Immunopreventive Effects against Murine H 22 Hepatocellular Carcinoma in vivo by a DNA Vaccine Targeting a Gastrin-Releasing Peptide

Hepatocellular carcinoma (HCC) is reported to be the fifth most frequently diagnosed cancer in the world and the second leading cause of cancer related-death worldwide (Jemal et al., 2011). However, notwithstanding great advances, no systemic chemotherapeutic protocol have yet been successful in HCC treatment (Ozturk and Oter, 2011). Potential surgical or local ablative therapies ever applied are having poor prognosis (Ray, 2013). Therefore, there is a pressing need for novel strategies to impact development and recurrence of HCC. Great efforts are being made towards developing targeted therapies that may contribute in cancer control; in that respect construction of nucleic acid vaccines targeting hormone and/or growth factor have recently emerged as a promising approach (Luigi and Gennaro, 2011; Bele 2012). DNA vaccines have become an attractive approach in generating antigen-specific immunity. The naked plasmid DNA is safe, has low immunogenicity, and can be repeatedly administered. In addition, DNA vaccines can be easily prepared on large scale with high purity and are highly stable relative to proteins and other biological polymers (Donnelly et al., 1997). Extensive studies by many investigators established that angiogenesis has a central role in the invasion, growth and metastasis of


Introduction
Hepatocellular carcinoma (HCC) is reported to be the fifth most frequently diagnosed cancer in the world and the second leading cause of cancer related-death worldwide (Jemal et al., 2011).However, notwithstanding great advances, no systemic chemotherapeutic protocol have yet been successful in HCC treatment (Ozturk and Oter, 2011).Potential surgical or local ablative therapies ever applied are having poor prognosis (Ray, 2013).Therefore, there is a pressing need for novel strategies to impact development and recurrence of HCC.
Great efforts are being made towards developing targeted therapies that may contribute in cancer control; in that respect construction of nucleic acid vaccines targeting hormone and/or growth factor have recently emerged as a promising approach (Luigi and Gennaro, 2011;Bele 2012).DNA vaccines have become an attractive approach in generating antigen-specific immunity.The naked plasmid DNA is safe, has low immunogenicity, and can be repeatedly administered.In addition, DNA vaccines can be easily prepared on large scale with high purity and are highly stable relative to proteins and other biological polymers (Donnelly et al., 1997).Extensive studies by many investigators established that angiogenesis has a central role in the invasion, growth and metastasis of solid tumors.Thus, the inhibition of tumor growth by attacking the tumor vascular supply offers a primary target for anti-angiogenesic intervention (Matejuk et al., 2011;Zhu et al., 2013).Rinaldi et al. (2009) revealed a prominent preventive effect of dendritic cell-based tumor vaccines against HCC.Gastrin-releasing peptides (GRP) are neuroendocrine peptides that have been previously implicated as regulators of angiogenesis and therefore can be potential target for anti-angiogenic therapies.
Previous investigations of gene therapy approaches for liver cancer have been targeting endothelial cells (Greten et al., 2006;Butterfield, 2007).Our approach is based on a vaccine taking interest in both endothelial cell and tumor cells.In this work, we reported a preventive strategy based on application of a DNA vaccine targeting gastrinreleasing peptide.The efficacy of the anti-GRP DNA vaccine was tested on the murine H22 hepatocarcinoma model.In addition, the inhibition of tumor-induced angiogenesis induced by active immune responses was evaluated in an intradermal tumor model in vivo.
Plasmid DNA used for animal immunization were purified using Qiagen Plasmid Mega Kit (Qiagen) and suspended in sterile saline at a concentration of 0.5 µg/µl.

Mice and hepatocarcinoma tumor cell line
Male BALB/c mice (5 to 6 weeks old) were purchased from the Chinese Academy of medical Sciences (Beijing, China) and housed in pathogen-free conditions.All animal procedures were performed according to approved protocols and in accordance with recommendation for the proper use and care of laboratory animals.H22 cell line was obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Beijing, China).Tumor cells were transferred into the abdomen cavity of mice.The ascites were taken from the mouse in the super clean bench and then diluted with physiological saline to 1510 7 ml-1.A total of 2510 6 ml-1 H22 cells in 0.1 ml were subcutaneously inoculated at the right armpit of mouse to establish a solid tumor model.

Immunization procedure
Purified plasmids DNA were resuspended in sterile saline at a concentration of 0.5 mg/ml.A total of six BABL/c male syngeneic mice were used for each experimental group (one group for each of the three DNA vaccine and saline control).Mice were first anesthetized via i.m. injections in the anterior thigh with 100 µl of 0.25% bupivacaine hydrochloride solution.72 hours later, mice received i.m. injections of 100 µl each of the plasmid DNA vaccine or saline solution.Booster administrations were given every other week (week 3 and 5) using the same protocol.Sera were taken at various time points for later analysis of antibodies titers.

ELISA for serum anti-GRP antibodies
The formation of antibodies against GRP in immunized mice was tested using ELISA as described by Yankai et al. (2006).Briefly, 96-well flat-bottomed ELISA plates (Costar, USA) were coated with 100 µl/well of recombinant hVEGF-GRP18-27 proteins (2 µg/well) in 0.1 mM carbonate-bicarbonate buffer and kept overnight at 4 o C. Plates were blocked with PBS containing 5% (w/v) bovine serum albumin (BSA; Sigma, USA) for 1 h and then incubated at 37 o C for another hour with 100 µl/ well 1:100 dilution of serum collected from immunized animals in PBS containing 2% BSA.Thereafter, wells were washed three times by PBST (PBS containing 0.1% Tween 20), and then incubated 1 h at 37 o C with 100 µl/ well HRP-conjugated goat anti-mouse IgG (Sigma, USA) diluted 1:20,000 in PBS containing 1% BSA.Wells were washed intensively six times in PBST and followed by 20 min incubation at 37 o C with 100 µl/well of the peroxidase substrate 0.01% 3,3',5,5'-tetramethylbenzidine (TMB) and 0.24% (w/v) H 2 O 2 -urea solubilized in 0.2M Na 2 HPO 4 0.1M citrate buffer (pH 5.5).The reaction was stopped with 50 µl/well of 2M H 2 SO 4 .The ELISA plate was read with a standard ELISA reader at 450 nm.Each measurement was carried out in duplicate.To determine the antibody titer, sera collected at week 10 with different dilutions were tested using ELISA method.Pre-immune serum was used as negative control.Antibody titers was defined as the maximum serum dilution that manifested an ELISA optical density (OD) reading that was at least 5 standard deviations greater than the mean OD obtained in wells that contained pre-immune serum.

In vivo tumor protection experiments
In order to study the antitumor effect of the anti-GRP, mice (six per group) were immunized four times every 2 weeks followed by subcutaneous injections of 1510 6 H22 cells into the left flank.After 14 days, animals were sacrificed, and solid tumors were excised and weighed.

In vivo angiogenesis assay
As shown in Figure 3, a vaccination protocol was designed for each experimental group (four mice per group).To visualize the induction of angiogenesis by a tumor in vivo, an intradermal tumor model was used.In this model, neovasculature observed predominantly at the tumor periphery can be quantified by vessel counting (Kreisle and Ershler, 1988).At day 0, mice were anesthetized (inhalant 4% Halothane-vet), and 2.10 6 H22 viable cells in 100 ml of PBS were implanted intradermally at two sites in the abdominal region.Two additional injections of PBS (50 ml) were performed on each mouse as a control.All implant sites were marked with indelible ink to aid identification at the end of the experiment.When the intradermal tumor grew up to 5 mm in diameter, a section of the abdominal wall skin encompassing all implant sites was removed and spread onto filter paper.Sections were examined by light microscopy (510 magnification), and the total number of blood vessels (major vessels and branching points) was determined within a 1 cm 2 area around each implant site.Vessel counts from implants of PBS were consistent irrespective of treatment; the mean value was therefore subtracted from control and vaccines immunized vessel counts to give a more accurate indication of tumor-induced blood vessel formation and to ascertain the effect of treatment.

Statistical analysis
Data were expressed as means (±SD) and the differences between groups were analyzed by Student's t test.Findings were regarded as significant if P values were <0.05.

Induction of anti-GRP antibodies in immunized mice
An ELISA was performed to detect the GRP-specific immunoglobulin G (IgG) level in sera from immunized mice as described in section 2.4.Samples were collected on week 2, 4, 6, 8 and 10 after the initial immunization.TP-GRP6-M2 and TP-GRP6 DNA vaccines, compared with the control vaccine (TP-M2) or the saline control, greatly increased titers of specific anti-GRP antibodies 4 weeks post-inoculation, and the antibody levels remained high for up to 10 weeks post-inoculation (Figure 2).Among the two GRP-based vaccines, the anti-GRP antibody titers in mice immunized with the plasmid containing two copies of the mycobacterial HSP70407-426 gene (M) were highest at 10 weeks post immunization.
These data clearly demonstrated that two copies of HSP70407-426 gene were highly potent in augmenting humoral response and immunogenicity of the anti-GRP vaccine.

The anti-GRP vaccine suppressed the growth of subcutaneous H22 tumors
We determined whether vaccination with TP-GRP6-M2 could protect mice against hepatocarcinoma.H22 cells injected subcutaneously formed large solid tumors in nonimmunized mice (saline) or in mice injected   The total number of blood vessels (major vessels and branching points) was determined within the precise 1 cm 2 area around each implant site with a non-GRP-containing plasmid (TP-M2).The size and weights of tumors removed from all immunized mice on day 14 after tumor cell challenge of the three experimental groups were measured.The tumor sizes decreased progressively in mice immunized with the anti-GRP vaccine (TP-GRP6-M2) (Figure 3).The average weight of solid tumors in mice immunized with TP-GRP6-M2 was significantly lower than that for mice in the saline group (0.099±0.085 g vs 1.609±0.99g, p=0.03).However, tumor weight in mice injected with TP-GRP6 (p=0.466) or TP-M2 (p=0.828) was not significantly lower than that of the saline (Figure 3).A highly inhibition was achieved in TP-GRP6-M2 group.It was concluded that this DNA vaccine had strongly elicited anti-tumor immunity against the growth of H22.

Intradermal tumor angiogenesis
To assess the effect of the immune response on tumorassociated angiogenesis induced by anti-GRP DNA vaccine TP-GRP6-M2, H22 tumor cells were implanted intradermally at two sites in the abdominal region.Four groups of mice were immunized with saline, vaccine control TP-M2, TP-GRP6 or anti-GRP DNA vaccine TP-GRP6-M2 respectively, as described earlier.As shown in Figure 4, tumor cells implanted intradermally were found to induce significant angiogenesis when the intradermal tumor grows up to 5 mm in diameter.

Discussion
Cancer mortality is related to the spread of neoplastic cells to distant loci where the cells, supported by existing blood vessels and angiogenesis, proliferate and give rise to secondary tumors.Tumor angiogenesis is upregulated by a number of conditions including hypoxia, hypoglycemia, mechanical disruption, and genetic and inflammatory alterations (Rosen, 2009) that lead to activation of growth factors and pro-angiogenic genes (Gordan and Simon, 2007;Gruber and Simon, 2006).Angiogenesis has a critical role in physiologic and disease processes.For the growth of tumors, angiogenesis must occur to carry sufficient nutrients to the tumor.In addition to growth, development of new blood vessels is necessary for invasion and metastases of the tumor.A number of strategies have been developed to inhibit tumor angiogenesis and further understanding of the interplay between tumors and angiogenesis should allow new approaches and advances in angiogenic therapy.One such promising angiogenic approach is to target and inhibit angiogenesis with vaccines (Matejuk et al., 2011).Targeting the tumor vasculature, over targeting tumor cells, with vaccines as well as other immunotherapies may have several potential advantages namely a more accessibility to the immune system; the genetic stability and as a result the reduction of the risk of resistance to developing immunotherapies (Boehm et al., 1997;Huang et al., 2004) a more potent CD8+-mediated response favored by less frequency of down-regulation of MHC I in tumor endothelial cells; finally, since inhibition of a single endothelial cell can inhibit up to 100 tumor cells (Folkman, 1996;Folkman, 1997), immunotherapies directed toward tumor endothelial cells have the potential of an amplifying inhibitory effect.
Over the past 20 years, abundant evidence has been collected to suggest that GRP and its receptors play an important role in the development of a variety of cancers (Hohla and Schally, 2010).These observations have inspired researchers to find a suitable approach to treat cancers by taking advantage of blocking the GRP to GRPR signal pathway.GRP is a strong mitogen that augments the growth of several types of tumors including breast (Ni et al., 2012), prostate (Beer et al., 2012), lung (Egloff et al., 2012), anal cancer (Martins et al., 2013), and neuroblastomas (Pirtha et al., 2013, Lee et al., 2013).By activating its receptors (GRPR) located on both tumor and endothelial cells, GRP is also a direct angiogenic factor as shown by in vitro and in vivo angiogenic assays (Martinez et al., 2005).In addition, GRP is able to contribute indirectly to tumor angiogenesis by augmenting expression of pro-angiogenic factors such as IL-8 and VEGF (Kang et al., 2007;Lee et al., 2013).The GRP antagonist, 77427, markedly inhibits endothelial cell cord formation in vitro and inhibits angiogenesis in vivo (Levine et al., 2003).Several therapeutic strategies targeting GRP/GRPR, such as peptide antagonists and monoclonal antibodies, have demonstrated their antitumor efficacy in vitro and in vivo (Cornelio et al., 2007).Recently, vaccines targeting GRP displayed an effective anti-tumor response by inducing humoral and cell mediated immune responses.A recombinant chimeric protein vaccine (HG6) containing six tandem repeats of a GRP fragment (18 to 27 amino acids) fused to the 65-kDa heat shock protein (HSP65) was used to treat mice challenged with breast cancer (Wu et al., 2008).Results from this treatment revealed significant prophylactic and therapeutic activities in term of reduction of tumor size, along with a marked anti angiogenic effect in mice administered with the GRP fusion HG6 protein.In an analogous approach, a DNA vaccine encoding the GRP-HSP65 fusion product showed anti-tumor activities in a prophylactic setting for subcutaneous implanted tumors and a pulmonary metastasis model (Fang et al., 2009).Furthermore, proofs have been given that introduction of two T-helper epitope (HSP70406-426) downstream the immunogen in anti-GRP DNA vaccine augment humoral response and immunogenicity of the vaccine against murine prostate carcinoma (Lu et al., 2009).Our results demonstrated efficient protection against growth of H22 liver tumor cells injected subcutaneously along with a reduced neoangiogensis.
The mechanism underlying the ability of the anti-GRP vaccine to inhibit the proliferation of H22 cells in vivo is yet to be elucidated.It is clear that the humoral immune response has been elicited by vaccination of DNA vaccine with a high titer of GRP antibodies detected.This phenomenon suggests that the GRP antibodies neutralize the self-epitope GRP and lower the concentration of GRP DOI:http://dx.doi.org/10.7314/APJCP.2014.15.20.9039Immunopreventive Effects against Murine H22 HCC Cells in Vivo of a DNA Vaccine Targeting Gastrin-Releasing Peptide around the tumor, whereas the activation of GRPR may be interrupted and thus blocking the signaling pathways.
On the other hand, Gastrin-releasing peptide is known to stimulate pro-angiogenic gene expression as well as the expression of various angiogenic markers such as vascular endothelial growth factor (Kang et al., 2007).In addition, it has been shown that GRP or GRPR silencing significantly suppress tumor progression and vascularization (Cornelio et al., 2007).HCC is known as a highly angiogenic cancer, with critical implication of VEGF (Zhu et al., 2011).The results from this study clearly demonstrate that vaccination with the DNA vaccine pCR3.1-VS-HSP65-TP-GRP6-M2leads to significant reduction of the tumor-associated angiogenesis and vascularization of H22 solid tumor.This effect can contribute to prophylactic approach in developing immunotherapy in liver cancer.DNA vaccine targeting GRP have also shown therapeutic effect (Wu et al., 2008); and due to some immune escape events reported, it will be interesting that our vaccine be tested against well formed H22 tumors.
In conclusion, in researching new alternatives in immunotherapy of liver cancer, we relied on a DNA vaccine targeting Gastrin-Releasing peptide (GRP).The vaccine was applied for its immune preventive effects against H22 hepatocarcinoma cells, suggesting that it could inhibit tumor growth through anti-angiogenic effects.The strategy offers a novel approach to be developed in the treatment.However, further research need to be carried out, such as therapeutic activity, to assess the efficiency of the vaccine in the control of liver cancer.

Figure 4 .
Figure 4. Effects of Anti-GRP Vaccine on the Angiogenesis of Intradermal Tumors.(Left): Light microscopical picture of H22 tumor cells implanted intradermally in the anterior abdominal wall and the development of new blood vessels.Tumor-associated angiogenesis (2/mouse, 4 mice/group) in mice injected with saline A) and pCR3.1-VS-HSP65-TP-M2control vaccine B) appeared to be higher than in mice immunized with the anti-GRP vaccine pCR3.1-VS-HSP65-TP-GRP6-M2D).Representative images were taken at 10x objective.(Right):The total number of blood vessels (major vessels and branching points) was determined within the precise 1 cm 2 area around each implant site