[CANCER RESEARCH 61, 7875–7877, November 1, 2001] Prevention and Inhibition of Nasopharyngeal Carcinoma Growth by Antiviral Phosphonated Nucleoside Analogs 1 Shigeyuki Murono, Nancy Raab-Traub, and Joseph S. Pagano 2 Lineberger Comprehensive Cancer Center [S. M., N. R-T., J. S. P.], and Departments of Microbiology and Immunology [N. R-T., J. S. P.] and of Medicine [J. S. P.], University of North Carolina, Chapel Hill, North Carolina 27599-7295 ABSTRACT Nasopharyngeal carcinoma (NPC) is universally associated with EBV infection. We have shown that the phosphonated nucleoside analog, (S)- 1-[3-hydroxy-2-(phosphonylmethoxy)-propyl]cytosine (HPMPC) strongly inhibits growth of NPC xenografts in nude mice by causing apoptosis (J. Neyts et al., Cancer Res., 58, 384 –388, 1998). We, therefore, tested two additional members of this drug family that have different degrees of antiviral activity, 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) and 9-2-(R)-(phosphonomethoxy)propyladenine (PMPA). Intratumoral injec- tion of PMEA (75 l of 2% solution) in C15 NPC xenografts, which are latently infected with EBV, slowed tumor growth moderately, whereas PMPA (75 l of 2% solution) slowed tumor growth only marginally. Compared with the previous results showing complete regression of tu- mor, PMEA had less antitumoral effect than HPMPC, and PMPA had the least. After 4 weeks of preventive treatment, tumors formed in 12.5, 50, and 100% of mice treated with HPMPC, PMEA, and PMPA, respectively, in contrast to the development of tumors in all of the PBS-treated control mice. We also investigated the effect of each drug on the EBV-positive epithelial cell line NPC-KT in vitro. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe- nyltetrazolium bromide (MTT) assay showed inhibition of growth of NPC-KT cells by HPMPC and PMEA, but not by PMPA, which correlates with the results observed in tumor xenografts. Growth inhibition was attributable to induction of apoptosis in NPC-KT cells as indicated by a DNA fragmentation assay. Cleavage of poly(ADP-ribose) polymerase after treatment of NPC-KT cells with HPMPC was observed, which suggested that the apoptosis may be mediated by caspase(s). The apoptotic effects of the drugs are independent of any effects on EBV DNA polymerase, which is not expressed in these latently infected NPCs. These results suggest that HPMPC as well as PMEA could provide an adjunctive treatment for NPC. INTRODUCTION The acyclic nucleoside phosphonates [HPMPC 3 (cidofovir), PMEA (adefovir), and PMPA] have proved to be effective in cell culture sys- tems, animal models, and clinical studies against a wide variety of DNA virus and retrovirus infections. HPMPC has efficacy against herpesvirus (herpes simplex virus types 1 and 2, varicella-zoster virus, cytomegalo- virus, EBV, human herpesvirus 6, 7, and 8), polyomavirus, papillomavi- rus, adenovirus, and poxvirus (vaccinia and molluscum contagiosum viruses) infections; PMEA has efficacy against herpesvirus, hepadnavirus (human hepatitis B virus), and retrovirus (human immunodeficiency virus types 1 and 2, and simian and feline immunodeficiency viruses) and EBV infections; and PMPA has efficacy against both hepadnavirus and retrovirus infections (1–3). These molecules are characterized by a stable phosphonate linkage between the acyclic nucleoside and the phosphate moiety. In contrast to antiviral drugs such as acyclovir and ganciclovir, these drugs bypass the first phosphorylation step by herpesvirus-encoded kinases. Cellular kinases phosphorylate these drugs, and the diphospho- rylated metabolite selectively inhibits the viral DNA polymerases re- quired for viral replication (4). Quite distinct from these antiviral effects, two of these drugs have proved to be effective for tumors in which virally encoded DNA poly- merases are not expressed. HPMPC has a dramatic inhibitory effect on severe recurrent laryngeal papillomatosis caused by human papillomavi- rus (5, 6). HPMPC has also been reported to inhibit growth of xenografts in nude mice that were derived from papillomavirus-positive human cervical cancer cells (7). Moreover, clinically, HPMPC in a gel, applied topically, inhibits cervical dysplastic lesions (8). Finally, PMEA has been shown to inhibit the growth of choriocarcinoma in rats (9). NPC, which is endemic in southern China and Taiwan, is univer- sally associated with EBV infection (10). The EBV genomes in NPC are monoclonal (11), which suggests that NPC develops from a single EBV-infected cell rather than a secondary infection of a proliferating tumor and is consistent with a causal role for EBV in NPC. In NPC, EBV infection is mainly latent, and the tumors contain episomal viral DNA rather than linear genomes. Episomal DNA is replicated by host DNA polymerase, whereas the linear genomes produced in cytolytic infection are replicated by EBV DNA polymerase. NPC is usually treated by irradiation and chemotherapy. We have demonstrated that HPMPC has pronounced apoptotic effects against xenografts of NPC latently infected with EBV and grown in nude mice (12). Here we report that two other less potent members of the phosphonated nucleoside analog family, PMEA and PMPA, also have activity, although less so, against the growth of NPC xenografts. We also demonstrate growth inhibition by induction of apoptosis by HPMPC in an EBV-positive epithelial cell line derived from NPC. MATERIALS AND METHODS NPC Xenografts Grown in Nude Mice. The NPC xenograft C15 was passaged in nude mice as before (12). Intratumoral Treatment. HPMPC was prepared as a 2% solution in PBS. PMEA and PMPA were prepared as 2% solutions in distilled water and were sterilized through filtration. The NPC xenograft C15 was grown s.c. until the size of the tumor reached 0.5–1.0 cm 3 , followed by intratumoral injection with 75 l of drug or PBS per day for 28 consecutive days at almost the same dose used previously (12). Preventive Protocol. Two days after s.c. implantation of the C15 tumor, each mouse was treated daily systemically with 75 l of drug or of PBS by s.c. inoculation. Tumor formation was evaluated after 4 weeks’ treatment. Salvage Treatment with HPMPC. After 4 weeks under the preventive protocol with PMEA or PMPA, tumors formed in some mice (as described in “Results”). These tumors were inoculated directly with 75 l of HPMPC or PBS daily. Tumor volume was evaluated weekly during 21 days of treatment. Cells. NPC-KT (13) is an EBV-positive epithelial cell line derived from fusion between EBV-negative nasopharyngeal epithelial Ad-AH cells and EBV-positive NPC tissue. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics. MTT Assays. NPC-KT cell proliferation was analyzed with the MTT assay (14). Cells were treated with PBS or each drug (1 mM); then the MTT assay was performed on days 1, 3, and 5. Administration of PBS or each drug was Received 4/20/01; accepted 8/27/01. 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. 1 This work was supported by NIH Grant P01CA19014. S. M. was supported in part by the Sumitomo Life Enterprise Group for Social Welfare, Osaka, Japan. 2 To whom requests for reprints should be addressed, at Lineberger Comprehensive Cancer Center, CB #7295, University of North Carolina, Chapel Hill, NC 27599. Phone: (919) 966-8644; Fax: (919) 966-9673; E-mail: Joseph_Pagano@med.unc.edu. 3 The abbreviations used are: HPMPC, (S)-1-[3-hydroxy-2-(phosphonylmethoxy)- propyl]cytosine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NPC, nasopharyngeal carcinoma; PARP, poly(ADP-ribose) polymerase; PMEA, 9-[2- (phosphonomethoxy)ethyl]adenine; PMPA, 9-2-(R)-(phosphonomethoxy)propyladenine; EBER, EBV-encoded small RNA. 7875 Research. on December 15, 2021. © 2001 American Association for Cancer cancerres.aacrjournals.org Downloaded from