Oncolytic reovirus shows potentially anticancer effect against CT26 cell lines CURRENT STATUS: POSTED

Background: Reovirus type 3 Dearing (T3D) is a segmented double-stranded RNA that selectively replicates and triggers anticancer responses in the KRAS mutant cancer cells. Here, we examine the anticancer activity of ReoT3D against murine colon carcinoma cell line (CT26) with deferent in vitro experiments. Results : Our results showed that ReoT3D significantly induced oncolysis in the CT26 cells by induction of apoptosis, and cell cycle arrest in the G0/G1 and G2/M checkpoints and reduction of cell migration and colony forming ability. Conclusion : Present study confirmed that ReoT3D has strong anticancer impacts on CT26 cells which can be considered as a new approach against KRAS mutant cancer cell lines in laboratory investigation.


Background
Therapeutic usage of viruses as a cancer treatment option has been increasingly explored over the past year [1].The results of these studies demonstrated that the oncolytic viruses selectively replicate in the tumor cells [2,3].
Oncolytic reovirus (Respiratory Enteric Orphan viruses) is a small non-enveloped virus, which commonly, isolated from respiratory and gastrointestinal tract without any known and severs disease [4]. Reovirus has three strains (Dearing, long, Jones) with anti-tumor properties, but most clinical trials have recently focused on the T3D strain (Reolysine) [5,6]. The viral ligand in all strains is the Sigma 1 protein attached to the Junction adhesion molecule A (JAM-A) receptor in the long and Jones strains, but T3D strain beside this receptor (JAM-A), can use from Nogo receptor1 (NgR1) for virus attachment and entrance to the host cells [5].
Normally, penetration of dsRNA viruses into the untransformed cells triggered cellular defense mechanisms by activation of PKR (dsRNA dependent protein kinase). So, dimerization, auto phosphorylation, and activation of PKR prevent the virus transcription [7]. In contrast, ReoT3D is a wild type reovirus with innate ability to inhibit KRAS signaling pathway in transformed cells [8]. KRAS mutant cells promote viral replication by inhibition of PKR and led to the selectively replication of 3 reovirus in the KRAS transformed cells [9].
KRAS mutations contribute to the development of different cancers [10], 90% pancreatic [11], 50% colorectal [9], and 29% lung carcinoma [12]. The publish data demonstrated that reovirus may be a useful option in the treatment of several cancers with activated KRAS signaling pathway. Also, reovirus application in the treatment of some cancers was reported in the recent publications [13][14][15].
Colorectal cancer (CRC) is the third most common cancer among United States population with around 500000 death annually [16]. Surgery, chemotherapy and radiation therapy are common methods for CRC treatment that they do not have remarkable impacts on the patients survival [17].
Therefore, developing of new approaches for treatment of colon cancer is urgently needed. Distinct features of oncolytic reovirus including: high infectivity and virus production rate, proper cell cultures and hosts, easily virus manipulation, selectively infection and apoptosis induction in the cancer cells caused introduce this agent as promising antitumor candidate [18][19][20].
In current study, we inoculated CT26 cells with ReoT3D and investigated its antitumor impacts in murine colorectal cancer cells. Our results showed that ReoT3D infection markedly persuaded apoptosis induction, arrested cell cycle, inhibited migration and colony forming in CT26 cells. Our findings suggested that ReoT3D can be considered as promising agent for the treatment of colorectal cancer.

ReoT3D infection causes cytotoxic effects with dose-dependent manner:
ReoT3D stock propagated in the L929 cells and cytopathic effect was followed by light microscopy.
To determine the susceptibility toward the ReoT3D at 24, 48, and 72 hpi, cells were infected at MOI of 0.05-40 pfu/cells. Our results demonstrated that the proliferation of CT26 cells significantly was inhibited and ReoT3D infection led to cell shrinkage and cell detachment in the CT26 cells that all of these changes efficiently destroyed infected cells by time and dose-dependent manner within 24-72 h as compared to the mock uninfected cells. Minimum toxicity was seen at 24 hpi with lowest dose of 4 reovirus (MOI: 0.05) and maximum toxicity appeared at 72 hpi with MOI of 40 (Fig. 2). So that, we observed MOI>1 of reovirus was toxic in all of the time exposure groups (24-

ReoT3D reduces migration and invasion of CT26 cells:
Subsequently, we examined the potential effect of ReoT3D in CT26 cell migration and invasion.

ReoT3D reduces colony forming ability of CT26 cells:
We next desired to investigate the colony-forming ability of CT26 cells in the presence of ReoT3D.
Noteworthy, CT26 cells were infected with ReoT3D and incubated until colony formation (for 10days).
Number and area size of stained colonies analyzed by ImageJ software. ImageJ data demonstrated ReoT3D infection resulted in a significant reduction (from 1308 ± 10 to 303 ± 7, **p<0.01) in the number of survived colonies (Fig. 6A). So that, colony area significantly diminished (from 85.37% ± 1.24% to 10.7% ± 0.98%, **p<0.01) from ReoT3Dto ReoT3D + plates (Fig. 6B). Beside the remarkable reduction in the number of colonies, the sizes of colonies were decreased in the ReoT3D cured sample (Fig. 6A). Altogether, our results confirmed the inhibitory effect of ReoT3D infection on the colony forming ability of CT26 cells.

ReoT3D enhances expression of apoptotic genes and reduces anti-apoptotic genes:
To evaluate the anticancer effect of ReoT3D on the apoptotic and anti-apoptotic genes expression (P53, P21, Caspase8, Caspase3, Bid, and Bax) and anti-apoptotic genes (Bcl2, and Bcl-xL), we applied qRT-PCR assay in the treated and no-treated mock cells at 48 and 72 hpi. Also, S16 (Rps16) gene with relatively stable expression rate in the treated and non-treated samples was considered as a benchmark for comparing the difference in gene expression between ReoT3D + and ReoT3D -.
Generally, qRT-PCR findings showed that apoptotic and anti-apoptotic genes expressions levels in the CT26 cells were affected by ReoT3D infection (Fig 7). Subsequently, at 48 hpi, expression level of apoptotic genes including P53, and p21 significantly (p*<0.05) elevated by ReoT3D. But any distinct increase was not observed in the expression level of Bid, and Bax (p>0.05) genes at 48 hpi ( Fig 7A).
We also examined the effect of ReoT3D on the anti-apoptotic Bcl-2 and Bcl-xL proteins that are responsible for the prevention of apoptosis-activating factors releasing from mitochondria [23]. As shown in Fig7, The relative mRNA expression level of Bcl-2 and Bcl-xL significantly suppressed in the ReoT3D + than ReoT3D -CT26 cells at 48 hpi (p*<0.05) and 72 hpi (p** < 0.01).
Moreover, we assessed the anti-cancer potential of ReoT3D on the expression level of EGFR and KRAS genes expressions. As shown in Fig 7, EGFR and KRAS were expressed in the both of experimental groups, but we did not observe significant effect (p>0.05) on KRAS and EGFR expression rate in the ReoT3D + cell compared with ReoT3D -.

Discussion
Oncolytic reoviruses due to selectively replication and triggering anti-cancer responses into the cancer cells with low pathogenicity in normal cells, was considered as an anti-cancer option by scientists [2,3]. Recently most clinical trials have focused on T3D strain (Reolysine) because of its maximum oncolysis effect among the other reovirus strains against several cancers [5].
ReoT3D performed its anti-cancer capability by inhibiting KRAS signaling pathway in transformed cells [8]. KRAS activation rate is variable in the different cancer cells and contributes to the development of 50% colorectal cancer [9][10][11]. Also, Patients with KRAS mutations are precluded from anti-EGFR targeted therapy [24]. On the other hand, epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that overexpressed in most cancers especially in CRC and seems to reflect a significant role during CRC progression [25,26]. So, the lack of alternative option for these patients treatment and preferential oncolysis effect of ReoT3D against KRAS mutant cancer cells prompted us to assess oncolytic reovirus as an anti-cancer choice in the present study [7,8].  [29,30]. Therefore, our results from in vitro toxicity assay demonstrated that lower 8 concentrations of ReoT3D were tolerable for CT26 cells, but infected cells were significantly susceptible to ReoT3D infection and destroyed at >1pfu/cell concentrations in the both time and dose depended manners (Fig. 2).
Different experiments were performed to determine the anticancer mechanisms of ReoT3D in the CT256 cell lines. At the first, AnnexinV-FITC staining demonstrated early apoptosis distinctly inducted in the CT26 cells at both 48 and 72 hpi of ReoT3D inoculation. Also, we observed late apoptosis rate was increased (Fig. 3). In addition to flow cytometry data, our results from gene expression analysis showed that P53, P21, Caspase8, Caspase3 levels were markedly increased compared with Bid and Bax at 48 and 72 hpi (Fig. 7). Furthermore, our results revealed that anti-apoptotic genes expressions levels such as Bcl-2 and Bcl-xL significantly suppressed in the ReoT3D+ cells at 48 and 72 hpi in timedependent manner (Fig. 7).
There is some conflict about cell death caspase-dependent or/and independent manner in the ReoT3D infected cells. Some studies suggest that cell death by ReoT3D infection in the cancer cells is caspase-3 dependent manner [31], but in contrast, another study suggested reovirus mediated cell death by independent of caspase activation that is named necroptosis [32].
Moreover, some studies suggested that ReoT3D-induced apoptosis involves both death receptor-(exogenous) and mitochondrial-caspase-dependent (endogenous) pathways that our apoptosis results were consistent with these results [5,30,33]. We showed that cell death induction in infected-CT26 cells by inducing both endogenous and exogenous apoptosis pathways that mediated by both caspase-independent and dependent manner and inhibition of anti-apoptotic responses. Also, a study reported that Caspase-dependent (endogenous) apoptosis led to sensitization of cancer cells to TRAILinduced apoptosis (exogenous pathway) following reovirus-infection that emphases reovirus infection induced both apoptosis manners in cells [34]. However, the precise apoptosis mechanisms in the cancer cells by reovirus are not fully known. So, it seems that ReoT3D mediated CT26 cell death in multiple ways and further investigations are essential to determine the exact cell death induced by reovirus in the cancer cells.
T3D strains could arrest cell cycle and induce apoptosis with variable range that is reovirus strain-9 specific and this difference determined by viral S genes products in the strains [35][36][37]. Also, the viral S1 gene is responsible for induction of cell cycle arrest at the G2/M phase in Reo T3Dinfected cells and may be also affected on apoptosis induction [23,38,39]. Previous reports demonstrated that ReoT3D induced cell cycle arrest at G1/S and G2/M at cancer cells [21,22]. Our cell cycle distribution analysis showed a remarkable G0/G1 and G2M phase arrest with noted S-phase ablation in ReoT3D infected-CT26 cells at 48 hpi, this inhibitory effect significantly increased by time depended manner.
Also, the ReoT3D infected CT26 cells showed a remarkable increase of sub-G1 apoptotic fraction suggesting that ReoT3D plays a crucial role in increasing of sub-G1 apoptotic cells populations (Fig.   4). So, our cell cycle distribution results suggest that ReoT2D-infected CT26 cells led to G0/G1and G2M arrest that are consistent with mentioned reports and could be increasing at sub-G1 apoptotic and G2 phase CT26 cell populations.
Also, it has been demonstrated that ReoT3D infection led to oncolysis and reduction of colony forming ability of cancer containing KRAS mutations. We observed a significantly reduction in the cells migration and invasion rate of ReoT3D infected CT26 cells by time (Fig. 5). Furthermore, our colonyforming results showed that the number and colony area covered by the CT26 cells in ReoT3D treated petri dishes significantly diminished in comparison with ReoT3Dsamples (Fig. 6). These results confirmed that ReoT3D could be a promising anti-cancer option against CRC by apoptosis induction, cell cycle arrest, colony forming, migration, and invasion suppression in the cancer cells.

Conclusions
Present results clarified that higher concentration (>1pfu/cell) of ReoT3D was significantly toxic for and DMSO (50 ul) (Sigma, USA) added into each well following 30 min incubation. Subsequently, Optical density (OD) was taken at 24, 48, and 72 h using a spectrophotometer at a wavelength of 570nm. All experiments were performed in triplicate.

CT26 treatment with ReoT3D:
For assessing the in vitro anticancer effect of ReoT3D against murine colorectal cell line, CT26 cells were cultured, rinsed with PBS and 3 x 10 5 cells/well were seeded in the 6 well plates and incubated at 37 ˚C with 5% CO2. Then, wells were infected withT3D (MOI: 1pfu/cell) and incubated at 37 ˚C with 5% CO2 for 48 and 72 h. For this purpose, apoptosis, cell cycle, colony formation, scratch, and Realtime PCR assay were applied after 48 and 72 hpi. Also, mock (untreated) cells were used as a control groups. All of the experiments were performed in triplicate. The mean of three values has been shown in each test.

Annexin/PI cell death assay:
ReoT3D induced apoptosis in CT26 cells was measured by flow cytometry assay. Briefly, at 48 and 72 hpi with T3D, treated and mock cells harvested, rinsed, and stained with the AnnexinV-FITC

Cell migration by scratch test:
The monolayer of CT26 was harvested from 25cm tissue culture flasks and about 3 x 10 5 cells/well transferred in 2cc of DMEM with 10% FBS into 6 well plate. The plate gently moved from side to side to ensure the cells equally spread in the wells and were placed in a conventional incubator 5% CO2 at 37 °C overnight. The wells at 85-90% confluency manually scratched with a new pipette tip in each well, then media removed and respectively replaced with media containing MOI 1 of ReoT3D and media, in the treated and mock cells. The light microscopic images were taken in the 0, 12, 24, 48 and 72 h hpi. Finally, wound closure of test samples were measured by Image J software Colony formation assay: CT26 cells colony formation assay in the presence of ReoT3D were performed by plating the cells (1×10 3 ) into10 cm tissue culture Petri dishes [40]. After 10 days of incubation at 37 ˚C with 5% co2, colonies were grown; dishes were rinsed with PBS and fixed by adding 4% cold paraformaldehyde.
Survived colonies were stained by Gimsa (1%). Then, dishes with colonies were left to dry in the room atmosphere. At the end, number of colonies and the total area covered by the CT26 cells measured by Image J software in the treated dishes and compared with mock.

RNA extraction, cDNA synthesis and Real-time PCR:
Total RNA was extracted by TRIzol (Invitrogen) and reverse-transcribed using the HyperScript™ firststrand synthesis kit according to the manufacturer's instructions. Real-time PCR (qRT-PCR) was conducted with Applied Biosystems StepOnePlus PCR machine (Applied Biosystems) by applying SybrGreen Master Mix Kit (Amplicon, Denmark). The cycling parameters were 95˚C for 10 min followed by 40 cycles of 95˚C (5 sec) and 60˚C (30 sec), followed by melting curve analysis. Primers which were used for qRT-PCR listed in Table 1. S16 (Rps16) was defined as a reference gene and median Ct (cycle threshold) used for analysis.

Statistical analysis:
All experiments were performed in triplicate and presented as mean ± SEM. The cytotoxicity impacts of ReoT3D on CT26 cells were analyzed by applying of Graph pad prism software and P values <0.05 were considered as statistically significant. 13 This document has been edited with the instant web content composer. The online instant HTML editor tools make a great resource that will help you a lot in your work. Save this link or add it to your bookmarks.

Ethics approval and consent to participate
Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests HS has designed the work plan, supervised the whole study and edited the original draft. EA was adviser for present study that has done bioinformatics works and designed primers for molecular experiment. All authors read and approved the final manuscript. synthesis: role of the S1 and M2 genes. J Virol, 1996. 70 (11)