LI Yajiao， LEI Wen， QIN Yun， YING Chang， ZHOU Xiumei*

(1.Xinyuan Institute of Medicine and Biotechnology， College of Life Sciences，Zhejiang Sci-Tech University， Hangzhou 310018;2.Department of Hematology， the First Affiliated Hospital， College of Medicine，Zhejiang University， Hangzhou 310003)

Synergisticanti-tumoreffectsofoncolyticvirustherapycombinedwithsorafenibonhumanhepatocellularcarcinoma

LI Yajiao1， LEI Wen2， QIN Yun1， YING Chang1， ZHOU Xiumei1*

(1.Xinyuan Institute of Medicine and Biotechnology， College of Life Sciences，Zhejiang Sci-Tech University， Hangzhou 310018;2.Department of Hematology， the First Affiliated Hospital， College of Medicine，Zhejiang University， Hangzhou 310003)

Human hepatocellular carcinoma is one of the most common and high fatal cancer in the world. In China， the liver cancer rates is the highest in the world. Many researchers put into the research of liver cancer and tried many ways and methods， but still couldn’t find effective ways to cure hepatocellular carcinoma. In the recent years， oncolytic vaccinia virus become a promising novel way in cancer treatment because of its efficient anti-tumor effect， low side effects and good safety， and has been widely used in the treatment of cancer. Sorafenib， a new tumor-targeting kinase inhibitor， has shown its effective and safe chemotherapy drugs in treatment of HCC， and has already used in clinical trials. In the present study， we combined sorafenib with VV to explore its anti-tumor effects. Through in vitro experiments like MTT， Hoechst， Flow cytometry， Western blot andinvivotransplantation of tumor model and immunohistochemical analysis， it’s demonstrated that combination of sorafenib and vaccinia virus significantly inhibited the growth of tumor cells and promoted apoptosis of tumor cells. According to the result of mice transplanted tumor model， the date showed that with the extension of time， the combined group tumor size has a tendency to decline. Thus， the combination use of sorafenib and vaccinia virus might be a novel approach for liver cancer therapy．

oncolytic vaccinia virus； sorafenib； combination therapy； liver cancer

Liver cancer is one of the most common and fatal types of cancer in the world[1]. It is especially common in Asia， where its morbidity and mortality are significantly higher than any other continent[2]. Chinese patients alone account for 55% of new HCC(human hepatocellular carcinoma) cases worldwide[3]. Traditional treatment for HCC include surgery， chemotherapy and radiotherapy alone or in combination[4]. While these treatments are effective for the patients in the early phases of the disease， they are not as effective for those who are advanced liver cancer[5]. The ineffectiveness of standard therapies， in addition to the rising mortality of liver cancer， has made development of novel strategies to treat liver cancer imperative．

Oncolytic viruses are an emerging strategy for cancer therapeutics[6]. Oncolytic viruses preferentially infect tumor cells with minimal spread into normal tissues[7-9]. Virus replication leads to lysis of the tumor cell and eventual destruction of the tumor[10]. Over the past decade， clinical trials using oncolytic adenovirus， vaccinia virus (VV)， and herpes simplex virus (HSV) have shown encouraging treatment effects against various types of cancer.

One of the most promising oncolytic viruses is oVV. There is mounting evidence that oVV is safe in vivo， displays rapid replication， and exhibits the highest oncolytic virus anti-tumor activity in vitro and in vivo. oVV is a member of the well-studied Poxviridae family， and has been modified to express various antigens， cytokines， and immunostimulatory molecules[11-14]. The oVV used in our study lacks the thymidine kinase (TK) gene which is necessary for virus replication. There tends to be high expression of TK in cancer cells but little or no expression in normal cells[15]. Thus， vaccinia virus constructs that lack the TK gene replicates in tumor cells but not in normal cells. Vaccinia virus infects cells via membrane fusion rather than receptors on the surface of the host cell. Virus release begins 8 h after infection and within 72 hours there will be rapid virus replication and cell lysis allow vaccinia virus to evade the host immune response．

Another emerging cancer therapeutic is sorafenib. Sorafenib is a multi-kinase inhibitor capable of inhibiting tumor growth[16]. Sorafenib inhibits several cell surface kinases implicated in cancer， including the RAF kinase， vascular endothelial growth factor receptor 2 and receptor 3 (VEGFR-2; VEGFR-3)， platelet-derived growth factor receptor beta (PDGFR-beta)， KIT and PLT[17-18]. Thus， sorafenib is capable of inhibiting multiple pathways that contribute to different tumor promoting processes. In addition， sorafenib is able to inhibit several tyrosine kinase receptor that related to new blood vessels and tumor development， thus blocking tumor angiogenesis. Sorafenib is the only FDA-approved systemic chemotherapeutic agent for the treatment of HCC and is currently recognized as a standard first-line therapy for advanced HCC[19-21]．

In the present study， we examined the effect of combined oVV and sorafenib therapy on anti-tumor activity in vitro and in vivo. The combinatorial treatment is demonstrated to increase liver cell death in vitro and reduce tumor growth in vivo significantly. Thus， this combinatorial treatment might provide a novel effective strategy for future liver cancer therapies．

1 Materials and Methods

1.1 Materials

The human embryonic kidney cell line HEK293， human HCC cell lines PLC， Huh7， Hep3B were purchased from Shanghai cell collection (Shanghai China). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM， GIBCO， Carlsbad， USA) supplemented with 10% fetal bovine serum (FBS， GIBCO). All cells were incubated at 37°C humidified air atmosphere with 5% CO2. The oVV were previously constructed and stored in our laboratory. The amplification of oVV was carried out by infecting HEK293 cells integrated the viral replication related genes. The purification of vaccinia virus was through sucrose gradient centrifugation．

1.2 Methods

1.2.1 Cell viability assay Human liver cell lines Hep3B， Huh7， PLC were seeded in 96-well plates at a density of 5 000 cells / well， incubated for 12 h at 37°C before infected with oVV and sorafenib. Cells were treated with different MOI of virus and different concentration of sorafenib. After 48 h， 3-(4， 5-dimethylthiazol-2-yl)-2， 5-diphenyltetrazolium bromide (MTT) solution in PBS (5 mg/mL， 20 μL) was added into each well. Then cells were incubated at 37°C for 4 h， and the supernatant was removed with 150 μL dimethyl sulfoxide added to each well. Cell viability was assessed by measuring the absorbance at 490 nm with a micro-plate reader after mixing thoroughly．

1.2.2 Hoechst 33342 staining Human liver cancer cell line Hep3B cells were seeded in 6-well plate， and then infected with oVV (10 MOI)， sorafenib (20 μmol)， and oVV (10 MOI) plus sorafenib (20 μmol)， respectively， with uninfected cells served as control. 48 h later， cells were stained with Hoechst 33342 (Beyotime， Shanghai) at 1 mg/mL for 30 min at 37°C and observed under the inverted fluorescence microscope．

1.2.3 Western blot analysis Cells were seeded in 6-well plates at a density of 3×105cell， and then infected with oVV (10 MOI)， sorafenib (20 μmol)， oVV (10 MOI) plus sorafenib (20 μmol)， respectively， with uninfected cells served as control. Two days later， cells with different treatments were washed three times with ice-cold PBS， and lysed in buffer (50 mM Tris-HCl， pH8.0， 150 mM NaCl， 100 μg/mL PMSF， 1%TritonX-100) for 30 min at ice. Cells were collected and cell debris was removed by centrifugation. The protein concentration of cell lysates was measured by bicinchoninic acid (BCA) protein assay kit (Beyotime， Shanghai). Protein samples were subjected to SDS-PAGE (12%) separation and transefered to PVDF membranes. Cleaved-caspase-3， cleaved-caspase-9 and pro-PARP antibodies were purchased from Cell Signaling Techology (Danvers， MA)， β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz， CA). The secondary antibodies used were anti-rabbit and anti-mouse which purchased from Vazyme-Biotech (China，Nanjing)．

1.2.4 Flow cytometry analysis Hep3B cells were seeded in 6-well plates at a density of 3×105cell， after cell attached， treated with oVV (10 MOI)， sorafenib (20 μM)， oVV (10 MOI) plus sorafenib (20 μmol)， uninfected cells served as control. After 48 h， PI staining was added into each well (500 μL/tips)， and incubated at 37°C for 30 min. Cell apoptosis was examined using the FACStar (BD Biosciences， Franklin Lakes)．

1.2.5 Animal experiments All procedures for animal experiments were followed according to the guide for regulations and standards of experimental animal. 4-week old female nude mice were purchased from the Shanghai Experimental Animal Center of Chinese Academy of Science (Shanghai). Hep3B tumor xenografts were established by subcutaneously inoculating 5×106Hep3B cells (suspended in 100 μL PBS) into right flank of each nude mouse. When the tumors reached 80-120 mm3， mice were randomly divided into four groups (n=8). Subsequently， mice were injected with oVV， sorafenib， combination of the virus and the drug， or PBS， respectively. oVV (1×107plaque-forming units-PFUs， per mouse) was injected in an intratumor injection manner ， while sorafenib (solvent in PBS) intraperitoneally injected into mice at a dose of 30 mg/kg body weight or 100 μL PBS as control for two times of continuous injection once every other day. Tumor growth was monitored by periodic measurement using calipers， and measured every 4 days. Tumor volume (mm3)=(A×B2)/2， A and B are the tumor length and width (in mm)， respectively. Then tumor volume growth curve were draw．

1.2.6 HE staining and immunohistochemistry assay Tumor tissues were fixed in 4% formaldehyde， dehydrated with gradient ethanol， and embedded in paraffin wax. The sections from tumors were stained with hematoxylin and eosin (HE) for histological analysis. For immunohistochemistry (IHC)， tumor sections were treated with rabbit monoclonal anti-vaccinia virus antibodies at 1∶500 dilutions. The slides were then washed with PBS and incubated with goat anti-rabbit IgG antibodies-HRP polymers with 1∶100 dilution. The slides were washed with PBS and incubated with the avidin-biotin-peroxidase complex reagent (Vector Laboratories， Burlingame， CA) detected with diaminobenzidine tetrahydrochloride solution. All sections were counter stained with hematoxylin and examined with bright-field microscopy. 1.2.7 Statistical Analysis The experimental statistical significance were expressed as mean ± standard deviation (SD) and analyzed by GraphPad 6.0 software and student’s t test. WhenP<0.05， it is considered statistically significant.

2 RESULTS

2.1 Sorafenib and oVV synergistically inhibited tumor cell proliferation

To determine whether the combination of oVV and (Fig.1A) sorafenib (Fig.1B) inhibit tumor cell growth， we performed MTT assay on human liver cell lines infected with oVV alone， sorafenib alone， and oVV plus sorafenib， respectively. For each cell line there was a significant difference in the viability of cells treated that received single or multiple treatments. However， the significant difference was only noticed at the highest levels of sorafenib and oVV application. The largest treatment effect was found in Hep3B cells with 2% viability compared with 20% and 4% viability for Huh7 and PLC cell lines， respectively (Fig.2)．

Notes: (A) Schematic of oVV construction. The PCB cassette was inserted in the TK region of vaccinia by homologous recombination. (B) Schematic structure of sorafenib．Fig.1 Characterization of sorafenib and vaccinia virus

Notes: HCC cell lines Hep3B， Huh-7 and PLC were treated with oVV (0.5， 1， 5， 10 MOI)， Sorafenib (1， 2， 10， 20 μmol)， or combination for 48 h， respectively. The image showed three independent experiments representatively. Cell viability was evaluated by MTT assay. The image showed three independent experiments representatively．Fig.2 Sorafenib enhances oVV-mediated growth inhibition in HCC cells

2.2 Sorafenib and oVV synergistically induced apoptosis in liver cancer cells

To investigate whether combinatorial treatment with sorafenib and oVV induces tumor cell apoptosis， Hoechest 33342 staining was observed using a fluorescence microscope (Fig.3A). Compared with PBS group， other group cell morphology significantly changed. Sorafenib and oVV together group treated cells displayed more cell nuclear fragmentation and cell death．

To further examine the effect of sorafenib and oVV on cell death， PBS， sorafenib (20 μmol)， oVV (10 MOI) and sorafenib+oVV treated cells underwent PI staining 48 h after treatment. Cell apoptosis rates are detected by flow cytometry， the result show that each group cell apoptosis rates is 3.08%， 4.1%， 14.7% and 38.1% (Fig.3B)， The result demonstrated that the apoptosis rate of Hep3B cells combination of sorafenib and vaccinia virus was 10 times more than control group．

2.3 Sorafenib and oVV influenced cell signal pathway

To determine the mechanisms underlying sorafenib and oVV enhanced cell apoptosis， we treated Hep3B cells with PBS， sorafenib， oVV or sorafenib+oVV. Total protein was collected and expression of anti-apoptotic and pro-apoptotic proteins was examined 48 h later. Expression of pro-apoptotic proteins cleaved caspase-3 and cleaved caspase-9 were significantly upregulated in the combinatorial group. pro-PARP was also downregulated， suggesting that the pro-apoptotic protein cleaved-PARP was upregulated in sorafenib+VV exposed Hep3B cell (Fig.3C). Taken together， the data indicated that sorafenib and VV induced apoptosis by increasing expression of pro-apoptotic proteins．

Notes: (A) Hoechst 33342 staining was used to detect apoptosis. The white arrows indicate positive apoptotic cells. Original magnification， ×200. (B) Cell apoptosis were detected by flow cytometry assay. (C) Western blot analysis of the apoptosis associated proteins. β-actin was used as a loading control．Fig.3 Sorafenib enhances oVV-induced apoptosis in HCC cells

2.4 Sorafenib and oVV significantly inhibited tumor growth in liver cancer cell-derived xenografts

To examine the effect of sorafenib and oVV on tumor growth， Hep3B cells at a density of 5×106were injected subcutaneously into the right flank of the nude mice. When the tumors grew to 80-100 mm3， PBS， sorafenib， oVV or sorafenib+oVV was injected into the mice intraperitoneally， respectively. Compared with the PBS injected mice， mice injected with sorafenib alone or oVV alone significantly inhibited tumor growth. However， the combination of sorafenib and oVV significantly inhibited tumor growth even more compared to mice treated with each therapy alone (Fig.4A)．

2.5 Cytopathic effect of combination therapy on tumor growth inhibition in vivo

To determine the effect of sorafenib and oVV on tumor histology， liver cell derived-xenograft tumors of mice treated with PBS， sorafenib， oVV or sorafenib+oVV were removed 25 days after cell injection and sectioned. Hematoxylin and eosin (H&E) staining of the sections revealed that tumors from mice treated with the combinatorial therapy displayed cell death at deeper levels in the tumor and signs of necrosis compared to tumors from mice receiving one therapy or PBS. Immunohistochemical staining with anti-vaccinia virus antibodies revealed that the combination therapy and vaccinia virus group the virus relatively scattered， indicating that the virus did played an anti-cancer role in tumor tissues (Fig.4B)．

Notes: (A) Tumor volumes( means±SD， n=8， **， P<0.01) were measure with caliper and estimated using the following formula: tumor volume (mm3)=(length×width2)/2. (B) Histological analysis of tumor sections. The upper rows are hematoxylin and eosin staining analysis for animal liver and tumor tissues， indicating that hepatotoxicity or cell necrotic area in tumors. The lower row showed anti-vaccinia virus expression by IHC analysis in tumor tissues. Fig.4 Synergistic effects of Sorafenib and oVV in vivo

3 Discuss

The incidence of HCC has increased worldwide in recent years. Mortality has doubled from 1990 to 2010[22]. Despite development in cancer therapies advanced liver cancer remains incurable. The discovery of sorafenib is an important breakthrough when used molecular targeted drugs treatment of liver cancer， and is the only drug that can be administered orally[23-25]. Compared with other virus， vaccinia virus replication in the cytoplasm do not integrated DNA to cellular genome， so has good security. Also vaccinia virus as medicine can through a long distance， by the blood cycle to the tumor tissue. Combination therapy is a promising anti-cancer strategy because it targets multiple factors that contribute to cancer progression[26]. This is essential given the diversity and chemoresistance of liver cancer[27]. Previous studies have shown that combining oncolytic virus with chemotherapy synergistically enhances viral burst size and apoptotic cell death[28-29].

In our study， we combined sorafenib and vaccinia virus to treat liver cancer， the results show that the combined sorafenib and oVV treatment has a stronger anti-tumor effect than either treatment alone. MTT， Hoechst 33342 and flow cytometry test found that combined sorafenib and oVV not only can inhibit the growth of tumor cells but also can promote the apoptosis of tumor cells. Western blot results show that compared with the other group， combined group cleaved-caspase-9 and cleaved-caspase-9 obviously raised， pro-PARP significantly decreased， illustrating cleaved-PARP raised obviously. When apoptosis pathway starts， the caspase-9 as a more upstream in the process of cell apoptosis signal transduction caspase， its activation can activate other caspase， for example， activated caspase-9 can activate caspase-3 which is a very key enzyme in the process of cell apoptosis， thus promote the subsequent apoptosis signaling pathways. PARP locates in the nucleus as a signal of cell apoptosis. Increase of cleaved caspase-3 is able to promote the shear of PARP， caused between Asp124 and Gly215 cut into 31 kD and 85 kD， which lost its normal function. At the established of mice transplanted tumor models， through the analysis of tumor size and pathology test， show that the therapeutic effect of combined group was obviously higher than that of other group， but the killing mechanism need further discuss. It’s reported that sorafenib induced autophagy in prostate cancer， liver cancer， gastric cancer cells[30-32]. However， deeper discussion such as autophage inducing mechanism of sorafenib remained to be done in further work.

In summary， this is the first time to report the combinational effect of vaccinia virus and sorafenib on liver cancer. This study suggests that combining sorafenib with vaccinia virus might be a promising strategy for anti-liver carcinoma therapy．

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索拉非尼聯(lián)合溶瘤痘苗病毒對肝癌細(xì)胞抑制作用及其機(jī)制探討

李亞姣1， 雷 文2， 秦 云1， 應(yīng) 暢1， 周秀梅1

(1.浙江理工大學(xué) 生命科學(xué)學(xué)院 新元醫(yī)學(xué)與生物技術(shù)研究所， 杭州 310018；2.浙江大學(xué) 醫(yī)學(xué)院 第一附屬醫(yī)院, 杭州 310003)

人類肝細(xì)胞癌是全球最常見和具有高致命性的癌癥之一.中國的肝癌患者在全球居于首位，目前仍然沒有找到有效的方法來治療肝癌.近年，溶瘤痘病毒(vaccinia virus)由于其高效的抗癌效應(yīng)，副反應(yīng)較低和良好的安全性已成為一種癌癥治療的新方法，且已經(jīng)廣泛應(yīng)用于癌癥的治療中.索拉非尼，一種新型的腫瘤激酶抑制劑，作為化療藥物可安全有效地治療肝癌，且已應(yīng)用于臨床試驗(yàn)中.本實(shí)驗(yàn)將索拉非尼與溶瘤痘病毒聯(lián)用并探究其在肝癌細(xì)胞中的抗腫瘤的效應(yīng).通過體外MTT，Hochest，流式細(xì)胞術(shù)，Western blot等細(xì)胞實(shí)驗(yàn)和體內(nèi)小鼠移植瘤模型的建立以及免疫組化的分析，發(fā)現(xiàn)索拉非尼聯(lián)合溶瘤痘病毒，可顯著的抑制腫瘤細(xì)胞的生長，促進(jìn)腫瘤細(xì)胞的凋亡.小鼠移植瘤模型結(jié)果表明，隨著時間的延長，聯(lián)合處理組的腫瘤大小有下降的趨勢.聯(lián)合使用索拉非尼和溶瘤痘病毒可能為一項(xiàng)肝癌治療的新途徑.

溶瘤痘病毒； 索拉非尼； 聯(lián)合治療； 肝癌

[Q291]

A

2017-05-25.

國家自然科學(xué)基金青年科學(xué)基金項(xiàng)目(81602706);國家自然科學(xué)基金項(xiàng)目(81172137);浙江省自然科學(xué)基金項(xiàng)目(LY13H080005);浙江省公益性技術(shù)應(yīng)用研究計(jì)劃項(xiàng)目(2015C37035) ;浙江省中醫(yī)藥科學(xué)研究基金項(xiàng)目(2017ZB006).

1000-1190(2017)05-0638-08

*通訊聯(lián)系人. E-mail: 183026077@qq.com．

10.19603/j.cnki.1000-1190.2017.05.014