Hrk Mediates 2-Methoxyestradiol–Induced Mitochondrial Apoptotic Signaling in Prostate Cancer Cells Free

2-Methoxyestradiol (2-ME) is produced in vivo by catechol-O-methyltransferase–mediated O-methylation of 2-hydroxyestradiol (1). Although it is initially considered to be an inactive end product of estrogen metabolism, 2-ME has emerged as a potential antitumor agent for several types of cancer including prostate cancer (2). The molecular network of 2-ME action is a complex process that involves various pathways, and despite extensive investigation, the precise mechanisms for antitumor activity of 2-ME are not fully elucidated (2, 3).

c-Jun-NH₂-kinase (JNK) activation plays an important role in the mitochondrial apoptotic pathway induced by 2-ME (3). Following 2-ME exposure, activated JNK is translocated to the mitochondria, where it initiates a decrease in membrane potential and subsequent release of cytochrome c (cyt c) into the cytosol leading to caspase activation (4–6). Although the mechanism by which JNK mediates cyt c release is not fully understood, several lines of evidence indicate that the Bcl-2 gene family may be the potential targets of JNK (7). In the unstimulated state, the antiapoptotic proteins Bcl-2 and Bcl-x_L neutralize the function of proapoptotic proteins Bax and Bak, which constitute the pore or channel that permeabilize mitochondria (8). 2-ME–mediated JNK activation regulates the inhibitory function of antiapoptotic proteins by phosphorylation in prostate cancer cells (9, 10). JNK also modulate the activities of proapoptotic BH3-only proteins of the Bcl-2 family, especially Bid, Bim, Bmf, and Bad, at the posttranslational level and by doing so, BH3-only proteins can engage Bax and Bak to release cyt c by inactivating the antiapoptotic proteins (7).

Harakiri (Hrk) belongs to the human BH3-only protein subgroup of the proapoptotic Bcl-2 family and was identified by its ability to bind Bcl-2 and Bcl-x_L in a yeast two-hybrid screen (11). Exogenous expression of Hrk activates cell death and this is repressed by overexpression of Bcl-2 and Bcl-x_L (11, 12). Bax and mitochondrial protein p32 are suggested as direct effector molecules for Hrk action (13, 14). However, despite these studies, the precise molecular mechanism involved in Hrk-mediated cell death remains largely unknown.

It has been suggested that Hrk downregulation contributes to the development and progression of cancers (15). Hrk expression is frequently lost in colorectal and gastric cancer (16), glioblastoma (17), primary central nervous system lymphoma (18), and prostate cancer (19) due to aberrant methylation of its promoter region. Hrk inactivation is also inversely correlated with apoptosis indices in tumors (17–19). Hrk is located in chromosome 12q13 (20) where loss of heterozygosity is often observed in several types of human tumors (21–23). Moreover, Hrk overexpression suppresses cell growth in prostate, breast, and ovarian cancer cells (24). Despite its potential significance, functional role of Hrk has not been defined in cancer.

In this study, we found that 2-ME induces Hrk in a JNK-dependent manner in prostate cancer cells. We also show that Hrk is involved in the 2-ME–induced apoptotic pathway by activating caspase through Bak-mediated cyt c release. Therefore Hrk, a newly identified target of 2-ME action, is a critical downstream effector of JNK-dependent mitochondrial apoptotic signaling pathway of 2-ME in prostate cancer cells.

Human prostate carcinoma cell lines (LNCaP, PC-3, and DU-145) were obtained from the American Type Culture Collection. All cell lines were authenticated at ATCC before purchase by standard DNA typing. Eagle's Minimum Essential Medium (EMEM), RPMI-1640, Opti-MEM, and penicillin/streptomycin mixtures were obtained from the University of California San Francisco Cell Culture Facility. FBS was a product of Atlanta Biologicals. 2-ME and SP600125 were obtained from Sigma and chemical structures are shown in Fig. 6.

The LNCaP and PC-3 cell lines were grown in RPMI-1640. The DU-145 cell line was cultured in EMEM. All culture medium contained 10% FBS and 100 μg/mL penicillin/streptomycin mixture. All cell lines were maintained at 37°C in a humidified atmosphere composed of 5% CO₂/95% air.

Cells were stained with an Annexin V-fluorescein isothiocyanate (FITC)/7-amino-actinomycin D (7-AAD; BD Biosciences) as described by the manufacturer and analyzed by a Cell Lab Quanta SC MPL (Beckman Coulter). Both early (Annexin V-positive, 7-AAD–negative) and late (Annexin V-positive, 7-AAD–positive) apoptotic cells were included in cell death determinations.

Total RNA was isolated using the RNeasy Mini Kit (Qiagen) and was converted into cDNA by using the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer's instructions. To assess gene expression, cDNAs were amplified with the TaqMan Gene Expression Assays and TaqMan Fast Universal PCR Master Mix using the 7500 Fast Real-Time PCR System (Applied Biosystems). To investigate the expression of genes key to apoptosis, the Human Apoptosis RT² Profiler PCR Array (SABiosciences) were used as per manufacturer's instructions.

Whole-cell extracts were prepared using radioimmunoprecipitation assay buffer (Thermo Scientific) containing protease inhibitor cocktails (Roche). For subcellular fractionation, cytosolic and membrane proteins were isolated with a Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific). Immunoblotting was carried out according to standard protocols with antibodies against Hrk (Sigma), cyt c, phospho-JNK, JNK, c-Jun, Bcl-x_L, Bcl-2, Bak, X-linked inhibitor of apoptosis (XIAP; Cell Signaling Technology), phospho-c-Jun (Ser 63/73), JNK1 (Santa Cruz Biotechnology), and FLAG (OriGene). Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin (Santa Cruz) were used to confirm equal loading.

Caspase-3 activation was measured using a Caspase-3 Assay Kit as described by the manufacturer's instructions (BD Biosciences).

Cells were transfected with pCMV6-ENTRY vector expressing the C-terminally FLAG-tagged human Hrk cDNA and empty pCMV6-ENTRY vector as a control (OriGene) using Fugene HD Transfection Reagent (Roche) according to the manufacturer's protocol. For small interfering RNA (siRNA) transfection, siRNA duplexes (20 nmol/L) or universal scrambled negative control (OriGene) was transfected using Lipofectamine 2000 Transfection Reagent (Invitrogen) as described by the manufacturer's instructions. Target specificity and knockdown efficiency were evaluated by real-time (RT) PCR with 3 different sets of siRNA duplexes at different concentrations.

Cell lysates were prepared in lysis buffer (20 mmol/L Tris-pH 7.4, 135 mmol/L NaCl, 1.5 mmol/L MgCl₂, 1 mmol/L EGTA, 10% glycerol) containing 1% CHAPS (Cell Signaling Technology), supplemented with protease inhibitor cocktails (Roche). Immunoprecipitation was conducted with a Pierce Direct IP Kit (Pierce) according to the manufacturer's instructions.

2-ME–Treated cells were stained with CMXRos (Mitotracker Red; Molecular Probes) in PBS for 20 minutes at 37°C, and analyzed by a Cell Lab Quanta SC MPL (Beckman Coulter).

Values are presented as the mean ± SEM based on results obtained from at least 3 independent experiments. Statistical significance was evaluated by conducting a 2-tailed unpaired Student t test using GraphPad PRISM Software. A P < 0.05 was regarded as statistically significant.

Dose-dependent apoptosis assays show that 2-ME was effective at a concentration of 1 μmol/L (Fig. 1A), and modest degrees of apoptotic cell death were noted after 12 hours of 2-ME exposure reaching maximal levels after 72 hours (Fig. 1B). On the basis of these results, androgen-dependent LNCaP and androgen-independent PC-3 cells and 1 μmol/L of 2-ME were used in subsequent experiments.

To identify 2-ME signaling pathways, we looked for changes in gene expression in LNCaP cells exposed to 2-ME using the Human Apoptosis RT² Profiler PCR Array. Among several affected genes, we detected an increase of TP53 gene expression similar to a previous report showing its induction by 2-ME (25). Also, Hrk was induced 6.7-fold over controls (Table 1). A significant increase in Hrk mRNA expression was detected as early as 1 hour after 2-ME exposure in both LNCaP and PC-3 cells (Fig. 1C). An increase in Hrk protein expression was also noted after 6-hour exposure of 2-ME reaching maximum levels after 36 hours in LNCaP and 24 hours in PC-3 cells (Fig. 1D).

We also evaluated whether mRNA expression of other BH3-only proteins such as Bim, Puma, and Noxa are influenced by 2-ME. Induction of Puma and Noxa was at modest levels compared with Hrk in LNCaP cells (Supplementary Fig. S1). As Puma and Noxa are p53-dependent genes, we examined induction of these genes in PC-3 and DU-145 cells, which have mutated p53. Unlike LNCaP cells, which have wild-type p53, Puma and Noxa were not increased by 2-ME in PC-3 and DU-145 cells. Bim was induced only in PC-3 cells (Supplementary Fig. S1).

2-ME caused an increase in the level of phospho-JNK without detectable changes in total JNK levels (Fig. 2A). This was followed by c-Jun phosphorylation, paralleling the increase in JNK phosphorylation. The c-Jun protein level was significantly increased, the probable consequence of autoregulation (Fig. 2A).

Although JNK regulation of Hrk levels has been studied in cultured neurons and pancreatic β-cells (26–28), it is unknown in cancers. Therefore, we examined whether JNK activation is required for Hrk upregulation in prostate cancer cells using JNK inhibitors. SP600125, a potent JNK inhibitor, suppressed Hrk induction by 2-ME (Fig. 2B). As a further test of specificity, we conducted selective knockdown of the JNK pathway using a siRNA. JNK1 siRNA significantly reduced the level of endogenous JNK1 protein (Fig. 2C) and resulted in a dramatic decrease in Hrk mRNA induction (Fig. 2D). In addition, c-Jun siRNA efficiently suppressed endogenous c-Jun (Fig. 2E) and led to inhibition of Hrk mRNA induction in prostate cancer cells (Fig. 2F). These results indicate that Hrk is regulated by JNK signaling in prostate cancer cells.

To investigate functional significance of Hrk in 2-ME–mediated apoptosis of prostate cancer cells, we conducted knockdown experiments using Hrk-specific siRNA. Transfection with 2 different Hrk siRNAs resulted in a dramatic reduction in endogenous levels of Hrk mRNA (Fig. 3A) and suppressed 2-ME–mediated induction of Hrk mRNA and protein compared with a control siRNA (Fig. 3A and B). Hrk knockdown did not affect cell viability under basal conditions, but significantly reduced the cytotoxic effect of 2-ME (Fig. 3C and Supplementary Fig. S2). Also Hrk siRNA-1, which had a more significant knockdown effect than siRNA-2, caused less 2-ME–induced cell death than siRNA-2 (Fig. 3A–C).

As 2-ME potently induces apoptosis through the mitochondrial apoptotic pathway (29) and Hrk is predominantly located in the mitochondria (Supplementary Fig. S3), we examined the effect of Hrk knockdown on the mitochondrial apoptotic events triggered by 2-ME. As shown in Fig. 3D, 2-ME caused a significant increase of CMXRos-negative cells indicating the reduction of mitochondrial membrane potential but Hrk knockdown reduced this change. In a parallel experiment, no difference was observed in total cell number by 2-ME treatment (Supplementary Fig. S4). This result excludes the possibility that the decrease in CMXRos-stained cells is not a secondary result caused by a reduction in mitochondrial number but rather the result of a decrease in membrane potential following 2-ME exposure. Hrk knockdown also diminished 2-ME–induced cyt c release into the cytosol from mitochondria (Fig. 3E) and subsequent caspase-3 activation (Fig. 3F). Collectively, these results indicate that Hrk plays an important functional role in 2-ME–induced prostate cancer cell apoptosis by targeting mitochondria.

Hrk physically interacts with Bcl-2 and Bcl-x_L, but not Bax or Bak (11). Hrk-induced cell death is repressed by overexpression of Bcl-2 and Bcl-x_L (11, 12). Therefore, we hypothesized that Hrk triggers apoptosis by neutralizing Bcl-2 and/or Bcl-x_L rather than directly activating Bax and/or Bak. Reduction of 2-ME–induced cell death with knockdown of Bak (Fig. 4A and B) but not Bax (Supplementary Fig. S5) indicates that Bak activation is a 2-ME downstream apoptotic signal. Moreover, Bak knockdown diminished 2-ME–induced cyt c release into cytosol (Fig. 4C) and subsequent caspase-3 activation (Fig. 4D) consistent with the results obtained by Hrk knockdown (Fig. 3E and F). In prostate cancer cells, Bak binds with Bcl-x_L, but not with Bcl-2 (Supplementary Fig. S6). These results led us to examine whether Hrk affected the association between Bak and Bcl-x_L.

While the levels of Hrk interacting with Bcl-x_L were increased by 2-ME, the level of Bak associated with Bcl-x_L was diminished (Fig. 4E). This change is abolished by Hrk knockdown (Fig. 4F). We subsequently evaluated whether overexpression of Hrk affects the interaction of Bak with Bcl-x_L. Consistent with 2-ME treatment, the level of Bak associated with Bcl-x_L was reduced in Hrk transfectants compared with the vector control (Fig. 4G). Together, these data show that Hrk contributes to 2-ME–mediated apoptosis by liberating Bak from the complex with Bcl-x_L.

BH3 domain of BH3-only proteins mediates its association with antiapoptotic proteins and is required for cell death induction (30). To determine whether the BH3 domain of Hrk is critical for 2-ME–mediated prostate cancer cell apoptosis, we engineered a mutant form of Hrk lacking the BH3 domain (Hrk ΔBH3). Substantial amounts of both wild-type and mutant Hrk proteins were expressed in transiently transfected prostate cancer cells (Supplementary Fig. S7A). Absence of the BH3 domain dramatically reduced the ability of Hrk to induce apoptotic cell death (Supplementary Fig. S7B) and to interact with Bcl-x_L (Supplementary Fig. S7C). As a consequence, there was no change in the level of Bak associated with Bcl-x_L by Hrk ΔBH3 overexpression (Supplementary Fig. S7D). These results indicate that the BH3 domain-mediated interaction with Bcl-x_L is critical for the proapoptotic activity of Hrk in prostate cancer cells.

To investigate additional downstream target molecules of Hrk activation, we evaluated gene mRNA levels altered by 2-ME (Table 1) in cells overexpressing Hrk (Fig. 5A). From the genes analyzed, we found that only XIAP mRNA was significantly decreased by 40% compared with cells transfected with vector control (Fig. 5B). We also verified the decrease in XIAP mRNA expression by 2-ME (Fig. 5C). Furthermore, XIAP protein expression was significantly reduced by both 2-ME exposure (Fig. 5D) and Hrk overexpression (Fig. 5E). Next, we evaluated XIAP levels in cells transfected with Bak siRNA after 2-ME treatment to assess whether Hrk activation of Bak is required for the regulation of XIAP level. As shown in Fig. 5F, Bak knockdown abolished the reduction of XIAP protein in cells treated with 2-ME. Therefore, these data indicate that Hrk participates in the 2-ME-mediated downregulation of XIAP by activating Bak in prostate cancer cells.

In this study, we identified the BH3-only protein, Hrk, as a new 2-ME target gene, acting downstream of the JNK signaling pathway in prostate cancer cells. Hrk is a particularly interesting candidate because: (i) it belongs to the proapoptotic Bcl-2 gene family (11, 12), (ii) it is downregulated in prostate cancer and loss of Hrk expression is closely associated with decreased apoptosis in high-grade prostate tumors (19), (iii) its expression is regulated by JNK (Fig. 2) which is a downstream molecule of 2-ME (3, 26), (iv) unlike other BH3-only proteins such as Bim, Puma, and Noxa, 2-ME–mediated Hrk induction is not affected by androgen dependency or p53 status of cells (Supplementary Fig. S1).

Although it has been well described that JNK activation is involved in the 2-ME–mediated cytotoxic effect in cancer cells, the upstream components regulating JNK activation are still unknown. Davoodpour and Landstrom suggest that Smad7, which is stabilized by 2-ME, is required for 2-ME–induced JNK activation (6). In addition, death-associated protein kinase 1 (DAPK1), which was induced by 2-ME in our study (Table 1) also regulates JNK signaling through protein kinase D1 as described by other investigators (31). Therefore, DAPK1 and Smad7 may be downstream signaling components of 2-ME that activate the JNK cascade. Further investigation regarding the interaction between DAPK1 and Smad7 will be needed to delineate the precise molecular mechanisms of 2-ME–mediated JNK activation.

JNK activation occurs rapidly following 2-ME treatment and plays an important role in the 2-ME–mediated mitochondrial apoptotic pathway (3). Although JNK-induced cyt c release is a critical step for downstream apoptotic signaling, the mechanism is unclear. 2-ME–induced JNK phosphorylation and subsequent translocation to the mitochondria was proposed as a possible explanation (4). On the basis of the findings that JNK mitochondrial signaling modulated the activities of BH3-only proteins at the posttranslational level in other cell types by different apoptotic stimuli (7), it is highly possible that BH3-only proteins might also be involved in 2-ME activation of JNK-mediated cyt c release. In this study, we have shown that the BH3-only protein Hrk is a key molecule that links JNK signaling to mitochondria targeted by the interaction of Bak with Bcl-x_L to induce apoptosis accompanied by cytosolic release of cyt c.

How BH3-only proteins trigger the activation of Bax or Bak has been a central issue in the regulation of apoptosis by the Bcl-2 gene family (8). BH3-only proteins could promote activation of Bax or Bak via their ability to inactivate antiapoptotic Bcl-2 family members. Alternatively, Bax or Bak may be activated via direct association with certain BH3-only proteins. We observed that in addition to Hrk, 2-ME upregulates other BH3-only proteins Bim, Puma, and Noxa (Supplementary Fig. S1). Bim and Puma can directly activate Bax and Bak, and they also sequester all antiapoptotic Bcl-2 family members (8). Noxa neutralizes only Mcl-1 and A1 and promotes Mcl-1 degradation (32). Despite the idea that Hrk might promote cell death by inhibiting the protective activity of Bcl-2 and Bcl-x_L (11), the precise mechanism of Hrk has not been extensively studied. The results of this study suggest a mechanism for Hrk-induced apoptotic cell death by regulating the interaction of Bak with Bcl-x_L. In untreated prostate cancer cells, Bcl-x_L sequesters active forms of Bak; upon 2-ME treatment, transcriptionally activated Hrk displaces Bak from the heterodimer with Bcl-x_L, thus releasing Bak (Fig. 4E and G). This promotes Bak oligomerization on the mitochondrial membrane and results in cyt c release followed by induction of apoptosis. Thus, our data support the indirect activation or displacement model to explain how Hrk activates Bak in addition to the results showing no association of Bim and Puma with Bak after 2-ME treatment in prostate cancer cells (I. Chang; unpublished data). However, this does not entirely rule out the possibility that the direct activation model might also be involved in this mechanism. For instance, Bim and Puma upregulated by 2-ME may independently cause Bax or Bak activation. It is also possible that Bim and Puma may interact with Bax instead of Bak. Kuwana and colleagues observed that Hrk seemed to activate Bax directly although it was modest and the significance of the activation was uncertain (33). Therefore, whether 2-ME–mediated induction of BH3-only proteins activates Bak or Bax directly or indirectly remains a matter of discussion and needs further clarification.

On the basis of previous findings, Hrk may regulate apoptotic cell death via several different mechanisms. Rizvi and colleagues reported that C6 ceramide induces not only Hrk expression through JNK phosphorylation but also its translocation to mitochondria where Hrk interacts with the mitochondrial protein p32 and the BH3-only protein Bad. These protein interactions eventually lead to mitochondria dysfunction and cell death in human corneal stromal fibroblasts (34). It will therefore be of interest to determine whether 2-ME also induces translocation of Hrk to mitochondria to interact with p32 and Bad, and plays a role in Hrk proapoptotic activity in prostate cancer cells. Unlike Bim, Puma, and Bid, other BH3-only proteins selectively neutralize only a subset of antiapoptotic Bcl-2 proteins. Thus, a combination of several BH3-only proteins binding to complementary subsets is required to promote apoptosis; for example, Bad binding to Bcl-2, Bcl-x_L, and Bcl-w, plus Noxa binding to Mcl-1 and A1. In vitro competitive binding assays have shown that Hrk has high binding affinity to Bcl-w and A1 in addition to Bcl-x_L but not to Mcl-1 (35). Although binding to Bcl-2 is controversial (35), Hrk was originally identified as a Bcl-2 binding protein (11) and we have also verified its interaction with Bcl-2 in prostate cancer cells (I. Chang; unpublished data). Therefore, in addition to Bad, Hrk and Noxa can completely neutralize antiapoptotic proteins. Intriguingly, antiapoptotic proteins Bcl-2, Bcl-x_L, and Mcl-1 are significantly expressed and confer resistance to apoptotic signaling in prostate cancer cells (36). Hrk depletion causes partial reduction of 2-ME–induced apoptosis (Fig. 3C). This can be explained by the incomplete knockdown of Hrk but also indicates the possibility that Hrk activation is not entirely responsible for induction of apoptosis and other mechanisms are involved. Although 2-ME causes diverse apoptotic signaling, it is an interesting possibility that both Hrk and Noxa are involved in 2-ME–mediated prostate cancer cell death.

XIAP is often overexpressed in malignant cells and elevated levels of XIAP increase resistance to apoptosis (37). It has been shown that 2-ME reduces the levels of XIAP in prostate cancer cells (38, 39). However, the underlying mechanism is largely unknown. In this study, we found that Bak activation is essential for 2-ME–mediated XIAP reduction and Hrk plays an important role in the process by Bak activation and subsequent cyt c release. It has been reported that 2-ME also induces a second mitochondrial-derived activator of caspase (Smac) (4). Therefore, in addition to cyt c-mediated caspase activation, the release of Smac from mitochondria presumably mediated by Bak activation may facilitate the processing of caspase-3 by competitive binding to XIAP and liberate the bound caspases. The increased caspase activation could further inactivate XIAP by proteolysis.

It is well known that antiapoptotic proteins Bcl-2 and Bcl-x_L are substantially expressed and confer resistance to apoptotic signaling in prostate cancer cells (36). 2-ME activation of JNK phosphorylates Bcl-2 and Bcl-x_L to suppress their antiapoptotic function (9, 10). Aside from this mechanism, Hrk induction is a newly discovered inhibitor of Bcl-x_L and an initiator of apoptosis in human prostate cancer cells. As Hrk expression is downregulated in prostate cancers through promoter methylation (19), a combination of 2-ME with a DNA methylation inhibitor may enhance the Hrk-mediated cell death pathway. According to Lin and colleagues, Hrk is the most effective suppressor of cancer cell growth compared with p53, BRAC1, and PTEN in prostate, breast, and ovarian cancer cells which are estrogen-associated cancers expressing high levels of Bcl-2 and Bcl-x_L (24). Moreover, breast and ovarian cancer cells are very sensitive to 2-ME (2). Therefore, it would be of interest to determine whether Hrk is involved in the 2-ME–mediated apoptotic pathway in these types of cancer.

In summary, we have identified Hrk as a critical mediator of 2-ME–induced apoptosis in prostate cancer cells. Our study shows that Hrk links 2-ME activation of JNK signaling to the mitochondrial apoptotic pathway and contributes to 2-ME–mediated XIAP reduction by releasing Bak from the complex with Bcl-x_L. This study provides useful insights into the molecular mechanisms underlying Hrk-mediated apoptosis and also into the future development of 2-ME–based therapeutic strategies for prostate cancer treatment.

No potential conflicts of interest were disclosed.

Conception and design: Y. Tanaka, I. Chang, R. Dahiya

Development of methodology: Y. Tanaka, I. Chang, S. Majid, G. Deng, R. Dahiya

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Tanaka, I. Chang, V. Shahryari, S. Fukuhara

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Tanaka, I. Chang, S. Majid, S. Saini

Writing, review, and/or revision of the manuscript: Y. Tanaka, I. Chang, M.S. Zaman, R. Dahiya

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Tanaka, S. Saini, S. Yamamura, T. Chiyomaru, R. Dahiya

Study supervision: Y. Tanaka, R. Dahiya

The authors thank Dr. Roger Erickson for critical reading of the manuscript.

This study was supported by the Veterans Affairs Merit Review grant and Research Enhancement Award Program (Y. Tanaka).

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.