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,1
* State Key Laboratory of Biotherapy, West China Hospital, and
College of Life Science, Sichuan University, Chengdu, Sichuan, China; and
Pharmacological Department of Medical College, Yan’an University, Yan’an, shanxi, China
2Correspondence: State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China. E-mail: julio_wy{at}sohu.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: apoptosis • cellular immune response • plasmid DNA • cationic liposome • cytotoxic T lymphocytes • CTLs
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Our previous data also confirmed its oncolytic activity and furthermore showed that the activity may be largely due to its capability of inducing tumor cell apoptosis (3)
.
But biosafety concerns have discouraged the development of clinical applications involving replication-competent VSV. Many species of wildlife can also be affected by VSV and develop flu-like signs such as fever, headache, muscular aches, and oral blisters similar to herpes virus infection (4)
. VSV may even cause a fatal meningoencephalitis in experimentally infected mice (5
, 6)
. Virus infection usually triggers powerful host antiviral response and immune responses that will abolish the virus soon and diminish its oncolytic effect. Therefore, it is necessary to develop a new strategy to take advantage of VSV while avoiding the use of replication-competent intact virus.
The VSV genome codes for the assembly of only 5 proteins (N, P, M, G, and L), and of these, the M protein plays a remarkable role in virus infection, including inhibition of host gene expression and induction of cell apoptosis (7)
. Expression of M protein alone causes similar effects as infection with VSV by inhibiting transcription of all three host RNA polymerases (8
9
10
11)
. Moreover, M protein (as a virus antigen) may modify tumor cells and enhance host systemic immune responses, which could favor tumor eradication (12)
. In this study, an M protein-expressing plasmid was generated, and cationic liposome was used as carrier for the treatment of various tumor cell lines (in vitro and in vivo) to evaluate the antitumor effect of M protein.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasmid construction
The study protocol was approved by the institutional review board of Sichuan University (Chengdu, Sichuan, China). VSV M protein cDNA was amplified from total RNA extracted from virus-infected BHK-21 cells by reverse transcriptase-polymerase chain reaction (RT-PCR) with upstream primer 5'-CGCGGATCCATCATGAGTTCCTTAAAGAAG-3' and downstream primer 5'-CGGAATTCTCATTTGAAGTGGCTGATAGAATCC-3'. The cDNA was digested with BamHI and EcoRI and was inserted into eukaryotic expression vector pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA). Verification of the recombinant construct was performed by DNA sequencing. The plasmid was termed as pcDNA3.1-M (p-m). The empty vector pcDNA3.1(+) was used as a control (e-p). All plasmids used were purified by two rounds of passage through Endo-Free columns (Qiagen, Chatsworth, CA, USA), as described elsewhere (13
, 14)
.
Preparation of cationic liposome and liposome-DNA complex
Liposome-containing DOTAP (Sigma D6182; Sigma-Aldrich, St. Louis, MO, USA) in a 1:1 M ratio with DOPE (dioleylphosphatidyl-ethanolamine) (Sigma P1223) was prepared by solubilizing the lipid in 3:1 (v/v) chloroform:methanol. The lipid mixture was then dried as a thin layer in a round-bottomed flask under a stream of N2. Residual chloroform and methanol were removed under high vacuum. The resulting lipid film was hydrated at 2 mg/ml in 5% dextrose, and sonicated until completely solubilized. The liposome was extruded through 100 nm polycarbonate membrane to generate small unilaminar vesicles, and the recovered liposome reagent was stored at 4°C. Before use, plasmid DNA was mixed with liposome in 1:5 ratio (w/w) to form a complex (15)
. This complex was used in all of the experiments below to evaluate M protein activity.
MTT colorimetric assay
Cell viability was monitored using 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay, which is based on the ability of living cells to reduce MTT, a yellow, water-soluble monotetrazolium salt, to an insoluble purple formazan (16)
. To validate the influence of M protein on cell growth, A549, LLC, SW480, and CT26 cells were seeded in 96-well flat-bottomed plates at a concentration of 2000–5000 cells/well in 100 µl medium. The next day, cells were transfected with p-m or e-p, and cultivated in a CO2 incubator at 37°C for 24–96 h. Empty liposome and nontransfected cells were used as controls. At the time of analysis, each well was supplemented with 50 µl of MTT solution (2 mg/ml) in complete medium and incubated at 37°C for 4 h; then the medium and MTT solution were removed, and 150 µl of dimethyl sulfoxide (DMSO) was added into each well. Absorbance was read at 540 nm using a microplate reader, and the experiment was repeated 3 times. Cells treated only with medium served as the indicator of 100% cell viability.
Quantitative assessment of apoptosis
Flow cytometric analysis was performed to identify sub-G1 or apoptotic cells. The percentage of sub-G1 cells was determined after propidium iodide (PI) staining in hypotonic buffer, as described elsewhere (17
18
19)
. Briefly, 1 x 106 LLC cells transfected with p-m or e-p (as described above) or untreated cells were collected and resuspended in 1 ml hypotonic fluorochrome solution containing 50 µg/ml PI in 0.1% sodium citrate plus 0.1% Triton X-100, the cells were analyzed by flow cytometer (ESP Elite, Beckman Coulter, Fullerton, CA, USA). Cells appearing in subG1 stage were considered as apoptotic cells.
Morphological analysis
1–3 x 105 LLC and CT26 cells were seeded in 6-well plates and incubated at 37°C with 5% CO2. Twenty-four hours later, cells were transfected with plasmids (p-m or e-p), with empty liposome and untransfected cells as controls. The morphological analysis was performed 48 h post-transfection. The medium was removed; cells were collected and rinsed with PBS, then stained for 30 min with 50 µg/ml propidium iodide in 0.1% sodium citrate plus 0.1% Triton X-100, and then examined by fluorescence microscope.
Murine tumor models and treatment
Animal studies were performed in accordance with institutional guidelines concerning animal use and care. All studies involving animals in connection with the present investigation were approved by the Animal Care and Use Committee of Sichuan University (Chengdu, Sichuan, China).
MethA fibrosarcoma and LLC models were established in 6- to 8-wk-old female BALB/c and C57BL/6 mice, respectively. Each type of cell (2x105 to 1x107) suspended in 100 µl of PBS was injected into the right flank of mice. To explore the therapeutic efficacy of p-m, mice were treated at 7–10 days after the implantation of tumor cells (when tumor size ranged between 15 and 20 mm3 in cross section) and were randomly assigned to four groups (10 mice in each group). Tumor-bearing mice were injected intratumorally with different doses of pcDNA-M (p-m), pcDNA3.1-null (e-p), empty liposome, or PBS. Injections were distributed equally into each of the four tumor quadrants. Mice were treated every 3 days over a 3-wk period. The tumor volume was determined by the following formula: tumor volume (mm3) = 
/6 x length (mm) x [width (mm)]2.
Tumor cell rechallenge
In the MethA tumor model, mice treated with pcDNA-M displayed complete tumor regression and survived for longer than 50 days. To determine whether surviving mice had developed long-term immunological memory, the animals were divided into two groups and rechallenged subcutaneously at the other flank with either parental MethA or CT26 tumor cell.
Evaluation of T-cell response
Adaptive cellular immune response was evaluated using cytotoxic lymphocyte (CTL) assay. A 4-h 51Cr release assay was performed as described elsewhere (20
, 21)
. Briefly, splenocytes obtained from the treated or control mice were depleted of erythrocytes with ammonium chloride Tris buffer. A T-enriched cell fraction was prepared. All 5–10 x 106 target cells were labeled with 51Cr for 2 h at 37°C. After washing several times, 1–3 x 104 51Cr-labeled target cells and serially diluted effector cells, at varying E:T ratios (40:1 to 5:1), were incubated in 200 µl of RPMI 1640 with 10% FBS in each well of 96-well V-bottomed plates. The plates were centrifuged at 500 g for 3 min and incubated at 37°C for 4 h. The supernatant (50 µl) was harvested, and the activity was calculated by the formula: % cytotoxicity = [(experimental release–spontaneous release)/(maximum release–spontaneous release)] x 100%.
Histological analysis
Paraffin sections (3–5 µm) of embedded tissues from each group were stained with hematoxylin and eosin (H&E) (22)
. The largest diameter of the tumor was scanned in the low field using the computer-aided image analysis system Quantimet 600 and Qwin software (Leica, Benshaim, Germany). The total tumor area and necrotic tumor area were subsequently marked by the examiners on the screen. The areas were evaluated by the image analysis system, and the ratio of tumor necrosis area to total tumor area was given in percentages. Heart, liver, spleen, lung, kidney, and brain from the mice were fixed in 4% neutral buffered formalin solution and embedded in paraffin for observation of potential side effects in treated mice.
Terminal deoxynucleotidyltransferase-mediated nick end labeling (TUNEL) assay was performed for detecting fluorescence of apoptotic cells in situ using the TUNEL kit (Boehringer Mannheim, Indianapolis, IN, USA) following the manufacturer’s protocol.
Immunofluorescence staining was used for detection and analysis of lymphocytes infiltrated into tumor in situ. Anti-CD3 [fluorescein isothiocyanate (FITC) conjugate; eBioscience, San Diego, CA, USA] and CD8 (Cy5PE conjugate; eBioscience) monoclonal antibodies were used to determine cytotoxic T lymphocytes. Tumors were snap-frozen, and 8-µm sections were prepared in Tissue Tek (Sakura Finetek, Torrance, CA, USA) for immunofluorescence analysis, as described elsewhere (23)
. Sections were blocked (10% FBS, 3% BSA) for 30 min before staining with anti-CD3 or CD8 monoclonal antibody. Sections were washed 3 times between each reagent. Fluorescence was visualized, and images were captured with Olympus BX60 (Olympus, Tokyo, Japan).
Data analysis and statistics
For comparison of individual time points, analysis of variance (ANOVA) and an unpaired Student’s t test were used. Survival analysis was computed by the Kaplan-Meier method and compared by the log-rank test. P < 0.05 was considered statistically significant (24)
.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The p-m also showed cytotoxicity to these tumor cells.
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The type of cell death induced by M protein was also determined. Morphological changes characterized as apoptosis were observed in LLC, A549, CT26, and SW480 tumor cells transfected with p-m after PI staining. Bright red fluorescent condensed nuclei (intact or fragmented) and apoptotic bodies were observed by fluorescence microscopy focused on PI-stained nuclei (Fig. 2A
). The numbers of apoptotic cells were quantitatively estimated by observing sub-G1 cells using flow cytometry. The cells treated with p-m displayed many more sub-G1 cells (apoptotic cells) compared with the control groups (48.7 vs. 12.3–14.0%, P<0.05, Fig. 2B
). Furthermore, a ladder-like pattern of DNA fragments consisting of multiples of 
180–200 base pairs was demonstrated by agarose gel electrophoresis of chromosomal DNA extracted from M protein-treated cells; this is consistent with internucleosomal DNA fragmentation (data not shown).
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In vivo growth inhibition of the established tumors in mice
To determine the optimal dose of M-protein-plasmid:liposome complex in therapeutic treatment, a pilot study was performed. A ratio of 1:5 (50 µg plasmid:250 µg liposome) was found to be most effective and was used in subsequent experiments.
Mice bearing LLC or MethA tumors were treated with M protein plasmid every 3 days for 18 days, beginning 7 days after tumor cell implantation, when the tumors were palpable. Retarded progression or complete regression of the established tumors was observed in p-m-treated mice in the LLC or MethA models, respectively (Fig. 3a, b
; P<0.05). The survival time of the tumor-bearing mice treated with p-m was also significantly longer than that of the untreated mice or other control groups (Fig. 3c, d
; P<0.05).
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Gross changes, such as weight loss, ruffling of fur, and changes in behavior were not seen in M protein plasmid-treated mice. In addition, no obvious pathological changes of liver, lung, kidney, spleen, or brain were found by microscopic examination.
Tumor cell rechallenge
To determine whether there is a protective antitumor immune response after p-m treatment, the long-term surviving mice were rechallenged subcutaneously, 50 days after first injection of MethA, with 2 x 105 CT26 cells or 1 x 106 MethA fibrosarcoma cells in the other flank (Fig. 4
, arrowhead). Tumor growth in these long-term survivors was compared with that in naive (untreated) BALB/c mice injected with the same number of tumor cells. After 8 days, all untreated mice and those rechallenged with CT26 cells had developed large flank tumors, while none of the previously p-m treated mice had detectable flank tumors after rechallenge with MethA cells. In the MethA model, animals treated with p-m were "cured" and exhibited long-term protection against the same tumor model rechallenge. These observations suggest that the intratumoral treatment of established MethA tumor model with M protein plasmid resulted in the development of a specific long-term protective immune response.
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Induction of specific cytotoxic T lymphocyte in response to M protein treatment
To determine whether M protein could induce tumor-specific CTL activity in vivo, tumor cell cytotoxic activity of CTLs was examined. T cells isolated from spleens of p-m treated mice exhibited higher cytotoxicity to LLC tumor cells than those from control groups (Fig. 5a
). This cytotoxicity could be blocked by anti-CD8 or anti-major histocompatibility complex (MHC) class I mAbs, but not by anti-CD4 in vitro (Fig. 5b
), suggesting that the killing activity observed may result from MHC class I-dependent CD8+ CTL activity. Furthermore, the adoptive transfer of CD4-depleted (CD8+) T lymphocytes isolated from mice treated with p-m showed antitumor activity against LLC tumor cells, but not against other tumors. The transfer of T-lymphocyte subsets from mice treated with e-p or liposome alone or saline had no such effect. Further, CD8-depleted (CD4+) T lymphocytes isolated from mice treated with p-m showed little effect (Fig. 5c
).
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Histological evaluation of T-cell migration and apoptosis
Histological examination of tumor sections by H&E staining at day 14 after the beginning of treatment showed that PBS group tumor cells had well-defined cell borders and hyperchromatic nuclei. The cytoplasm of these cells was vesicular and eosinophilic, with evidence of mitosis. In contrast, tumors from mice treated with M protein gene showed extensive necrosis, characterized by loss of nuclear staining, increased cytoplasmic eosinophilia, and loss of cellular detail and cell borders (Fig. 6a-d
). Such observations support the idea that the remaining tumor mass at day 14 consisted largely of dead or dying tumor cells.
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In view of the apparent antitumor activity in LLC and in MethA models, it was decided to investigate the mechanism underlying liposome:p-m mediated cancer cell death. Toward this goal, we examined apoptosis-related molecular markers on tumor sections 14 days after the beginning of treatment. The TUNEL method was utilized to detect early DNA fragmentation associated with apoptosis. In comparison with control groups, apoptotic cells could be easily detected within the residual tumors treated with VSV-M before complete tumor regression (Fig. 6e-h
). To better understand the relation between the antitumor effect in vivo and apoptosis, a scattergram was used to plot the percentage of inhibition of tumor growth against the apoptosis index, linear regression analysis suggested that inhibition of tumor growth was strongly correlated with apoptosis in the p-m-treated group and that this was in a dose-dependent manner. Similarly, the inhibition was also related to the apoptosis index by the treatment with e-p or liposome alone (data not shown).
To detect the infiltrated lymphocytes in tumors and determine their type, Anti-CD8 and CD3 monoclonal antibodies were used in immunofluorescence staining. The results showed that many more infiltrated lymphocytes were detected in the M protein-treated tumor than in control groups and that these cells mainly consisted of CD8+ cytotoxic T lymphocytes (Fig. 7
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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26
27)
. Studies of apoptosis induction triggered by anticancer agents may provide insights into facilitating the development of novel anticancer therapies. It has been reported that the introduction of new antigens into tumor cells by virus modification, drug treatment, or allogeneic MHC gene transfer approaches were shown to stimulate immune responses against autologous malignant cells (28
29
30
31)
. Thus, the combination of apoptosis induction and active immunotherapy may be a new and potent strategy for cancer therapy. In this study, we cloned cDNA of VSV M protein, the main apoptosis-inducing gene of VSV, and investigated its antitumor activity in vitro and in vivo. Cationic liposome-mediated delivery of M protein-expressing plasmid was introduced into several cultured tumor cell lines in vitro and was found to efficiently inhibit cellular growth and induce apoptosis. After intratumoral injection into mouse MethA fibrosarcoma and LLC tumors, VSV M protein plasmid clearly inhibited tumor growth and prolonged the survival of the mice bearing the tumors. For MethA fibrosarcoma, the tumor was completely regressed, and the mice acquired long-time protection against rechallenge by the same tumor cell line. Several approaches have been applied concerning the induction of apoptosis and the activation of immune responses by M protein. Many more apoptotic and necrotic cells could be visualized in the tumors treated with M protein when compared with control groups by in situ TUNEL assay and H&E staining. In addition to inducing tumor cell apoptosis, M protein appeared to initiate a potent cellular immune response, which included both higher MHC class I-dependent CD8+ CTL activity from splenocytes and increased levels of infiltrated CD8+ T lymphocytes in tumor sections of treated mice, as detected by 51Cr release assay and immunofluorescence staining, respectively. To our knowledge, most cancer therapy agents that are used in clinical practice or in research usually show only one major antitumor activity. For example, known chemotherapeutic agents or monoclonal antibodies or immunotoxins may kill cancer cells or induce cell apoptosis, but the former are unable to activate immune responses, and the latter trigger undesirable immune responses that clear the agents themselves from the body and reduce the antitumor effect. On the other hand, most therapeutic tumor vaccines do initiate immune responses against the tumor but cannot induce cell apoptosis directly. An agent that is able to concomitantly initiate both of these two effects, such as the M protein plasmid described here, is rarely found.
Unlike the replication-competent VSV, M protein-expressing plasmid cannot replicate in vivo, and this may partly limit its antitumor effect. However, M protein plasmid has many advantages over VSV. First, live VSV can cause a mild febrile illness in a few individuals or cause epidemic diseases in various domestic animals and lead to disastrous economic loss. Consequently, VSV is a biohazardous agent and unlikely to be utilized in clinical applications. In contrast, treatment with M protein plasmid is very safe. Second, VSV eventually triggers strong humoral and cellular immune responses against VSV itself within 1 or 2 wk of infection (32
33
34)
, which makes the cancer treatment incomplete and is undesirable. In contrast, cationic liposome-mediated delivery of plasmid can activate an innate immune response that includes the induction of proinflammatory cytokines and immune cell activation (35
36
37)
and lead to further induction of cell immune responses after cellular penetration and expression into protein. Induction of immune response by the liposome-plasmid complex itself also explained why the control empty plasmid group of our study also induced increased levels of apoptotic cells and conferred a partially protective effect in tumor-bearing mice. However, the liposome-plasmid complex itself is unlikely to be neutralized by antibody and could be used in repeated treatment. Therefore, the immune responses will favor tumor cell clearance, while having no influence on the plasmid itself.
In our previous study, the antitumor effect of VSV M protein was preliminarily evaluated in rat C6 glioma model (38)
. M protein plasmid was transfected into cultured C6 glioma cells and shown to inhibit the C6 intracranial tumorigenicity in vivo. Intracranial injection of 150 µg of M protein plasmid complexed with liposome into rats preimplanted with C6 glioma, resulted in inhibition of tumor growth and the survival time of rats was prolonged. The antitumor efficacy of M protein on glioma is 50%. In this work, the antitumor efficacy of M protein plasmid was reaffirmed in different tumor models, mainly LLC in C57BL/6 mice and MethA fibrosarcoma in BALB/c mice. The overall efficacy is 70–100%. A possible mechanism of action was explored in both studies. A potent apoptosis induction effect by M protein was observed in both studies and may be the main contributor to its antitumor effect. In previous research, CD31 was stained and fewer microvessels were observed in M protein-treated C6 glioma, which may mean that M protein has an additional antiangiogenesis function. In the present work, CD31 staining was not carried out, but the immune response was considered. It has been shown here that as a viral protein, M protein did induce cell responses, which also contributed to its antitumor activity. Consequently, M protein is a multifunctional protein and may become a promising cancer gene therapy agent. This present finding is in accordance with our previous work and includes the exploration of additional immune responses.
Taken together, the findings of the present study demonstrate that the introduction of VSV M protein into tumor cells not only induces cell apoptosis but also enhances the immune response specifically targeting the tumor cells, and this dual activity resulted in a better therapeutic effect. This study provides a basis for further study into the establishment of a novel and safe cancer therapeutic strategy based on the apoptosis-inducing genes of oncolytic viruses.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication March 26, 2008. Accepted for publication July 24, 2008.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T cells and heat shock protein 70. Cancer Res. 56,1104-1110
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 50 µg e-p/250 µg liposome complex (
), 250 µg liposome alone (
), or PBS (•), respectively, once every 3 days for 6 times. It was evident that, compared with control groups, p-m-treated mice showed significant tumor volume decrease and survival time prolongation both in LLC model (a, c) and MethA fibrosarcoma model (b, d) (P<0.05).
) appeared to be protected and survival was prolonged, whereas the mice rechallenged with CT26 did not appear to be protected and soon died (
). B) Tumor growth in these long-term survivors was compared with that in naive (untreated) BALB/c mice. Naive mice were injected s.c.with 2 x 105 CT26 (
), and the p-m-treated long-term survivors were also injected s.c. with the same number of CT26 (
) into the other flank. All naive mice and the mice rechallenged with CT26 developed large flank tumors, whereas none of the previously treated mice rechallenged with MethA had detectable flank tumors.
