Gonadotropin-Releasing Hormone Promotes Ovarian Cancer Cell Invasiveness through c-Jun NH₂-Terminal Kinase–Mediated Activation of Matrix Metalloproteinase (MMP)-2 and MMP-9

The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) plays a key role in the ovary and ovarian cancer (reviewed in ref. 1). GnRH receptor expression has been shown in 80% of human ovarian tumors and numerous cancer cell lines, and the analogues inhibit proliferation of the GnRH receptor-bearing tumor cells both in vivo and in vitro, supporting evidence for a direct antiproliferative effect (1–3). Of particular interest, levels of GnRH receptor seem to be associated with cancer grading and have been reported to be elevated in advanced-stage (stages III and IV) ovarian carcinomas (4). This opens the possibility that GnRH could directly regulate tumor progression of ovarian cancer cells. However, a role of GnRH in ovarian epithelial cell migration/invasion has not been established and the mechanism by which GnRH contributes to ovarian cancer invasiveness is also unknown.

Ovarian cancer is the most lethal gynecologic malignant tumor in the Western world. This is in part due to the fact that most (60%) of the patients are diagnosed with already widespread intraperitoneal metastasis and ascites and the lack of effective therapies for advanced-stage disease (5). Thus, there is a need for new therapeutic targets and a better understanding of the mechanisms involved in the spread of ovarian carcinoma. The factors that regulate the metastatic process of ovarian cancer, however, are poorly understood.

Matrix metalloproteinases (MMP), a family of secreted or transmembrane enzymes, can collectively digest almost all extracellular matrix (ECM) and basement membrane components (6, 7). Thus, MMPs are largely implicated in promoting angiogenesis and tumor metastasis. Above all, MMP-2 and MMP-9, which can degrade collagen IV, the major ECM component of the basement membranes, have been suggested to be critical for the invasive and metastatic potential in ovarian carcinoma. Elevated expression of MMP-2 and MMP-9 has been detected in ovarian cancer ascites, tissues, and cancer cells in culture (8–11), and experimental metastasis is suppressed by a synthetic MMP inhibitor (12). MMP activity is often controlled by a specific inhibitor known as the tissue inhibitor of metalloproteinases (TIMP). TIMP-1 selectively binds pro-MMP-9, whereas TIMP-2 associates with pro-MMP-2 in a crucial step in the cell-mediated activation of MMPs (13). By inhibiting active MMPs, TIMPs inhibit cell invasion in vitro and tumorigenesis and metastasis in vivo (14–16).

In the present study, we show for the first time a role for GnRH in the invasive phenotype and motility of human ovarian cancer cells. We also present evidence suggesting that the activation of metastasis-related proteinases, especially MMP-2 and MMP-9, through the c-Jun NH₂-terminal kinase (JNK) mediated signaling pathway in this process.

Cell culture. The human ovarian epithelial carcinoma cell lines Caov-3 and OVCAR-3 (kindly provided by Dr. N. Auersperg, University of British Columbia, Vancouver, British Columbia, Canada) were grown in medium 199:105 (Sigma, St. Louis, MO) supplemented with 5% fetal bovine serum and 100 units/mL penicillin-streptomycin (Invitrogen, San Diego, CA) in a humidified atmosphere of 5% CO₂ at 37°C.

Antibodies, reagents, and plasmids. GnRH agonist (GnRHa; D-Ala⁶) was purchased from Sigma. PD98059, SB203850, SP600125, MMP-2 inhibitor (OA-Hy), MMP-9 inhibitor (SB-3CT), and monoclonal antibodies to MMP-2 and MMP-9 were purchased from Calbiochem (San Diego, CA). Anti-TIMP-1 and anti-TIMP-2 were from Chemicon International, Inc. (Temecula, CA). Anti-extracellular signal-regulated kinase 1/2 (ERK1/2), anti-p38 mitogen-activated protein kinase (MAPK), anti-JNK, anti-phosphorylated ERK1/2, anti-phosphorylated p38 MAPK, and anti-phosphorylated JNK were obtained from Cell Signaling Technology, Inc. (Beverly, MA). The dominant-negative mutant of human JNK was kindly provided by Dr. R. Davis (University of Massachusetts Medical School, Worcester, MA). The MMP-2 promoter region between positions −1,659 and +57 was cloned into promoterless luciferase reporter vector pGL3-Basic as described previously (17). The MMP-9 promoter plasmid was a generous gift of Dr. D. Boyd (M. D. Anderson Cancer Center, Houston, TX).

Western blot analysis. Equal amounts of protein (50 μg) were subjected to SDS-PAGE (7.5%) and then transferred to nitrocellulose membrane. Membranes were blocked using 5% nonfat dry milk in PBS containing 0.05% Tween 20 and incubated overnight at 4°C with the appropriate primary antibody. The immunocomplex was detected with horseradish peroxidase–conjugated IgG as secondary antibody (Bio-Rad, Hercules, CA), and blots were developed with an enhanced chemiluminescence kit (Amersham Biosciences, Little Chalfont, United Kingdom). The experiment was repeated at least twice with independent protein samples.

Small interfering RNA transfection. The pSUPER vector encoding 19-mer hairpin small interfering RNA (siRNA) duplex specific to GnRH receptor sequence 5′-AGATCCGAGTGACGGTTAC-3′ (nucleotides 107-125 downstream of the start codon) has been described elsewhere (18). As a nonspecific siRNA control, scrambled siRNA duplex was used. Transfection was done using LipofectAMINE 2000 reagent (Invitrogen) following the manufacturer's instruction.

Gelatin zymography. The activities of MMP-2 and MMP-9 in the conditioned medium were determined by gelatin zymography. Briefly, the media were collected and clarified by centrifugation to remove cells and debris. Samples (80 μg) were loaded under nonreducing conditions onto SDS-polyacrylamide gel polymerized with 1 mg/mL gelatin. Following electrophoresis, the gels were washed with 2.5% Triton X-100 to remove SDS and then incubated in a developing buffer [50 mmol/L Tris-HCl buffer (pH 7.4), 10 mmol/L CaCl₂] overnight at 37°C. Gels were stained with 0.25% Coomassie Brilliant Blue R-250 and destained in the same solution without dye. Gelatinase activity was visualized as clear bands against the blue-stained gelatin background. Molecular sizes were determined from mobility using gelatin zymography standards (Chemicon International). Three individual experiments were conducted with independent protein samples.

Semiquantitative reverse transcription-PCR. Total RNA was isolated from cultured cells using the Trizol reagent (Invitrogen) according to the manufacturer's procedure. First-strand cDNA synthesis was done from 2.5 μg of total RNA using SuperScript Reverse Transcriptase (Life Technologies). The cDNA was used for subsequent PCR using primers specific for human GnRH receptor, MMP-2, MMP-9, TIMP-1, and TIMP-2 as reported previously (19, 20). β-Actin was used as internal control to normalize the relative amount of cDNA in each reaction. The logarithmic phase of PCR amplification for each target gene had been determined in initial experiments. The reproducibility of the quantitative measurements was evaluated by three independent cDNA syntheses and PCR ran from each preparation of RNA.

Reporter gene assay. Cells were transiently transfected with 1 μg of the construct using LipofectAMINE (Invitrogen) as directed by the manufacturer. After 6 hours, the cells were incubated with or without GnRHa (0.1 nmol/L) for an additional 24 hours. The pSV-β-galactosidase plasmid was cotransfected as internal control. Luciferase units were calculated as luciferase activity/β-galactosidase activity and are presented as the mean ±SD of three individual experiments with triplication. The fold change was calculated by comparison with the promoterless luciferase vector (pGL3-Basic).

Invasion and migration assays. Twenty-four-well Transwell inserts with 8-μm pores coated with Matrigel (50 μL/well; BD Biosciences, Palo Alto, CA) were used to assess cell invasion. Cells (1 × 10⁵) in serum-free medium, with or without GnRHa, were seeded in triplicate in the upper chamber. Serum-free medium was placed in the lower wells. The chambers were incubated for 24 hours at 37°C in a 5% CO₂ atmosphere. Cells that had not penetrated the filter were wiped out, and invaded cells on the lower surface of the filter were fixed with ice-cold methanol and stained with 0.5% crystal violet. Results are presented as the mean number of invaded cells of five fields ±SD of three independent experiments. To assess cellular migration potential, the protocol described above was used, except that Matrigel was omitted and 3T3 fibroblast conditioned medium was placed in the lower chamber as a chemoattractant.

Statistical analysis. Statistical analysis was carried out using ANOVA followed by Tukey's post hoc test (GraphPad Software, San Diego, CA). P < 0.05 was considered statistically significant.

GnRH stimulates ovarian tumor cell migration and invasion. During metastasis, invasion of basement membrane by tumor cells is thought to be a critical event (21). To study whether expression of GnRH receptor is associated with metastasis of ovarian cancer cells, the effect of GnRH on cellular motility and invasive activity was evaluated. Because native GnRH has a relatively short half-life both in vivo and in vitro, GnRHa, in which Gly6 has been substituted by a D-amino acid, have been commonly used to resist degradation and to increase potency (22). The GnRHa D-Ala⁶ used in this study exhibits 3.5 to 4.5 times higher potency and 3 to 8 times higher stability than the native hormone in vitro (22, 23). Caov-3 and OVCAR-3 cells, which express functional GnRH receptors (24), were treated with increasing concentrations of the GnRHa (D-Ala⁶), and their invasive capacities were assessed using Transwells with filters coated with Matrigel. Our results showed that GnRHa induced a dose-dependent biphasic regulation on invasion: cells treated with GnRHa at low doses of 0.1 to 10 nmol/L showed significantly higher numbers of invaded cells (P < 0.05), whereas the response to higher doses of GnRHa (100 nmol/L to 1 μmol/L) was insignificant (Fig. 1A).

We also assessed in vitro migration in response to GnRH stimulus using a Transwell migration assay. GnRHa dose dependently stimulated the migration of ovarian cancer cells through uncoated porous filter, with no significant effect at a concentration of 100 nmol/L and 1 μmol/L and a maximal effect at 0.1 nmol/L (P < 0.05; Fig. 1B).

GnRH receptor siRNA inhibits ovarian tumor cell invasion and migration. Numerous studies have shown that alteration of GnRH receptor mRNA levels is involved in the regulation of ovarian responsiveness to GnRH (19, 25, 26). We therefore examined whether the effect of GnRH on ovarian cancer invasion was associated with altered GnRH receptor gene expression. As shown in Fig. 1C, treatment with the GnRHa induced a biphasic regulation pattern for GnRH receptor mRNA in both Caov-3 and OVCAR-3. Low doses of GnRHa (0.1-10 nmol/L) increased GnRH receptor mRNA levels, with a maximal effective dose of 0.1 nmol/L (80.5% increase); thereafter, the levels decreased in cells treated with GnRHa at 100 nmol/L to 1 μmol/L. Cotreatment of cells with the GnRH antagonist antide prevented the biphasic effects of GnRHa, whereas antide alone had no effect, indicating the specificity of the response (data not shown).

To further confirm the role of GnRH receptor on the invasive properties of ovarian cancer cells, we inhibited the expression of GnRH receptor using vector-based RNA interference assay. The effectiveness of this siRNA to deplete GnRH receptor expression was confirmed by semiquantitative reverse transcription-PCR (RT-PCR) and Western blot analysis (data not shown) and was consistent with the previous study (18). Importantly, siRNA-mediated depletion of GnRH receptor, but not nonspecific siRNA, reduced invasion to near basal levels (80.8% inhibition; Fig. 1D,, left). Similarly, we showed that depletion of GnRH receptor inhibited the cell migration by 85.2% (Fig. 1D , right), confirming a direct involvement of GnRH receptor in the acquisition of a migratory and invasive phenotype in ovarian cancer cells.

GnRH induces MMP-2 and MMP-9 expression and enhances their activities. We next determined whether GnRH-stimulated cell invasion resulted from elevated levels of MMPs, which are well-documented ECM-degrading enzymes and whose activity is associated with tumor invasiveness (27). MMP protein and enzymatic activities were measured by Western blotting and gelatin zymography using conditioned medium from Caov-3 (Fig. 2) and OVCAR-3 (data not shown). As shown in Fig. 2A, the effect of GnRHa on MMP production was dose dependent because low concentrations (0.1-10 nmol/L) of GnRHa resulted in an up-regulation of MMP-2/MMP-9 expression (P < 0.05), whereas high concentration (100 nmol/L to 1 μmol/L) had minimal response (Fig. 2A). Consistently, gelatin zymography showed stronger lytic zones at the molecular mass corresponding to latent and active forms of MMP-2 (72-kDa and 66-kDa forms) and MMP-9 (92-kDa and 86-kDa forms) in cells treated with 0.1 nmol/L GnRHa with respect to untreated cells, showing their elevated secretion and enzyme activities by GnRHa (Fig. 2B). These results matched well with those observed in cellular invasion (Fig. 1), suggesting that GnRH-mediated induction of MMP-2 and MMP-9 may play a role in GnRH-enhanced cell invasion/migration. To ascertain whether GnRH plays a critical role in the induction of MMP-2 and MMP-9, we monitored the regulation of these proteinases after transfection of a GnRH receptor-targeted siRNA. As shown in Fig. 2C, both the activity and expression of MMP-2 and MMP-9 were clearly reduced in the presence of the GnRH receptor siRNA. In contrast, control experiments with nonspecific siRNA showed negligible effect.

MMP expression is often coordinately regulated with production of their endogenous inhibitors, TIMPs (21). However, there was no change in the expression of TIMP-1 and TIMP-2 irrespective of the presence of different concentration of GnRHa as shown by Western blot analysis (Fig. 2D).

GnRH modulates transcription activities of MMP-2 and MMP-9. To determine whether the observed alteration in MMP-2 and MMP-9 was the result of changes in gene expression, RT-PCR was done. In accordance with changes in protein expression, MMP-2 and MMP-9 mRNA expression were found to be significantly enhanced with 0.1 nmol/L GnRHa (5.3-fold for MMP-2 and 2.5-fold for MMP-9 compared with untreated control cells; P < 0.05; Fig. 3A).

To test whether the effect of GnRH on MMP-2 and MMP-9 mRNA expression was the result of increased mRNA stability, we did actinomycin D (ActD) chase experiments to determine the half-life of MMP-2 and MMP-9 mRNA. Cells were preincubated with 0.1 nmol/L GnRHa for 3 hours. Then, ActD (4 μg/mL) was added to stop transcription and RNA was isolated after 0, 1, 2, 4, and 6 hours. The amount of gelatinase mRNA compared with β-actin was determined by RT-PCR. As shown in Fig. 3B, GnRHa did not affect the decay rate of the MMP-2 and MMP-9 mRNA.

To assess whether GnRHa causes transcriptional activation at the MMP-2 and MMP-9 promoters, human MMP-2 or MMP-9 promoter-driven luciferase constructs were transiently transfected into Caov-3 and OVCAR-3 cells in either the presence or the absence of 0.1 nmol/L GnRHa for 24 hours. The results showed that exposure of cells to GnRHa increased the activities of MMP-2 and MMP-9 promoters by 10- and 3-fold, respectively (P < 0.005; Fig. 3C). Moreover, these increases in promoter activities were completely abolished by GnRH receptor siRNA treatment but not the nonspecific siRNA (Fig. 3C). These results collectively show a role for GnRH in the transcriptional activation of MMP-2 and MMP-9.

Inhibition of MMP blocks GnRH-enhanced invasion and migration in ovarian cancer cells. To evaluate the potential contribution of MMP-2 and MMP-9 in the GnRH-induced invasion of ovarian cancer cells, we blocked the gelatinase activities using potent MMP-2 (OA-Hy, 15 μmol/L) or MMP-9 (SB-3CT, 10 μmol/L) specific inhibitors or MMP-9 neutralizing antibody (15 μg/mL) in the presence or absence of 0.1 nmol/L GnRHa. Specific inhibition of MMP-2 by OA-Hy reversed the stimulatory effect of GnRH on cell invasion (by 81.8% in Caov-3 and 82.9% in OVCAR-3), whereas concomitant treatment with MMP-9 inhibitor SB-3CT or anti-MMP-9 significantly blocked the increase in invaded cells after GnRHa treatment (by 75.6% and 72.7%, respectively, in Caov-3 and 70.3% and 73.1%, respectively, in OVCAR-3; Fig. 4A). Similar results were obtained in migration assays as shown in Fig. 4B. Together, these experiments show a crucial role for both MMP-2 and MMP-9 in the GnRH-dependent invasion and migration.

Prolonged activation of JNK by GnRH. Because studies have shown that the MAPK pathway is critical for the activation of gene expression by GnRH in some cell types (28, 29), we asked if they also play a role in GnRH-induced human ovarian cancer cell invasion and/or migration. We first examined the levels of phosphorylated (active) forms of MAPK family members (ERK1/2, JNK, and p38 MAPK) in Caov-3 and OVCAR-3 cells treated with 0.1 nmol/L GnRHa for increasing time course of 0, 5, 15, 30, 60, and 120 minutes. As shown, addition of GnRHa to the cells significantly increased the phosphorylation of ERK1/2, JNK, and p38 MAPK (Fig. 5). Interestingly, whereas prominent (15-fold) and sustained activation (60 minutes) of JNK was shown in GnRH-activated cells (P < 0.005), activation of ERK1/2 and p38 MAPK was rapid and transient, reaching a maximum of approximately 2- to 3-fold 15 minutes after GnRHa addition and decline thereafter (Fig. 5).

GnRH-induced invasion and motility are JNK dependent. To assess the functional significance of GnRH-activated ERK1/2, JNK, and p38 MAPK in invasion and motility, we asked if interfering with their activation by specific inhibitors diminishes GnRH-mediated in vitro invasiveness and motility. As shown, treatment of 50 μmol/L SP600125, a specific JNK inhibitor, significantly reduced the number of both invaded (Fig. 6A) and migrated (Fig. 6B) cells to near basal levels. In contrast, inhibition of GnRH-stimulated ERK1/2 and p38 MAPK activation by 50 μmol/L PD98059 or 10 μmol/L SB203580, respectively, had no effect (Fig. 6A and B). These results indicate a critical role for JNK, but not ERK1/2 or p38 MAPK, in GnRH-induced invasive phenotype and migration in ovarian cancer cells.

To additionally investigate the role for JNK in cellular invasion and migration, we transfected Caov-3 and OVCAR-3 cells with a dominant-negative JNK (DN-JNK) construct in which the dual phosphorylation motif Thr-Pro-Tyr was mutated to Ala-Pro-Phe (30). Specific inactivation of JNK in DN-JNK-transfected cells was confirmed by Western blot analysis (Fig. 6A,, inset). Both the invasive phenotype and cell migration were clearly reduced in DN-JNK transfectants as shown in Fig. 6A and B, showing the significance of the JNK pathway in GnRH-induced invasion and migration in Caov-3 and OVCAR-3 cells.

GnRH-mediated up-regulation of MMP-2 and MMP-9 involves JNK. Next, we asked whether JNK mediates GnRH induction of MMP-2 and MMP-9 activities. As shown in Fig. 6C, both basal and GnRH-stimulated proform and active form of the MMP-2 and MMP-9 production were inhibited with 50 μmol/L SP600125. GnRH-induced MMP-2 and MMP-9 promoter activities were also inhibited by targeted inhibition of JNK using SP600125 or DN-JNK (Fig. 6D). Consistent with the effects on invasion and cell migration, pretreatment with either PD98059 or SB203580 showed no inhibition on GnRH-mediated MMP induction (data not shown). These results suggest that activation of JNK is required for enhanced MMP-2 and MMP-9 expression.

There is much evidence that implicates an important role for GnRH in regulating the proliferation for a wide range of hormone-dependent malignancies, including ovarian cancer (1). However, little is known about its role in tumor invasion, metastasis, and other aspects of cancer progression. In this study, we report for the first time that GnRH may contribute to the metastatic dissemination of ovarian cancer cells by promoting the expression and/or processing of metastasis-related MMP, with subsequent downstream changes in migratory and invasive behavior, providing an insight into the prospect of developing targeted therapy for ovarian carcinoma.

The present study showed a biphasic effect of GnRH on cellular migration and invasion. Whereas lower concentrations of the GnRHa stimulated cellular migration and invasion in a dose-dependent manner, high concentrations were not as efficient. Alteration of GnRH receptor numbers or gene expression is involved in mechanisms underlying the biphasic effect of GnRH in the pituitary and ovary (19, 25, 26, 31). In ovarian carcinoma cells, we showed previously (25) and in this study that low doses of GnRH up-regulated its receptor, whereas high doses decreased it. This also argues for a role for GnRH receptor to activate intracellular signals necessary for cellular responses. Notably, the effects of GnRH on JNK and MMP-2/MMP-9 activation were consistent with changes in the GnRH receptor mRNA levels. When GnRH receptor mRNA was up-regulated, GnRH enhanced JNK and MMP-2/MMP-9 activities; however, when GnRH receptor mRNA was unaffected or down-regulated, GnRH had no effect on JNK³

Metastasis is a complex phenomenon that requires several specific steps, such as decreased adhesion, increased motility, and proteolysis. In prostate cancer, GnRH has been shown to regulate cell motility through its interaction with the small GTPases Rac1, Cdc42, and RhoA, which are involved in the regulation of actin polymerization (32). Our data show that another mechanism by which GnRH may promote tumor cell migration and invasion is through increased expression and proteolytic activities of MMP-2 and MMP-9 that specifically degrade the basement membrane. MMP-2 and MMP-9 have been suggested to play critical roles in ovarian cancer metastasis, and up-regulation of MMP-2 and MMP-9 is associated with increased invasion and poor prognosis in late-stage or invasive ovarian cancer patients (8–11). In addition to their enzymatic activities, MMP-2 and MMP-9 can also promote tumor cell migration by influencing cytoskeletal organization through their association with different families of adhesion receptors (33). They have also been reported to promote ascites formation in ovarian cancer cells via vascular endothelial growth factor production (34). Surprisingly, activation of MMP-2 and MMP-9 was not related to a decrease in the specific inhibitor of TIMP-1 and TIMP-2 in the GnRH-induced ovarian cancer cell invasion and motility. There was, however, an increase in MMP/TIMP ratios, which also in favor of ECM degradation.

One of the novel findings of the present study is the demonstration that GnRH is a direct transcriptional activator of MMP-2 and MMP-9 synthesis. MMP-2 and MMP-9 expression are reported to be regulated at many levels, including gene activation, mRNA stability, proenzyme activation, and inactivation by endogenous inhibitors in a complex fashion by numerous oncogene and tumor suppressor pathways and conditions of hypoxia (35–38). In this study, we showed that GnRH increased MMP-2 and MMP-9 mRNA expression, and such expression was not due to changes in mRNA stabilities. We further showed that GnRH directly induced MMP-2 and MMP-9 promoter activities. This is the first demonstration that MMP-2 and MMP-9 are target genes of GnRH receptor activation and adding GnRH as a new member of MMP-2 and MMP-9 transcriptional modulators.

Our results identify a JNK signaling pathway that mediates GnRH-stimulated MMP expression, secretion, and cell invasion. The kinetic profile revealed differential regulation of ERK1/2, p38 MAPK, and JNK by GnRH with a sustained signaling through the JNK pathway. The duration of kinase activation is a major determinant for signal outcome in different cellular contexts (39), and its influence may have included invasiveness. In support of this hypothesis, GnRH-stimulated MMP expression and cell invasion were attenuated by specific inhibition of JNK but not ERK1/2 and p38 MAPK. Consistently, a recent report shows that activated JNK levels are much higher in the malignant pleural and peritoneal effusions of patients with advanced-stage ovarian carcinomas, suggesting that activation of JNK may contribute to a more invasive phenotype (40). However, unlike ERK1/2 and p38 MAPK, a role for JNK in tumor invasion or migration has not been widely reported. JNK signaling was shown recently to regulate MMP-2 production and invasiveness in human gliomas (41). The JNK pathway targets multiple transcription factors, including c-Jun and c-Fos (42), ATF (43), and PEA (44), and putative binding sites for these DNA-binding proteins are present in the MMP promoters (45). Whether these putative regulatory elements participate in the GnRH-dependent activation of the MMP-2 and MMP-9 genes remains to be determined.

In summary, our findings emphasize the potential role of GnRH in promoting cellular migration and metastasis-related proteinase activities in GnRH receptor-expressing ovarian tumors. This information provides a mechanistic rationale for the observed GnRH receptor overexpression in advanced-stage ovarian carcinomas. The promising role for GnRH in invasion of ovarian cancer cells in vitro merits further investigation in vivo, which may provide additional insight into its potential as a therapeutic molecular target to decrease metastasis.

Grant support: Hong Kong Research Grants Council grants 7484/04M and 7599/05M (A.S.T. Wong) and Canadian Institutes of Health Research grant 42441 (P.C.K. Leung).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.