Radiation-induced VEGF-C expression and endothelial cell proliferation in lung cancer

Background The present study was undertaken to investigate whether radiation induces the expression of vascular endothelial growth factor C (VEGF-C) through activation of the PI3K/Akt/mTOR pathway,subsequently affecting endothelial cells. Materials and methods Radiotherapy-induced tumor micro-lymphatic vessel density (MLVD) was determined in a lung cancer xenograft model established in SCID mice. The protein expression and phosphorylation of members of the PI3K/Akt/mTOR pathway and VEGF-C secretion and mRNA expression in irradiated lung cancer cells were assessed by Western blot analysis, enzyme-linked immunosorbent assays (ELISAs), and reverse transcriptase–polymerase chain reaction (RT-PCR). Moreover, specific chemical inhibitors were used to evaluate the role of the PI3K/Akt/mTOR signaling pathway. Conditioned medium (CM) from irradiated control-siRNA or VEGF-C-siRNA-expressing A549 cells was used to evaluate the proliferation of endothelial cells by the MTT assay. Results Radiation increased VEGF-C expression in a dose-dependent manner over time at the protein but not at the mRNA level. Radiation also up-regulated the phosphorylation of Akt, mTOR, 4EBP, and eIF4E, but not of p70S6K. Radiation-induced VEGF-C expression was down-regulated by LY294002 and rapamycin (both p < 0.05). Furthermore, CM from irradiated A549 cells enhanced human umbilical vein endothelial cell (HUVEC) and lymphatic endothelial cell (LEC) proliferation, which was not observed with CM from irradiated VEGF-C-siRNA-expressing A549 cells. Conclusions Radiation-induced activation of the PI3K/Akt/mTOR signaling pathway increases VEGF-C expression in lung cancer cells, thereby promoting endothelial cell proliferation.


Introduction
About 1.2 million new cases of lung can cer occur annually worldwide. Approxi mately 35 % of patients with nonsmall cell lung cancer (NSCLC) are diagnosed with stage III disease [1]. Of the stage III subgroup, only about 20 % of patients can be treated with surgery; the majori ty of patients may choose radiotherapy (RT) for local treatment, and metaanal ysis indicates that combined chemother apy and radiotherapy (CCRT) has be come the standard of care for this group of patients, with good outcomes [2]. Spe cifically, among patients who under went CCRT with a standard dose of ra diation (> 60 Gy), the complete remission rate ranged from 10 to 60 %, with a 5year survival rate of 5-23 % [3]. Patients sur viving for 6-12 months had a statistically significantly increased survival rate when the intrathoracic tumor was controlled. Patients treated with 50-60 Gy showing tumor control had a 3year survival rate of 22 %, compared with 10 % for patients with intrathoracic failure [4], suggesting that local intrathoracic control may be predictive of patient survival. Therefore, it is important to enhance the radiosensi tivity of lung cancer cells, which may af fect local control and subsequent surviv al. In this study, we attempted to analyze the environmental changes after RT that increase the likelihood of the resistance of cancer cells to RT. Blood and lymphatic vascular system development and maintenance are cou pled through multiple growth factors and receptors expressed on blood and lym phatic endothelial cells (BECs and LECs, respectively). Vascular endothelial growth factors A and C (VEGFA and VEGFC, respectively) are two major growth fac tors that promote endothelial cell prolif eration, migration, and induction of per meability. VEGFC binding to VEGF re ceptor 3 (VEGFR3) induces LEC prolifer ation and lymphatic vascular hyperplasia [5,6]. VEGFC can also bind to VEGFR2, eliciting a response that is similar to that induced by VEGFA but less potent [7].
The expression of VEGFC and VEG FR3 in lung cancer tumor cells was sig nificantly associated with more advanced regional lymph node metastasis [8][9][10]. Previous studies also demonstrated that radiation not only induced angiogenesis but also caused lymphangiogenesis [11]. For example, Nathanson et al. reported that subtherapeutic hyperthermia or ra diation increased lymphatic flow from tumors, which was associated with an in crease in metastasis of melanoma [12]. Talmadge et al. also showed that sublethal dose irradiation activated genes or signal pathways that induced functional chang es that increased the survival or metastasis of cancer cells [13]. Moreover, radiation of tumor cells induced vascular or micro environment changes that enhanced cap illary invasion capacity [14,15]. In the present study, we investigated the mech anism and consequences of irradiation induced VEGFC expression in lung can cer cells.

Lung cancer cells
The human lung cell lines A549 and H1299 were obtained from the Ameri can Type Culture Collection (Manassas, Va.) and were maintained in Dulbecco's modified Eagle medium (DMEM) or RP MI supplemented with nonessential ami no acids, lglutamine, a twofold vitamin solution, sodium pyruvate, 10 % fetal bo vine serum, penicillin, and streptomycin. Cells were cultured at 37 °C in a humidi fied atmosphere of 5 % CO 2 and 95 % air.

Establishment of lung cancer xenografts and irradiation
Male SCID mice (5-6 weeks old) were ob tained from the National Taiwan Univer sity Animal Center, Taipei, Taiwan. To es tablish the xenografts, 1 × 10 6 A549 cells were injected subcutaneously into the right hind limb. At 10 days postimplanta tion, mice were immobilized in a custom ized harness that exposed the right hind leg. The remainder of the body was shield ed by five times the halfvalue thickness of lead. A cobalt60 unit was used to irradi ate the primary tumor with 25 or 50 Gy (5 Gy or 10Gy daily fractions, at a dose rate of 1 Gy/min).
On postimplantation day 9, tumor vol umes were calculated every 3 days using a standard formula: width 2 × length/2. Tu mors were harvested at the time of sac rifice 14 days after completion of the ra diotherapy and fixed in 10 % neutral buff ered formalin and processed for immu nohistochemical evaluations. All mice were grouphoused under a fixed lightdark cycle (12 h of light and 12 h of dark ness) with ad libitum access to sterilized food and water. All experimental proce dures were performed in accordance with protocols approved by the Committee of Experimental Animal Management at the College of Medicine, National Taiwan University.

Immunohistochemistry analysis
All tissues were fixed in 10 % neutral buff ered formalin and embedded in paraffin using standard protocols. Immunohisto chemistry (IHC) analysis was performed on a single representative block from each case. Tissue sections (5 μm) were dewaxed, and antigen retrieval was per formed in a citrate buffer (pH 6) using an electric pressure cooker set at 120 °C for 5 min as previously described (Choi et al. 2005). Sections were incubated for 5 min in 3 % hydrogen peroxide to quench en dogenous tissue peroxidase. The sections were incubated in antibodies specific for the lymphatic endothelial marker, D240 antibodies (1:200 dilution; Biocompare, San Francisco, Calif.), for 60 min at room temperature. After washing unbound primary antibody, sections were treated with biotinylated secondary antibodies followed by avidin coupled to biotinylat ed HRP at room temperature according to the manufacturer's instructions (DA KO, Carpinteria, Calif.). Immunohisto chemical reactions were developed with diaminobenzidine as the chromogen ic peroxidase substrate. Sections were counterstained with hematoxylin. Speci ficity was verified by negative control re actions that did not contain primary anti bodies as well as by appropriate cytoplas mic reactions for each antigen in positive control tissues.

Microlymphatic vessel density assessment
Immunohistochemical reactions for D2 40 were analyzed at low magnification (× 40), and lymphatic vessels were count ed in five representative highmagnifica tion (× 400; 0.152 mm 2 ; 0.44mm diam eter) fields. Single immunoreactive lym phatic endothelial cells, or lymphatic en dothelial cell clusters separate from oth er microlymphatic vessels, were counted as individual microlymphatic vessels. The mean visual microlymphatic vessel densi ty (MLVD) for D240 was calculated.

Culture of HUVECs and LECs
Human umbilical vein endothelial cells (HUVECs) were purchased from the Food Industry Research and Develop ment Institute and cultured in M199 me dium (Life Technologies, Carlsbad, Ca lif.) supplemented with 20 % fetal bovine serum, endothelial cell growth supple ment (Millipore, Billerica, Mass.), hepa rin, lglutamine, penicillin, and strepto mycin. Human lymphatic endothelial cell (LECs) were isolated from human lymph nodes (ScienCell, Carlsbad, Calif.) and cultured in endothelial cell medium (Sci enCell). The cells were cultured at 37 °C in a humidified atmosphere of 5 % CO 2 and 95 % air.

Irradiation protocol
Cell culture plates were irradiated with different doses of radiation (0-10 Gy) us ing a cobalt60 unit. The source-skin dis tance technique was set at 80 cm at the bottom of the flask. The dose rate was 1 Gy/min. Dosimetry was performed with an ionization chamber.
Enzyme-linked immunosorbent assay to detect secreted VEGF-C Cell culture supernatant was collected af ter culturing A549 cells in completed cul ture medium for 24 h after irradiation. VEGFC secretion was measured in the cell culture supernatant using a commer cial enzymelinked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, Minn.) according to the manufacturer's protocol. Color intensity was measured by a plate reader at 450 nm. Data repre sent the average of three different assays.

Reverse transcriptionpolymerase chain reaction
Reverse transcription (RT) of RNA was performed in a final reaction volume of 20 µl containing 5 µg of total RNA in Mo loney murine leukemia virus (MMLV) re verse transcriptase buffer that contained 10 mM dithiothreitol, dNTPs (2.5 mM each), 1 mM (dT) primer, and 200 U of MMLV reverse transcriptase (Prome ga, Madison, Wis.). The reaction mix ture was incubated at 37 °C for 2 h, and was terminated by heating at 70 °C for 10 min. A portion of the reaction mix ture was then amplified by PCR with the following primer pairs: VEGFC, sense 5′CAGTTACGGTCTGTGTCCAGT GTAG3′ and antisense 5′GGACA CACATGGAGGTTTAA3′; βactin, sense 5′ ATCCGCAAAGACCTG TACGC3′ and antisense 5′TGTGTG GACTTGGGAGAGGA3′. A total of 30 cycles were performed. The prod ucts were separated on 2 % agarose gels, stained with 1 mg/ml ethidium bromide, and visualized using a UVP GDS7900 digital imaging system. The results were confirmed by conducting at least three in dependent experiments.

Transfection of small interfering RNA
Small interfering RNA (siRNA) duplex es specific for VEGFC or Akt were pur chased from Santa Cruz Biotechnology (Santa Cruz, Calif.); control siRNAs with sequences that do not target any gene product were obtained from Invitrogen (Carlsbad, Calif.). Cells were transfect ed with 25 nM VEGFC siRNA, Akt siR NA, or control siRNA in serumfree Op tiMEM using the oligofectamine method (Invitrogen) for 1 h at 37 °C. After chang ing the culture medium, cells were cul tured for 24 h at 37 °C prior to further ex periments.

Conditioned media collection
A549 (1 × 10 5 cells) cells expressing con trol or VEGFC siRNA were cultured in a 6cm dish overnight, washed two times with PBS, and then incubated in medi um before exposure to 5Gy irradiation.

Radiation-induced VEGF-C expression and endothelial cell proliferation in lung cancer
Abstract Background. The present study was undertaken to investigate whether radiation induces the expression of vascular endothelial growth factor C (VEGF-C) through activation of the PI3K/Akt/mTOR pathway,subsequently affecting endothelial cells. Materials and methods. Radiotherapy-induced tumor micro-lymphatic vessel density (MLVD) was determined in a lung cancer xenograft model established in SCID mice. The protein expression and phosphorylation of members of the PI3K/Akt/mTOR pathway and VEGF-C secretion and mRNA expression in irradiated lung cancer cells were assessed by Western blot analysis, enzyme-linked immunosorbent assays (ELISAs), and reverse tran-scriptase-polymerase chain reaction (RT-PCR). Moreover, specific chemical inhibitors were used to evaluate the role of the PI3K/ Akt/mTOR signaling pathway. Conditioned medium (CM) from irradiated control-siRNA or VEGF-C-siRNA-expressing A549 cells was used to evaluate the proliferation of endothelial cells by the MTT assay. Results. Radiation increased VEGF-C expression in a dose-dependent manner over time at the protein but not at the mRNA level. Radiation also up-regulated the phosphorylation of Akt, mTOR, 4EBP, and eIF4E, but not of p70S6K. Radiation-induced VEGF-C expression was down-regulated by LY294002 and rapamycin (both p < 0.05). Furthermore, CM from irradiated A549 cells enhanced human umbilical vein endothelial cell (HUVEC) and lymphatic endothelial cell (LEC) proliferation, which was not observed with CM from irradiated VEGF-C-siRNA-expressing A549 cells. Conclusions. Radiation-induced activation of the PI3K/Akt/mTOR signaling pathway increases VEGF-C expression in lung cancer cells, thereby promoting endothelial cell proliferation.

Schlüsselwörter
Strahlung · Vaskulärer endothelialer Wachstumsfaktor C · Lungenkrebszellen · Endothelzellen · PI3K/Akt/mTOR-Signalweg Cell proliferation assay Cells were cultured in 96well plates at a density of 5,000 cells/well. The quanti ty of viable cells was estimated by a col orimetric assay using 3(4,5dimethylthi azol2yl)2,5diphenyltetrazolium bro mide (MTT). MTT (10 µl of 5 mg/ml so lution, Sigma, Germany) was added to each well and incubated for 4 h at 37 °C. The cells were then treated with 40 µl/well dimethyl sulfoxide (DMSO) and incubat ed for 60 min at 37 °C. The absorbance of each well was determined in an ELI SA plate reader using an activation wave length of 570 nm and a reference wave length of 630 nm. The percentage of vi able cells was determined by comparison with untreated control cells.

Statistical analysis
All data presented are the mean ± stan dard deviation of experiments repeated three or more times. A paired ttest was used to evaluate statistically significant differences between indicated groups in specified tests; p values < 0.05 were con sidered statistically significant.

Results
Sublethal RT dose significantly increased tumor MLVD in SCID mice bearing ectopic lung cancer xenografts As shown in . Fig. 1a, 5 fractions of 10 Gy significantly suppressed the tu mor growth, and 5 fractions of 5 Gy had a mild therapeutic effect. Analysis of tu mor MLVD by examining the expression of the lymphatic endothelium vesselspe cific marker D240 revealed stronger ex pression in the tumors irradiated with 5 fractions of 5 Gy (. Fig. 1b). Howev er, in tumors irradiated with 5 fractions of 10 Gy, severe tissue necrosis was ob served (. Fig. 1b). As shown in . Fig. 1c, the MLVD was significantly higher in tu mors irradiated with 5 fractions of 5 Gy. However, no MLVD was found in severe tissue necrosis of tumors irradiated with 5 fractions of 10 Gy.

Radiation induced a dose-dependent increase in VEGF-C expression by lung cancer cells
Due to the proteolytic processing of VEGFC in cancer cells, a 58kDa VEGF C propeptide as well as 31 and 29kDa secreted VEGFC peptide levels were evaluated by Western blot analysis [16]. As compared with the nonirradiated cells, those treated with 5Gy irradiation for 24 h had increased VEGFC expres sion in A549 cells (. Fig. 2a). In A549 and H1299 lung cancer cells, radiation in creased VEGFC expression in a dosede pendent manner (. Fig. 2b). At 24 h after exposure to radiation, VEGFC expres sion increased 1.75, 1.98, and 2.01fold with 2.5, 5, and 10 Gy, respectively, rela tive to the nonirradiated group in A549 cells (p < 0.05 for cells treated with 2 and 5 Gy ; . Fig. 2c). In addition, the effects of 5Gy irradiation on VEGFC expression increased over time relative to the non irradiated control cells (. Fig. 2d and  e). Moreover, assessment of the soluble VEGFC peptides by ELISA and West ern blot analysis revealed their upregu lation after radiation treatment over time (. Fig. 2f). The effects of radiation on the PI3K/Akt/ mTOR signaling pathway were deter mined by Western blot analysis. After 5Gy radiation, protein samples were col lected at 5, 15, 30, 60, and 120 min. Radi ationinduced Akt phosphorylation was significantly increased by 1.2fold from the control at 15 min (p < 0.01; . Fig. 3a).
To confirm the relationship between PI3K/Akt and mTOR as well as the role of Akt in irradiationinduced VEGF C expression, we manipulated Akt ex pression using Akt siRNA. As shown in . Fig. 4b, Akt phosphorylation was aug mented by irradiation and blocked by Akt siRNA. Furthermore, irradiationinduced mTOR phosphorylation and VEGF up regulation in A549 cells were inhibited by Akt siRNA (. Fig. 4b), indicating that PI3K/Akt/mTOR signaling mediates irra diationinduced VEGFC expression.
Analysis of VEGFC mRNA expres sion by RTPCR revealed no obvious changes in VEGFC mRNA expression between the sham and irradiated groups (. Fig. 4c), indicating that the effects of irradiation on VEGFC expression were at the translational level, but not the tran scriptional level.

Irradiation-induced VEGF-C expression promotes the proliferation of HUVECs and LECs
The effect of irradiationinduced VEGF C expression on HUVEC and LEC prolif eration was assessed using siRNA specif ic for VEGFC. The inhibition of VEGF C siRNA on the VEGFC levels in the cell culture supernatant was first confirmed by ELISA. As shown in . Fig. 5a, VEGF C, but not control siRNA, significantly reduced the irradiationinduced VEGF C expression in A549 cells.

| Strahlentherapie und
The effect of CM on HUVEC and LEC proliferation was next determined using the MTT assay. As shown in . Fig. 5b, HUVEC proliferation was significantly increased by 1.30fold after culturing HU VECs in CM from irradiated A549 cells as compared with that from nonirradiated A549 cells (p < 0.01). This effect was sig nificantly inhibited when HUVECs were cultured in CM from VEGFC siRNAex  Fig. 5b).
The CM from irradiated A549 cells also increased LEC proliferation by 1.28fold, which was significantly suppressed when LECs were cultured in CM derived from irradiated VEGFCsiRNAexpressing A549 cells (p = 0.03; . Fig. 5c).

Discussion
Tumor cells can produce multiple growth factors to create a microenvironment con ducive for BEC and LEC growth. Tumor cell progression and metastasis may ben efit from crosstalk between tumor cells, VECs, and LECs [17,18]. Radiation has antiangiogenic as well as antilymphogen ic effects by damaging cell membranes, DNA, and endothelial cells, resulting in apoptosis. However, radiation also has proangiogenic and prolymphogenic ef fects either through direct mechanisms or through signals that are released by irra diated cancer cells and the tumor micro environment. Recent studies also demon strated a close association between radio sensitivity, proangiogenic growth factors, and endothelial cell survival; VEGFR in hibitors enhance the therapeutic efficacy of irradiation in NSCLC by hindering the repair of sublethal radiation damage [19].
In the current study, radiation in creased VEGFC expression in a dosede pendent manner over time. Furthermore, CM from irradiated A549 cells enhanced proliferation of HUVECs and LECs, which was ameliorated in irradiated A549 cells expressing VEGFC siRNA, suggest ing that VEGFC may play an important role in the angiogenic or lymphogenic ef fect of radiation in lung cancer. These re sults were consistent with those of other studies, which reported that radiation can both stimulate and inhibit angiogenesis through regulation of the proangiogenic and antiangiogenic balance [20,21]. For example, Sonveaux et al. [20] found that irradiation stimulated migration and tu bulogenesis of HUVECs accompanied by upregulation of endothelial nitric oxide synthase (eNOS). In human tumor sam ples, the clinical relevance of irradiation induced angiogenic growth factor expres sion has been observed [21]. For example, in a study of rectal cancer patients receiv ing neoadjuvant RT followed by surgery, a significant increase in VEGF expression was observed in postradiation specimens compared with primary tumor samples [21]. Therefore, radiation might induce a defense mechanism against radiation damage through the activation of a poten tial proangiogenic reaction. Most studies demonstrated radiationinduced VEGF A expression but the novel finding in our study is VEGFC overexpression after ir radiation in lung cancer cells.
Several studies have demonstrated that radiation can activate various signal ing pathways, including the PI3K, MAPK, JNK, and NFκB signaling pathways [22,23]. The PI3K/Akt/mTOR signaling path way is involved in tumorigenesis, transla tion of proteins for cell cycle progression, and lung cancer cell proliferation [24,25]. In addition to killing tumor cells, radia tion could induce radioprotective signal ing. Several studies analyzing the mecha nism of radioresistance have demonstrat ed that it may be associated with activation of the PI3K/Akt/mTOR pathway [26,27]. SunavalaDossabhoy et al. [26] observed that radiation increased the expression of a radioprotective protein, Tousledlike kinase 1B (TLK1B), which was preced ed by an increase in pAkt and p4EBP. Irradiation also induced phosphoryla tion of Akt and mTOR in a breast cancer cell line, which was attenuated by mTOR and PI3K inhibitors. These findings sug gest that the mTOR inhibitor attenuates radiationinduced Akt/mTOR prosur vival signaling and enhances the cyto toxic effects of radiation in breast cancer cells [27]. Moreover, Dionysopoulos et al. [28] demonstrated that clinicopathologi cal parameters as well as high mTOR and CCND1 mRNA expression were associat ed with poor patient outcome in localized laryngeal cancer. The present study fur ther supported that radiation can induce phosphorylation of Akt, mTOR, 4EBP, The conditioned media (CM) from irradiated A549 cells also promoted HUVEC and LEC proliferation, which was significantly suppressed when they were cultured in CM from irradiated A549 cells expressing VEGF-C-siRNA. Data are presented as mean ± SE, n = 3. (*p < 0.05) and eIF4E, indicating that the PI3K/Akt/ mTOR/eIF4E signaling pathway can be activated by irradiation. mTOR, p38, and JNK play important roles in the upregulation of VEGFC ex pression, and inhibition of mTOR by ra pamycin has an antilymphangiogenic ef fect, which inhibited lymphatic metasta sis [29]. Also, activation of the PI3k/Akt signaling pathway upregulated VEGFC expression [30]. These observations were supported by the present study, which found downregulation of radiationin duced VEGFC expression after inhibi tion of PI3K/AKT by LY294002, sup pression of mTOR signaling by rapamy cin, and expression of AKT siRNA. More over, our study observed that radiation induced VEGFC expression was at the translational level but not the transcrip tional level. The result is similar to the finding of Morfoisse et al. [31]. They dem onstrated that transcriptionindependent but translationdependent upregulation of VEGFC in hypoxia stimulates lym phangiogenesis in tumors.

Conclusion
In conclusion, in NSCLC cells, radiation induced VEGF-C expression at least in part through activation of the PI3K/Akt/mTOR pathway. In addition to the tumor cell itself, the irradiated supernatant promoted HUVEC and LEC proliferation, which was inhibited by VEGF-C-siRNA expression in cancer cells. The clinical implication of this study is the critical need to suppress radiation-enhanced microenvironment changes possibly with VEGF-C inhibition during treatment.