Abstract
Acyclic nucleoside phosphonates PMEDAP and PMEG modulate expression of selected proangiogenic genes in SD-lymphoma bearing rats. Antiangiogenic efficacy of PMEDAP is relatively weak and is manifested mainly by down-regulation of vascular endothelial growth factor (VEGF) and its receptor VEGFR detectable 24 hours after treatment. Compound PMEG (an active metabolite of the prodrug GS-9219) down-regulates selected proangiogenic genes EGF, FGF, PDGF, VEGF, EGFR, FGFR, PDGFR and VEGFR much more efficiently. Its antiangiogenic potency persists and is more intensive 48 hours after treatment. Findings show that in vivo antitumour efficacy of both antimitotic acyclic nucleoside phosphonates PMEDAP and PMEG consequently affect the angiogenesis in T-cell lymphoma.
Abbreviations: Acyclic nucleoside phosphonates (ANP): 9-[2-(phosphonomethoxy)ethyl]-guanine (PMEG), 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP), 9-[2-(phosphonomethoxy)ethyl]-N6-(2-dimethylaminoethyl)-2,6-diaminopurine (Me2NEt-PMEDAP), 9-[2-(phosphonomethoxy)ethyl]-N6-allyl-2,6-diaminopurine (allyl-PMEDAP), 9-[2-(phosphonomethoxy)ethyl]-N6-dimethyl-2,6-diaminopurine (Me2-PMEDAP) and 9-[2-(phosphonomethoxy)ethyl]-N6-cyclopropyl-2,6-diaminopurine (cypr-PMEDAP); SD-lymphoma, spontaneous transplantable T-cell lymphoma of Sprague-Dawley rats; SD/Cub, Sprague-Dawley inbred rats/Charles University Biology; RQ-RT-PCR, real-time quantitative reverse transcriptase-polymerase chain reaction.
The anticancer effects of compounds PMEDAP, PMEG and PMEDAP N6-substituted derivatives (Me2Net-PMEDAP, allyl-PMEDAP, Me2-PMEDAP and cypr-PMEDAP) have been studied in detail in a spontaneous T-cell lymphoma model in inbred SD/cub rats (1) which is relevant to human pathology (non-Hodgkin's lymphoma) (2). A significant therapeutic effect has been recorded after treatment with PMEDAP (3-5). Decrease of the lymphoma weight during PMEDAP administration was accompanied by suppression of mitotic activity of neoplastic cells and increased chromatin condensation as well as by increased nuclear DNA fragmentation resulting in induction of apoptosis in in vivo growing lymphomas (4). Another study has shown that the therapeutic effect of PMEDAP depends on the phenotype of the individual neoplasm (5). Unlike PMEDAP, its N6-mono- and disubstituted congeners were less effective or exhibited the same antitumor efficacy as PMEDAP (6). PMEG and cypr-PMEDAP were also potent but significantly reduced the survival of treated lymphoma-bearing rats due to toxicity (7).
Recent studies have shown that the antiproliferative effect of PMEDAP N6-substituted derivatives is evoked by intracellular transformation to PMEG catalyzed by methylAMP aminohydrolase (8, 9). PMEG is the active compound of a novel prodrug GS-9219, which possesses an excellent therapeutic efficacy in vivo against spontaneous non-Hodgkin's lymphoma in dogs (10). PMEG and PMEDAP are phosphorylated by cellular kinases to form diphosphates (analogs of nucleoside 5′ triphosphates) (11, 12), which inhibit replicative DNA polymerases (13, 14) while the prodrug GS-9219 is first hydrolyzed to cyprPMEDAP and thereafter deaminated to PMEG (10). This ability is believed to account for the cytostatic activity of the parental compound. Moreover, the latest findings show that the diphosphates of PMEG and PMEDAP inhibit activity of human telomerase in vitro (15). These results are consistent with cytostatic properties of both compounds. It is highly probable that DNA damage will affect signaling pathways associated not only with cell proliferation and apoptosis but also with angiogenesis.
Tumor angiogenesis is a complicated process stimulated by the secretion of various signaling molecules to promote neovascular formation (16, 17). Angiogenic factors and their activity are well described in solid tumors but have not yet been completely established in hematopoietic neoplasms (18). Increasing evidence suggests that neovascularization is also important in hematological malignancies (19).
The purpose of the present study was to examine an expression of selected genes associated with angiogenesis in the spontaneous SD-lymphoma during in vivo treatment with anticancer acyclic nucleoside phosphonates PMEDAP and PMEG. Expression of genes coding epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF) and their receptors was examined using quantitative real time PCR.
Materials and Methods
Experimental animals. Two- to three-month-old males of the Prague subline of Sprague-Dawley inbred rats (SD/Cub) were used for the experiments. The rats were bred under conventional conditions with commercial granule food (Bergman TM2-CH) and tap water ad libitum. Fifteen untreated rats served as an independent group for evaluation of lymphoma growth dynamics. Six rats were included in each treatment and control group for evaluation of the effect of PMEDAP and PMEG (for treatment; see Drug administration). Animals of the same age were used in treatment and control groups. Animal handling and experimental protocols were undertaken as per guidelines of the Institutional Ethical Committee for Animal use.
Tumor. Spontaneous transplantable T-cell lymphoma is a highly genetically defined model of spontaneous hematological malignancy (2). In this study, lymphoma SD 10/96 (74th passage) was used. In all experiments, a suspension of 106 lymphoma cells in PBS (saline) was injected subcutaneously (s.c.) into the right flank of the anesthetized rat.
Lymphatic nodes. Submandibular lymphatic nodes of young healthy SD/Cub rats were used for isolation of total RNA, which served as a calibrator rat sample.
Angiogenesis. Samples of lymphoma tissue were scrutinized by (real-time) RQ-RT-PCR for expression of genes EGF (Rn 00563336_m1), FGF (Rn 00563362_m1), PDGF (Rn 00709363_m1), VEGF (Rn 00582935_m1), EGFR (Rn 00580398_m1), FGFR (Rn 00577234_m1), PDGFR (Rn 00709573_m1) and VEGFR (Flt4; Rn 00586429_m1), which could be associated with angiogenesis in lymphoma tissue. Three rats were killed and autopsy performed at three-day intervals, starting 14 days after the inoculation of tumor cells and continued over 12 days (26 days after lymphoma cell inoculation). Lymphoma tissue weight was measured at autopsy. Submandibular lymphatic node cells from the autopsied animals were also examined. Expression of the afore mentioned genes was examined after PMEDAP or PMEG treatment of SD/Cub lymphomas (see Drug administration).
Compounds. Acyclic nucleoside phosphonates 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP) and PMEG - 9-[2-(phosphonomethoxy)ethyl]guanine, were prepared according to Holý et al. (20).
Drug administration. In the in vivo experiments PMEDAP was injected intraperitonealy (i.p.) in each animal in the treated group at a dose of 5 mg kg-1; PMEG at a dose of 0.3 mg kg-1. Five doses were administered at three-day intervals, once daily (1 × d). Administration of the drugs started 4 days after the inoculation of tumor cells and continued during the next 12 days. An autopsy was performed 24 hours and 48 after the last injection of PMEDAP and PMEG, respectively; lymphoma weight was determined and the sample of each lymphoma tissue was scrutinized by RQ-RT-PCR for expression of genes associated with tumor angiogenesis.
Relative quantification of gene expression - evaluation by RQ-RT-PCR. Total RNA was extracted from rat tissues by RNeasy Mini Kit (Qiagen, MD, USA) according to the manufacturer's recommendations. RNA integrity was determined by gel electrophoresis in 2% agarose gel stained with ethidium bromide. The purity of the RNA was assessed by the ratio of absorbance at 260 nm and 280 nm. The purity was in the range of 1.8 to 2.2. Total RNA concentration was estimated by spectrophotometric measurements at 260 nm assuming that 40 μg of RNA per milliliter was equivalent to one absorbance unit. RNA was stored in aliquots at -20°C until used for reverse transcription. Isolated mRNA was converted to cDNA using reverse transcription reagents (Applied Biosystems, CA, USA), according to the manufacturer's instructions.
Real-time PCR was performed in the 7300 real-time PCR system (Applied Biosystems) using standard conditions. Every 25 μL reaction mix for real-time PCR was made with 12.5 μL TaqMan Universal PCR master mix (Applied Biosystems), 100 ng of cDNA, RNase free water and specific TaqMan probes for EGF, FGF, PDGF, VEGF, EGFR, FGFR, PDGFR and VEGFR (all Applied Biosystems). Beta-2-microglobulin was used as an endogenous control (Applied Biosystems). Expression of all genes was normalized to mRNA loading for each sample and used as an internal standard. The quantity of mRNA was given as 2-ΔΔct. ΔΔct was calculated as follows: ΔΔct=Δct (sample of interest) - Δct (calibrator) and Δct=ct (target gene) - ct (endogenous control). Mixed sample of mRNA from healthy SD/Cub rats was used as the calibrator (its value was assumed to equal 1). The gene expressions in rats after treatment were given as relative values referred to the value of the calibrator. Levels of the gene transcripts were quantified in the quadruplex PCR reaction.
Statistics. The Student's t-test was used for the statistical comparison and p-values ≤0.05 were considered statistically significant.
Results and Discussion
Relative expression of genes EGF, FGF, PDGF, VEGF, EGFR, FGFR, PDGFR and VEGFR was examined in SD-lymphoma cells in relation to the tumor weight of the experimental animals during a period of 26 days (Figures 1, 2). Samples for RQ-RT-PCR were recovered on days 14, 17, 20, 23 and 26 after s.c. inoculation of tumor cells.
The data show that the expression of both fibroblast growth factor (FGF) and its receptor (FGFR) genes was significantly lower in the lymphoma cells compared to the expression in lymph node cells of healthy animals (FGF - day 14: p<0.002; day 17: p<0.02; day 20: p<0.03; day 23: p<0.03; FGFR - day 14: p<0.04; day 17: p<0.02; day 20: p<0.007; day 23: p<0.03; day 26: p<0.005) (Figure 2A). By contrast, VEGF expression increased significantly, with a well-defined maximum on day 20 (day 14: p<0.03; day 17: p<0.03; day 20: p<0.02; day 26: p<0.03) while the expression of the corresponding receptor gene VEGFR significantly decreased in lymphoma cells during the lymphoma tissue progression (day 17: p<0.03; day 20: p<0.03; day 26: p<0.002) (Figure 2B).
On the other hand, relative expression of PDGF gene persisted at normal values compared with PDGF expression in control tissue. However, the expression of PDGFR gene significantly increased during the growth of the lymphoma (day 20: p<0.05; day 23 p<0.04; day 26: p<0.0006) (Figure 2C). On the contrary, the expression of both EGF and EGFR genes did not change in the course of the 26 days when the expression of these genes was followed (data not shown).
Very significant VEGF and VEGFR gene expression enhancement observed in SD-lymphoma conforms to the findings in many different types of tumors. Both genes are considered to initiate neovascularization in tumor tissue (19, 21, 22).
In the second experiment, the relative expression of previously mentioned angiogenesis-associated genes were followed in the PMEDAP and PMEG treated SD-lymphoma bearing animals with significantly lower lymphoma weights compared to untreated SD lymphoma rats (Table I). The results show (Figure 3A) that 24 h after the last injection of the drug, the PMEDAP treatment induced non-significant changes in the expression of EGFR, EGF, FGFR, FGF, PDGFR and PDGF genes, while that of VEGFR was significantly down-regulated (p<0.0006) and that of VEGF was still up-regulated (p<0.02) when compared with the lymph node of healthy animals. Forty-eight hours after the last PMEDAP administration, the regulation of VEGF showed an entirely reverse course and its expression returned towards a normal level (p<0.01) while by that time the expression of PDGFR had risen (p<0.03) (Figure 3B).
Surprisingly, after the treatment of SD-lymphoma suffering rats with PMEG, the selected angiogenesis-associated genes did not maintain the same profile as in the case of PMEDAP therapy. Twenty-four hours after the last PMEG injection, the EGFR and EGF genes were significantly up-regulated (p<0.05 and p<0.007 respectively) while FGF and VEGF genes were down-regulated (p<0.0005 and p<0.001 respectively, Figure 4A). Twenty-four hours later, all studied genes were significantly down-regulated compared to the lymph node of healthy animals and also when compared with untreated lymphomas, with the exception of a relative expression of FGF and VEGFR genes which persisted unchanged (Figure 4B).
VEGF, FGF, EGF and PDGF and their receptors are angiogenesis-modulating molecules. Their mode of action is mostly paracrine or autocrine. VEGF is one of the most important proangiogenic factors and acts as a mitogen for vascular endothelial cells. VEGF increases vascular permeability and contributes to endothelial cell survival in blood and lymphatic vessels. Linkage of VEGF with VEGFR initiates a cascade of intracellular signals through increased receptor tyrosine kinase activity, thus resulting in endothelial cell proliferation and formation of new blood vessels (21-23). Angiogenic growth factor FGF also induces endothelial cell growth and microvascular permeability (16, 24). EGF and its receptor EGFR are involved in pleiotropic signaling pathway in cancerogenesis. Activation of EGFR stimulates tumor growth, angiogenesis, invasion, metastasis and inhibition of apoptosis (25). Up-regulation of these genes is a marker of poor prognosis in malignant disease and hence advanced therapy of cancer is focused on an EGF blockade (26). The importance of PDGF consists in its direct tumor growth-promoting effect. On the one hand, it increases survival and proliferation of endothelial cells and moreover, it regulates vessel growth via pericyte recruitment and association with newly formed vessels. Pericytes in tumor vessels are critical for vessel integrity and function. Targeting pericytes actually provides additional benefits in antiangiogenic treatment (27, 28).
The antiangiogenic efficacy of PMEDAP is relatively weak and is manifested mainly by down-regulation of VEGF and its receptor VEGFR detectable 24 hours after treatment. On the other hand, compound PMEG down-regulates selected proangiogenic genes much more efficiently. Its antiangiogenic potency persists and is more intensive 48 hours after treatment. It is highly possible that the novel prodrug of PMEG, compound GS-9219, which has a substantial antineoplastic activity in dogs with spontaneous non-Hodgkin's lymphoma (29) exerts antiangiogenic potential, as well.
In conclusion, we presented a study which shows that the in vivo antitumor efficacy of both ANPs PMEDAP and PMEG is not only caused by the inhibition of DNA synthesis but that these compounds consequently affect angiogenesis. This process plays an important role in tumor growth, since tumor tissue located distant from a blood vessel needs neovascular formation to ensure a sufficient supply of nutrients and oxygen.
Acknowledgements
This work was supported by the Project VZ 0021620808 of the Ministry of Education, Youth and Sports of the Czech Republic, and partially by research project OZ40550506 of the IOCB and Centre of New Antivirals and Antineoplastics 1M0508 by the Ministry of Education, Youth and Sports of the Czech Republic.
- Received September 10, 2008.
- Revision received December 11, 2008.
- Accepted December 22, 2008.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved