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Volume 8, Issue 4 (November 2021)                   IJML 2021, 8(4): 275-283 | Back to browse issues page


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Maleki S, Soleimanjahi H, Abdoli A, Karimi H, Banijamali R S, Bamdad T. The Effect of Autophagy Induction in Oncolytic Reovirus Replication in Mesenchymal Stem Cells. IJML. 2021; 8 (4) :275-283
URL: http://ijml.ssu.ac.ir/article-1-401-en.html
Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
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Introduction
Autophagy is an essential intracellular degradative process known as type II programmed cell death through a lysosomal-dependent degradation mechanism. Autophagy plays several roles in adaptation to stress conditions, cell death, and tumor suppression [1]. Beclin-1 and LC3 are connected with the initial steps of autophagosome formation and maturation during the autophagy process, respectively. Beclin-1 is an important, primary molecule that acts as a platform for autophagy establishment [2-4] to start autophagy.
Many drugs, such as metformin, are currently approved by the Food and Drug Administration (FDA) that induce autophagy [5]. Metformin is an AMP-activated protein kinase (AMPK) activator and activates autophagy via a reduction in the mammalian target of rapamycin (mTOR) signaling pathway [6].
Oncolytic viruses are novel anti-cancer therapeutics that induce an immune response and demonstrate minimal human toxicity [7]. Oncolytic reovirus has shown oncolytic potential against over 80% of human cancer cell lines. Clinical trials demonstrated that therapies based on oncolytic reovirus are safe and tolerated in patients with a wide variety of cancers [8].
Autophagy is one of the probable mechanisms of oncolysis by oncolytic reovirus. It has been suggested that oncolytic reovirus induces autophagy to kill cancer cells by exploiting p17 protein in the PI3K-Akt-mTORC1 pathway [9]. Several studies have suggested a novel vehicle system for virus delivery by mesenchymal stem cells (MSCs) to obtain optimum delivery and maximum antitumor effects of these oncolytic viruses [10, 11]. Before using MSCs as a carrier for oncolytic reovirus, the relationship between the effects of induction of autophagy on the replication of oncolytic reovirus in the MSCs should be examined. This study evaluated the effect of autophagy induction using metformin on the replication of reovirus T3D in the adipose derived mesenchymal stem cells (AD-MSCs) and L929 cell as control cell lines.
Materials and Methods
AD-MSCs cultures
The AD-MSCs were isolated from abdomen adipose tissues of C57BL/6 mice (Pasteur Institute of Iran) and were characterized and maintained as previously described [12]. The cells expressed CD29, CD90, and CD105 and were negative for CD34 and CD45. AD-MSCs were also examined for their ability to differentiate between adipocyte and osteocyte.
Cell lines and culture conditions
Murine L929 fibroblasts cell lines were provided by Dr. Soudi (Tarbiat Modares University, Iran). The cells were maintained at 37 °C with 5% CO2 and cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented by 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA).
Virus preparation
Reovirus T3D was propagated in L929 cell culture in serum-free DMEM. Briefly, L929 cells were infected at multiplicities of infection (MOI) of 0.1 and allowed to incubate at 37°C until a complete cytopathic effect was observed, at which point the virus was collected. Viruses were harvested by three freezes (-70 °C)-thaw cycles and clarified by low-speed centrifugation for 15 min. Large stocks of viruses are kept frozen at -70 °C in aliquots. Virus titer was determined by cell culture infectious dose 50% (CCID50) assay as previously described [13].
Chemicals
Metformin (Sigma-Aldrich, USA) was diluted in sterile water to make the 1M stock solution and used at a concentration of 5 mM in all the experiments, except cell viability assay, in which concentrations ranging from 2.5 to 20mM were tested.
Cytotoxicity assay
Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay based on the manufacturer's protocol (Bioidea, Iran). Briefly, L929 cells were seeded into the 96-well plates. After overnight incubation at 37 °C, these cells were treated with metformin at different concentrations (2.5 to 20 mM). Three wells were remained untreated as a control. At 24 hours after incubation at 37 °C and 5% CO2, 10 μl of MTT reagent was added to each well and incubated for four hours; the cell-free wells were considered blank controls. Then, the MTT solution was removed, and 50 μl of dimethyl sulfoxide was added to each well. The plate was maintained for 15 min at 37°C, the wells' optical density was determined at 570 nm through a spectrophotometric microplate reader.
Cell viability assay
The viability of treated AD-MSCs and L929 cells was determined using MTT assay, which evaluates the percentage of viable cells. AD-MSCs and L929 cells were seeded into a 96-well plate and treated with 5mM of metformin for 12 hours. After that, the cells were infected with reovirus T3D at MOI of 0.01. Twenty-four hours after infection, cell viability was measured by MTT assay, similar to the previous section. Experimental groups are shown in Table 1.
 
Table 1. Experimental groups
MRA Metformin/Reovirus /AD-MSCs AD-MSCs which treated with metformin and reovirus
MA Metformin/AD-MSCs AD-MSCs which treated with metformin
RA Reovirus /AD-MSCs AD-MSCs which infected with reovirus
MRL Metformin / Reovirus / L929 cells L929 cells which treated with metformin and reovirus
ML Metformin / L929 cells L929 cells treated with metformin
RL Reovirus/L929 cells L929 cells which infected with reovirus
 
 
 
RNA extraction and real-time polymerase chain reaction (PCR)
AD-MSCs and L929 cells were seeded in 48-well plates. After 24 hours of incubation, the cells were treated with 5 mM of metformin. At 12 hours after treatment, the cells were infected with reovirus T3D at an MOI of 0.01. According to the manufacturer's protocol, total RNA was isolated from cultured cells using YTA Total RNA Purification Mini kit (Yekta tajhiz azma, Iran) 24 hours after infection. A total of 3 μl of RNA was employed to synthesize complementary DNA (cDNA) by RT series kit (BioFact™, Korea) with Random Hexamer and oligo dT primers.
Real-time PCR was performed using EvaGreen® qPCR mix plus [7] and step one plus real-time PCR system (Applied Biosystems, USA). Primer sequences were as follows: L3 (major capsid protein lambda 1) gene segment of Reovirus:
5′-CGCGTCCTCAATTTTGGGTAAAC-3′(F) 5′-CCGCCGTCTTTTGGATATGAACTA-3′(R) [12]; Beclin-1: 5′-ACAGCCCAGGCGAAACCAG-3′(F) 5′-CCTCCCCGATCAGAGTGAAGC-3′(R); HPRT1: 5′-TCCCAGCGTCGACTTAGACT-3′(F) 5′-CGAGCAAGTCTTTCAGTCC-3′(R).
RT-PCR was conducted with the following amplification conditions: 1 cycle of 95 °C for 15 min, 40 cycles of 95 °C for 15 sec, 62 °C for 20 sec and 72 °C for 30 sec. The expression revealed by each assay was normalized using the HPRT1 expression level as a control. This study was approved by the Research Ethics Committee of Tarbiat Modares University, Tehran, Iran (Code: IR.TMU.REC.1395.468).
Statistical analysis
All of the experiments in this research were performed three times in three replicates. The data are presented as mean ± standard deviation and analyzed by GraphPad Prism 7.04. statistically, significance was compared between groups using one-way ANOVA analysis of variance followed by Dennett's test for real-time PCR and Tukey's test for viability assay. Asterisks indicated groups are significantly different from each other (*: P ≤ 0.05; **: P ≤ 0.01; ***: P ≤ 0.001).
Results
MTT assay
L929 cells were treated with various concentrations of metformin ranging from 2.5-20 mM for 24 hours. With the increasing amount of metformin, the cell viability was decreased using MTT assay. The half-maximal (50%) inhibitory concentration (IC50) values were 5 mM for the cells incubated for 24 hours, and this concentration was used in all experiments (Fig. 1).
AD-MSCs and L929 cells were measured with 5 mM of metformin for 12 hours to measure viable cells. Then, the cells were infected with reovirus T3D at MOI of 0.01. Twenty-four hours after infection, the percentage of viable cells was measured by MTT assay. As illustrated in Figure 2, the percentage of viable cells in AD-MSCs after treatment with metformin and infection with reovirus (MRA group) reduced significantly compared to MA and AD-MSCs which infected with MOI:0.01 of reovirus (RA groups). Furthermore, co-treatment of metformin and infection with reovirus in L929 cells (MRL group) significantly reduced cell viability compared to metformin (ML group) or reovirus infection (RL group) alone. However, at a steady-state, there was no significant difference between MRA and MRL groups.
 
 
Real-time PCR analysis
The changes in the relative expression level of Beclin-1 and reovirus L3 gene were observed to determine the effect of metformin and reovirus infection on induction of autophagy and its relation to reovirus replication. As shown in Figure 3A, in the MRA group, the relative expression level of the Beclin-1 gene increased significantly compared to the RA group. However, the relative expression level of the Beclin-1 gene in the MRA group decreased significantly compared to the AD-MSCs treated with 5 mM of metformin (MA group). In the MRL group, co-treatment of metformin and reovirus infection significantly increased the relative expression level of the Beclin-1 gene compared to the RL group. On the other hand, the relative expression level of the Beclin-1 gene in the MRL group decreased significantly compared to the ML group. However, at a steady-state, there was no significant difference between their effect in primary and line cells (MRA and MRL groups).
As demonstrated in Figure 3B, the relative expression level of the reovirus L3 gene in AD-MSCs after treatment with metformin and infection with reovirus (MRA group) increased significantly compared with reovirus infection alone (RA group). Also, in the MRL group, co-treatment with metformin and reovirus infection significantly increased the relative expression level of the reovirus L3 gene compared to infection with reovirus. However, the relative expression level of the reovirus L3 gene in the MRL group increased significantly compared to the MRA group.
Discussion
Given that cancer is a complex multistage process and that autophagy performs its effects in different ways, its role in tumor treatment with virotherapy should be evaluated [1]. Autophagy is an essential, conserved process by which cytoplasmic components are degraded by lysosomes [14]. Beclin-1, the mammalian orthologue of yeast Atg6, has a central role in autophagy initiation [2]. In this way, metformin, an anti-diabetic drug, can trigger autophagy [15]. However, other studies demonstrated that reovirus induces autophagy in tumor cells to destroy them [9]. However, to enhance the quality of treatment with an oncolytic virus, many studies suggested using MSCs as a vehicle [16]. Also, Banijamali et al. [17] suggested that AD-MSCs can be used as effective carrier cells candidates for reovirus T3D to maximize their anti-cancer cell activity.
Nevertheless, a better understanding of the reovirus-host interaction is crucial to improving therapeutic strategies for oncolytic virotherapy. This study demonstrates the viability of AD-MSCs and L929 cells after treatment with metformin and reovirus infection. Furthermore, the relationship between the autophagy induction with metformin in the replication of reovirus was evaluated. The results of this study revealed that combined treatment of metformin and reovirus infection in both AD-MSCs
and L929 cells (MRA and MRL groups) significantly increased cell death compared to treatment with metformin (MA and ML groups) or reovirus infection (RA and RL groups) alone. So, treatment with metformin and reovirus T3D infection promoted the cell death of AD-MSCs as a primary cell and L929 line cells. Cell death may occur through active mechanisms, for example, cellular suicide or active or programmed cell death. At a steady-state, there was no significant difference between results from AD-MSCs and L929 cells. Some reports have shown that metformin treatment can induce apoptosis [18] and autophagy [19] in some types of cancer. Metformin exhibited significant synergy with chemotherapeutic drugs models via broad-spectrum action mechanisms. The combination of metformin and sorafenib prevents proliferation and triggers autophagy of hepatocellular carcinoma through targeting the mTOR pathway [20]. Nazim et al. [21] found co-treatment of metformin with tumor necrosis factor-related apoptosis-inducing ligand significantly induced cell death of human lung adenocarcinoma A549 cells. Yang et al. studied the efficiency of PEGylated liposomes comprising metformin and epirubicin against CD133+ cancer stem-like cells [22]. Their results exhibited that liposomes-encapsulated metformin and epirubicin induce cell death compared to the metformin and epirubicin alone. Buzzai and colleagues [23] pointed that metformin induces autophagy in HCT116 colon cancer cells.
Beclin-1 has a key role in autophagy because it is involved in the triggering of autophagosomes formation [24]. Our real-time PCR results demonstrated that treatment with metformin alone significantly increased the relative expression level of the Beclin-1 in AD-MSCs and L929 cells (MA and ML groups) compared with MRA and MRL groups. In contrast, co-treatment of metformin with reovirus (MRA and MRL groups) significantly increased the relative expression level of the Beclin-1 compared to treatment with reovirus infection alone (RA and RL groups). However, at a steady-state, there was no significant difference between results from AD-MSCs and L929 cells. It seems that coadministration of the metformin and reovirus has a synergistic effect on the increase of autophagy compared with virotherapy alone, which mutually reinforces the virus replication.
Parallel with this study, Xie and colleagues [25] have shown that metformin dramatically enhanced Beclin-1 protein expression in OVE26 mice. On the other hand, it has been demonstrated that viruses have evolved ways to suppress or induce the autophagy machinery to facilitate their replication and survival [26].
This in-vitro study examined metformin-induced autophagy in AD-MSCs and L929 cells at the early stage of infection with reovirus T3D. Our data suggest that autophagy induced via metformin enhances viral replication in AD-MSCs and L929 cells at the early stage of infection. Nevertheless, the reovirus L3 gene expression level in the MRL group increased significantly compared to the MRA group because  L929 cells are a susceptible host cell line for reovirus T3D. Niu and colleagues [27] reported that avian reovirus induces autophagy, which benefits its replication and dissemination in chicken tissues. Similarly, Chi et al. [28] reported that autophagy induction through rapamycin treatment increased avian reoviral protein synthesis and viral yield.
Conclusions
Overall, autophagy can be stimulated by metformin and reovirus T3D infection and plays a positive role in reovirus T3D replication. Also, the oncolytic effect of reovirus was increased by simultaneous application of metformin. Still, the interplay between autophagy and cell death, leading to oncolysis in reovirus T3D infection, needs further study to understand the crosstalk between autophagy and reovirus oncolytic activity deeply.
Conflict of Interest
The authors declared no conflict of interest.
Acknowledgments
The results described in this paper were part of the student thesis, supported by the grant number Med-62290 from the Research Deputy of Tarbiat Modares University, Faculty of Medical Sciences.
 
 
 
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Type of Study: Research | Subject: Virology
Received: 2021/02/11 | Accepted: 2021/07/25 | Published: 2021/12/15

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