Pegylated Interferon-α2a Inhibits Proliferation of Human Liver Cancer Cells In Vitro and In Vivo

Purpose We investigated the effects of pegylated interferon-α2a (PEG-IFN-α2a) on the growth of human liver cancer cells. Methods The effect of PEG-IFN-α2a on the proliferation of 13 liver cancer cell lines was investigated in vitro. Cells were cultured with medium containing 0–4,194 ng/mL of PEG-IFN-α2a, and after 1, 2, 3, or 4 days of culture, morphologic observation and growth assay were performed. After hepatocellular carcinoma (HCC) cells (HAK-1B and KIM-1) were transplanted into nude mice, various doses of PEG-IFN-α2a were subcutaneously administered to the mice once a week for 2 weeks, and tumor volume, weight, and histology were examined. Results PEG-IFN-α2a inhibited the growth of 8 and 11 cell lines in a time- and dose-dependent manner, respectively, although the 50% growth inhibitory concentrations of 7 measurable cell lines on Day 4 were relatively high and ranged from 253 ng/mL to 4,431 ng/mL. Various levels of apoptosis induction were confirmed in 8 cell lines. PEG-IFN-α2a induced a dose-dependent decrease in tumor volume and weight, and a significant increase of apoptotic cells in the tumor. Subcutaneous administration of clinical dose for chronic hepatitis C (3 μg/kg, 0.06 μg/mouse) was effective and induced about 30-50% reduction in the tumor volume and weight as compared with the control. Conclusions Although in vitro anti-proliferative effects of PEG-IFN-α2a were relatively weak, PEG-IFN-α2a induced strong anti-tumor effects on HCC cells in vivo. The data suggest potential clinical application of PEG-IFN-α2a for the prevention and treatment of HCC.


Introduction
Interferons (IFNs) are types of cytokine that are produced by host cells, such as leukocytes, in response to inflammation. Since IFNs possess antiviral activity, antiproliferative activity and various immunoregulatory activities, IFN therapy is used to treat patients with chronic viral hepatitis or certain types of cancer including malignant melanoma, acquired immunodeficiency syndrome-related Kaposi's sarcoma and some hematopoietic malignancies [1,2]. Lai et al also showed that recombinant IFNα is useful in prolonging survival among patients with inoperable hepatocellular carcinoma (HCC) [3]. In addition, some studies showed IFN therapy might prevent either occurrence or recurrence after initial curative therapy of HCC, such as liver resection and radiofrequency ablation, in patient with chronic viral hepatitis [4][5][6][7]. This cancer preventive effect of IFNs is regarded mainly as results of their antiviral effect and the consequent suppression of inflammation, and might be due to their direct antitumor effect against clinically undetectable HCC as well. The detailed mechanism of the antitumor effect of IFNs, however, remains obscure.
Pegylated interferon-α2a (PEG-IFN-α2a) and pegylated interferon-α2b (PEG-IFN-α2b), which are used to treat patients with chronic hepatitis C virus (HCV) or B virus (HBV) infection, are modified IFNs that have longer serum half-life in body than non-pegylated forms of IFNs, therefore they can be given to patients only once a week, whereas a standard IFN without pegylation used to be injected up to three to five times a week. This once-a-week injection of pegylated IFNs in combination with daily oral dosing of the nucleoside analogue ribavirin has substantially improved the rate of sustained virological response in patients with chronic HCV infection and got a position as the first line therapy [8,9]. We previously reported that PEG-IFN-α2b which contains 12 kDa polyethylene glycol (PEG) has stronger antitumor effects in vivo than nonpegylated IFNs and this result might be indicating that continuous IFNs exposure to cancer cells in body is more effective than continual injection [10]. On the basis of abovedescribed background, we examined the growth inhibitory effects of PEG-IFN-α2a which contains two chains of 20 kDa PEG and has the longest serum half-life among clinically available IFNs on liver cancer cell lines in vitro and in vivo.

Effects of PEG-IFN-α2a on the Proliferation of HCC and CHC Cell Lines in vitro
The effects of PEG-IFN-α2a on the growth of the cultured cells were examined with colorimetry using 3-(4,5dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay kits (Chemicon, Temecula, CA) as described elsewhere [18,21]. Briefly, the cells (1.5~8 X 10 3 cells per well) were seeded on 96-well plates (Nunc, Inc, Roskilde, Denmark), cultured for 24 hours, and the culture medium was changed to a new medium with or without PEG-IFN-α2a (

Morphological Observation
For morphological observation under a light microscope, cultured cells were seeded on Lab-Tek tissue culture chamber slides (Nunc, Inc.), cultured with or without PEG-IFN-α2a (262, 1,048 or 4,194 ng/mL) for 72 hours, fixed for 10 min in Carnoy's solution, and stained with hematoxylin-eosine (HE).

Effects of PEG-IFN-α2a on HCC Cell Proliferation in Nude Mice
All animal experiments were approved by the institutional committee for animal experiments in Kurume University School of Medicine (Permit Number: 1334), and conducted according to the Guide for the Care and Use of Laboratory Animals of the National Institute of Health and the Regulations for Animal Experimentation of Kurume University School of Medicine. Mice were killed by cervical dislocation under diethyl ether anesthesia, and all efforts were made to minimize suffering. Cultured HAK-1B or KIM-1 (10 7 cells/mouse) was subcutaneously (s.c.) injected into the backs of 5-week-old female BALB/c athymic nude mice (Clea Japan, Inc., Osaka, Japan). Five to seven days later when the largest diameter of the tumor, which was measured by using caliper, reached approximately 5~10 mm (Day 0), tumor volume (mm 3 ) was calculated in the equation 'the largest diameter X (the smallest diameter) 2 X 0.5', and then the mice were divided into 5 groups (n=8 each). Tumor volume was measured on Day 0, 1, 2, 4, 6, 8, 10, 12, and 14. Mouse body weight was measured on Day 0, PEG-IFN-α2a Inhibits HCC Growth PLOS ONE | www.plosone.org 8, and 14. After 2-week treatment, mice were killed on Day 15 and the actual tumor weight was also measured. In experiment 1, the 5 groups of 8 mice received either phosphate-buffered saline (PBS) (Control) or PBS with the different dosages of PEG-IFN-α2a (0.06-60 μg) once a week for 2 consecutive weeks (Day 1 and Day 8). The clinical dose of PEG-IFN-α2a in chronic hepatitis C treatment is about 3 µg/kg and is equivalent to the lowest dose (0.06 µg/mouse=840 IU/mouse) in this experiment. After killing, resected tumors were used for morphological studies (e.g., HE staining and immunohistochemistry) and Enzyme-linked immunosorbent assay (ELISA) analysis. Every mouse received an intraperitoneal injection of 1 mg of BrdU 30 min before killing. In experiment 2, to examine the difference between nonpegylated and pegylated IFNs, 5 groups of 8 mice received either PBS (Control), PBS with 0.0042 or 0.042 µg of IFN-α2a (840 or 8,400 IU, respectively), or PBS with 0.06 or 0.6 µg of PEG-IFN-α2a (840 or 8,400 IU, respectively). In this experiment, tumor weights on Day 15 and numbers of apoptotic cells were compared among the groups.

Morphological Examination of the Subcutaneous Tumors of Nude Mice
The number of cells showing the characteristics of apoptosis (e.g., cytoplasmic shrinkage, chromatin condensation, and nuclear fragmentation) was counted in at least three 0.25 mm 2areas within an HE-stained specimen, and the average number per area was obtained. The TUNEL technique (ApopTag ® Peroxidase In Situ apoptosis Detection Kits, CHEMICON International, Inc, CA) was used to detect apoptotic cells, and the average number of TUNEL-positive cells per area was obtained, as described above. The specimens were also immunostained for incorporated BrdU using BrdU Staining Kits (Oncogene Research Products, Boston, MA), and the average number of positive cells per area was obtained as described above. In addition, double-immunostaining was performed with anti-mouse endothelial cell antibody, anti-human α-SMA antibody, Histofine simple stain mouse MAX-PO (Rat) kits (Nichirei, Tokyo, Japan), and HistoMouse™-plus kits to detect artery-like blood vessels as described in our previous report [21,22]. The number of double-immunostaining-positive blood vessels in the tumor was counted on each specimen. Granulation tissue within the tumor were excluded in counting of blood vessels. The size of the counted area was measured by tracing the outline displayed on a computer monitor using Mac SCOPE (MITANI Corp., Chiba, Japan). From the obtained number of vessels per unit area (mm 2 ), the group mean was obtained for group comparison.

Enzyme-linked immunosorbent assay (ELISA)
Portions of the resected xenograft tumors were homogenized in 500 μl of ice-cold Ca 2+ and Mg 2+ -free PBS containing 100 mg/ml phenymethylsulfonyl fluoride using a pellet pestle. The mixture was centrifuged for 10 min (12,000 g, 4°C), and the supernatant was stored at -20°C until use. After the determination of the amount of the tissue protein in the supernatant using a BCA protein assay reagent (Pierce, Rockford, IL), the amount of basic fibroblast growth factor (bFGF) and IL-8 was measured by using commercially available ELISA kits (R&D Systems, Minneapolis, MN).

Statistics
Comparisons of estimated tumor volume and colorimetric cell growth were performed using two-factor factorial ANOVA and Student's t-test, respectively. The other data comparisons were performed using the Mann-Whitney U test.

Effects of PEG-IFN-α2a on Liver Cancer Cell Proliferation in vitro
Twenty-four hours after the addition of 4,194 ng/mL of PEG-IFN-α2a, mild increase in the relative viable cell number occurred in 9 cell lines (all cell lines except KYN-2, HAK-1A, HAK-6, and KMCH-1). However, after 72 hours or later, a 10% or more decrease in the cell number occurred in all cell lines ( Figure 1A). In HAK-2, HAK-3, and HAK-4, HAK-6, and KMCH-2, proliferation was suppressed up to 72 hours and the cell number reached a plateau or slightly increased thereafter.
In the other 8 cell lines, proliferation was suppressed to varying degrees up to 96 hours.

Effects of PEG-IFN-α2a on HCC Cell Proliferation in Nude Mice
Chronological changes in estimated tumor volume after subcutaneous injection of cultured HAK-1B or KIM-1cells to nude mice are summarized in Figure 3. Dose-dependent suppression of tumor volume was observed in mice receiving PEG-IFN-α2a. In the experiment of HAK-1B tumors, a significant difference in the changes in tumor volume and tumor weight was observed between the Control mice and the mice PEG-IFN-α2a Inhibits HCC Growth  PEG-IFN-α2a Inhibits HCC Growth PLOS ONE | www.plosone.org that received 0.06, 0.6, 6 or 60 μg of PEG-IFN-α2a (P < 0.0001 by two-factor factorial ANOVA; and P < 0.001~0.02 by the Mann-Whitney U test, Figure 3A and Table 2). In the experiment of KIM-1 tumors, a significant reduction of tumor volume was also observed with the use of PEG-IFN-α2a (P < 0.001 by two-factor factorial ANOVA, Figure 3B). There were significant differences in the actual tumor weight between the Control group and the PEG-IFN-α2a groups, except for the PEG-IFN-α2a (0.06 μg) group ( Table 2). The actual tumor weight at the end of the experiment 2 was summarized in Table  3. Subcutaneous injection of 0.6 μg of PEG-IFN-α2a induced the significant reduction of tumor weight, compared with the Control group and the group that received the same international unit of non-pegylated IFN-α2a (P<0.005 and P<0.03, respectively). In this experiment, there was no significant difference between the Control group and the PEG-IFN-α2a (0.06 μg) group (P=0.078).
Histological examination of the HAK-1B tumor specimens stained with HE revealed that the numbers of apoptotic cells in the mice treated with PEG-IFN-α2a (0.06 or 0.6 μg) were significantly higher than that of the Control, and the number increased dose dependently (Figure 4, A and B; Table 4). The incidence of apoptosis in TUNEL-stained sections showed the same tendencies as those obtained in HE-stained sections ( Figure 4C and Table 4). Immunohistochemical examination of BrdU uptake in HAK-1B tumors revealed that there was no significant difference in BrdU labeling index between the Control and PEG-IFN-α2a (0.06 or 0.6 μg) groups ( Table 4). As for apoptosis, similar findings were observed in experiment 2 in which KIM-1 was used. The group treated with 0.6 μg of PEG-IFN-α2a showed increased number of apoptotic cells than the control group. There was no significant difference between the control and IFN-α2a group. In addition, the group treated with Table 1. Quantitative analysis of apoptosis in HAK-1B or KIM-1.  The resected tumor of the PEG-IFN-α2a group showed granulation tissue at the middle of the tumor to various degrees ( Figure 5). Arteries that appeared in the granulation tissue were excluded in blood vessel count within tumor. There was no significant difference in the number of blood vessels per unit area within the HAK-1B tumor and the expression of bFGF and IL-8 in the tumors between the PEG-IFN-α2a group and the Control group ( Figure 5; Table 5).

Discussion
In the in vitro study, we showed that PEG-IFN-α2a inhibit the growth of 8 and 11 out of 13 cell lines in a time-and dosedependent manner, however, PEG-IFN-α2a was apparently less active on an IC50 basis, compared with either PEG-IFN-α2b or IFN-α2b or consensus IFN-α or BALL-1 lymphoblastoid IFN-α which was tested in the same experimental condition in our previous reports [10,18,21]. For example, IC50 for HAK-1B cells was approximately 253 ng/ml of PEG-IFN-α2a, 13.1 ng/ml of PEG-IFN-α2b, 2.4 ng/ml of IFN-α2b, 0.7 ng/ml of consensus IFN-α and 1.1 ng/ml of BALL-1 lymphoblastoid IFN-α. On the other hand, in the in vivo study, s.c. injection of PEG-IFN-α2a once a week showed better antitumor effect on a tumor volume or weight basis, compared with that of non-pegylated IFN-α2a. These results might support our hypothesis that continuous contact with IFNs induces strong in vivo antitumor effects, and are not surprising because it was reported that PEG-IFN-α2a showed less active in vitro antiviral activity and but had much more in vivo antitumor activity than non-pegylated IFN-α2a [23]. We also showed that PEG-IFN-α2a can inhibit the proliferation of CHC cell lines as well as HCC. In MTT assay, the growth of KMCH-1 was well suppressed although another CHC cell line, KMCH-2 was not. One possible explanation for the different sensitivity between KMCH-1 and KMCH-2 is that the origin of KMCH-1 is CHC, classical type and that of KMCH-2 is CHC with stem-cell features, intermediate-cell subtype according to the latest WHO classification [24]. Such a stem-cell properties of the tumor might be the reason for IFN resistance. Another interesting finding in the in vitro study is the discrepancy between the results of MTT assay and apoptosis detection assay. When HAK-1B or KIM-1 was cultured with PEG-IFN-α2a, IC50 for HAK-1B was much lower than that for KIM-1 although HAK-1B showed lower rate of apoptotic cells than KIM-1. These findings suggest that there might be some  Table 3. The actual weight and numbers of apoptotic cells of subcutaneous tumors at killing (Experiment 2).

Treatment group a activity of interferon (IU) Tumor weight (g) Apoptosis (Number of cells/0.25mm 2 )
Control 0 IU 0.726 ± 0.09 b, c 7.6 ± 0. mechanisms other than apoptosis, which affect the sensitivity to antitumor effects of PEG-IFN-α2a. We previously reported that both pegylated and non-pegylated IFN-α inhibited the proliferation of cultured HCC cells by inducing the cell-cycle arrest [10,18]. The expression of interferon receptor on tumor cells might be a possible factor related to antitumor effect. For instance, Nagano et al reported that the expression of this type I IFN receptor on HCC tissue might be a useful predictor to find potential responder to INF-α/5-fluorouracil combination therapy [25]. Immunomodulation by IFNs has also been well studied as a factor related to antitumor effect. In this study, we used athymic mice, which lack mature T-cell, and human IFNs. Since IFNs are species-specific [26], we surmise that this immunomodulatory effect is limited in our study, but this should be confirmed in the future study using mouse IFN.
Morphological observation of the subcutaneous tumors of nude mice revealed that s.c. injection of PEG-IFN-α2a induce the significant increase of apoptotic cells compared with Control group. This result in the in vivo study is consistent with that in the in vitro study showing characteristic changes of apoptosis after adding PEG-IFN-α2a. Although the inhibition of angiogenesis as well as the induction of apoptosis is regarded as one of the biological effects of IFNs, there was no significant difference in the number of artery-like blood vessels of the subcutaneous tumors between the control and treatment groups. There are two possible explanations of this finding. Firstly, PEG-IFN-α2a was less effective for mouse endothelial cells compared with human cancer cells due to the species specificity of human IFNs. Secondly, it might be difficult to visualize the alteration in the number of vessels in order to examine the efficacy of drugs that possess antiangiogenic    [27]. We had observed similar findings in our previous report in which human HCC tumors subcutaneously transplanted in nude mice showed much apoptosis in either PEG-IFN-α2b or IFN-α2b treatment group compared with the Control group, but no significant difference in the number of blood vessels [10]. Kojiro et al also showed that s.c. injection of BALL-1 lymphoblastoid IFN-α increase the number of artery-like blood vessels and the protein expression of bFGF within HCC xenograft tumors in spite of the significant decrease of actual tumor weight [28]. In contrast, Dinney et al showed that IFN-α2a decreases the blood vessel density and the expression of bFGF in orthotopic xenograft model of bladder tumor [29]. The reason for these contrary findings remains unclear and further evaluation with caution is needed by using different doses and types of IFNs and different cell lines, not only in subcutaneous tumor model but also in orthotopic model. The association between IFN therapy and occurrence or recurrence of HCC has been investigated in some reports. HALT-C trial group showed in their randomized control trial in a large cohort that long-term PEG-IFN-α2a therapy does not reduce the incidence of HCC among patients with chronic HCV infection who have previously failed to achieve a sustained virologic response to therapy [30]. Among only patients with cirrhosis, long-term PEG-IFN-α2a therapy reduced a risk of HCC after a long-time observation [31]. EPIC study group also showed long-term PEG-IFN-α2b therapy does not prevent HCC [32]. On the other hand, Nishiguchi et al reported that long-term IFN-α therapy after curative resection of HCV-related HCC prolongs the survival rate, although preventive effect of intrahepatic recurrence was marginal [33]. Sakaguchi et al also showed that among patients who underwent radical  Table 5. Numbers of artery-like blood vessels, and Enzyme-linked immunosorbent assay (ELISA) of angiogenesis factors in human HCC tumors subcutaneously transplanted in nude mice. radiofrequency therapy for HCV-related HCC, long-term IFN-α2b therapy reduced the recurrent rate of HCC [4]. These reports with conflicting results may be suggesting that IFN therapy is effective only after the initial curative treatment of HCV-related HCC. In addition, there are several reports that support that IFN therapy prevents the development of HCC among patients with chronic HBV infection or those underwent curative resection of HBV-related HCC [5,7]. Thus the chemopreventive effect of IFNs against HCC are still controversial, and mechanisms behind that remain unclear. Antiviral effect against HBV and HCV, which are risk factors for HCC, and immunomodulatory effect of IFNs are regarded as main mechanisms. Another possible mechanism is that IFNs may suppress the growth of clinically undetectable HCC due to their direct antitumor effect. Our finding in the current study provide the evidence that PEG-IFN-α2a possesses the direct antitumor effect against HCC.
In conclusion, we demonstrated antitumor effect of PEG-IFN-α2a for human liver cancer cells in vitro and in vivo and our results suggest that longer contact to IFNs may induce stronger antitumor effect in body. PEG-IFN-α2a might be a possible treatment option for HCC as well as chronic viral hepatitis. Further studies are needed from both molecular and clinical view points in order to find out particular patient group those respond to this therapy.