The phosphatase and tensin homolog gene inserted between NP and P gene of recombinant New castle disease virus oncolytic effect test to glioblastoma cell and xenograft mouse model

Background Glioblastoma is one of the most serious brain cancer. Previous studies have demonstrated that PTEN function disorder affects the causing and exacerbation of glioblastoma. Newcastle disease virus (NDV) has been studied as a cancer virotherapeutics. In this study, PTEN gene was delivered to glioblastoma by recombinant NDV (rNDV) and translated into protein at the cytoplasm of the glioblastoma. Methods We did comparison tests PTEN protein expression efficiency and oncolytic effect depend on the PTEN gene insertion site at the between NP and P genes and the between P and M gene. PTEN protein mRNA transcription, translation in glioblastoma cell, and functional PTEN protein effect of the rNDV in vitro and in vivo test performed using western blotting, RT-qPCR, MTT assay, and Glioblastoma xenograft animal model test. Results The result of this study demonstrates that rNDV-PTEN kills glioblastoma cells and reduces cancer tissue better than rNDV without the PTEN gene. In molecular immunological and cytological assays, PTEN expression level was high at located in the between NP and P gene, and PTEN gene was successfully delivered to the glioblastoma cell using rNDV and PTEN gene translated to functional protein and inhibits hTERT and AKT gene. Conclusions PTEN gene enhances the oncolytic effect of the rNDV. And our study demonstrated that NP and P gene site is better than P and M gene site which is commonly and conventionally used. PTEN gene containing rNDV is a good candidate virotherapeutics for glioblastoma. Supplementary Information The online version contains supplementary material available at 10.1186/s12985-022-01746-w.


Introduction
Glioblastoma or glioma, one of the most serious brain cancer originates from glial cells that is a fatal tumor in the human central nervous system [1,2]. The routine treatments for glioblastoma patients are surgical operations combined with radiotherapy, chemotherapy, and other effective treatments [3,4]. Commonly observed symptoms of glioblastoma are persistent headaches, vomiting, double or blurred vision, loss of appetite, change in mood and personality, seizures, cerebral hemorrhage, and symptoms related to specific parts of the brain suspected of having glioblastoma [5]. According to case studies show 75% of all malignant brain tumors Open Access *Correspondence: vacchyun@naver.com 1 Libentech Co. LTD, C-722 Daedeok BIZ Center, Techno 4-ro, 17 Yuseong-gu, Daejeon 34013, Republic of Korea Full list of author information is available at the end of the article are glioblastoma [6]. Malignant grades of glioblastoma are diverse in appearance depending on the variations in infiltrative growth [7][8][9]. Thus, while glioblastoma is of clinical interest, developing effective treatments therapeutics for patients remains unsolved.
Oncolytic virotherapy is a perspective form of new biological therapeutics for various cancer treatments. The current oncolytic viruses are constructed using cancer cell killing or immune response electing gene to increase the efficacy of the virotherapeutics. Other virotherapeutics using intrinsic oncolytic virus or cancer cell killing effective gene containing intrinsic oncolytic virus were tried to using cancer treatment. Several results show utilizes viral intrinsic oncolytic properties and specific genes more effectively destroy malignant tumor cells than utilize oncolytic virus or specific gene [10]. Since the 1950s several research has been tried using NDV for cancer treatment [11]. NDV could be developed as a perspective anticancer virotherapeutics for cancer patients but remain how could increase the oncolytic effect of lentogenic strain using safety-guarantied technology. The research about the basic mechanism of oncolytic effect has been studied by the velogenic NDV strains and that show strong tumor suppression result from many brain cancer patients during the clinical test [12]. But velogenic strain has safety issue as a pathogen of the avian infectious disease, the lentogenic strain used instead of velogenic strain. Intrinsic oncolytic effect of the lentogenic strain less than that of the velogenic strain, several studies were performed increase oncolytic effect using several tumor suppression gene containing rNDV has been constructed, which was show cancer cell killing and tumor suppression effect to various cancer. NDV can not propagate in the normal cell but in cancer. Because in cancer cell IFN-α mediated innate immune response is not elect by virus infection. Most of cancer cell do not produce IFN-α and not properly work the cytokine-producing signal pathway [13][14][15][16]. Newcastle disease virus (NDV) is one of the old history cancer viral therapeutics. In the 1950s velogenic strain of NDV has been used to cancer-killing effect test even some of the oncolytic effects test was doing in Human patients. Several research and clinical studied show perspective to cancer treatment because NDV does not infect mammalian and no side effect by repeat inoculation and cancer cell killing effect and tumor suppression effect also good to patients who participated in clinical trial survive more than five years [17]. But NDV velogenic strain is a serious pathogen of poultry disease it can be a big damage to the poultry industry. So, this is one of the obstacles of NDV develop viral cancer therapeutics. In the 2000s many scientists start using a lentogenic strain of NDV used to developing as an oncolytic virus. But this lentogenic strain also has defects not strong cancer cell killing effect or tumor suppress effect compare with velogenic strain and the reason is lentogenic strain show lysogenic virus-like does not occur reinfection to neighbor cancer cell after the first infection. To overcome this defect, several cancer cell killing and tumor suppress gene containing recombinant NDV virus has been constructed and used to test anticancer effect test include clinical trial. This kind of progress successfully makes good results comparing to just attenuated NDV vaccine strain used previous test [18]. One more advantage of NDV compare with the currently used viral vector, NDV can pass the brain-blood barrier (BBB) and be transmitted to the human brain. It is very suitable as a viral vector for glioblastoma treatment [19]. Now increasing NDV oncolytic effect several related area researches were performed such as the most efficient foreign gene insertion site and expression of a functional molecule using recombinant NDV. A most important study is the development of NDV using cancer therapeutics that NDV or recombinant NDV was how they get the selectively infect to a specific cancer cell and not infected to a normal cell.
PTEN protein is a well-known tumor suppressor protein that acts as a phosphatase enzyme that catalysis the dephosphorylation of the 3′ phosphate of the inositol ring which is in PIP3 and makes the biphosphate product PIP2. This dephosphorylation act a key role in regulating cellular behaviors such as migration, survival, and cell growth. Deletions and mutations of PTEN inactivate its enzymatic activity during tumor development leading to reduced cell death and increased cell proliferation. Frequent genetic disorder of PTEN occurs in prostate cancer, endometrial cancer, and glioblastoma; and decreased expression is found in a lot of other tumor types such as breast and lung cancer. Current studies show about 50% of GBMs have a somatic change in the phosphatidylinositol 3-OH kinase pathway [20,21]. Weak protein phosphatase activity is also important to tumor suppression. Protein phosphatase activity of PTEN may be involved in cell cycle regulation, preventing cells from dividing and growing too promptly.
In the present study, newly constructed recombinant NDV(rNDV) based on the VG/GA lentogenic strain [22] which has the PTEN gene and Kozak sequence in the rNDV genome and tests the oncolytic effect in vitro and in vivo.

cDNA construction of NDV containing PTEN CDS
The 1212 base pair DNA sequence encoding human PTEN CDS (NM_000314.8) was synthesized and provided from Bioneer (Daejeon, Republic of Korea). In this study, PTEN gene was inserted between NP and P genes or between P and M genes. Two sites are designated Position "1" (NP-P) and Position "2" (P-M) (Fig. 1A)

Transfection and recovery of recombinant NDV
NP, P, and L genes which form the NDV transcriptase complex were cloned into a pBR322 vector and used as a helper plasmid (pBR322-NP, pBR322-P, pBR322-L). HEp-2 cells were prepared in a 6-well plate at 5 × 10 5 cells/well. After 24 h of culture, Vaccinia virus expressing T7 RNA polymerase infected HEp-2 cells (MOI 1). 5 μg of rNDV-PTEN cDNA vector was transfected into HEp-2 cells with 2.5 μg of NP helper plasmid, 1.5 μg of P helper plasmid, and 0.5 μg of L helper plasmid by Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. After 72 h of transfection, 100 μl HEp-2 cell supernatant was harvested and inoculated in the allantoic cavity of a 10-dayold specific-pathogen-free (SPF) embryonated chicken egg. After 96 h, the allantoic fluid was harvested. The harvested viruses were adapted to the Vero76 cell. Using TCID50 assay by Reed-Muench method, NDV titers and MOI are determined. In order to confirm the existence of the NDV and the inserted PTEN gene, reverse transcription PCR (RT-PCR) was performed with primers of 'NDV check' to amplify an rNDV L gene fragment, primers 'NP-PTEN-P check' to confirm inserted PTEN gene fragment in Position "1" and primers 'P-PTEN-M check' to confirm inserted PTEN gene fragment in Position "2". (Table 2) ( Fig. 1) RT-PCR conditions to amplify an rNDV L gene fragment and confirm inserted PTEN gene were as follows: 1 cycle at 42 °C for 1 h, 94 °C for 5 m, followed by 35 cycles at 94 °C for 60 s, 60 °C for 60 s, and 72 °C for 60 s. Sequencing was performed to confirm that the rescued rNDV-PTEN virus has proper PTEN CDS (Additional file 1: S1, S2). And another sequencing was performed whether the virus has intact PTEN CDS even when subcultured to Passage10 (Additional file 1: S3, S4).

MTT assay
An MTT assay is used to accurately measure live cells and evaluate changes in cell proliferation or death.

Statistical analysis
All data are presented as the mean ± the standard deviation of the means (SD), as indicated. Statistical comparisons were made by using Student's t-test or one-way ANOVA with SPSS (Version 23, SPSS, Inc., Chicago, IL, USA). p Values < 0.05 were considered statistically significant.

Growth kinetics and selective oncolytic effect of recombinant NDV infection to cancer cell
To evaluate the rNDV replication efficiency, a growth dynamics curve in Vero76 cells was constructed (MOI 0.1) (Fig. 2A). The growth dynamics curve shows that the two rNDV viruses, one is having PTEN gene Position "1", but both of the recombinant viruses' growths have no significant difference. Both rNDV increase titer following the culture time and when 48 through 72 h post-infection show highest virus titer 10 6.7 TCID 50 /ml rNDV and 10 6.5 TCID 50 /ml rNDV-PTEN Position "1". Real-time quantitative PCR was used to assess interferon-α mRNA transcription levels (Fig. 2B). rNDV infected normal (CCD18-Co) cells have 4.64, 5.12, and 5.76 times higher interferon-α mRNA transcription levels than not infected normal (CCD18-Co) cells, rNDV infected glioblastoma (T98G) cells, and not infected glioblastoma (T98G) cells.
These results demonstrate that even when a foreign gene is inserted into rNDV, it proliferates as well as rNDV and the foreign gene is well expressed. And according to the expression of interferon-α, rNDV does not show apoptotic effect in normal cells but shows in glioblastoma cells.

Effects of PTEN gene delivered by rNDV to apoptosis suppressor genes of T98G glioblastoma cells
Western blotting was used for comparing PTEN protein, P-Akt protein, and hTERT protein expression between PTEN inserted rNDV Position "1" and PTEN inserted rNDV Position "2" (Fig. 3A) (MOI1). Both PTEN inserted rNDV infected glioblastoma cells showed increased PTEN protein expression than rNDV infected glioblastoma cells and not infected glioblastoma cells (negative control). PTEN protein expression level of PTEN inserted rNDV Position "1" infected glioblastoma cells was slightly higher than PTEN inserted rNDV Position "2" infected glioblastoma cells. Both PTEN inserted rNDV infected glioblastoma cells showed reduced P-Akt and hTERT protein expression than rNDV infected glioblastoma cells and not infected glioblastoma cells (negative control). P-Akt and hTERT protein expression level of PTEN inserted rNDV Position "1" infected glioblastoma cells were lower than PTEN inserted rNDV Position "2" infected glioblastoma cells.
Western blotting was used for evaluating PTEN protein expression as time goes on (Fig. 3C). rNDV-PTEN Position "1" infected glioblastoma cells have significantly increased protein expression than rNDV infected glioblastoma cells and not infected glioblastoma cells in all times of this experiment. Quantification of Results of Western blotting was also performed (Additional file 1: S5).
These results demonstrate that rNDV-PTEN infected glioblastoma cells have increased PTEN expression and decreased P-Akt and hTERT activity due to increased PTEN expression. And PTEN inserted rNDV Position "1" is more effective than rNDV Position "2" to expression PTEN gene.
These results demonstrate that rNDV-PTEN inhibits glioblastoma and removes tumor cells in vitro. And PTEN inserted rNDV Position "1" is more effective than rNDV Position "2" to remove tumor cells in vitro.

PTEN gene containing rNDV induces size reduction of glioblastoma tumor on T98G cell planted xenograft mouse
Using mice, in vivo assay has been performed (Fig. 5, Table 4). At all injection sites, the tumor repression effect of rNDV-PTEN Position "1" was better than that of rNDV. Tumor injection was more effective than tail vein injection to reduce the volume of the tumor. rNDV-PTEN Position "1" injected tumors showed a steep decrease of tumor volume on day 2 of virotherapy and rNDV injected tumors showed a steep decrease of tumor volume on day 3 of virotherapy. On all days of virotherapy, the volume (%) of rNDV-PTEN Position "1" injected tumors was always smaller than rNDV injected tumors. rNDV-PTEN Position "1" effectively reduced the volume of glioblastoma tumors compared to rNDV and PBS groups. Especially, the gaps of reducing the volume of glioblastoma tumors between rNDV-PTEN Position "1" groups and rNDV groups or PBS groups were larger in tail vein injection. Orthotropic glioblastoma model in vivo test was also performed (Additional file 1: S6).
This result demonstrates that rNDV-PTEN Position "1" is more effective than rNDV to reduce the size of glioblastoma in the xenograft mouse model.

Discussion
The mutation of PTEN in various cancer is observed [25][26][27][28]. Therefore restore of PTEN expression in various cancer is important. Especially, PTEN mutation accounts for 34-44% of the cause of glioblastoma [29][30][31]. Elucidating the PTEN functions and the consequences of PTEN mutations allows the further understanding of cancer etiology and neoplasia. In glioblastoma, PTEN regulates glioblastoma oncogenesis by histone H3.3 and chromatin-associated complexes of DAXX [32] and monoubiquitination [33]. There is study indicated that PTEN inhibits lung cancer growth by promoting G0/G1 arrest and cell apoptosis, so rNDV-PTEN could be applied to lung cancer [34]. Immunotherapy which includes antigens on the cancer cell surface was used in various types of cancer [35][36][37]. Immunotherapy of tumors needs specific biomarkers of cancer cells such as specific receptors, recognition molecules, or antigen domains. Immunological response against occurred specifically against therapy and anti-neoplastic agents is effective [38][39][40][41]. As a result, restoration of normal PTEN expression in glioblastoma is very important in remove or therapy glioblastoma.
There have been disadvantages in glioblastoma virotherapeutics using virus vectors such as adenovirus, vaccinia virus, and adeno associated virus. In the case of the vaccinia virus, there is a limitation in repeated inoculation to humans because pre-existing immunity to the vaccinia virus is a problem in humans [42]. In the case of adenovirus or adeno-associated virus, glioblastoma removal or treatment is impossible because they cannot pass through the brain-blood barrier (BBB) and thus cannot access the glioblastoma [43]. However, NDV can be inoculated repeatedly because pre-existing immunity is not a prohibit cancer cell killing effect when inoculated into the human body, and NDV can pass through the brain-blood barrier (BBB), it is possible to infect glioblastoma cancer cells in the brain [44][45][46][47]. In addition, since NDV has an intrinsic oncolytic effect, it is possible to safely perform cancer treatment with a relatively small dose compared to viral vectors which were no intrinsic oncolytic effect. Therefore, NDV virus or tumor suppressor gene containing recombinant NDV has many advantages in effective treatment method to glioblastoma as a virotherapeutics.
rNDV has an intrinsic oncolytic effect but this study show rNDV-PTEN has more strong oncolytic effect against glioblastoma cells and T98G cell-induced glioblastoma tumor tissue. PTEN gene expression by rNDV-PTEN produced increased anti-tumor effects by mediating cell death and PTEN-mediated apoptosis. In glioblastoma cells treated with rNDV-PTEN, antiapoptotic protein expression was strongly increased, suggesting that the apoptosis-resistance in glioblastoma had been decreased. NDV has been previously used for delivery of several genes [19,[48][49][50][51][52]. In addition, it has been confirmed that these rNDV-PTEN viruses have intact PTEN CDS even after 10 passages of subculture (Additional file 1: S3,S4). In conclusion, this study shows that rNDV-PTEN has a beneficial effect on the NDV oncolytic function by expressing PTEN in treated glioblastoma. Currently, several researches used foreign gene insertion site using between P and M genes (Position "2"). This site was known not seriously affect recombinant NDV growth and stable expression of an inserted gene and showed a good effect on cancer suppression but until now any kind of direct comparison test between P and M site (Position "2") with other four more possible foreign gene insertion sites [53]. In this study, the PTEN gene was inserted between NP and P gene (Position "1") which position is not used before inserting anticancer gene to make oncolytic virus using NDV. Real-time qPCR and Western blotting results show PTEN gene transcription level by PTEN inserted NDV Position "1" is higher than by PTEN inserted rNDV Position "2". Because of higher PTEN gene expression of PTEN inserted rNDV Position "1" than of rNDV Position "2", the oncolytic effect of PTEN inserted rNDV Position "1" also better than the oncolytic effect of PTEN inserted rNDV Position "2". It means NDV has six major structural genes each gene post-infection in the host cell and mRNA transcription level depends on the distance from the 3′ end of the NDV genome and NP gene mRNA transcription level highest compares with other structural genes. Because of this mechanism of NDV, the closer the foreign gene is inserted to the NP gene, the higher the transcription level of foreign gene mRNA. But still is not clear that mRNA transcription level is not directly correlated with translation level. In addition, further researches are needed to determine whether the difference in inserted foreign gene expression level is dependent on the insertion site or the type of the inserted foreign gene or whether there are other factors.

Conclusions
In this study show PTEN gene enhance glioblastoma cell killing effect of recombinant NDV, which is one of an intrinsic oncolytic virus. NDV known as a virus pass the brain blood barrier, therefore recombinant NDV containing cancer suppressor genes will be good candidate for virotherapeutics for brain cancer treatment.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Declarations
Ethics approval and consent to participate Animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee of Libentech and all experiments conform to all relevant regulatory standards.