Recently, the use of oncolytic viruses against cancer has attracted considerable attention. We studied the potential of the US3 locus-deficient herpes simplex virus (HSV), L1BR1, for oncolytic virus therapy. Its high specificity and potency indicate that L1BR1 is a promising candidate as a new oncolytic virus against pancreatic cancer. Moreover, the virus exhibited the unique characteristic of increasing apoptosis when used in combination with anticancer drugs. We assessed the feasibility of using the US3 locus-deficient HSV named L1BR1 as a new replication-competent oncolytic virus for the treatment of pancreatic cancer. The US3 locus of HSV has been shown to be a key gene in producing a multifunctional protein kinase that inhibits apoptosis induced by viral infections, chemicals and ultraviolet (UV) light. L1BR1 has been reported to be more than 10 000-fold less virulent than the parental virus in mice. In this study, we examined the tumor specificity and oncolytic effect of this attenuated replication-competent virus, L1BR1, in pancreatic cancers derived from SW1990, Capan2 and Bxpc-3cells compared with the parent virus and other well-known oncolytic herpes viruses (R3616 and hrR3). We also studied the efficacy of L1BR1 for the induction of apoptosis as an attribute of this virus in combination with the anticancer drugs 5FU and cisplatin. The combined treatment of the pancreatic cancer cells with L1BR1 and these anticancer drugs enhanced apoptosis significantly. More importantly, L1BR1 showed the lowest replication capacity in normal human hepatocytes, but the highest tumor-reducing effect in vivo among the oncolytic herpes viruses tested. In addition, L1BR1 significantly increased the induction of apoptosis of cancer cells when treated in combination with anticancer drugs although the parental virus inhibited the induction of apoptosis. These results suggest that L1BR1 is promising as a new anticancer oncolytic virus.
The prognosis of patients with pancreatic cancer is poor, with or without treatment. In the United States, there are about 27 000 new cases of pancreatic cancer with approximately 26 000 deaths each year. The incidence of this cancer has been increasing over the past 20 years, ranking it as the fourth most common cause of cancer death. Even after surgery for pancreatic cancer, the 1-year survival rate is approximately 10% for patients with stage III or IV. (Unfortunately, most patients belong to the advanced stage at the time of diagnosis.) Chemotherapy has been used for advanced pancreatic cancer, but systematic drug infusion has proved ineffective in curing patients.
Recently, cancer gene therapy using a variety of viral vectors has been studied intensively, and several viruses have been tested in clinical trials. Some of the viral vectors include an artificial therapeutic gene that can induce cancer cell death, but viral vectors often have anti-apoptotic genes to evade apoptotic cell death.1, 2, 3, 4 Although several laboratories have shown that herpes simplex virus (HSV) US3 exhibits anti-apoptotic activity, the mechanism by which US3 functions in the regulation of apoptosis is still controversial.5 It is known that eukaryotic cells respond to certain cellular stresses by stimulating the c-JUN N-terminal kinase (JNK), and that p38 mitogen-activation of JNK results in apoptosis. We previously reported that the expression of US3 PK suppresses the activation of JNK, and hence US3 may prevent apoptosis through the attenuation of JNK signaling.6 Munger and Roizman7, 8 recently reported that HSV-1 US3 PK prevents the apoptosis induced by over-expression of the Bcl-2-inhibitor of cell death protein (BAD) in rabbit skin cells. BAD, a pro-apoptotic member of the Bcl-2 family, promotes apoptosis, suggesting that the aberrant BAD phosphorylation mediated by US3 PK is involved in the suppression of apoptosis.
Although L1BR1 replicates as efficiently as its parental virus in a number of cultured cells, the intraperitoneal injection of L1BR1 into mice does not result in systemic viral proliferation, and fails to kill mice, even at high doses. In our previous study, we demonstrated that L1BR1 is more than 10 000-fold less virulent (LD50) than the parental virus after intraperitoneal injection. Numerous focal necrotic lesions were produced on the surface of the liver by infection with the parental virus but not with L1BR1.9 We also reported that L1BR1 could not propagate even when it was inoculated directly on to the surface of mice cornea. Microscopically, it was observed that the surface of the corneal cells had pealed away due to apoptotic death induced by the invading viral particles. This results in the restriction of viral propagation and the subsequent viral infection of neighboring cells.10
Moreover, the US3 gene has the ability to inhibit the apoptosis induced by various stimuli, including treatment with cycloheximide (CHX),11 ceramide, tumor necrosis factor, anti-Fas antibody,12 osmotic shock using sorbitol13 or ethanol,14 hypothermia and thermal shock15, 16 and ultraviolet (UV) light.17 In this paper, we attempt to show the potential of US3-inactivated HSV mutant L1BR1 as a promising new oncolytic virus for cancer treatment, and the benefit of the combined use of this US3 gene-deficient oncolytic virus with the anticancer chemodrugs 5FU and cisplatin.
Materials and methods
The SW1990 cell line, derived from human pancreatic cancer, was provided by Dr T. Sawada (First Department of Surgery, Osaka City University, Osaka, Japan).18 CAPAN-2, another human pancreatic cancer cell line, was obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan), while BxPC-3 was from the American Type Culture Collection (Manassas, VA). SW1990 and CAPAN-2 carry a K-ras gene with a point mutation, while BxPC-3 has no K-ras mutations. BxPC-3 and CAPAN-2 have a mutation in the p53 gene.19 Cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (Sigma, Tokyo, Japan). We previously reported the dissemination of SW1990 in the abdominal cavity after intraperitoneal inoculation.20
Human hepatocytes (hNHeps, Cambrex, Charles City, IA, USA), hepatocyte media and supplement (HCM Bullet kit, Cambrex) were purchased from Takara Bio Co. (Otsu, Japan). The normal liver cells were cultured in human hepatocyte media with HCM supplement.
Wild-type HSV-2 (strain HSV186) was prepared in Vero cells, a stable line of African green monkey kidney cells, by infecting them at a low multiplicity. L1BR1 is a protein kinase (US3)-inactivated HSV-2 mutant, in which the ICP8 promoter-LacZ gene cassette inserted into the US3 sequence of the wild-type HSV-2 strain 186. The plasmid and construction of L1BR1 were described in a previous paper9 (Figure 1). ICP6 locus-deficient HSV, hrR3, was derived from a wild-type HSV-1, KOS virus (kindly provided by Sandra K. Weller, University of Connecticut, CT). Duplicate γ34.5 locus-deficient virus, R3616, was derived from the wild-type HSV-1 F-strain (kindly provided by Dr Bernard Roizman, University of Chicago, IL).
In vivo viral replication assay
The viral replication assays were performed as described previously.21 Briefly, 1 × 106 cells were infected with 2 × 106 pfu (plaque forming units) of the virus for 2 h, after which the unabsorbed virus was removed by washing with a glycine–saline solution (pH 3.0). The supernatant and cells were harvested 40 h after infection, exposed to three freeze–thaw cycles to release virions and titered on Vero cell monolayers. The results were divided by the cell number (1 × 106) and were reported as the mean of three independent experiments.
Viral cytotoxic assays were performed as described previously.22 Briefly, cells were plated onto 96-well plates at 5000 cells per well for 36 h. Viruses were added at multiplicity of infection (MOI) values ranging from 0.001 to 10 and incubated for 6 days. The number of surviving cells was quantitated by a colorimetric MTT assay, and the percent cell survival was calculated by comparison with the control (mock-infected) cells. Tests were performed in quadruplicate.
Percent apoptotic caused by virus in vitro
A total of 1 × 106 SW1990, BxPC-3 and CAPAN-2 cell lines were infected with strain HSV186 or L1BR1 at an MOI of 0.5. At the indicated time after infection, the cells were washed once with phosphate-buffered saline (PBS). The apoptotic percentages were assessed by an ApoAlert mitochondrial membrane sensor kit (Clontech Laboratories, Palo Alto, CA). Determinations were based upon fluorescence under green light. The MitoSensor label accumulates in the mitochondria of healthy cells, where it aggregates and fluoresces red. In apoptotic cells, since the mitochondrial membrane potential is altered, MitoSensor labels, which cannot accumulate in mitochondria, remain in the cytoplasm as monomers that fluoresce green. This experiment was performed in triplicate.
Percent apoptosis caused by virus with chemodrugs in vitro
The pancreatic cell lines (SW1990, BxPC-3, CAPAN-2) were infected with viral strains HSV186 or L1BR1 at a MOI of 0.5. After 1 h, the cells were washed once with PBS, and then the chemodrugs (5FU or cisplatin) were added to yield a final concentration of 500 μ M. The apoptotic percentages were assessed using an ApoAlert mitochondrial membrane sensor kit. Determinations were based upon fluorescence under green light 24 h after infection. This experiment was performed in triplicate.
Animal studies (tumor burden)
Animal studies were performed in accordance with the guidelines issued by the Nagoya University Animal Center. Adult female BALB/c-nu/nu mice were obtained from SLC (Hamamatsu, Japan). Ten mice were used per group. A total of 1 × 107 human pancreatic SW1990 cancer cells were injected subcutaneously into the back of each mouse. A total of 10 tumors were induced in the group. Fourteen days after tumor injection, they reached an approximate diameter of 5 mm, after which either 5 × 106 pfu/100 μl of L1BR1, hrR3 or R3616 or the same volume of PBS were injected once into the subcutaneous tumor. The size of the tumor was estimated by determining their longest diameter (L) and width (W), and calculating the volume (0.5L × W2). Next, 2 days after the viral inoculation of 5 × 106 L1BR1 or 5 × 104 HSV186, the subcutaneous tumors were killed for an evaluation of virus selectivity by polymerase chain reaction (PCR).
Animal studies (abdominal tumor)
After the mice (BALB/c-nu/nu) were anesthetized with diethyl ether, the peritoneal cavity was opened, and the pancreas was identified by the fatty tissue surrounding the intestine. Typically, within 1 week of the intraperitoneal inoculation of 1 × 107 SW1990 cells into BALB/c nude mice, multiple white nodules approximately 0.5 mm in diameter develop on the surface of the intestine and mesenterium fatty tissue surrounding the pancreas. Mice were inoculated with either replication-conditional 5 × 106 L1BR1 or 5 × 104 HSV186 15 days after inoculation of the SW1990 cells. Two days after viral inoculation the mice were killed, and the tissues were harvested for histochemical X-gal staining.
Histopathological and histochemical study
Tissues from the mice that had disseminated abdominal tumors were harvested and rapidly frozen in liquid nitrogen. Cryostat sectioning of the tissue was performed at 10 μm thickness. Sections were then fixed and stained histochemically for X-gal to assess lac Z expression.
PCR amplification of L1BR1 specific sequences was used to investigate its biodistribution following its injection into mice. The forward oligonucleotide primer 5′-IndexTermGGAGGCGCCCAAGCGTCCGGCCG-3′ and the reverse oligonucleotide primer 5′-IndexTermTGGGGTACAGGCTGGCAAAGT-3′ were used to amplify a 229-bp fragment of the viral DNA polymerase gene. Mouse tissues were incubated at 56°C overnight in digestion buffer (10 mM Tris–HCl, pH 7.4; 5 mM ethylenediaminetetraacetic acid (EDTA); 0.5% sodium dodecyl sulfate (SDS); and 200 μg/ml proteinase K, pH 8.0). Following phenol and chloroform (1:1) extraction, the DNA was precipitated in 70% ethanol, lyophilized and resuspended in distilled water. An aliquot of the DNA (0.1 μg) was then subjected to PCR amplification. The PCR amplification was performed in a 25 μl volume using Taq DNA polymerase for 35 cycles of 1 min each at 94, 60 and 72°C. Negative controls were the tumors from mice not injected with the virus.
Student's t-test was employed to analyze the data (SAS Institute Inc. StatView for Mac (R), version 5.0).
Time-course study of the percent apoptosis caused by virus in vitro
We first examined the difference in the apoptosis-inducing ability between the parent virus (strain 186) and the US3-deficient virus (L1BR1) in three human pancreatic cell lines without any stimulus. From previous observations, we expected that the deletion of US3 would raise the percentage of apoptotic cells after viral infection. However, L1BR1 did not induce a higher percentage of apoptosis than did the parent virus in the three cell lines; the apoptotic percentage induced by L1BR1 was very similar to that induced by the parent virus in SW1990, and considerably lower in CAPAN-2 and BxPC-3. No lines of L1BR1 ever intersected the lines of the parent virus (Figure 2). In these experiments, we observed that the plaque-forming time of the parent virus was faster than L1BR1 in the plates, and that the parent virus exhibited higher cytotoxicity than did L1BR1. There was no statistical significance of the difference between L1BR1 and HSV186, in SW1990, P<0.4916; in Capan 2, P<0.3146; and in BxPC-3, P<0.0734.
Dose escalation study of the percentage of apoptosis caused by virus with chemotherapy in vitro
We next studied the anticellular effect of the US3-deficient virus in combination with anticancer chemotherapy. In this experiment, 5-FU and cisplatin were used as the stimuli for apoptosis. The combination of drugs with L1BR1 generally caused the highest percentage of apoptosis among these three groups. The drugs given with wild-type virus were definitely lower than the drugs with L1BR1 (Figure 3). The parent virus exerted a greater anti-apoptotic effect than did L1BR1 in this stimulation study. There were statistically significant differences between L1BR1 and HSV186, in the presence of cisplatin: SW1990: P<0.001, BxPC-3: P<0.0058, and Capan 2: P<0.0014. The corresponding values in the presence of 5FU are SW1990: P<0.0004, BxPC-3: P<0.0021, and Capan 2: P<0.0002.
Viral replication assay in vitro
To assess the safety and potency of L1BR1 as on oncolytic virus, the replication of L1BR1 was examined in a normal cells (human hepatocyte) and three pancreatic cell lines. In Figure 4, greater than log 0 means that the virus propagated, while less than log 0 indicates that the virus did not propagate. The viral replication assay revealed that the mutant viruses did not propagate in normal cells, while the parent virus did. Among the mutant viruses, L1BR1 also exhibited poor viral replication in human liver cells. However, all the viruses tested replicated well in all pancreatic cancer cell lines tested, and L1BR1 had somewhat lower replication rate in SW1990 and Capan 2 cells but nearly the same rate in BxPC-3 cells (Figure 5). This experiment was performed in triplicate.
Cytotoxic assay in vitro
We also investigated the cytotoxicity of L1BR1 in comparison with other viruses in the three pancreatic cancer cell lines. The cytotoxicity generally reflects the efficacy of the tumor-reducing effect caused by a virus. L1BR1 was less cytotoxic than were R3616 and hrR3 in all three pancreatic cancer cell lines (Figure 6). Thus, L1BR1 failed to generate a strong cytotoxicity against pancreatic cancer cell lines, and it was not expected to exhibit a strong tumor-reducing effect against tumor-bearing mice in vivo, compared with the other mutant viruses. This experiment was performed in triplicate.
Animal studies (tumor burden)
The tumor-reducing effect induced by the direct intratumoral injection of HSV mutants was examined in an animal model using nude mice bearing SW1990. The result was quite different from what we had expected. L1BR1 caused the highest tumor-reducing effect among the mutant viruses tested in vivo. Over the 2 weeks after virus injection, R3616 and hrR3 had curves similar to that of L1BR1, but over the next 2 weeks, the tumor size of R3616 and hrR3 gradually increased. On the other hand, the curve of L1BR1 continued to decrease. In the L1BR1-treated mice, two of the 10 visible tumors disappeared 4 weeks after the virus injection (Figure 7). These results showed that L1BR1 has the strongest anti-tumor effect among the mutant HSV viruses.
Animal studies (abdominal tumor)
Two days after virus inoculation, a histopathologic study was performed on disseminated multiple intraperitoneal tumors (SW1990). X-gal staining disclosed many blue-stained tumor nodules in the virus-treated mice (indicating viral infection), and cancer cells floating in the ascites also stained blue. Virus-induced cell damage and viral inclusion bodies were observed in the region of the stained cells. LacZ-positive tumors displayed inflammatory infiltrates with a few neutrophils and a small number of lymphocytes, as well as degenerative changes. Neither blue-stained cells nor significant cell damage was observed in any normal tissues or organs including the liver, spleen, pancreas, gastrointestinal tract and the mesentery (Figure 8).
Selectivity of L1BR1 by PCR
We tried to prove the virus selectivity by PCR. We compared the biodistribution of L1BR1 to the parent strain HSV 186 by the presence of the viral DNA polymerase gene in mouse tissues. When the mice were killed 2 days after the virus was injected into subcutaneous tumors, virus DNA was detected in all tissues (tumor, brain, lung, spleen, liver and colon) in the HSV 186 group. On the other hand, no viral DNA was detected in the L1BR1 group except for the tumor samples themselves. Based on this result, the biodistribution of virus L1BR1 is strictly restricted to the tumor (Figure 9).
The purpose of this study was to evaluate the suitability of L1BR1 as a new oncolytic virus. Many physicians and scientists have attempted myriad studies to find a cure for cancer, but no truly effective form of treatment is available for most tumors. There are great expectations from gene or virus therapy. In the present study, we investigated the suitability of the US3-deficient HSV virus, L1BR1, which has a favorable characteristic regarding the induction of apoptosis, as a new candidate for oncolytic virus therapy. We attempted to show that L1BR1 could replicate in pancreatic cancer cells and reduce tumor growth through its oncolytic effect as an apoptosis inducer. In our previous study, we reported that L1BR1 was more than 10 000-fold less virulent than its parental virus.9 We also showed that, even if this virus is inoculated directly on to the corneal surface of immune competent mice, it could not propagate and did not cause any neurovirulent disease.23 This study showed that L1BR1 can replicate well in three human pancreatic cancer cells but not in human hepatocytes. Moreover, we confirmed that L1BR1 was detected only within the tumor, as evidenced by the blue stains inside the tumor and floating cancer cells in the ascites. No blue staining was seen in normal tissue, and the absence of virus was confirmed by PCR. Judging from these results, L1BR1 seems to be a highly promising candidate for a new oncolytic virus. Although the cytotoxicity of L1BR1 was rather weak compared with the other mutant viruses in vitro, it had the strongest anti-tumor effect among the mutant viruses against subcutaneous tumors in vivo. This is indeed a remarkable characteristic of L1BR1 as a new candidate for an oncolytic virus.
The US3 region has been shown to be a key gene with an anti-apoptotic function in infected cells. The expression of US3 prevents apoptosis induced by various stimuli (as mentioned earlier in the Introduction). However, Jerome suggested that the anti-apoptotic effect of HSV is complex. Several HSV genes including US3 and US5 have been shown to be involved in the suppression of apoptosis in infected cells. These gene products may play distinct roles in preventing apoptosis.17
Regarding the number of apoptotic cells caused solely by viruses, the apoptosis caused by L1BR1 was the same or lower than those of the parent virus in three pancreatic cancer cell lines (CAPAN2, SW1990 and BxPC-3). This might be due to the fact that the growth rate of the parent virus is generally faster than that of the mutant.24 In the presence of an anticancer agent, however, L1BR1 accelerated the extent of apoptosis more than did the parent virus in all three pancreatic cancer cell lines. These results suggest that the US3 gene is actually involved in the regulation of host-cell apoptosis in the presence of anticancer drugs. Both its selectivity for neoplasm and its effectiveness with anticancer agents are of primary importance for human gene therapy and viral therapy. The apoptosis-inducing activity of L1BR1 should also be studied in combination with radiation therapy.
Basically, the role of the anti-apoptotic function of a virus is to prolong the survival of infected cells so as to protect the virus until it reaches maturity so that the production of the viral progeny can thus be maximized. This phenomenon has been observed with many types of virus. For example, the inhibition of apoptosis enhances the production of human immunodeficiency virus,25, 26, 27 baculovirus28 and African swine fever virus.29, 30, 31 Adenovirus, a representative oncolytic virus, is known to encode an anti-apoptosis gene that is called the E1B-19kD gene. This gene is a powerful inhibitor of the apoptotic pathways, and plays a role in maintaining viral propagation in the host cell.32 The E1B-19kD gene is an anti-apoptotic member of the Bcl-2 family of apoptosis regulators, which function upstream of caspase activation.33, 34 The effect of an E1B-19kD mutation on viral replication may be of concern, as premature death of the host cell and viral DNA degradation may compromise viral yield.35 It has been shown that, although there is less viral product, viral release occurs earlier than for the wild type, which is caused by early host-cell death.32, 36 Sauthoff32 has reported that early cell lysis and a large bolus of early viral release might accelerate cell-to-cell spread, which would be beneficial for cancer therapy when the host immune system is under attack, especially in cases of inefficient tumor cell infection. Worgell37 has indicated that efficient infection of a solid tumor will, therefore, depend on cell-to-cell viral spread and its clearance by the immune system and innate immune mechanisms.
The HSV-US3-deficient virus did not show sufficient propagation in pancreatic cancer cell lines in vitro, but unexpectedly had a potent tumor-reducing activity in vivo. The results with the solid tumor in vivo might be caused by the same early viral release and enhanced cell-to-cell spread seen in the adenovirus E1B-19kD mutant virus. However, this must be confirmed by further study.
Recently, many types of HSV mutants have been reported and used for cancer therapy. G207 has been in Phase 1 trials, and Phase II trials are under way for neural tumors.38 NV1020, which was derived from HSV-1 and includes a 5.2-kb fragment of HSV-2, has already been used in a Phase I trial against colon cancer liver metastases at the Sloan Kettering Cancer Center in New York.39 However, such mutants appear to be too attenuated to have a sufficient oncolytic effect. We have already completed a Phase I/II clinical trial using HF10 against breast cancer. The final pathological finding in this trial showed a 30–100% tumor-reducing effect against breast cancer.40 We are now preparing to conduct a clinical trial against pancreatic cancer using the HSV-1 mutant HF10.41 L1BR1 offers a new, beneficial and unique aspect of oncolytic virus therapy. We predict that virus therapy will be used as a form of combination therapy together with chemotherapy or radiation in the future. In our view, the regulation of the anti-apoptotic effect is indeed highly promising for virus therapy and gene therapy using virus vectors. Oncolytic virus therapy permits higher tumor selectivity and safety, but few, if any, reports have described the possibility of regulating the apoptosis effect caused by the deletion of an original viral gene. To our knowledge, this is the first paper to report the effect of an anti-apoptotic gene deletion-HSV mutant involving oncolytic viral therapy. Our data regarding the US3 gene may well contribute to all gene therapy using HSV mutants and provide considerable information for future studies. This promising gene of HSV should be studied further in greater depth.
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This study was supported by the Grant-in-Aid for Scientific Research in Japan, No. 16390358. There is no financial conflict of interest.
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Cite this article
Kasuya, H., Nishiyama, Y., Nomoto, S. et al. Suitability of a US3-inactivated HSV mutant (L1BR1) as an oncolytic virus for pancreatic cancer therapy. Cancer Gene Ther 14, 533–542 (2007). https://doi.org/10.1038/sj.cgt.7701049
- oncolytic virus
- herpes simplex virus
- pancreatic cancer
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