All procedures of animal research were conducted in accordance with the Laboratory Animals Welfare Act (protocol no. 8852), the Guide for the Care and Use of Laboratory Animals (protocol nos. 9025 and 21370), and the Guidelines and Policies for Rodent Experiment provided by the Institutional Animal Care and Use Committee (IACUC) of the School of Medicine, The Catholic University of Korea. The present study was reviewed and approved by the Catholic University of Korea’s IACUC (CUMC-2012-0054-07: Effects of BV on the inhibition of HPV E6 and E7 expression in cervical cancer cells). All rodents used for surgeries were initially anesthetized using isoflurane in desiccators then followed by isoflurane as required.

C33A (HPV-uninfected cervical cancer cell line), CaSki and HeLa (HPV-infected cervical cancer cell lines) were purchased from the Korean Cell Line Bank (KCLB; Seoul, Korea). TC-1 cells were prepared by transformation of C57BL/6 primary mouse lung cells with HPV16 E6/E7 oncogene and activated HRAS (21,22). All cell lines were incubated in RPMI-1640 cell culture medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (all from Gibco) at 37°C in a humidified 5% CO₂ atmosphere.

The cytotoxicity and sensitivity of BV was first determined by measuring the conversion of the tetrazolium salt 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; Sigma) to formazan. Briefly, CaSki, HeLa and C33A were seeded 1×10⁴ cells/well in 96-well culture plates and cultured overnight. The cells were then treated with 1–15 μg/ml of BV. The control group was treated with the same volume of phosphate-buffered saline (PBS). After 12 and 24 h of incubation, 20 μl of MTT stock solution (2 mg/ml in PBS) was added to each well. After 4 h incubation at 37°C, the supernatant was discarded and the precipitate was dissolved with 200 μl of dimethyl sulfoxide (DMSO). The absorbance of the wells was measured at 570 nm using Soft Max ELISA (Molecular Devices, Sunnyvale, CA, USA). The optical density (OD) was calculated as the difference between the reference wavelength and the test wavelength. Percent of cell viability = [A570 nm absorbance of drug-treated cells/A570 nm absorbance of control cells] × 100.

Total RNA was extracted from CaSki, HeLa and C33A cells or TC-1 tumors treated by BV using TRIzol (Invitrogen, Carlsbad, CA, USA) and purified using RNeasy columns (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. After processing with DNase digestion, clean-up procedures, RNA samples were quantified and stored in 10 μl aliquot at −80°C until use. For quality control, RNA purity and integrity were evaluated by denaturing gel electrophoresis, OD 260/280 ratio, and analyzed on Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The cDNA was synthesized using PrimeScript reagent kit (Takara, Japan) according to the manufacturer’s instructions. Briefly, 1 μg of RNA was reverse-transcribed to cDNA using 200 U moloney murine leukemia virus (M-MLV) reverse transcriptase and oligo(dT) primer in a total reaction volume of 20 μl for 1 h at 37°C. The cDNA was amplified using HPV16 E6, HPV16 E7, HPV18 E6 and HPV18 E7 primers. The E6 primers were 5′-GAGAACTGCAATGTTTCAG GAC-3′ and 5′-CCACCGACCCCTTATATTATGG-3′ for HPV-16, and 5′-AATACTATGGCGCGCTTTGA-3′ and 5′-CTGGATTCAACGGTTTCTGG-3′ for HPV-18. The E7 primers were 5′-GCAACCAGAGACAACTGATCTCTAC-3′ and 5′-GGTCTTCCAAAGTACGAATGTCTACG-3′ for HPV-16, and 5′-TGCATGGACCTAAGGCAA-3′ and 5′-GCT GGGATGCACACCA-3′ for HPV-18. Amplified products were analyzed using an image documentation system (GelDoc 2000) with image analysis software (Quantity One) (both from Bio-Rad, Hercules, CA, USA). DNA size markers (Fermentas, Pittsburgh, PA, USA) were run in parallel to validate the predicted sizes of the amplified bands. For qRT-PCR analysis, cDNA was amplified using these specific primers and SYBR Premix Ex Taq (2X) kit (Takara, Japan) according to the manufacturer’s instructions. HPV E6/E7 expression analysis was carried out using LightCycler 480 II (Roche, Palo Alto, CA, USA) and gene expression raw data were extracted using the software provided by the manufacturer (BeadStudio v.3.0; Partek^® Genomics). HPV E6/E7 gene expression was determined compared to GAPDH gene expression.

For immunoblots, confluent monolayers of CaSki, HeLa and C33A or TC-1 tumors were lysed with cell extraction buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate) (BioSource International, Camarillo, CA, USA) in the presence of protease inhibitors. Protein concentration was determined using the BCA protein assay (Bio-Rad). Electrophoration was performed on 10–12% SDS-polyacrylamide gels for 2 h, and the gels were then transferred onto PVDF membranes (Millipore, Temecula, CA, USA). Membranes were blocked in Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% skim milk for 1 h at room temperature. After blocking, membranes were incubated for 1 h at room temperature or overnight at 4°C with polyclonal rabbit anti-HPV16/18 E6, anti-HPV16 E7 and anti-HPV18 E7, and monoclonal mouse anti-β-actin antibodies (both from Santa Cruz Biotechnology, Santa Cruz, CA, USA) in a 1:200–500 dilution buffer (5% skim milk in TBST). Membranes were washed three times with TBST and then incubated with goat anti-mouse IgG-HRP and goat anti-rabbit IgG (H+L) HRP-conjugated antibodies (Zymed, San Francisco, CA, USA) for 1 h at room temperature. Immunoblots were developed with enhanced chemiluminescence agents according to the manufacturer’s instructions (SuperSignal West Pico Chemiluminescent Substrate; Pierce, Rockford, IL, USA) and exposed to imaging film.

TC-1 tumors were implanted in the abdomens of 4- to 5-week old female C57BL/6 mice by subcutaneous injection of 5×10⁵ TC-1 cells in 100 μl of serum and antibiotic-free DMEM media (Gibco-BRL). When tumors reached a volume of 150–200 mm³, mice were randomly assigned to one of three groups to receive PBS, 1 mg/kg BV and 2 mg/kg BV (5 mice/group). The first day of treatment was designated as day 1. BV or PBS was administered three times via intra-tumoral injection (for TC-1 tumors, BV diluted in 100 μl of PBS) on days 1, 2 and 3. Tumor growth delay was assessed by taking measurements every day or every 2 days. Tumor volume was calculated by the following formula: Volume = 0.523 LW², where L is length and W is width. Tumor responses to each treatment were compared by use of the Mann-Whitney test.

The data were obtained by three or more independent experiments and are expressed as means ± standard error of the mean (SEM). Statistical comparisons were analyzed by Mann-Whitney test (non-parametric method) using StatView software (Abacus Concepts, Inc., Berkeley, CA, USA). Survival was assessed with the Kaplan-Meier method, and results were compared with a log-rank test SPSS software version 13.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was defined as P<0.05.

CaSki (HVP16-infected), HeLa (HPV18-infected) and C33A (HPV-negative) cells were treated by various concentrations of BV for 12 or 24 h. To find out the half maximal inhibitory concentration (IC₅₀) of BV, cell viability was determined by MTT assay using various BV concentrations (1, 2.5, 5, 10 and 15 μg/ml). The results showed that cell viability was affected in a dose-dependent manner (1–15 μg/ml). The IC₅₀ value of CaSki, HeLa and C33A was 9.8, 10.0 and 10.1 μg/ml after 12 h of BV treatment, respectively (Fig. 1A). After 24 h of BV treatment, the IC₅₀ value was 5.4, 9.7 and 14.3 μg/ml in CaSki, HeLa and C33A, respectively (Fig. 1B). The represented data are means ± SEM from three independent experiments and are expressed in terms of the control value. These data support the hypothesis that there is a significant difference in sensitivity to BV between HeLa and CaSki cells. In contrast, in the case of C33A, relatively less suppression of cell growth was observed, suggesting that inhibition of cell growth is mediated by antiviral effects of BV.

To examine whether BV treatment downregulates HPV E6/E7 mRNA expression in CaSki and HeLa cells, the levels of mRNA were analyzed by RT-PCR (Fig. 2). The mRNA expression of HPV16 E6 was gradually decreased until 24 h and that of HPV16 E7 was decreased at 24 h after 10 μg/ml BV was treated to CaSki cells. In the case of HeLa, the mRNA expression of HPV18 E6 was decreased at 24 h after 10 μg/ml BV treatments but that of HPV18 E7 was not altered by BV. We also evaluated the mRNA expression by qRT-PCR that analyzed HPV16/18 E6/E7 mRNA expression levels as compared with GAPDH in BV-treated CaSki and HeLa cells. The mRNA expression levels were obtained for the threshold cycle (Ct) for each gene and normalized using the average of the GAPDH gene and determined quantified relative folds. HPV16 E6 and E7 mRNA expressions were 0.90±0.01 and 0.77±0.01-fold after 12 h, and 0.91±0.01 and 0.59±0.01-fold after 24 h in 5 μg/ml BV-treated CaSki cells, respectively. In 10 μg/ml BV-treated CaSki cells, the mRNA expression levels of HPV16 E6 and E7 were significantly decreased by BV: 0.44±0.10 and 0.39±0.11-fold (after 12 h), and 0.35±0.06 and 0.44±0.07-fold (after 24 h) in BV-treated CaSki cells (Fig. 3A). HPV18 E6 and E7 mRNA expression levels were not significantly altered by 5 μg/ml BV-treated HeLa cells. In 10 μg/ml BV-treated HeLa cells, HPV18 E6 and E7 mRNA expressions were 0.68±0.02 and 0.66±0.02-fold after 12 h, and 0.73±0.03 and 0.81±0.04-fold after 24 h, respectively (Fig. 3B). When the HPV16/18 E6/ E7 mRNA expression levels at 5 μg/ml BV-treatment were compared between CaSki and HeLa cells, more HPV16 E7 was observed in CaSki cells, as compared to HeLa cells. Moreover, when the cervical cancer cells were treated with 10 μg/ml BV, the inhibition of HPV16/18 E6/E7 mRNA expression levels was significantly observed. Collectively, these data further suggest that BV treatment can induce these different cell lines in a different manner.

To investigate whether HPV E6/E7 protein expression by BV treatment displayed a different level, we performed western blot analyses that estimated HPV16/18 E6/E7 protein expression as compared with β-actin. The protein expression levels were also quantified by densities of blotted bands and were measured by relative folds compared to those of HPV16/18 E6/E7 of BV-untreated CaSki and HeLa cells. In BV-treated CaSki and HeLa cells, the levels of these protein expressions were significantly lower than those of BV-untreated cells. Protein expressions of HPV16 E6 and E7 were significantly decreased at 5 μg/ml BV-treated CaSki cells for 24 h, compared to the BV-treated CaSki cells for 12 h (Fig. 4A). In 10 μg/ml BV-treated HeLa cells, HPV18 E6 and E7 were less expressed at 12 and 24 h as compared to the expression of HPV16 E6 and E7 by BV-treated CaSki cells (Fig. 4B). These data support the theory that HPV16/18 E6 and E7 are downregulated by BV in the cervical cancer cells.

To determine the antitumor effects of BV in vivo, C57BL/6 mice were challenged s.c. with TC-1 cells and then treated with BV. As shown in Fig. 5, BV treatment was initiated on day 1 when tumors were relatively small in size. The growth of TC-1 tumors was significantly decreased compared with that of tumors treated with PBS. Specifically, by day 15 after treatment, PBS-treated tumors reached an average tumor volume of 2,797±215 mm³, while those treated with 1 mg/kg of BV reached 1,369±98 mm³, and those treated with 2.5 mg/kg of BV reached 839±122 mm³. In addition, at day 15 after treatment, the 2.5 mg/kg BV-treated TC-1 tumor group showed that the tumor growth was significantly suppressed as compared with the 1.0 mg/kg BV-treated group (P<0.0001) and with the PBS group (P<0.0001). The antitumor effects of BV in vivo were consistent with the MTT assays and protein expression analyses in vitro.

To analyze HPV16 E6/E7 mRNA and protein levels in vivo by BV treatment in TC-1 tumors, we evaluated mRNA and protein expression by qRT-PCR and western blotting, respectively. The mRNA expression levels of HPV16 E6 and E7 were decreased by BV. HPV16 E6 gene in the TC-1 tumors was 0.22±0.02-fold and E7 gene was 0.07±0.004-fold compared with PBS-treated tumor (Fig. 6A). Protein expression levels of HPV16 E6 and E7 in the TC-1 tumors were significantly decreased by BV (Fig. 6B). Overall, these data suggest that BV treatments induce downregulation of HPV16 E6 and E7 depending on the cervical cancer cell lines.