Potent Antiviral Activity of Topoisomerase I and II Inhibitors against Kaposi's Sarcoma-Associated Herpesvirus

Abstract

The lytic DNA replication of Kaposi's sarcoma-associated herpesvirus (KSHV) initiates at an origin (ori-Lyt) and requires trans-acting elements, both viral and cellular. We recently demonstrated that several host cellular proteins, including topoisomerases I and II (Topo I and II), are involved in KSHV lytic DNA replication (Y. Wang, H. Li, Q. Tang, G. G. Maul, and Y. Yuan. J. Virol. 82: 2867–2882, 2008). To assess the importance of these topoisomerases in viral lytic replication, shRNA-mediated gene silencing was used. Depletion of Topo I and II severely inhibited viral lytic DNA replication as well as virion production, suggesting essential roles of these cellular proteins in viral DNA replication. The discovery of Topo I and II as enzymes indispensable for KSHV DNA replication raises a possibility that these cellular proteins could be new targets of therapeutic approaches to halt KSHV replication and treat KSHV-associated diseases. In this report, we examined one Topo I inhibitor and several Topo II inhibitors (inclusive of Topo II poison and catalytic inhibitors) as potential therapeutic agents for blocking KSHV replication. The Topo II catalytic inhibitors in general exhibited marked inhibition on KSHV replication and minimal cytotoxicity. In particular, novobiocin, with the best selectivity index (SI = 31.62) among the inhibitors tested in this study, is effective in inhibiting KSHV DNA replication and virion production but shows little adverse effect on cell proliferation and cycle progression in its therapeutic concentration, suggesting its potential to become an effective and safe drug for the treatment of human diseases associated with KSHV infection.

INTRODUCTION

Kaposi's sarcoma (KS) is a multicentric malignant neoplasm of endothelial origin and the most common malignancy associated with HIV infection. About 20% of AIDS patients develop KS, and most cases (60%) manifest as oral lesions (1). Oral KS is often the first presenting sign of AIDS. With the development of AIDS-KS, KS lesions progress to skin and internal organs, including the lungs and gastrointestinal track. KS has proven to be a progressive, fatal disease, which contributes greatly to the morbidity and mortality of AIDS. In addition, it was found that patients with KS in their oral mucosa had a higher risk of death than those with KS appearing only on the skin. Patients with oral KS generally have a less than 10% 5-year survival rate. Despite its dramatic decrease in frequency since the advent of highly active antiretroviral therapy (HAART), KS remains the most common AIDS-associated cancer in the United States. In addition to this AIDS-associated (epidemic) form, other epidemiological forms of KS include the classic (sporadic), African (endemic), and immunosuppression-associated (iatrogenic) forms (18).

There is currently no definitive cure for KS. For classic KS, classic cancer therapies are generally used to treat patients, which include surgical excision and radiation therapy for patients with a few lesions in a limited area and chemotherapy for patients with extensive or recurrent KS (1). The chemotherapeutics which have been approved by the FDA and are often used include liposomal anthracycline products (liposomal doxorubicin or liposomal daunorubicin), paclitaxel, and alpha interferon (34, 49). However, the majority of these agents are associated with serious side effects, and the tumor response to any chemotherapeutic regimen is only transient.

Kaposi's sarcoma-associated herpesvirus (KSHV), also named human herpesvirus 8 (HHV-8), has been proven to be an etiologic agent of Kaposi's sarcoma. Irrespective of the source or clinical subtype (i.e., classic, AIDS-associated, African endemic, and iatrogenic KS), almost 100% of KS lesions are found to carry KSHV. KSHV is also unequivocally associated with two B-cell-associated lymphoproliferative disorders, namely, primary effusion lymphoma (PEL) and the plasma cell variant of multicentric Castleman's disease (MCD) (1, 18).

In KS lesions, most spindle cells of endothelial origin are latently infected with KSHV, but a small percentage of these cells undergo spontaneous lytic replication (39, 44, 51). Increasing evidence suggests that the small percentage of cells experiencing viral lytic replication plays an important role in viral pathogenicity. Unlike other DNA tumor viruses, latent infection with KSHV is not sufficient to sustain KS tumorigenesis. Studies have shown that the lytic replication cycle directly contributes to viral tumorigenesis by spreading viruses to target cells and providing paracrine regulation for KS development (5). It was found that infection of endothelial cells by KSHV transforms these cells into spindle-shaped KS cells. However, when these spindle-shaped cells proliferate, they quickly lose the KSHV genome and revert to normal cells. Therefore, lytic replication of KSHV in the spindle cells, release of viral particles, and reinfection of endothelial cells become critical in sustaining the population of latently infected cells and maintaining the KS lesion (19). Thus, blockade of KSHV lytic replication in the spindle cells and prevention of extracellular virions from reinfecting fresh endothelial cells should lead to regression of KS lesions and could be the strategies for treatment of KS or other KSHV-associated human diseases.

Since lytic replication of KSHV is crucial for viral propagation and pathogenicity, the study of viral lytic DNA replication will shed light on new strategies for halting viral replication and treating KSHV-associated human diseases. Lytic DNA replication initiates at an origin (ori-Lyt) and requires trans-acting elements, both viral and cellular (27, 47, 48, 50). Recently, we demonstrated that several host cellular proteins, such as topoisomerases I and II (Topo I and II), RecQL, MSH2/6, and poly[ADP-ribose] polymerase 1 (PARP-1), are involved in KSHV lytic DNA replication (48). Discovery of the involvement of these cellular proteins in viral DNA replication raises a possibility that these proteins could be new targets of therapeutic approaches to block KSHV replication and treat KSHV-associated human diseases.

In this report, we have been exploring the possibility of topoisomerases I and II to serve as anti-KSHV drug targets and the potential of their inhibitors to become drugs for treatment of KS and other KSHV-associated diseases. Topo I is an enzyme needed to release the topological stress created by DNA unwinding during DNA replication by nicking and religating DNA ahead of the replication fork (2). Topo II also modulates the topological state of DNA by making transient double-stranded breaks in DNA, which is needed for converting replicating intermediates into a mature replication product (36). By conducting shRNA-mediated silencing of Topo I and II gene expression, our results suggest that both Topo I and II are required for KSHV DNA replication and thus may serve as potential drug targets for the treatment of KSHV-associated disease. This result encouraged us to explore the potentials and values of inhibitors to Topo I and II to inhibit KSHV lytic DNA replication, to halt the viral production, and to treat KSHV-associated diseases.

MATERIALS AND METHODS

Cells.

The primary effusion lymphoma cell line BCBL-1, which carries latent KSHV and was established by Renne et al. (35), was obtained from the NIH AIDS Research and Reference Reagent Program. The cells were grown in RPMI 1640 medium (Gibco-BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Gibco-BRL), penicillin-streptomycin (50 units/ml), and fungizone (1.25 μg/ml amphotericin B and 1.25 μg/ml sodium deoxycholate). Human embryonic kidney (HEK) 293T cells were obtained from ATCC and were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, and antibiotics (penicillin-streptomycin and fungizone at the same concentrations).

Antibodies.

The anti-Topo I monoclonal antibody was provided by Daniel Simmons at the University of Delaware. The anti-Topo IIβ polyclonal antibody was provided by Gary Gorbsky at the Oklahoma Medical Research Foundation. The anti-K8 polyclonal antibody was obtained from Jae Jung at the New England Regional Primate Research Center. The anti β-actin polyclonal antibody was purchased from Cell Signaling Technology (Danvers, MA).

shRNA-mediated gene silencing technique.

Mission shRNA gene sets against human Topo I and IIβ, respectively, were purchased from Sigma-Aldrich. This shRNA system is a lentiviral vector-based RNA interference library against annotated human genes, which generates siRNAs in cells and mediates gene-specific RNA interference for extended periods of time. The Topo I set consists of four individual shRNA lentiviral vectors in pLKO.1-puro plasmids against different target sites of Topo I mRNA. The Topo IIβ set contains five clones targeting different sites of Topo IIβ mRNA. A nontargeting or control shRNA vector that activates the RNAi pathway without targeting any known human gene (SHC002) was also purchased (Sigma-Aldrich). Each of the shRNA vectors and the control vector were used to prepare lentiviral stocks by cotransfecting 293T cells with the shRNA vector and two packaging vectors (pHR′8.2DR and pCMV-VSV-G) at a ratio of 4:2:1, respectively. Three days posttransfection, the culture media that contained shRNA retroviruses were harvested, centrifuged (500 × g for 10 min at 4°C), and filtered through a 0.45-μm-pore-size filter to ensure removal of any nonadherent cells.

BCBL-1 cells were transduced with the shRNA-encoding lentivirus stocks in the presence of Polybrene (8 μg/ml). Transduced cells were selected with puromycin (2 μg/ml) for a week. Efficacies of these shRNAs in knockdown of the respective proteins were assayed by Western blotting with specific antibodies. BCBL-1 cells (4 × 10⁵ cells/ml of culture) stably expressing the Topo I/Topo IIβ/control shRNAs were treated with TPA (20 ng/ml) to induce KSHV lytic replication.

Western blotting.

Cells were lysed with ice-cold lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 30 mM NaF, 5 mM EDTA, 10% glycerol, 40 mM glycerophosphate, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1% Nonidet P-40, 1 mM sodium orthovanadate) supplemented with protease inhibitor cocktail (Roche). The cell lysates were homogenized and centrifuged at 13,000 rpm for 5 min at 4°C. The whole-cell extract was resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked in 5% dried milk in 1× phosphate-buffered saline (PBS) plus 0.2% Tween 20 and then incubated with diluted primary antibodies for 2 h at room temperature or overnight at 4°C. Anti-rabbit or anti-mouse immunoglobulin G antibodies conjugated to horseradish peroxidase (Amersham) were used as the secondary antibodies. An enhanced chemiluminescence system (Amersham) was used for detection of antibody-antigen complexes.

Analysis of viral replication in BCBL-1 cells.

For intracellular KSHV DNA, tetradecanoyl phorbol acetate (TPA)-induced and uninduced BCBL-1 cells were harvested 2 days postinduction, and DNA was purified using the DNeasy tissue kit (Qiagen) according to the manufacturer's protocol. KSHV genomic DNA was quantified by real-time PCR on a Roche LightCycler instrument using the LightCycler FastStart DNA Master^Plus SYBR green kit with primers directed to LANA (forward, 5′-CGCGAATACCGCTATGTACTCA-3′; reverse, 5′-GGAACGCGCCTCATACGA-3′). The intracellular viral genomic DNA in each sample was normalized with the amount of GAPDH determined also by real-time DNA PCR by using primers directed to GAPDH (forward, 5′-AGCCACATCGCTCAGACAC-3′; reverse, 5′-GCCCAATACGACCAAATCC-3′).

Extracellular virions were pelleted from the medium supernatant of BCBL-1 cultures 5 days postinduction as detailed earlier (37) and were resuspended in 1× phosphate-buffered saline (PBS) in 1/100 of the original volume. Concentrated viruses were first treated with Turbo DNase I (Ambion) at 37°C for 1 h to remove any contaminating DNA outside viral particles. Virion DNA was liberated by digestion with lysis buffer (AL) and proteinase K (supplied with the DNeasy tissue kit [Qiagen]) and extracted with phenol-chloroform. Extracted DNA was precipitated with ice-cold ethanol, and the final DNA pellet was dissolved in Tris-EDTA (TE) buffer. The KSHV genomic DNA in virions was measured by real-time DNA PCR with primers directed to LANA (ORF 73) as described above. Virion DNA copy numbers were calculated from a standard curve using BAC36 DNA. KSHV virion numbers were presented as the copy numbers of viral genomic DNA per milliliter of culture supernatant.

Chemicals and cell treatment.

Camptothecin, etoposide, ellipticine, and novobiocin were purchased from Sigma-Aldrich (St. Louis, MO) and merbarone was purchased from Calbiochem (Gibbstown, NJ). Novobiocin was prepared as aqueous stock, while the others were dissolved in dimethyl sulfoxide (DMSO).

Each inhibitor with different concentrations was added to BCBL-1 cells (4 × 10⁵ cells/ml of culture) 3 h after TPA induction. Two days postinduction, cells were collected from TPA-induced and uninduced cultures to determine the intracellular viral DNA content in cells as detailed above. For each treatment, the KSHV DNA content value from TPA-induced cells was subtracted by that from uninduced cells. These corrected values were divided by those from the control, non-drug-treated cells and then represented on the y axes of dose-response curves: y axis value = (TPA_X − no TPA_X)/(TPA₀ − no TPA₀), where X is any concentration of the drug and 0 represents nontreatment.

The 50% DNA replication inhibitory concentration (IC₅₀) for each compound was calculated from the dose-response curve with the aid of Graphpad Prism software.

Five days postinduction, cell culture media were collected and virion particles were cleared by passing through 0.45-μm-pore-size filters, and extracellular virions were then pelleted from the medium supernatant of the cultures. To remove any contaminating DNA outside viral particles, the concentrated viruses were treated with Turbo DNase I (Ambion) at 37°C for 1 h followed by proteinase K digestion. The amounts of virion particles from the media were determined by quantifying encapsidated viral DNA by real-time PCR, and values were corrected as described above. The 50% antiviral effective concentration (EC₅₀) for each compound was calculated from the dose-response curve with the aid of GraphPad Prism software.

Cytotoxicity assay.

The cell viabilities of BCBL-1 cells after treated or untreated with chemicals were assessed by the counting of Trypan blue-stained cells 2 or 5 days posttreatment using a light microscopy. Cell viabilities were defined relative to control cells (non-drug treated) and represented on the y axes of dose-response curves: y axis value = TPA_X/TPA₀, where X is any drug concentration and 0 represents nontreatment.

The 50% cytotoxic concentration (CC₅₀) for each compound was calculated from these dose-response curves with the aid of Graphpad Prism software.

Cell proliferation assay.

BCBL-1 cells (starting with 2 × 10⁵ cells/ml) were treated with topoisomerase inhibitors for 5 days at two different concentrations: IC₅₀ and an excess concentration (5× IC₅₀). Cell samples were daily collected, stained with Trypan blue, and counted. To provide a constant cellular growth, fresh medium (supplemented with or without the drug) was added to these cultures every 2 days.

Cell cycle assay.

BCBL-1 cells (starting with 4 × 10⁵ cells/ml) were treated with topoisomerase inhibitors for 48 h at two different concentrations: IC₅₀ and 5× IC₅₀. Cells were collected 48 h posttreatment, fixed with cold ethanol at 70% for 15 min, permeabilized, and stained with PI solution (50 μg/ml of propidium iodide, 0.1 mg/ml of RNase A, and 0.05% of Triton X-100) for 2 h. DNA content was measured using a FACStar PLUS cell sorter flow cytometer (Becton Dickinson).

Transient-transfection DNA replication assay.

Plasmid pOri-A was constructed by cloning an EcoRI-PstI fragment (nucleotides 22409 to 26491) of KSHV DNA in pBluescript at the EcoRI/PstI site as previously described. This EcoRI-PstI fragment contains the ori-Lyt sequence of the KSHV genome (27). pCR3.1-ORF50 was constructed by cloning the ORF50 gene sequence of KSHV into pCR3.1 vector (Invitrogen). These constructs are described in detail in Lin et al. (27).

To assay the effects of each compound on ori-Lyt-dependent DNA replication, BCBL-1 cells were transfected with plasmids pOri-A (2.5 μg) and pCR3.1-ORF50 (2.5 μg) by nucleoporation (Amaxa) and cultured in the media with each drug in a wide range of concentration. Seventy-two hours posttransfection, extrachromosomal DNAs were prepared from cells using the Hirt DNA extraction method as follows. Cells were lysed in 700 ml lysis buffer (10 mM Tris-HCl [pH 7.4], 10 mM EDTA, and 0.6% SDS). Chromosomal DNA was precipitated at 4°C overnight by adding 5 M NaCl to a final concentration of 0.85 M. Cell lysates were centrifuged at 4°C at 14,000 rpm for 30 min. The supernatant containing extrachromosomal DNA was subjected to phenol-chloroform extraction, followed by ethanol precipitation. The DNA was treated with RNase A at 25°C for 30 min and then with proteinase K at 50°C for 30 min. Five micrograms of DNA was digested with KpnI/SacI or KpnI/SacI/DpnI (New England BioLabs). The DNAs were separated by electrophoresis on 1% agarose gels and transferred onto GeneScreen membranes (Perkin Elmer, Boston, MA). The Southern blots were hybridized with a ³²P-labeled pBluescript plasmid in 5× SSC, 2× Denhardt's solution, 1% SDS, and 50 mg/ml denatured salmon sperm DNA at 68°C.

RESULTS

Evaluation of Topo I and II for their potential to serve as anti-KSHV drug targets.

Our recent study identified Topo I and II as components of the KSHV DNA replication complex that is formed on viral ori-Lyt DNA, suggesting their involvement in viral DNA replication (48). Therefore, we decided to explore these topoisomerases as potential anti-KSHV drug targets. To be an effec-tive drug target, the candidate protein has to be essential for viral DNA replication. To assess whether these two topoisomerases are essentially required for KSHV DNA replication, we attempted to knockdown Topo I and Topo II expression in cells through a short-hairpin RNA (shRNA)-based approach and examined the effects on KSHV DNA replication and virion production. Mission shRNA gene sets against human Topo I and Topo IIβ were purchased from Sigma-Aldrich. Each shRNA set consists of four or five individual shRNA lentiviral vectors against different sites of the target mRNA sequence. After introduction into BCBL-1 cells by lentiviral transduction, the expression of the target protein was evaluated by Western blotting. One Topo I shRNA was found to effectively downregulate the expression of topoisomerase I in comparison to the control. Two Topo IIβ shRNA clones effectively reduced the expression of Topo II, which was shown on the Western blot as the upper band. A previous study has identified the upper protein band to be Topo IIβ and associated with the KSHV ori-Lyt DNA (48). These shRNA clones, which were chosen for further studies, did not affect the expression of viral protein K8 (Fig. 1 and 2), indicating that the shRNAs knocked down their target mRNAs specifically rather than inhibiting the general transcription process in cells. BCBL-1 cells stably expressing the Topo I and II shRNAs were induced with TPA. Two days postinduction, intracellular viral genomic DNA content was determined by real-time PCR. In addition, the extracellular virion titers were also estimated 5 days postinduction. Results showed that silencing of Topo I expression brought about reduced levels of intracellular viral DNA (20% of the level of the cells with control shRNA) and virions (38% in comparison with the control) (Fig. 1). Depletion of Topo IIβ expression by clone 1 and 2 shRNAs resulted in drastic reductions of viral DNA replication (36% and 14%, respectively) and extracellular virion titers (7% and 18%) in comparison with the control (Fig. 2). Taken together, the results indicate that both Topo I and IIβ are essentially required for KSHV lytic DNA replication and have the potential to be effective drug targets for the treatment of KSHV-associated diseases.

Fig 1.

Effects of Topo I knockdown by short-hairpin (shRNA)-mediated silencing on KSHV DNA replication and virion production. An shRNA lentivirus which targets Topo I mRNA (NM_003286.x-391s1c1; Sigma-Aldrich), along with a nontargeting control shRNA lentivirus, was transduced into BCBL-1 cells. Intracellular levels of Topo I (top left), K8 (top right), and β-actin (both panels) proteins were determined by Western blot analysis. The cells stably expressing the Topo I and the control shRNA were induced by TPA for lytic viral replication. KSHV lytic DNA replication in the cells was evaluated at 48 h postinduction by real-time PCR with primers directed to the LANA gene. KSHV virion production was assessed by determining encapsidated KSHV genomic DNA in the culture media 5 days after induction, as described in Materials and Methods. The mean values of results from three independent experiments and standard deviations are presented on inferior panels.

Fig 2.

Effects of Topo II knockdown by short-hairpin (shRNA)-mediated silencing on KSHV DNA replication and virion production. Two shRNA lentiviruses, which target Topo IIβ mRNA (NM_001068.2-1249s1c1, designated Topo II-1, and NM_001068.2-1825s1c1, designated Topo II-2; Sigma-Aldrich), along with a nontargeting control shRNA lentivirus, were transduced into BCBL-1 cells. Intracellular levels of Topo IIβ (top left), K8 (top right), and β-actin (both panels) proteins were determined by Western blot analysis. The cells stably expressing the Topo II and the control shRNA were induced by TPA for lytic viral replication. KSHV lytic DNA replication in the cells was evaluated at 48 h postinduction by real-time PCR with primers directed to the LANA gene. KSHV virion production was assessed by determining encapsidated KSHV genomic DNA in the culture media 5 days after induction, as described in Materials and Methods. The mean values from three independent experiments and standard deviations are presented on inferior panels.

Effect of Topo I inhibitor camptothecin on KSHV DNA replication and its cytotoxicity.

Given that Topo I is required for KSHV lytic DNA replication, it can be predicted that a Topo I inhibitor may inhibit viral DNA replication. Camptothecin is a quinoline-based alkaloid isolated from the bark of the Chinese tree Camptotheca acuminata (46), of which Topo I is the only cellular target (4). Two camptothecin derivatives, namely, irinotecan (CPT-11) and topotecan, have been approved by the U.S. FDA for cancer therapies (33).

The antiviral activity of camptothecin was tested in TPA-induced BCBL-1 cell cultures. Three hours after the induction, the cells were exposed to camptothecin in a wide range of concentration, and 48 h postinduction, viral genomic DNA in these cells was determined by real-time PCR. Results showed that camptothecin is very effective in blocking KSHV DNA synthesis. Its effect is dose dependent with a 50% DNA replication inhibitory concentration (IC₅₀) of 116.3 nM (Fig. 3).

Fig 3.

Effects of the Topo I inhibitor camptothecin on KSHV replication and its associated cytotoxicity. Camptothecin of different concentrations ranging from 0.5 to 1,000 nM was added to BCBL-1 culture 3 h after the induction of lytic replication by TPA. Intracellular KSHV genomic DNA (blue), extracellular virion DNA (green), and cell viability (orange) were determined for each concentration point as described in Materials and Methods. These values were compared to those from the control cells (non-drug treatment). Mean values of results from three independent experiments and standard deviations are presented on the y axes of dose-response curves. Camptothecin doses are indicated on the x axis as a logarithmic scale.

The effect of camptothecin on progeny virion production was also determined. Five days postinduction, amounts of virion particles from the media were determined by quantifying encapsidated viral DNA in preparations (37). The 50% antiviral effective concentration (EC₅₀) calculated from the extracellular virion dose-response curve is 18.08 nM (Fig. 3).

As an anticancer agent, camptothecin kills tumor cells by accumulation of Topo I-DNA intermediate complexes, known as the cleavable complexes. Camptothecin causes DNA breakage in tumor cells but also in normal cells, therefore presenting cytotoxicity and side effects (20, 29). Cytotoxicity of camptothecin on BCBL-1 cells was examined in parallel to inhibition of KSHV DNA replication and virion production. Cells treated with camptothecin at differing concentrations were subjected to the trypan blue exclusion method for estimating the numbers of viable cells and nonviable cells present in culture. The 50% cytotoxic concentration (CC₅₀) was determined to be 233 nM (Fig. 3). The selectivity index (SI = CC₅₀/IC₅₀) is calculated to be 2.0 (Table 1).

Table 1.

Summary of antiviral activities of Topo I and Topo II inhibitors and their associated cytotoxicities^a

Inhibitor	IC50	R2	EC50	R2	CC50b	R2	SI	CC50c	R2
Camptothecin	0.1163	0.806	0.01808	0.951	0.233	0.775	2.0	0.00699	0.947
Ellipticine	2.92	0.816	3.13	0.7	6.26	0.924	2.14	3.197	0.975
Etoposide	10.31	0.563	15.45	0.787	36.79	0.914	3.57	6.466	0.921
Merbarone	19.54	0.687	27.07	0.679	212.9	0.931	10.9	110.5	0.959
Novobiocin	27.55	0.990	27.35	0.997	871	0.953	31.62	224	0.942

^aIC₅₀ represents the half maximal inhibitory concentration for KSHV DNA replication. EC₅₀ denotes the half maximal effective concentration for blocking KSHV virion production. CC₅₀ refers to the concentration of the drug which causes 50% cell death after specific exposure time. They were determined by nonlinear regression analysis of dose-response curves and are expressed as mean values of results from at least three independent experiments. All these parameters are presented in μM units. R², correlation coefficient.

^bCytotoxicity was measured after 2 days of drug treatment. SI (selectivity index) was calculated as the ratio of CC₅₀/IC₅₀.

^cCytotoxicity was measured after 5 days of drug treatment.

Evaluation of Topo II poisons and catalytic inhibitors for their effects on KSHV DNA replication and cytotoxicities.

Topo II inhibitors are split into two categories: Topo II poisons, which target the topoisomerase-DNA intermediate (cleavable complex), and Topo II catalytic inhibitors, which disrupt catalytic turnover of the enzyme. Topo II poisons ellipticine (VM26) and etoposide (VP16) have been approved for anticancer chemotherapies (25, 41). Furthermore, etoposide has been used for KS treatment, and the treatment has been demonstrated to be effective and to have an overall positive effect on the quality of life of responding patients (14). However, there are important negative consequences associated with the use of Topo II poisons for antitumor therapies, including cytoxicity and induction of secondary malignancies (31, 52).

The Topo II catalytic inhibitors are less explored for the use as an anticancer drug, with only a few preclinical attempts with dissatisfactory results (6, 16, 22, 45). Topo II catalytic inhibitors do not target the cleavable complex. They target other steps of the catalytic cycle of Topo II. Thus, merbarone inhibits the DNA cleavage and novobiocin blocks the binding of ATP necessary for catalytic turnover. Given that KSHV requires Topo II enzymatic activity for its lytic DNA replication, this class of Topo II inhibitor may be able to efficiently inhibit KSHV DNA replication and may be less cytotoxic than Topo poisons because of their different modes of action. To test this hypothesis, we examined two Topo II poisons, ellipticine (VM26) and etoposide (VP16), and two catalytic inhibitors, novobiocin and merbarone. The effectiveness in blocking KSHV DNA replications and virion production and the cytotoxicities of these Topo II inhibitors in these two categories are compared in parallel.

BCBL-1 cells were treated with various concentrations of ellipticine, etoposide, novobiocin, or merbarone 3 h after induction of lytic viral replication with TPA. The effects of these inhibitors on the viral DNA synthesis and on virion production were examined 2 days and 5 days postinduction, respectively. Ellipticine was effective in inhibiting viral DNA synthesis with an IC₅₀ of 2.92 μM as well as in virion production with an EC₅₀ of 3.13 μM (Fig. 4A). However, ellipticine showed a very strong cytotoxicity to BCBL-1 cells, with a CC₅₀ of 6.26 μM (Fig. 4A).

Fig 4.

Effects of Topo II inhibitors on KSHV replication and their associated cytotoxicity. Two Topo II poisons (ellipticine [A] and etoposide [B]) and two Topo II catalytic inhibitors (merbarone [C] and novobiocin [D]) were added to BCBL-1 culture 3 h after TPA induction for viral lytic replication. The concentration ranges tested for different inhibitors are: 1 to 10 μM (ellipticine), 0.1 to 200 μM (etoposide), 0.01 to 300 μM (merbarone), and 2 to 1,000 μM (novobiocin). Intracellular KSHV DNA (blue), extracellular virion DNA (green), and cell viability (orange) were determined for each concentration point as described in Materials and Methods. These values were compared to those from the control cells (non-drug treatment). Mean values of results from at least three independent experiments and standard deviations are presented on the y axes of dose-response curves. Topo II inhibitor doses are indicated on the x axes as logarithmic scales.

Etoposide also exhibited an anti-KSHV activity, with IC₅₀ and EC₅₀ values of 10.31 and 15.45 μM, respectively (Fig. 4B). Similar to ellipticine, etoposide also showed a strong cytotoxicity, with a CC₅₀ of 36.79 μM (Fig. 4B). Taken together, both ellipticine and etoposide share the same pattern, i.e., high efficiency and high cytotoxicity. The selectivity indices (CC₅₀/IC₅₀) are 2.14 for ellipticine and 3.57 for etoposide (Table 1).

In contrast to these Topo II poisons, the Topo II catalytic inhibitors merbarone and novobiocin exhibited little cytotoxicity to BCBL-1 cells, with CC₅₀ values of 212.9 μM for merbarone and 871 μM for novobiocin (Fig. 4C and D). Both catalytic inhibitors are effective in halting KSHV DNA synthesis, with IC₅₀ values of 19.54 μM and 27.55 μM, respectively. The virion productions were also dramatically reduced by these two inhibitors, giving the EC₅₀ values of 27.07 μM (for merbarone) and 27.35 μM (for novobiocin) (Fig. 4C and D). The low cytotoxicities and high inhibition rates for both viral DNA replication and virion production, represented by the selectivity indices (CC₅₀/IC₅₀) of 10.9 for merbarone and 31.62 for novobiocin (Table 1), suggest potentials of these two Topo II catalytic inhibitors to serve as effective anti-KSHV drugs for treatment of KS and other KSHV-associated diseases.

Evaluation of Topo I and II inhibitors for their effects on host cell proliferation and cell cycle progression.

To further investigate the potentials of Topo II catalytic inhibitors to become safe anti-KSHV drugs, we first assessed the effects of these inhibitors on cell proliferation. BCBL-1 cells were cultured in the presence and the absence of novobiocin and merbarone and counted over 5 days (Fig. 5). The two Topo II poisons (ellipticine and etoposide) and the Topo I inhibitor camptothecin were also included for comparisons. The rates of proliferation of the cells treated with the Topo II poisons and the Topo I inhibitor camptothecin in the concentrations of IC₅₀ and excess concentrations (5 × IC₅₀) were found to be dramatically decreased. In contrast, novobiocin did not affect cell proliferation in the concentration of IC₅₀ but exhibited an inhibitory effect on cell growth in an excess concentration (5× IC₅₀). Merbarone was found to inhibit cell growth in both IC₅₀ and excess concentration (5× IC₅₀) (Fig. 5).

Fig 5.

Effects of Topo I and Topo II inhibitors on BCBL-1 cell proliferation. BCBL-1 cells (starting with 2 × 10⁵ cells/ml) were exposed to different inhibitors as indicated at two concentrations (IC₅₀ and 5× IC₅₀) and counted every day for five days. Data were obtained from three independent determinations and presented as means with standard deviations.

Then, the effects of these Topo II inhibitors on cell cycle progression were also examined. It was previously reported that Topo II poisons including etoposide are able to compromise G₂ phase progression (3, 11). Consistent with these reports, we also observed increased G₂ populations in the cells treated with ellipticine and etoposide by 11% and 6.2%, respectively (Table 2). In contrast, treatment of cells with novobiocin at the concentration of IC₅₀ showed a minimal effect on cell cycle pattern in comparison to that of control cells (no drug treatment). At an excess concentration (5× IC₅₀) of novobiocin, there was an extended S phase (Table 2). Given that cell proliferation was inhibited in such concentrations of novobiocin, we believe that the increased S population reflects a slow progression through S phase. The cells treated with merbarone show extended S phases in both concentrations (IC₅₀ and 5× IC₅₀). Overall, these results clearly demonstrated that novobiocin has a minimal effect on both cell proliferation and cell cycle progression of host cells; merbarone exhibited a lesser effect on host cell function than Topo II poisons.

Table 2.

Effects of topoisomerase inhibitors on BCBL-1 cell cycle progression following 48 h of exposure to the inhibitors as indicated^a

Treatment	IC50			5× IC50
	G1	S	G2	G1	S	G2
No drug	26.57 ± 1.33	13.13 ± 0.26	60.3 ± 1.07	26.57 ± 1.33	13.13 ± 0.26	60.3 ± 1.07
Camptothecin	18.76 ± 3.9	22.38 ± 4.7	58.86 ± 0.79	23.94 ± 5.13	14.63 ± 4.21	61.41 ± 0.93
Ellipticine	19.34 ± 0.7	9.38 ± 0.22	71.27 ± 0.92	25.33 ± 2.21	7.69 ± 4.67	66.98 ± 2.46
Etoposide	25.65 ± 2.3	7.86 ± 0.91	66.49 ± 1.21	23.07 ± 0.54	1.68 ± 2.37	75.24 ± 1.83
Merbarone	11.95 ± 1.24	24.89 ± 4.36	63.06 ± 2.85	21.6 ± 1.39	30.63 ± 7.18	47.76 ± 6.37
Novobiocin	25.23 ± 0.38	12.58 ± 0.56	62.19 ± 1.15	20.39 ± 0.77	26.14 ± 1.93	53.47 ± 1.85

^aData show the percentages of cells in the indicated cell cycle phase. They are presented as mean values and standard deviations of results from at least three determinations.

Validation of Topo I and II inhibitors for their effects on KSHV ori-Lyt-dependent DNA replication.

The topoisomerase inhibitors tested in this study have been demonstrated to inhibit KSHV DNA synthesis and virion production. We asked whether these inhibitors indeed block viral ori-Lyt-dependent DNA replication. To address this question, BCBL-1 cells were cotransfected with an ori-Lyt-containing plasmid (pOri-A). Lytic DNA replication was induced by cotransfection with an RTA expression vector. Expression of RTA sufficiently drives latent KSHV into lytic replication cycle (28, 43). The transfected cells were cultured in the absence or the presence of each inhibitor at various concentrations. The ori-Lyt-dependent DNA replication and the drug effects were measured by a DpnI assay (27, 47). In brief, DNA was isolated from the treated cells 72 h posttransfection and digested with KpnI/SacI and KpnI/SacI/DpnI. Replicated plasmid DNA was distinguished from input plasmid by DpnI restriction digest, which cleaves input DNA that has been dam⁺ methylated in Escherichia coli but leaves intact the DNA that has been replicated at least one round in eukaryotic cells. Thus, only newly replicated plasmid DNA in BCBL-1 cells is resistant to DpnI digestion and can be detected in Southern blot analysis. Replicated DNA was detected in the cells that were cotransfected with pOri-A and RTA expression vector. All the Topo I and II inhibitors that we tested, including the catalytic inhibitors novobiocin and merbarone, were found to be able to inhibit the ori-Lyt-dependent DNA replication in a dose-dependent manner (Fig. 6), proving that all these inhibitors act in blocking KSHV ori-Lyt-dependent DNA replication.

Fig 6.

Inhibition of KSHV ori-Lyt-associated DNA replication with Topo I and Topo II inhibitors. BCBL-1 cells were transfected with an ori-Lyt-containing plasmid (pOri-A) and RTA expression vector (pCR3.1-ORF50). Transfected cells were cultured in the absence or presence of increasing concentrations of camptothecin or ellipticine (A), merbarone (B), and novobiocin (C). After 72 h of incubation, hirt DNAs were extracted from the cells and subjected to a viral DNA replication assay as described in Materials and Methods. DpnI-resistant products of DNA replication (Rep'd DNA) were detected by Southern blotting with ³²P-labeled pBluescript plasmid.

DISCUSSION

Lytic DNA replication initiates at an origin (ori-Lyt) and requires trans-acting elements, both viral and cellular. Recently, we demonstrated that several host cellular proteins are involved in KSHV lytic DNA replication, including topoisomerases I and II (Topo I and II), RecQL, MSH2/6, and PARP-1 (48). Discovery of these cellular proteins' involvement in viral DNA replication raises a possibility that these proteins could be new therapeutic targets for blocking KSHV replication and for treatment of KSHV-associated human diseases. Given that viruses have tendencies to mutate their genome and therefore develop drug resistance, targeting host cellular proteins that viruses rely on for their replication offers the advantage of minimizing drug resistance and hence constitutes an important, novel therapeutic strategy. The objective of the current investigation is to assess the feasibility of blocking KSHV replication and treating KSHV-associated human diseases by targeting these cellular proteins that are required for viral DNA replication. The salient features of this report are as follows.

(i) The discovery of Topo I and II in KSHV lytic DNA replication complexes and the observation that the expression of these two topoisomerases are significantly upregulated during KSHV lytic DNA replication suggest roles for these two topoisomerases in viral lytic DNA replication (48). This hypothesis has been proven in this study by using the shRNA-mediated gene silencing approach and specific inhibitors of these two topoisomerases. It is conceivable that viral DNA replication, like cellular DNA replication, also requires topoisomerases to resolve the DNA topological problem during its DNA replication. Some large DNA viruses encode their own topoisomerases for their DNA replication. However, KSHV as well as all the herpesviruses studied so far do not encode topoisomerases. Thus, it would not be surprising that these viruses use cellular topoisomerases for the viral DNA replication. The actions of these cellular enzymes in viral DNA replication and detailed mechanisms underlying the process warrant further investigation.

(ii) Given that the cellular topoisomerases I and II are required for KSHV DNA replication, both enzymes could serve as good drug targets for inhibition of KSHV replication and treatment of KS and other KSHV-associated human diseases. This notion is further supported by the result of the current study that the inhibitors of Topo I and II inhibit the synthesis of KSHV DNA, resulting in a decreased yield of KSHV virion particles.

(iii) Among the inhibitors tested, including a Topo I inhibitor, Topo II poisons, and Topo II catalytic inhibitors, two Topo II catalytic inhibitors, novobiocin and merbarone, exhibit an unique feature. Both inhibitors are effective to inhibit KSHV DNA replication at a relatively low molar concentration (as measured by IC₅₀), and the amounts needed to show cytotoxicity are much higher (as measured by CC₅₀) than those of poisons. Thus, this class of Topo II inhibitors, especially novobiocin with a high selectivity index, have great potentials to become effective and safe anti-KSHV agents. Interestingly, novobiocin was reported able to inhibit HSV-1 DNA synthesis but only marginally affected cellular DNA synthesis (17).

Novobiocin, also known as albamycin or cathomycin, is produced by the actinomycete Streptomyces niveus. It has been widely used as an antibiotic drug against staphylococcal infection. It is still used in the treatment of methicillin-resistant Staphylococcus aureus. Side effects associated with its clinical use have been rare and minimal (24). Novobiocin has also been used in combination with chemotherapeutical agents for the treatment of several cancers (12, 23). The maximally tolerated dose of novobiocin (2 to 4 g/day by mouth) has been estimated from these combination regimens, which tend to show synergistic cytotoxicity. This tolerated dose renders plasma concentrations of novobiocin of at least 160 μM, a much higher concentration than the IC₅₀ and EC₅₀ values for KSHV (27. 55 and 27. 35 μM, respectively) determined in this study.

Cytotoxicities of camptothecin, ellipticine, and etoposide were also determined in this study by counting viable cells that exclude trypan blue. Ellipticine and camptothecin were previously tested in other cell lines with the use of other methodologies. For these studies, CC₅₀ values of 0.3 to 3 μM for ellipticine (13, 42) and 7 to 330 nM for camptothecin (7, 21) were reported. Although there are variations in toxicity among the cell lines and the methods chosen in these studies, these CC₅₀ values basically agree with the results reported in this study, pointing out the validity of the trypan blue exclusion method employed in the current study to evaluate cytotoxicity.

Camptothecin and etoposide have been used in antitumor chemotherapy. They have been intravenously administrated at different high dosages (20 to 150 mg/m² per day). Severe toxicities are frequently associated with the administration of camptothecin or etoposide (14, 26, 30). Although ellipticine is also an antitumor agent, it was rarely administrated to patients because of its mutagenesis activity and poor solubility. However, its chemical derivatives have more applications in clinics (8, 10).

It is worthwhile to point out that Topo II poisons, such as etoposide (VP16), doxorubicin, and daunorubicin, have been applied for Kaposi's sarcoma therapies (14, 26, 38). These Topo II poisons usually induce high response rates against tumor progression, but relapses of this cancer are very frequent, and repeated administrations are necessary to palliate this disease. Severe side effects, such as bone marrow suppression (myelosupression) and abnormally low numbers of neutrophils (neutropenia), red blood cells (anemia), or platelets (thrombocytopenia), are often associated with these therapies, causing treatment interruptions and allowing malignancies to progress (9, 32, 40, 52). The principles for use of Topo II poison versus Topo II catalytic inhibitors inclusive of novobiocin and merbarone in treatment of KS could be completely different. The former is used for its antitumor activities that kill KS cells, while the latter could be utilized for their antiviral activities on the basis that constant lytic replication in KS plays a role in sustaining the population of latently infected cells that otherwise are quickly lost by segregation of latent viral episomes as spindle cells divide, and therefore inhibition of KSHV lytic replication may result in KS regression and remission (19). Both novobiocin and merbarone exhibit high anti-KSHV activities with low cytotoxicities and demonstrate great potentials to be effective anti-KSHV agents and promising drugs for treatment of KS and other KSHV-associated diseases.

Currently there is no available anti-KSHV drug which is safe and highly effective to be administrated to infected individuals. Intense research on new potential therapies is absolutely required in order to reduce morbidity and mortality associated with KS. According to our research, it is suggested that catalytic inhibitors should be embarked in further investigations.

Topo I inhibitors are also a class of antitumor agents with a mechanism of interrupting cancer cell DNA replication and causing cell death. Most of the Topo I inhibitor-based drugs are derivatives of the plant extract camptothecin, including irinotecan (CPT-11) and topotecan (15). Camptothecin was included in our current study. A unique feature was noticed with this inhibitor. Camptothecin inhibits KSHV DNA replication with an IC₅₀ of 116.3 nM. However, the inhibitor is able to block KSHV virion production at an extremely low concentration with an EC₅₀ of 18.1 nM. These results may suggest that in addition to inhibition of KSHV lytic DNA replication, this inhibitor may also have some effects on other steps of the process that leads to the virion production. Thus, further investigations on the mechanism underlying inhibition of KSHV virion production as well as on exploring the potential of camptothecin and its derivatives in treatment of KS are definitely warranted.