Bovine lactoferrin inhibits lung cancer growth through suppression of both inflammation and expression of vascular endothelial growth factor

BMC Biology

Bovine Llactoferrin Iinhibits Lung Cancer Growth

though Suppression of Inflammation and

VEGFgrowth Expression of lung cancer cells and solid

tumors in vascular endothelial growth factor

(VEGF)-overexpressing transgenic mice

†

,

Hsiao-Ling Chen2†_{, Ming-Feng Lin}3_{, C hih-C hing}

Y

en1,4_{, Chih-Jie Shen}1_{, Cheng-Wei Lai}1_{,Yi-Wen Lai}1_{, and Chuan-Mu Chen}1,5*

1_{Department of Life Sciences, National Chung Hsing University, Taichung 402,} Taiwan; 2_{Department of Internal Medicine and College of Medicine, China Medical}

University, Taichung 404; 4

Department of Bioresources and Molecular Biotechnology, Da-Yeh University, Changhwa 515, Taiwan; 3_{Taichung Hospital, Department of} Health, Taichung 403; 4_{Department of Internal Medicine and} 5_{School of Chinese}

Medicine, China Medical University, Taichung 404, Taiwan.

Running title:

KEYWORDS:

†

These authors contributed equally to this work.

*

Correspondence: Chuan-Mu Chen, Ph.D.

Professor and Dean

Department of Life Sciences 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

National Chung Hsing University, Kuo Kuang Rd., Taichung 402, Taiwan,

Phone: 886-4-22856309 Faxx :: 886-4-22874740 E-mail: [email protected] 1 2 3 4 5 6 7 8

Abstract

Background: Lung cancers are among the most common cancers in the world. Angiogenesis is an important factor in the formation of tumors, tumor growth, invasion, and metastases. The Vvascular endothelial growth factor (VEGF) promotes the initial formation of blood vessels (vasculogenesis) and plays a vital role in the growth and expansion of existing blood vessels (angiogenesis). Bovine lactoferrin (bLF) is a major active component of milk, and it has exhibits exhibited remarkable effects on cancers, including such as tongue, esophagus, stomach, colon and bladder cancers. Previous researchers pointed have demonstrated out that the potential cancer mechanisms of anti-cancer function treated with bLF are include theprevention of angiogenesis and,the induction of programmed cell death. Additionally, bLF has an,

innate ability to bind iron and to regulation regulate of cell cycle protein expression.

Results: In this study, we used both in vivo and in vitro approaches to investigate the

anti-lung cancer activity of bLF. In the an animal model, we used hVEGF-A165 overexpressed transgenic mice overexpressing hVEGF-A165 carrying via the

mccsp-Vegf-A165-sv40 poly(A) transgene for to researching examine the mechanism of lung tumorigenesis. We found that bLF decreased cell proliferation of A54949h human lung cancer cells, A549, through by decreasing the expression of vegf mRNA and VEGF 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

protein. This effect occurred in a dependent manner and this effect is

dependent manners. Furthermore, we found that oral administration of bLF at 300

mg/kg b.w. three times a week for 1.5-month1.5 months, 10.5-month-old hVEGF-A165-overexpressing transgenic mice significantly eliminated hVEGF-A165 overexpression.

The treatment specifically led to normal VEGF expression to normal, specifically in

claraClara cells of the lungs of transgenic mice, and suppressed the formation of

tumors. In additionAdditionally, treatment with bLF significantly could decreased the

levels ofsignificantly proinflammatory cytokines, such as VEGF-A, TNF-α and IL-12,

and anti-inflammatory cytokines, such as IL-4 and IL-10. The levels of IL-6, which

isacts as both a , as well as both proinflammatory and anti-inflammatory cytokine,

were also reduced such as IL-6 when compared with Tg mice alone. These cytokines

could were be inhibited by bLF, thus resulting to result in limited inflammation, which thenand restricted the growth of the lung cancer.

Conclusions: bLF, an angiogenesis inhibitor, exhibits a considerable potential in the treatment of lung cancer because of its effects on angiogenesis. Therefore, the bLF protein and may be of therapeutic potential use a potential protein drug forin therapeutic purposes. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Background

According to statistics supplied by the Taiwanese Department of Health, cancers have

been ranked as the leading causes of death in Taiwan since 1982. There are more than 30,000 people diagnosed with cancer every year, and thus cancer has become the largest health threat. Of the top ten lethal cancers, lung cancer ranked has been the first leading cause of mortality in the past ten years in Taiwan for the last ten years [1].

Worldwide, lLung cancer has been the most common cancer in the world for several

decades. The majority of new the cases now occur in the developing countries (55%). Lung cancer is still the most common cancer type in men worldwide (1.1 million cases, 16.5% of the total), with high rates in Central, -Eastern and Southern Europe, Northern American and Eastern Asia [2]. Although the treatment of lung cancer has improved, the mortality rate of lung cancer patients remains high. To reduce these high rates of mortality, many researchers have focused on methods for tumor prevention as well

asin addition to more effective treatments [3]. Recently, many researchers have found

natural food components or products of their digestion that could mediate the process of angiogenesis and metastasis. A study by de Mejia and Dia [4] showed that the anti-cancer potential of dietary proteins, peptides and amino acids, which were either natural products of fermentation and, enzymatic hydrolysis, or products of

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

gastrointestinal digestion. These compounds mediated by mediating apoptosis and angiogenesis, which is a vital step to in controlling tumor metastasis.

Bovine lactoferrin (bLF), an 80 kDa siderophilic protein which that has two iron- binding sites. This protein, has a wide range of biological activities, including such as anti-cancer effects, antimicrobial effects, anti-inflammatory activity, improvement of iron status, promotion of cell proliferation promotion, and improvement of immunomodulatory functions [4]. Chemopreventive and cell growth inhibitory activities of bLF have been demonstrated in esophagealus [5], lung [6], colon [7-9], bladder [10], mammary [11], stomach [12], breast [13] and tongue [14, 15] cancers. González-Chávez et al. [16] pointed outdemonstrated that the anti-cancer functions of bLFf is are through its innate ability to bind iron. This iron , and due to iron cancould otherwise promote oxidation, thereby disrupting nucleic acid structure. Furthermore, other potential mechanisms of anti-cancer functions are include prevention of angiogenesis, induction of programmed cell death and regulation of cell

cycle protein expression [16-18].

In the early stages of cancer, the out-of-controlunregulated proliferation of cancer cells leading leads to a deficiency of both nutrients and oxygen, which causes a large degree of cell death. ThereforeThe cell death triggers , an inflammation inflammatory

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response occurs, and activates HIF-1α is activated. HIF-1α activation induces the secretion of a large quantity of VEGF-A165. The Ssecreted VEGF-A165 binds to VEGFR2 and activates a downstream signal that induces vasculogenesis [19, 20]. When cancer cells secrete a large amount of VEGF-A165, vasculogenesis is induced so as to provide sufficient nutrition nutrients and oxygen to the tumor, thus increasing the tumor growth rate. It is well-known that theThe expression level of VEGF-A165 is positively related to the growth and spread of cancer cells [21]. Therefore, the development of medicines that target VEGF-A165 is a popular topic of study. In this study, we used both in vivo and in vitro approaches to investigate the VEGF expression of lung cancers treated with bLF. In the animal model, Wwe used an animal model of hVEGF-A165- overexpressed overexpressing transgenic mice carrying the mccsp-Vegf-A165-sv40 poly(A) transgene for to examine researching the mechanism of bLF on lung

tumors. 1 2 3 4 5 6 7 8 9 10 11 12 13

Results

Effects of bLF on cancer cell viability, morphological changes and growth curve

A549 and CL1-0 cells, which are human lung adenocarcinoma cell lines, were treated with bLF (4.5, 2.25, 1.125, 0.5625 and 0.28125 mg/well) for 48 h. This treatment

decreased the cell viability in a concentration-dependent manner when compared to with controls (Figure 1a). In additionAdditionally, an MTT assay revealed that concentrations up to 4.5 mg/well produced no significant cytotoxic effects on Beas 2B

normal lung cells, Beas 2B cells, treated with bLF. The LD50 values of bLF treatment

were 0.96 and 1.68 mg/well for A549 and CL1-0 cells, respectively, treated with bLF.

Of these cancer cells, Among them, bLF has was able to successfully inhibita good

performance on inhibiting the growth of A549 cells.

After the A549 cells were treated with bLF (100, 50, 25, 12.5 and 6.25 mg/10-cm dish) for 48 h, the A549 cells underwent significant morphologic changes which that were examined and photographed under a microscope, as shown in Figure 1b. This result ruled out the possible cytotoxic effects of bLF, supporting a negative impact of bLF on cell proliferation. To determine the effect of bLF on the growth of A549 cells,

the cells were On culturing, A549 cells treated with or without bLF (3 mg/well) in

24-well microtiter plates for various time points (1, 4, 7, 8, 9 and 10 days), and and plotting the total number of cells against were plotted against time (Figure 1c). From 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

the log phase of the curve, the population doubling time of A549 cells treated with or without bLF was calculated which was found to be 23.5 and 59.3 h, respectively, indicating that A549 cells treated with bLF grew more slowly compared with A549 cells alone.

bLF suppressed vegf mRNA and VEGF protein expression in A549 lung cancer cells

In this study, the effect of bLF on vegf gene expression was also investigated. As shown in Figure 2a, 48 h of incubation resulted in a large increase in vegf mRNA expression, and bLF inhibited the expression of the vegf gene in a dose-dependent manner. Approximately 12 and 75% reductions were observed at 12.5 and 50 mg per 10-cm dish, respectively, as determined by with densitometry analysis. At concentrations of up to 100 mg per 10-cm dish, bLF could almost completely inhibit the expression of vegf mRNA in A549 cells. Additionally, in A549 cells treated with bLF (100, 50, 25, 12.5 and 6.25 mg/10-cm dish) for 48 h, and the expression of VEGF protein decreased in a concentration-dependent manner when compared to with

controls (Figure 2b). The result analysis of of VEGF protein expression was yielded the same with as that for vegf mRNA expression.

An Eearly staged cancer cells will keep continue to proliferation proliferateing in

vivo, and thus leading to the deficienciesy in both the nutritionnutrients and oxygen in

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vivo. In order toTo imitate the growth environment of the cancer cells in vivo, cells were cultured in DMEM supplemented with 10% FBS or 0.1% FBS in the normal or hypoxica conditions (95 % N2 and 5% CO2). In the present study, the vegf mRNA

expression in A549 cells was increased from the a ratio of 0.22 to 0.36 by after hypoxia treatment. Among the three conditions, the addition of 10% FBS in the hypoxia condition exhibited caused the highest the higher expression of the vegf gene

(Figure 2c). Figure 2c shows that, at the given dose of bLF, bLF markedly suppressed

the vegf mRNA expression in DMEM supplemented with 10% FBS in both the normal

or and hypoxia conditions. Thus, bLF warrants further development as a cancer-prevention agent.

Effects of bLF on pathological histology and immunohistochemical analysis in vascular endothelial growth factor (VEGF)-overexpressing transgenic mice

In this study, we used hVEGF-A165- overexpressed overexpressing transgenic mice

which withexpressing the constructed mccsp-Vegf-A165-sv40 poly(A) transgene for to researching examine the mechanisms of bLF on of lung tumors. After oral administration of bLF at 300 mg/kg b.w. three times a week for 1.5-month1.5 months,

the 10.5-month-old hVEGF-A165-overexpressing transgenic mice showed a dramatic

decrease in solid tumor formation compared to with untreated transgenic mice. The

exterior the lungs of transgenic mice and wild- type mice with the age overthat were

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

older than 10.5 months are demonstrated in , the exterior of its lung with that of the

wild type mouse was shown in Figure 3a and 3b. Figure 3b shows that the lung tumors

were found in the transgenic mice and primarily included neoplasms growing on the

peripheryal of the lung alveolus and adenomas growing on the site near the lung bronchus. In the a lung alveolus on the a lung bronchus of a transgenic micemouse, some obvious, and large-grained, pink cells were found. Such These pink cells were are macrophages, and they are indicative of an inflammation inflammatory response. These results suppose suggest that hVEGF-A165 is capable of promoting vascular permeability and the effectiveness of the inflammationan inflammatory response. Furthermore, oral administration of bLF at 300 mg/kg b.w. three times a week for 1.5

-months reduced the growth of neoplasmsic growth on the periphery of the lung alveolus and adenomas growing on the site near the lung bronchus (Figure 3c). In additionAdditionally, this histological examination revealeds that bLF has potential anti-cancer and anti-inflammatory effects in the lungs of hVEGF-A165-overexpressing transgenic mice.

Angiogenesis is an important factor in the formation of tumors as well asand in tumor growth, invasion, and metastasis. VEGF promotes the initial formation of new blood vessels (vasculogenesis) and plays a vital role in the growth and expansion of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

these new blood vessels (angiogenesis). Immunohistochemical analysis showed that VEGF was overexpressed in the clara Clara cells of lung tissues from the Tg mice compared with wild- type mice (Figure 3a and 3b). In and the Tg mice, treatment with bLF significantly blocked VEGF overexpression in Cclara cells when compared to with untreated Tg mice alone (Figure 3c). These results indicate that transgenic mice treated with bLF can reduce VEGF overexpessionoverexpression compared with transgenic mice, which in turn could eliminate the formation and growth of new blood vessels. The anti-vasculogenic and anti-angiogenic properties of bLF may thus represent a possible cause mechanism for the previously observed suppression of tumor growth, invasion, and metastasis.

bLF downregulated decreased vegf, IL-6 and TNF-α mRNA transcriptss

expression in vascular endothelial growth factor (VEGF)-overexpressing

transgenic mice

In our previous study, we found that the vegf mRNA expression treated with bLF in A549 cells treated with bLF line could bewas significantly decreased. Due It is

expected due to its previously shown association between bLF and with the formation

of lung cancer, it was expected that bLF could inhibit the vegf mRNA expression that was responsible for cell proliferation. The mRNA expression levels patterns of vegf,

IL-6 and TNF-α were markedly downregulated decreased in the Tg/bLF group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

compared with the to Tg/Placebo group (Figure 4a). The expression of vegf, IL-6 and TNF-α mRNAsexpression in the of Tg/bLF group were dramatically decreased to 4.3, 0.4 and 1.7, respectively, while these ratios were 42.1, 17.5 and 2.8, respectively, in the Tg/Placebo group (P < 0.05). Additionally, the mRNA expression patterns of vegf, kdr,

nrp-1, myc, brca-1, mmp2, mmp9, egfr, erk2, survivin, cyclin a, cyclin b1, cyclin d, and cyclin e in the Tg/placebo and Tg/bLF groups were not significantly different (data not shown). Furthermore, western blotting showed that the expression of the VEGF protein was significantly decreased in the Tg/bLF group when compared with the Tg/Placebo group (Figure 4b). Consequently, these results suggest that angiogenesis maybe blocked by because of the inhibition inhibiting the expression of VEGF protein

expression when thebysupplementation treatment with of bLF.

bLF suppressed cytokine levels in vascular endothelial growth factor (VEGF)-overexpressing transgenic mice

In this study, the levels of proinflammatory cytokines (VEGF-A, TNF-α and IL-12), anti-inflammatory cytokines (IL-4 and IL-10) and a both proinflammatory and anti-inflammatory cytokine (IL-6) in the serum of Tg/Placebo and Tg/bLF groups are shown in Figure 5. Previous reports have shown that the imbalance between proinflammatory and anti-inflammatory cytokines can influence neoplastic outcome (19 JAFC T1). The VEGF-overexpressing transgenic mice that underwent oral 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

administration of bLF at 300 mg/kg b.w. three times a week for 1.5 -months showed a significant reduction in the serum VEGF (30 ng/mL), TNF-α (466 ng/mL), IL-4 (410 ng/mL), IL-6 (377 ng/mL), IL-10 (696 ng/mL) and IL-12 (767 ng/mL) levels when compared with the Tg/Placebo group, which showed showing values of 332 ng/mL (P < 0.01), 643 ng/mL (P < 0.01), 812 ng/mL (P < 0.01), 2651 ng/mL (P < 0.01), 2007 ng/mL (P < 0.01) and 987 ng/mL (P < 0.05), respectively. These results showed

demonstrate that oral administration of bLF at 300 mg/kg could decreased both

pro-inflammatory and anti-pro-inflammatory cytokines, which that may balance

proinflammatory and anti-inflammatorythe cytokines and lead to restrictions , and thus

lead to restrict in the growth of the lung cancer.

Discussion

In the initial stages, the cancer cells will keep proliferatingon and thus lead experience deficiencies to the deficiency in both the nutritionnutrients and oxygen, rendering a large amount of cells to die.leading to massive cell death. When the cancer cells secret a large amount of VEGF-A165, vasculoar genesis will be induced so as to provide sufficient nutrition nutrients and oxygen to the tumor, therefore increasing the tumor 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

growth speed [21]. In this study, we demonstrateed that bLF slowed the proliferation

of A549 human lung cancer cells by , A549, through decreasing the expression of vegf

mRNA and VEGF protein and this effectin a is dose-dependent manners (Figure 1).

As Because bLF exhibited no cytotoxicity on normal human bronchial epithelial cells

under the same conditions, bLF warrants further development as an anti-angiogenic agent for in the treatment of lung cancer.

Additionally, we used aan hVEGF-A165- overexpressed overexpressing

transgenic mice model which with the constructed mccsp-Vegf-A165-sv40 poly(A)

transgene for to researching examine a lung tumors. to further examine. We found that oral administration of bLF at 300 mg/kg b.w. three times a week for 1.5 -months to ,

10.5-month-old hVEGF-A165-overexpressing transgenic mice showed led to a dramatic decrease in solid tumor formation when compared with untreated transgenic mice. Histological analysis showed that treatment with 300 mg/kg of bLF reduced lung tumor formation and inflammation in hVEGF-A165-overexpressing transgenic mice (Figure 3). In additionAdditionally, immunohistochemical analysis showed that oral administration of bLF could reduce VEGF overexpessionoverexpression in clara Clara

cells of the lung tissues, which is are related to vasculogenesis and angiogenesis. These processes are and is a vital factors in the formation of tumors as well asand in tumor 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

growth, invasion, and metastasis. bLF, an angiogenesis inhibitor, exhibits a considerable potential in the treatment of lung cancer because angiogenesis is necessary for the growth of tumor beyond a few millimeters in diameter. Without

angiogenesis, the tumor cannot because of the need of tumor for the supplement of

access increased levels of oxygen and nutrients or remove waste., as well as waste

removal. Moreover, Norrby et al. [22] found that bLF inhibited carcinogenesis through

by mediating VEGF-A, which regulates angiogenesis mediated by VEGF-A. Yoo et al.

[23] showed that bLF inhibiting inhibition of lung metastaseis from the B16-BL6 melanoma and L5178Y-ML25 lymphoma cell lines has been considered attributed due to the anti-angiogenic properties of bLFesis. Li et al. [6] showed that bLF binds immunoglobulin (CD79A)-binding protein 1 (IGBP1), and that the binding complex interacted with the catalytic subunit of protein phosphatase 2A (PP2A), thus reducing the phosphatase activity of PP2A and triggering apoptosis. Kuhara et al. [24] reported that orally administered bLF exhibited antitumor activities by producing yi through production of interferon (IFN)-gamma and interleukin (IL)-18 in the intestinal mucosa. Shimamura et al. [25] suggested that bLF participates as a regulator of angiogenesis, possibly explained by blocking endothelial function and inducing IL-18 production.

and thus itsThe antitumor activity of bLF may be partly mediated by angiogenesis

inhibition. Lee et al. [26] reported that the molecular mechanism of the anti

-1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

proliferative effects of bLFf in Jurkat leukemia T cells is mediated through the

modulation of the JNK- associated Bcl-2 signaling pathway. Xu et al. [12] suggested that bLF inhibits Akt activation and modulates its the phosphorylation of downstream proteins phosphorylation involved in apoptosis of in SGC-7901 human stomach cancer cells. Wang et al. [15] pointed outdemonstrated that lactoferrin could inhibit the expression of VEGF and, bFGF mRNA and protein in Tca8113 cells, and theseis effects might be comprise one of the mechanisms for the anticancer functions of lactoferrin. Spadaro et al. [27] showed that the inhibition of distant mammary tumors by oral bhLF treatment seems to be mediated by an IFN-gamma-dependent enhancement of CD8(+) T- and NKT cell activitiesy initiated within the intestinal

mucosa.

Our previous studies pointed outdemonstrated that the serum of hVEGF-A165

overexpressed overexpressing transgenic mice contains proinflammatory cytokines,

such as VEGF-A, TNF-α and IL-12,; anti-inflammatory cytokines, such as IL-4 and IL-10, and cytokines that are; both proinflammatory and anti-inflammatory, cytokine such as IL-6 (Figure 5). These proinflammatory cytokines could be inhibited by bLF, which might be regulated by NF-κB signaling pathway. In additionAdditionally, treatment with bLF reduces the levels ofse these cytokines, resulting to result in 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

limited inflammation and restricted lung cancer the growth of lung cancer. There This may be a potential use for exploiting the anti-lung cancer activity of bLF for

therapeutic purposes.

Conclusion

In this study, we found that bLF could inhibit the expression of vegf mRNA and VEGF protein, which regulated angiogenesis. Norrby [28] showed that bLF suppression ofes angiogenesis is mediated by VEGF-A. Shimamura et al. [29] and Norrby et al. [22] also pointed outdemonstrated that bLF inhibits tumor-induced angiogenesis as well asand tumor metastasis in mice [23]. These effects might be comprise one of the mechanisms for the anticancer functions of bLF. bLF is a multifunctional glycoprotein due to its anti-inflammatory and anti-cancer properties, and it may have and thus result in therapeutical potential. Further study is under way to fully evaluate the fully mechanisms of bLF effects on tumorigenesis and to examine the potential for use of

bLF as a therapeutic agent.

Methods

Cell lines and cell culture

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Human lung adenocarcinoma cell lines, A549 and CL1-0 cells, and human bronchial epithelial cells, Beas 2B cells, were cultured in Dulbecco’s modified Eeagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells were incubated in a 5% CO2 incubator at 3737o_{C°C [30].}

Cell viability on tumor cells by MTT assay

To measure the cytotoxicity of bLF oin cell proliferation, the A549, CL1-0 and Beas 2B cells (2 × 105 _{cells/well) were seeded into a 96-well plate in triplicate and} pre-incubated for 3 h to allowfor cell adherence. First, 200 μL of fresh medium containing various concentrations (4.5, 2.25, 1.125, 0.5625 and 0.28125 mg/well) of bLF were was added into the cultures and incubated at 3737o_{C°C for 48 h under humidified air}

containing 5% CO2. Following the removal of the medium from the wells, 100 μL of tetrazolium salt solutions (1 mL MTT in 10 mL DMEM) were was added. After 4 h of incubation at 3737o_{C°C, the medium was removed and 100 μL of DMSO were was}

added to dissolve the formazan crystals. Absorbance was measured in an enzyme-linked immunosorbent assay (ELISA) reader at 570 nm [31]. The cell viability ratio (%) was calculated from the following equation: % viability = (absorbance of test sample/absorbance of control) x 100.

Growth curve and doubling time

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The A549 cell line was used to generate a growth curve using a 24-well microtiter plates. A seeding density of 1500 cells in a 1 mL volume of DMEM supplemented with 10% FBS was used per well. Growing cultures were trypsinized at days 1, 4, 7, 8, 9 and 10, and the number of live cells per well was determined in triplicate. The aAverage cell count at each time point was then plotted against time to generate a growth curve using Excel program [32]. The doubling time (in hours) was calculated as = h × ln (2)/ln (c2/c1).

RT-PCR analysis

A549 cells were seeded in a 10 cm dish at 5 × 106 _{cells/well for 2 days. After 2 days,} 10 mL of DMEM supplemented with 10% FBS or with 0.1% FBS and containing various concentrations (100, 50, 25, 12.5 and 6.25 mg/10-cm dish) of bLF were was added into the cultures and incubated at 3737o_{C°C for 48 h under humidified air}

containing 5% CO2. After three washeings with ice-cold PBS, the cells were harvested,, and total RNA was extracted with use of TRIzol reagent (Invitrogen), as specified by the manufacturer. Total RNA (2 μg) was resuspended in 9 μL of diethylpyrocarbonate (DEPC)-treated water, and the first strand of cDNA was synthesized with random primers and using ImProm-IITM _{reverse transcriptase in a} total volume of 20 μL [33]. The reaction was carried out at 42°C for 1 hour. For further 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

PCR amplification, an aliquot (1:10) of the RT product was adjusted to contain 0.1 μg of each primer, and additional buffer was added to a total volume of 20 μL. RT-PCR was performed in a Thermal Cycler 2720 [34]. Oligonucleotide primers for vegf were

5’-CAGAAGCAGAATGTGACCATC- 3’ (sense) and

5’-CTTCTGGTCGATGTCATGAGC-3’ (antisense). The cDNA sequence of β-actin as an internal control was also amplified using the following primers:

5’-CCGTCTTCCCCTCCATCGTGGG-3’ (sense) and

5’-CGCAGCTCATTGTAGAAGGTGTGG-3’ (antisense). The amplified PCR products were analyzed with 2% agarose gel electrophoresis and visualized with ethidium bromide staining [35].

Western blotting

The Ccells were homogenized in 300 μL of RIPA buffer (5 mM Tris–HCl pH 7.4, 0.15 M NaCl, 1% NP-40, 0.25% sSodium deoxycholate, 5 mM EDTA, and 1 mM ethylene glycol-bis(2-aminoethyl-ether)-N, N, N, N-tetra-acetic acid). The homogenates were centrifuged at 12,000g for 30 minutes at 4°C. Protein (40 μg) was then separated by SDS-PAGE in 10% polyacrylamide and electrotransferred to polyvinylidene difluoride membranes [36]. The membranes were incubated in blocking solution (5% BSA) at room temperature for 2 hours. The membranes were then incubated with primary 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

antibody (VEGF-A and GAPDH) overnight at 4°C. After washing, the membranes were incubated with a goat anti-rabbit IgG peroxidase-conjugated secondary antibody directed against the primary antibody. The membranes were developed by using an enhanced chemiluminescence western blot detection system [37].

Transgenic mouse production and validation

The mccsp-Vegf-A165-sv40 transgenic mice were generated by pronuclear microinjection [35]. To detect the hVEGF-A165 transgene in the transgenic mice with a homozygous (hVEGF-A165+/+_{) or heterozygous (hVEGF-A165}+/−_{) genotype, the mice} were rapidly screened for the foreign gene by PCR analysis of tail genomic DNA with the primer set VEGF94(+): 5’-AAGGAGGAGGGCAGAATCATC-3’ and

VEGF315(−): 5’-GAGGTTTGATCC GCATAATCTG-3’.

Animals

The transgenic mice were given a standard laboratory diet and distilled water ad

libitum and kept on a 12-hour light/dark cycle at 22 ± 2°C. This study was conducted

according to institutional guidelines and approved by the Institutional Animal Care and Utilization Committee of National Chung-Hsing, Taiwan (IACUC-98-3). The transgenic mice with the heterozygous (hVEGF-A165+/−_{) genotype were randomly} assigned to two groups for treatment: (n = 6): Tg/Placebo (transgenic mice treated with 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

placebo) and Tg/bLF (transgenic mice treated with bLF) groups. After oral administration of bLF at 300 mg/kg b.w. three times a week, . Mmice were sacrificed when 10.5 months old, following 1.5 months of bLF administration. Lung tissues were collected for pathological histology, immunohistochemistry staining as well asand extracting RNA and protein extractions.

Pathological histology

Lung tissue was fixed in 10% buffered formaldehyde (pH 7.0), embedded with paraffin, sectioned into 3 μm sections, and examined using hematoxylin and eosin

(H&E) staining [38].

Immunohistochemistry staining

Formaldehyde-fixed and paraffin-embedded sections were cut to a thickness of 5 μm,

departed and rehydrated through a gradient of alcohols to water, and then treated with boiling water for 15 min. The sections were incubated in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity and then incubated overnight at 4°C with primary rabbit monoclonal antibody against hVEGF-A usinga1:40 working dilution. For antigen retrieval, the sections were immunostained with the VECTASTAIN® ABC kit (UNIVERSAL, VECTOR, USA) in accordance with the manufacturer’s 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

specifications. Diaminobenzidine (DAB) was used for staining development, and the sections were counterstained with hematoxylin [39]. The negative control consisted of substituting normal serum for the primary antibody.

Real-time RT-PCR

Real-time RT-PCR was performed using SYBR Green in a Rotor-GeneTM _{6000. To} evaluate gene expression, real-time RT-PCR was performed on 3 genes (vegf, IL-6 and

TNF-α) using cDNA from lung tissue. The cDNA of β-actin was used as an internal control [40].

ELISA assay for cytokines

Blood samples were centrifuged at 1400g at 4°C for 15 min, and the VEGF, TNF-α, IL-4, IL-6, IL-10 and IL-12 in serum supernatants were determined using a commercial kit fromPeproTech, Inc. [32].

Statistical analysis

Experimental values are expressed as the mean ± standard error (SE) or mean ± standard deviations (SD). All data were analyzed using the t-tests. Statistical significances are presented as P < 0.05 (*) or P < 0.01 (**).

Acknowledgements

None of the authors declare any conflicts of interest. This research was supported in part by grant NSC-98-2313-B-005-012 from the National Science Council, grant COA-97-6.2.1-U1(9) from the Council of Agriculture, and the Ministry of Education, Taiwan, Republic of China, under the ATU plan.

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Figure legends

Figure 1 (a) Effects of bLF over a range of concentrations (0.28125, 0.5625, 1.125,

2.25 and 4.5 mg/well) on the cell viability of A549, CL1-0 and Beas 2B cells after 24 h incubation. Different letters are significantly different at the level of P < 0.05 according to the t-test. (b) A549 cells were treated without (A) or with (B: 4.5, C: 2.25, D: 1.125, E: 0.5625 and F: 0.28125 mg/well) bLF for 48 h. The cells were examined and photographed (× 100) under phase-contrast microscopy. (c) On culturing, A549 cells treated with or without bLF (3 mg/well) in 24-well microtiter plates for various time points (1, 4, 7, 8, 9 and 10 days) and were cultured, and plotting the total number

of cells was plotted against time.

Figure 2 The expression of vegf mRNA and VEGF protein treated with bLF in A549 cells treated with bLF after 48 h incubation. (a) The expression of vegf mRNA treated with or without various doses (100, 50, 25, 12.5 and 6.25 mg per 10-cm dish) of bLF. Different letters are significantly different at the level of P < 0.05 according to the t-test. (b) The expression of VEGF protein treated with or without various doses (100, 50, 25, 12.5 and 6.25 mg per 10-cm dish) of bLF as measured by using western blotting. C. A549 cells were cultured in DMEM supplemented with 10% FBS or 0.1% 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

FBS in the normal or hypoxica conditions (95 % N2 and 5% CO2). * _{10% FBS in the} hypoxia condition (95 % N2 and 5% CO2). Different letters are significantly different at

the level of P < 0.05 according to the t-test.

Figure 3 The exterior, histopathological slides and immunohistochemical analysis of the lung tissues of 10.5-month-old hVEGF-A165-overexpressing transgenic mice in the (a) Wild- type mice treated with PBS alone, (b) tTransgenic mice treated with PBS alone and (c) tTransgenic mice treated with bLF at 300 mg/kg b.w. three times a week for 1.5 -months.

Figure 4 (a) Real time-PCR validations of mRNA expression levels of vegf, IL-6 and

TNF-α in the lung tissues of the Tg/Placebo group and the Tg/bLF group. β-actin was used as an internal control. Mean ± SEM (n = 6). * P < 0.05 vs. the Tg/Placebo group.

(b) The expression of VEGF pProtein in the lung tissues of the Tg/Placebo group and

the Tg/bLF group as measured by using western blotting. GADPH was used an internal control.

Figure 5 The Ccytokine levels (VEGF, TNF-α, IL-4, IL-6, IL-10 and IL-12) in the serum of 10.5-month-old hVEGF-A165-overexpressing transgenic mice treated with or without bLF at 300 mg/kg b.w. three times a week for 1.5-month1.5 months.

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