End

Mean weight of tumor (g)

Inhibition rate (%)

Control

10

Vehicle

19.71 ± 1.84

21.18 ± 2.01

2.631 ± 0.46

CTX

10

25 mg/kg

20.96 ± 1.72

19.24 ± 1.20

1.087 ± 0.19a

58.68

21.65 ± 2.59

a

42.80

a

52.64

Ad-rhLTF

10 10

a

Treatment

8

10 pfu/mL 8

5 × 10 pfu/mL

20.17 ± 1.84 20.86 ± 1.74

p < 0.05 as compared with control group, values are mean ± S.D.

22.57 ± 2.04

1.505 ± 0.24 1.246 ± 0.27


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Fig. 2. Cell apoptosis-related morphological changes of tumors in the EMT6 model treated with ad-rhLTF. (A) Results of control group. (B and C) In vivo results from EMT6 breast cancer model treated with CTX (B) and ad-rhLTF (5 × 108 pfu/ mL) (C) for 14 days. White arrows indicate necrosis tumor cells and black arrows indicate apoptotic cells (H&E stain, ×400).

Fig. 3. Morphological and apoptotic index changes of apoptosis in vivo in the EMT6 breast cancer model treated with adrhLTF (TUNEL staining ×200). (A) Untreated EMT6 breast cancer model. (B and C) EMT6 tumor mice were treated with CTX (B) and ad-rhLTF (5 × 108 pfu /mL) (C) for 14 days. Apoptotic cells in sections of mouse tumor tissue were detected using a TUNEL assay and the apoptotic index was calculated. (D) The changes of apoptotic index in the EMT6 breast cancer model in vivo treated with CTX and ad-rhLTF (108 pfu/mL and 5 × 108 pfu/mL) for 14 days. *p < 0.05 vs control group. The data are generated from four fields per slide, with four slides analyzed from each tumor, and 10 tumors examined from each group (TUNEL stain, ×200).

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Fig. 4. Effect of ad-rhLTF on tumor cell cycle. The percentage of apoptotic cells was determined by a FCM assay. (A, B and C) display results from control group, CTX group and ad-rhLTF group (5 × 108 pfu/mL), respectively.

by ad-rhLTF, the expression of Bcl-2, Bax and caspase 3 in tumor tissues were tested by RT-PCR and Western blotting. The expression of Bcl-2 mRNA was reduced, while the expression of Bax and caspase 3 were increased compared with the control group (Fig. 5). The result of Western blotting analysis was similar to RT-PCR analysis. The down-regulation of Bcl-2 and up-regulation of Bax led to a decrease in the ratio of Bcl-2/Bax. However, compared with the control group, the expression of above apoptosis related gene was not significantly changed in the CTX group (Fig. 5).

DISCUSSION This study is the first investigation into the effects of ad-rhLTF on mice harboring breast cancer cells. The results demonstrated that the adenoviral vectors carrying human LTF cDNA can produce a high-level of expression of hLTF and play an important antitumor role. Compared with the control group, ad-

rhLTF administration can significantly decrease the tumor weight and increase the tumor inhibition rates; induce morphological changes indicative of apoptosis; increase tumor cell apoptosis (e.g. arrest the cell cycle in G0/G1 phase, increased proportion of tumor cells in G0/G1 phase and sub-G1 cells and reduce the proportion of tumor cells in S and G2/M phase); and regulate the expression of Bcl-2 and Bax, leading to a decrease of the ratio of Bcl-2/Bax and caspase 3 activation. Our findings are consistent with previous studies using bovine LTF which showed G1 arrest, caspase 3 activation, poly-ADP ribose polymerase cleavage and DNA fragmentation in oral, head and neck and colon cancers (Fujita et al., 2004; Xiao et al., 2004; Sakai et al., 2005). Many anticancer drugs induce tumor cell apoptosis and induction of apoptosis is an obvious strategy for cancer therapy. The mechanisms of apoptosis induced by drugs are complex due to the differences in cell types and drugs (Sun et al., 2008). However, mitochondrial and cell-surface death receptor


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Fig. 5. Expression of apoptosis-related proteins in tumor tissues. (A) RT-PCR was performed to determine apoptosis related protein mRNA expression. β-actin was used as a control. (B) Western blot analysis was performed to detect apoptosis related protein expression. β-actin was used as a control to ensure equal loading. Data shown is representative of three independent experiments. (C) The intensities of the Bcl-2 and Bax bands were quantified and are shown as relative expression level after being normalized using β-actin (n = 3, mean ± S.D.); *p < 0.05, vs control group. (D) The intensity of caspase 3 was quantified and is shown as the relative expression level after being normalized by β-actin (n = 3, mean ± S.D.); *p < 0.05, vs control group.

-mediated apoptosis are the two principal pathways leading to programmed cell death. The mitochondrial pathway is thought to play a major role in response to cancer treatments and is mediated by the Bcl-2 family proteins, which are always over-expressed in many tumor cells (Reed, 2000; Sjöström et al., 2002; Ohtsuka et al., 2003) and they act as repressors of apoptosis by blocking the release of cytochrome-c, whereas pro-

apoptotic members (Bax) act as promoters. These effects are more dependent on the balance between Bcl-2 and Bax than on Bcl-2 quantity alone (Scorrano and Korsmeyer, 2003; Takahashi et al., 2004). Previous studies have shown that bovine LTF selectively induces apoptosis in human leukemia and carcinoma cell lines by down-regulating the expression of Bcl-2 and regulating the activity of caspases 3, 8 and 9

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(Mader et al., 2005), and induces apoptotic cell death by regulation of the apoptosis related-gene Bcl-2 and Bax expression in rat colon mucosa and human breast cancer cell lines (Sekine et al., 1997; Sjöström et al,. 2002; Fujita et al., 2004). These observations suggest that one of the apoptotic mechanisms induced by adrhLTF is the triggering of the mitochondrial-dependent pathway and associated caspase 3 activation. In the current study, the treatment with ad-rhLTF decreased the expression of Bcl-2 and increased the expression of Bax in mRNA and protein levels, which suggests that ad-rhLTF regulates the expression of Bcl-2 and Bax proteins by promoting the gene transcription of Bcl-2 and Bax. The up-regulation of Bax expression and the reduction of Bcl-2 expression in the treated groups leads to a decrease in the ratio of Bcl-2 to Bax, which might be responsible for the drug-induced apoptotic processes and which might be associated with better prognosis. Caspase-3 is an executioner caspase of the apoptosis pathway (Mlejnek, 2001; Cheung et al., 2002). In the present study, after administration with ad-rhLTF there was a considerable increase in caspase3 mRNA and protein levels, which indicated that adrhLTF also promotes caspase 3 gene transcription and subsequent increased expression of caspase 3 protein. The most likely explanation for the antitumor mechanisms of ad-rhLTF is that ad-rhLTF results in DNA damage of tumor cells and that in response a positive ratio between Bcl-2 and Bax leads to cytochrome c release from mitochondria, which triggers the mitochondrial-dependent pathway, finally leading to the activation of the caspase-3 and, eventually, to apoptosis. In conclusion, ad-rhLTF results in the growth inhibition of tumor cells by inducing apoptosis, and the mechanisms likely occur through the triggering of the mitochondrial-dependent pathway and caspase 3 activation. Based upon our in vivo tumorigenic data, ad-rhLTF might be a promising drug for breast cancer treatment. Finally, recombinant human LTF mediated by adenovirus vectors has similar biological activities to natural LTF with more economical and efficient procurement than natural LTF, which should greatly facilitate future research of hLTF in cancer therapy and other fields.

ACKNOWLEDGEMENTS The authors are grateful to Dr. Adam Paige for his careful proof reading, constructive comments and suggestions on this work.

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