Abstract
Introduction: Irinotecan, a topoismorase 1 inhibitor, has been used for the treatment of colorectal cancer. It was shown that monotherapy alone is largely ineffective. The combination therapy was used for antitumor activity. The synergistic anticancer effects of oncolytic reovirus-infected secretome in combination with irinotecan and metformin are evaluated in vitro. The aim of research was to assess anticancer impacts of ReoT3D, irinotecan, metformin in combination, against murine colorectal cancer cells (CT26). Methods: The L929 and the CT26 colorectal cancerous cell lines were treated in vitro with irinotecan, metformin, the Dearing strain of reovirus serotype 3 (ReoT3D) (V), and the secretome of intact (S) or reovirus-infected murine adipose-derived mesenchymal stem cells (SV). The cell viability was measured by MTT, and the apoptosis rate was analyzed by annexin V-FITC staining and flow cytometry 48 and 72 h after treatment. Results: We found that cells exposed to a combination of SV+Met+I had significantly lower cell viability and higher apoptosis rates as compared to cells exposed to Met+I, 48 and 72 h. These results suggest that metformin in combination with irinotecan and reovirus produces a synergistic effect on cell death, and adding reovirus-infected secretome (SV) to a Met+I regimen induces a higher apoptosis rate compared to Met+I alone. Based on the results, the combination of SV+Met+I has induced more apoptosis than S, SV, SV+I, and SV+Met. Also, all of the combined treatments induced apoptosis significantly versus secretome alone. Discussion: In this in vitro study, we found that the combination of T3D reovirus (oncolytic virus) and metformin with the anticancer drug irinotecan resulted in higher rates of growth inhibition and apoptosis induction in the colorectal cancer cell line. This synergistic effect was even more pronounced when using the combination of secretome derived from reovirus-infected AD-MSCs, metformin, and irinotecan. Conclusion: We highlight that the combination of ReoT3D-derived secretome with irinotecan and metformin showed a synergistic anticancer effect on the CT26 cell line, and this strategy may be considered as a new approach against colorectal cancer in the in vitro and in vivo in future studies.
Introduction
About two-million new cases of colorectal cancer are diagnosed annually worldwide. With half of these cases resulting in death, colorectal cancer has become the third cause of cancer-related death in the world and currently stands as the main hindrance for an increase in life expectancy [1‒3]. The use of ineffective conventional therapies for this cancer such as chemotherapy, radiotherapy, and surgery has led to partial elimination of tumors, recurrence of the disease, metastasis to other organs, and poor survival and quality of life. These outcomes have limited the use of monotherapies as a therapeutic strategy [4, 5].
Recently, researchers have focused on using targeted therapies along with a combination of different therapeutic methods to overcome this problem. The use of oncolytic viruses in this decade has become one of the most prevalently used strategies in treating cancers and has provided a new approach that has proven to be revolutionary in cancer treatment [6]. These relatively nonpathogenic viruses occur in two forms: natural and engineered. These viruses selectively proliferate in tumor cells, which is one of their discriminating features. Expression of specific surface markers and intracellular conditions of cancer cells are conducive factors for tropism of oncolytic viruses to these cells [7, 8]. These viruses induce cell death in a variety of ways after entering the cell, such as apoptosis, autophagy, necrosis, direct lysis, expression of toxic proteins, and stimulation of immune response against the tumor. It should be noted that these viruses do not affect normal cells [6, 9‒11]. Mammalian reovirus, which belongs to Reoviridae family is categorized into three serotypes based on neutralizing antibody and inhibitory activities of hemagglutinin: Lang type (1T1L or T1), Jones type (T2J2 or T2), and type 3 that includes two strains of Abney (T3A) and Dearing (T3D). The Dearing strain of reovirus serotype 3 (ReoT3D) is a non-engineered wild type virus, which has more oncolytic capability compared to other serotypes [12] and does not cause serious and noteworthy disease in humans [13]. This serotype was specifically chosen because it is a non-engineered and inherently oncolytic virus.
As a promising candidate for combination therapy in cancer, many studies have investigated combinations of different conventional chemotherapy drugs like irinotecan, 5-fluorouracil, and cisplatin with oncolytic viruses to alter the efficacy of both the viruses and drugs [14]. These studies showed that this combination of therapies augments proliferation and antitumor activity of oncolytic viruses by suppressing the innate and acquired immune responses against viruses, such as the complement system and CD8+ cytotoxic T cells [14]. Irinotecan resistance in CRC looks to progress by some mechanisms comprising low intratumor level of the active metabolite, a decrease in expression of topoisomerase I, and changes inactivity of downstream events such as suppression of apoptosis, cell cycle [15]. Irinotecan shows potent anticancer effect against several cancers by arresting topoisomerase I (Topo I) function [16]. Combining chemotherapeutic drugs like irinotecan, oxaliplatin, and 5-fluorouracil could be an intriguing option in the treatment of colorectal cancer because reovirus and irinotecan synergistically stop colorectal cancer cells and increase apoptosis through p21 protein [17‒19].
Irinotecan is a semi-synthetic analog of camptothecin that was first made in 1983. This Topo 1 inhibitor drug induces a permanent break in double-strand DNA, which leads to specified death in the S phase of the cell cycle [16, 20, 21]. Among the factors of tumor resistance to this drug, we can mention (1) changes in the levels of enzymes involved in the production of active irinotecan, (2) reduction in the expression of Topo 1, and mutations in the structure of this enzyme [16].
Metformin is known as the first choice for the treatment of type 2 diabetes and has drawn attention, recently, to be used as a new and promising option for the treatment of various cancers. According to recent findings, this drug could induce apoptosis, arrest the G1 phase of cell cycle, and prevent the proliferation of cancer cells [22‒26]. In addition to many other mechanisms, this drug reduces the increase in blood insulin production that increases the growth of colon cells and activates insulin receptors. It also sensitizes CRC to the toxicity of irinotecan and increase apoptosis induction and eventually increases amount of p21 [27].
Another new and promising therapeutic option for use against colorectal cancer that demands more investigation is the use of plastic adherent cells called mesenchymal stem cells (MSCs). These cells were first found in bone marrow [28] by Friendestein and colleagues [29] in 1970. Besides their unique properties, like self-renewal and differentiation to adipocyte, osteoblast, and chondroblast cells, MSCs have shown an inherent tendency toward becoming cancerous cells through the expression of different cytokines and receptors, which results in their ability to suppress cancerous cells [30, 31]. Despite significant potential benefits, there are disadvantages like embolism or even transmission of infection on their way to the target cell. Hence, researchers have recently used endocrine secretions of these cells, called secretome. These secretions, based on their size and origin are classified into exosomes, ectosomes, microvesicles, membrane particles, and apoptotic bodies. Secretomes contain serum proteins, growth factors, angiogenesis factors, hormones, cytokines, extracellular matrix, and extracellular matrix protease, which contrary to MSCs can exert their therapeutic potentials without the need for direct cell-cell adhesion and have the advantage of the possibility to be kept long-term [28, 32‒34].
The increasing prevalence of colorectal cancer and its high mortality rate have forced scientists to find more efficient therapeutic approaches. Here, we examine the in vitro efficacies of different combination therapies using secretomes of MSCs, reovirus as an oncolytic virus, as well as anticancer drugs irinotecan and metformin in colorectal cancer model.
In the current research, in vitro anticancer effect of combination regime of ReoT3D, irinotecan, and metformin against CT 26 cells have reported. Therefore, we explored the potential anticancer impacts of ReoT3D in KRAS activated pathway CT26 cells in combination with two available anti CRC effective drugs.
Molecular classification of colorectal cancer is an important issue, incorporating new knowledge into the classification system based on genomic and metagenomic signs. Global genomic status and chromosomal instability and epigenomic status play a significant role in defining clinical, pathological, and biological characteristics of colorectal cancer. New potent therapeutic strategy is needed to explore additional target for combating against CRC cancer statues in near future [35].
Materials and Methods
Chemicals and Reagents
The L929 and CT26 cell lines were purchased from Pasteur Institute of Iran and used in this study. L929 is a type of fibroblast cell isolated from the adipose tissue of 100-day old male C3H/An mice. CT26 is a type of adherent fibroblast cell an undifferentiated colon carcinoma cell line [36], which originates from colon carcinoma in BALB/c mice. These cells were passaged twice a week at 80–90% confluency with Dulbecco’s Modified Eagle’s Medium (DMEM/Gibco, USA) containing 10% fetal bovine serum (FBS/Gibco, USA) and the antibiotics penicillin (100 U/mL) (Gibco, USA) and streptomycin (100 μg/mL) (Gibco, USA). Cells were cultured at 37°C, 5% CO2, and 95% humidity in the incubator. Dimethyl sulfoxide was used for freezing the cells, and trypan blue (Bio-idea, Iran) was used for counting live cells.
Irinotecan and metformin were purchased from CinnaGen Medical Biotechnology Research Center (Alborz University of Medical Sciences, Karaj, Iran). Irinotecan was dissolved in sterile phosphate buffer saline (PBS) in the dark to prepare 2 mg/mL stock solutions. To prepare stock solutions of metformin (100 mm), this crystal-like drug was dissolved in sterile injection water, filtered through a 0.22 μm syringe filter, and kept at −20°C. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit (5mg/mL) was used to evaluate the viability of cells and was purchased from Bio-idea, Iran. The Annexin V-FITC Apoptosis Detection Kit was purchased from (eBioscience, Germany) and used according to the manufacturer’s instructions. The reovirus T3D (Dearing strain of reovirus serotype 3) stock with 107 titer was purchased in 1 mL volumes from Iran University of Medical Sciences, and it is cultured and confirmed as shown by Banijamali et al. [37]. The confirmed stock was cultured in L929 cells to extend the viral stock and was kept at −80°C.
Adipose-Derived Mesenchymal Stem Cells
A 6- to 7-week old BALB/c male mouse was purchased from the Pasteur Institute of Iran for the harvest of adipose-derived mesenchymal stem cells (AD-MSCs). With ethical considerations in mind, the mouse was put into a chloroform-containing glass bottle for euthanasia and was transferred into an alcohol-containing bottle immediately after death for avoiding contamination in preparation of MSCs. To isolate the adipose tissue, the abdominal skin was cut with scissors under sterile conditions, and the fat above the testicular and groin area was removed gently with forceps. The fat content was then transferred to a Petri dish containing PBS plus antibiotics and then was cut into smaller pieces with a surgical razor. Blood vessels and other useless tissues were removed. The fat pieces were then transferred to another falcon tube containing 500 mL of medium plus penicillin (100 U/mL) and streptomycin (100 μg/mL). Collagenase type I enzyme (500 mL) was added in order to lyse the intercellular components. After pipetting up and down for a short time, the tissue containing collagenase was incubated for 10 min at 37°C. At this point, it was brought out, pipetted again, and incubated for 10 more minutes. Next, 2.5 mL DMEM and 400 μL FBS (15%) were added, and the cells were centrifuged at 1,800 rpm/21°C for 5 min. The supernatant was discarded, and the pellet was pipetted with 1 mL of medium and then transferred to a T25 cell culture flask containing 4 mL medium with 20% FBS. The flask was incubated at 37°C, 95% humidity, and 5% CO2. The medium was changed after 48 h in order to remove non-adherent cells. The cells were subcultured after reaching 80% confluency. Once the cells reached passage 3 (P3), they were used for further investigations [38, 39].
Verification of MSCs
The surface markers of AD-MSCs at P3 were examined by flow cytometry to confirm the non-differentiated phenotype. Cells were checked for the presence of CD90, CD29 markers, and the absence of the CD45 marker using a Biolegend kit and subsequent analysis by a FACSCanto II flow cytometer (BD Biosciences, USA).
Inoculation of Reovirus T3D into AD-MSCs and Isolating the Secretome of Infected Cells
After reaching 80% confluency of AD-MSCs at P3, they were then washed with PBS and then inoculated with reovirus T3D at an infection multiplicity of MOI 1 PFU/cell. Cells were incubated for 1 h at 37°C. Thereafter, the medium was discarded and replaced by the serum-free medium. The morphology of the cells was checked every 12 h by microscope so that secretome could be collected before cytopathic effect (CPE) appeared. Accordingly, secretome derived from reovirus-infected MSCs was collected and categorized after 48 h and before the onset of CPE and then centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was kept at −80°C for further use. In addition to a virus-infected flask, a noninfected flask was cultured as well to isolate secretome from intact MSCs.
Treatment Groups
L929 and CT26 cells were exposed to varying doses of drugs (irinotecan and metformin) alone or combined with other factors such as reovirus inoculation and secretome derived from infected and intact AD-MSCs (Tables 1, 2). After counting live cells using trypan blue, the cells were seeded onto 96-well culture plates with a density of 10 × 103 cells/well. After 24 h, L929 cells were divided into 6 groups based on different regimens as presented in Table 1. Then CT26 cells were divided into 2 groups as presented in Table 2. Cells were assessed for cell viability 48 h and 72 h after treatment. The MTT assay and induction of apoptosis were performed in this study. Evaluation of cell cytotoxicity and apoptosis induction are used in anticancer cell therapy.
Monotherapy and combination therapy regimens used on L929 cells
Time . | 48 h and 72 h . | |||||
---|---|---|---|---|---|---|
Groups |
| I+V
| I+Met
| I+Met+V | Sec+I+Met
| Sec+V+I+Met
|
|
Time . | 48 h and 72 h . | |||||
---|---|---|---|---|---|---|
Groups |
| I+V
| I+Met
| I+Met+V | Sec+I+Met
| Sec+V+I+Met
|
|
I, irinotecan (μg/mL); Met, metformin (mmol); Sec, secretome derived from AD-MSCs; V, reovirus T3D (MOI 1);
AD-MSCs, adipose tissue mesenchymal stem cells.
Monotherapy and combination therapy regimens used on CT26 cells
Time . | 48 h and 72 h . | 72 h . | |
---|---|---|---|
Groups |
|
|
|
Time . | 48 h and 72 h . | 72 h . | |
---|---|---|---|
Groups |
|
|
|
I, irinotecan (μg/mL); Met, metformin (mmol); Sec, secretome derived from AD-MSCs; V, reovirus T3D (MOI 1);
AD-MSCs, adipose tissue mesenchymal stem cells.
MTT Assay
An MTT assay was done to evaluate the toxicity of different regimens of treatment on cells. After 48 and 72 h of treatment, cells were washed twice with PBS and then 100 μL of RPMI1640 as well as 10 μL of MTT solution (5 μg/mL) was added to each well. After incubating for 4 h, 50 μL dimethyl sulfoxide were added to each well to dissolve formazan crystals. Cells were incubated for another 10 min, thereafter. Absorbance values (OD) were read at 570 and 630 nm wavelengths, and the percent of cell viability was calculated. Experiments were conducted in triplicate wells.
Apoptosis Assay by Flow Cytometry
L929 and CT26 cells cultured in 12-well plates were checked for apoptosis 48 and 72 h after exposure to different regimens. Cells were washed with PBS and harvested by trypsinization, and apoptosis analysis was performed using the Annexin V-FITC Apoptosis Detection Kit (eBioscience, USA) according to the manufacturer’s instructions. Analysis was performed using a FACSCanto II flow cytometer (BD Biosciences, USA) and FlowJo software (BD Biosciences, USA).
Annexin V is an anticoagulant protein that has a high affinity for phosphatidylserine in the presence of calcium. Detection of this protein conjugated with FITC leads to recognition of early apoptosis. On the other hand, propidium iodide is a DNA-specific dye that can diffuse into the cells with altered membrane permeability and stain the DNA. The cells that were stained by both dyes had suffered from secondary death.
Statistical Analysis
Data were analyzed using GraphPad Prism software version 7.04. Mean ± standard deviation was calculated to summarize the data. Two-way ANOVA and post hoc multiple comparison testes were used to compare continuous variables of interest among different therapeutic regimens.
Results
MTT Assay Results
It is a colorimetric method that is used to measure cell metabolism. Its basis is the breakdown of the yellow tetrazolium crystals by the mitochondrial succinate dehydrogenase enzyme and the formation of insoluble purple crystals. With this test, the cell’s vitality can be measured after exposure to various substances such as drugs.
Determining the Optimal Dosage of Irinotecan
According to first diagram, L929 cells were treated with 3 different concentrations 80, 120, and 160 μg/mL of the irinotecan drug that the 80 μg/mL concentration was chosen as the optimal dosage. L929 as control cells were exposed to 80, 120, and 160 μg/mL of the irinotecan drug. The cells were examined by MTT assay after 48 and 72 h of exposure to the drug. We observed a decrease in percentage of cell viability from 48 to 72 h. After 48 h, the viability percentages for cells exposed to 80, 120, and 160 μg/mL of irinotecan were 66.1%, 53.5%, and 40%, respectively. After 72 h, corresponding viability percentages dropped to 48.7%, 30.3%, and 24.3%, respectively. These data show that incremental doses of irinotecan resulted in increased toxicity and decreased viability of cells in both periods, and IC50 value (50% cell growth inhibitory concentrations) of irinotecan was determined (L92: 75.39 μg/mL ± 1.877) (Fig. 1a). The 80 μg/mL concentration was chosen as the optimal dosage.
Cell viability determined by MTT assay for L929 and CT26 cells after 48 and 72 h of treatment. a The effect of irinotecan on L929 cell viability. The cells were treated with 3 different concentrations of irinotecan, and 80 μg/mL was chosen as the optimal dose. b The effect of irinotecan with reovirus (MOI = 1) on L929 cell viability. The combination of reovirus with 80, 120, and 160 μg/mL of irinotecan resulted in different inhibitory effects on cell viability compared to reovirus alone. c The effect of metformin alone and together with irinotecan on L929 cell viability. The viability of L929 cells exposed to 10 mmol metformin + 80 μg/mL irinotecan was significantly lower than cells exposed to irinotecan alone. The concentration of 5 mmol + 40 μg/mL (I) was chosen as the optimal dose. d The exposing of L929 cells to optimal doses of metformin and irinotecan combined with reovirus in two different conditions: (1) after onset of virus cytopathic effect (CPE); and (2) simultaneous with the virus. Cells exposed to metformin and irinotecan added after CPE onset had the lowest viability. e Investigating the effect of optimal doses of metformin and irinotecan combined with reovirus on CT26 cell viability. The combination of irinotecan, metformin, and virus has caused more death in cancer cells than other groups. f The effect of adipose-derived mesenchymal stem cell (MSC) secretome alone and together with optimal doses of other drugs on L929 cell viability. Using secretomes together with irinotecan and metformin had higher inhibitory effects on cell growth compared to secretome alone. g The effect of reovirus-infected adipose-derived MSCs secretome alone and together with optimal doses of other drugs on L929 cells viability. Treatment with reovirus-infected MSCs secretome combined with irinotecan and metformin caused twofold higher drop in L929 cell viability compared to the corresponding regimen containing intact MSCs secretome. h The effect of reovirus-infected and intact AD-MSCs secretome alone and together with optimal doses of other drugs on CT26 cancerous cells viability. Secretomes derived from reovirus-infected MSCs had higher inhibitory effects than secretomes derived from intact MSCs. The SV + 5 mmol + 40 μg/mL (I) regimen had the highest growth inhibitory effect on CT26 cells.
Cell viability determined by MTT assay for L929 and CT26 cells after 48 and 72 h of treatment. a The effect of irinotecan on L929 cell viability. The cells were treated with 3 different concentrations of irinotecan, and 80 μg/mL was chosen as the optimal dose. b The effect of irinotecan with reovirus (MOI = 1) on L929 cell viability. The combination of reovirus with 80, 120, and 160 μg/mL of irinotecan resulted in different inhibitory effects on cell viability compared to reovirus alone. c The effect of metformin alone and together with irinotecan on L929 cell viability. The viability of L929 cells exposed to 10 mmol metformin + 80 μg/mL irinotecan was significantly lower than cells exposed to irinotecan alone. The concentration of 5 mmol + 40 μg/mL (I) was chosen as the optimal dose. d The exposing of L929 cells to optimal doses of metformin and irinotecan combined with reovirus in two different conditions: (1) after onset of virus cytopathic effect (CPE); and (2) simultaneous with the virus. Cells exposed to metformin and irinotecan added after CPE onset had the lowest viability. e Investigating the effect of optimal doses of metformin and irinotecan combined with reovirus on CT26 cell viability. The combination of irinotecan, metformin, and virus has caused more death in cancer cells than other groups. f The effect of adipose-derived mesenchymal stem cell (MSC) secretome alone and together with optimal doses of other drugs on L929 cell viability. Using secretomes together with irinotecan and metformin had higher inhibitory effects on cell growth compared to secretome alone. g The effect of reovirus-infected adipose-derived MSCs secretome alone and together with optimal doses of other drugs on L929 cells viability. Treatment with reovirus-infected MSCs secretome combined with irinotecan and metformin caused twofold higher drop in L929 cell viability compared to the corresponding regimen containing intact MSCs secretome. h The effect of reovirus-infected and intact AD-MSCs secretome alone and together with optimal doses of other drugs on CT26 cancerous cells viability. Secretomes derived from reovirus-infected MSCs had higher inhibitory effects than secretomes derived from intact MSCs. The SV + 5 mmol + 40 μg/mL (I) regimen had the highest growth inhibitory effect on CT26 cells.
Effects of Reovirus Alone and in Combination with Irinotecan on L929 Cells
In diagram 2, effects of reovirus alone and in combination with irinotecan on L929 cells were assessed. Combinationtherapy by irinotecan and reovirus had an additive toxicity rather than reovirus alone.
The toxicity of reovirus on L929 cells was evaluated by MTT assay. We observed significant sensitivity of L929 cells to inoculation with reovirus (MOI = 1). Cell viability decreased to 71.1% (p < 0.01) and 52.3% (p < 0.0001) at 48 and 72 h after inoculation, respectively. Inoculation with the virus had an additive toxicity effect when combined with different doses of irinotecan. After 48 h, the combination of reovirus with 80, 120, and 160 μg/mL of irinotecan resulted in cell viabilities of 63.5%, 55.3%, and 51.7%, respectively. Corresponding viabilities were 44.01%, 35.8%, and 33.8% after 72 h (Fig. 1b).
Combining Metformin with Irinotecan to Select the Optimal Dosage
The effects of metformin alone and in combination with the irinotecan were investigated by MTT assay. Group of 40 μg/mL (I) + 5 mmol was chosen as the final optimal dosage. L929 cells were treated with 5 different regimens: 80 μg/mL (I), 10 mmol, 80 μg/mL (I) + 10 mmol, 40 μg/mL (I) + 5 mmol, and a control group. Corresponding cell viabilities were 76.8%, 77.6%, 59.4%, and 75.4% after 48 h and 55.52%, 66.83%, 51%, and 68.9% after 72 h. Treatment for 72 h resulted in a more profound drop in viability compared to 48 h. Furthermore, combination of 80 μg/mL (I) with 10 mmol had a higher inhibitory effect on cell growth compared to 80 μg/mL (I) alone, both in 48 h and 72 h time points (p < 0.0001 for both timepoints). This implies a synergistic toxic effect of this combination therapy (Fig. 1c). For further investigations in order to reduce the drugs toxicity, a concentration of 40 μg/mL (I) + 5 mmol was chosen as the final optimal dosage.
Effects of Reovirus with Irinotecan and Metformin on the Inhibition of L929 Cell Growth
The exposing of L929 cells to optimal doses of metformin and irinotecan combined with reovirus in two different conditions and choosing the appropriate group. Given the positive synergistic effects of both reovirus with irinotecan, we attempted to examine the combination of reovirus, metformin, and irinotecan as the next step. The final chosen dosage of 40 μg/mL (I)+5 mmol (M) was combined with reovirus (MOI = 1) in 2 groups: (1) after onset of virus CPE and (2) simultaneous with viral infection (before CPE). According to the MTT assay results, addition of 40 μg/mL (I)+5 mmol (M) to the cells after CPE resulted in higher toxicity compared to using only 40 μg/mL (I)+5 mmol (M). This triple-agent combination decreased L929 cell viability to 34.2% and 13.15% 48 h and 72 h after treatment, respectively. Cells exposed to 40 μg/mL (I)+5 mmol (M) had significantly different viability compared to 40 μg/mL (I)+5 mmol (M) groups both before and after CPE (p < 0.0001) (Fig. 1d).
Using Optimized Dosages of Reovirus, Irinotecan, and Metformin on CT26 Cells
CT26 a murine colon carcinoma cells were exposed optimized dosages of reovirus, irinotecan, and metformin, and this group was able to induce more cell death.
CT26 colon carcinoma cells were exposed to 5 different regimens: reovirus (V), 40 μg/mL (I), 5 mmol, 40 μg/mL (I)+ 5 mmol, and addition of 40 μg/mL (I)+5 mmol after the onset of CPE. According to MTT assay results, corresponding cell viabilities for 5 regimens were 57.82%, 72.2%, 74.4%, 71.08%, and 55.59% after 48 h and 53.84%, 69.2%, 70.17%, 59.49%, and 46.87% after 72 h (Fig. 1e). These data show that the cancerous cells were affected by the treatment groups and the combination of irinotecan, metformin, and reovirus induced more death on CT26 cell. The comparisons between the control and treatment groups after 48 and 72 h are shown in Figure 1e. The significance of data is determined by a p < 0.0001.
Combining Irinotecan, Metformin, and Secretome Derived from MSCs on L929 Cells
In this group, the uninfected secretome was added to the optimal dosage and the effect. It was checked on cell viability.
After observing synergistic inhibitory effects of the triple-drug regimen on the growth of L929 and CT26 cells in vitro, we next investigated the effects of AD-MSCs secretome alone and in combination with three other drugs on L929 cells. Accordingly, L929 cells were exposed to the following regimens: secretome (S), (S)+5 mmol, (S)+40 μg/mL (I), and (S)+5 mmol + 40 μg/mL (I). Exposure to secretome alone could induce an 8% and 22% drop in cell viability after 48 h and 72 h, respectively. Combining secretome with metformin or irinotecan resulted in a further decline in cell viability. Finally, triple-drug treatment with secretome, irinotecan, and metformin resulted in a 37% and 42% drop in cell growth after 48 h and 72 h, respectively (Fig. 1f).
Combining Irinotecan, Metformin, and Secretome Derived from Reovirus-Infected MSCs on L929 Cells
In this group, the effect of infected secretome alone and in proximity with the optimal dosage on cell viability was investigated. Combination of infected secretome with drugs was able to exert acceptable effects on cells.
Due to promising inhibitory effects of secretome on cell growth in vitro, we next exposed L929 cells to secretome derived from reovirus-infected MSCs (SV) alone and in combination with previously determined doses of irinotecan and metformin. Accordingly, cells were exposed to four regimens: SV, 40 μg/mL (I) + SV, 5 mmol + SV, and 40 μg/mL (I)+5 mmol + SV. Exposure to SV alone significantly decreased cell viability (Fig. 1g). Corresponding percentage of death of cells for four regimens were 39.2%, 47.36%, 42.74%, and 51.93% after 48 h and 70.18%, 87.15%, 86.11%, and 88.19% after 72 h. An acceptable difference between the inhibitory effects of SV alone compared to 40 μg/mL (I)+5 mmol + SV is obvious after 48 and 72 h (p < 0.001). Combining SV with irinotecan and metformin led to a twofold decrease in viability of L929 cells compared to 40 μg/mL (I)+5 mmol +S in Figure 1f.
Effects of Secretomes Derived from Intact or Reovirus-Infected MSCs Combined with Irinotecan and Metformin on CT26 Cells
In the last group, the effect of infected and uninfected secretome alone and in combination with drugs was measured on CT26. And a significant effect was observed between the groups containing infected and uninfected secretome.
After observing the significant inhibitory effects of monotherapy and combination therapy using secretome derived from either intact or reovirus-infected MSCs on L929 cells, we examined inhibitory effects of similar regimens on CT26 cells considering 72 h timepoint. Thus, CT26 cells were exposed to 8 different combinations of drugs as follows: SV, 40 μg/mL (I)+SV, 5 mmol +SV, 40 μg/mL (I)+5 mmol +SV; S, 40 μg/mL (I)+S, 5 mmol +S, and 40 μg/mL (I)+5 mmol +S. As expected, all 8 regimens decreased viability of CT26 cells (Fig. 1h). Interestingly, regimens containing secretome derived from reovirus-infected MSCs resulted in higher inhibition of growth compared to regimens containing secretome derived from intact MSCs. The 40 μg/mL (I)+5 mmol +SV regimen was shown to be the most effective by its induction of a 66.3% drop in cell viability. In this study, we did not perform co-culture.
Apoptosis Assay
The basis of this method is based on the fact that the investigation of apoptosis is done using double staining (annexin V/PI) so that during the early stages of apoptosis, many changes are made in the cytoplasmic membrane, which includes changes in permeability and changes in membrane lipids. Phosphatidylserine, which is naturally present in the inner half of the membrane, is transferred to the outer half of the membrane due to the disruption of the ATP-dependent translocase enzyme activity. On the other hand, annexin V is an anticoagulant protein that binds to phosphatidylserine with high affinity in the presence of calcium, and the conjugation of this protein with FITC dye detects apoptosis in the early stages, and propidium iodide dye, which is specific to DNA, by changing the permeability of the membrane during apoptosis, it can go inside the cell and identify the living cell from the dead and the stages of apoptosis.
Annexin V/PI staining assay was used for L929 and CT26 cells to investigate the type and extent of cell death (Table 3; Fig. 2). Depending on the intensities of annexin V and PI staining, cells were categorized into four groups by flow cytometry: live cells, cells with early apoptosis, cells with secondary apoptosis, and cells with necrosis (Fig. 3a–c). The results of flow cytometry tests are summarized in Table 3 and Figure 2 to compare the apoptosis rates of L929 cells 48 and 72 h after treatment as well as CT26 cells 72 h after treatment.
Summary of apoptosis rates by flow cytometry for L929 and CT26 cells
Treatment regimen . | Apoptosis rate . | ||
---|---|---|---|
after 48 h for L929, % . | after 72 h for L929, % . | after 72 h for CT26, % . | |
Control | 1.1 | 1.1 | 3.6 |
Virus (MOI = 1) | 38.8 | 91.3 | 60.8 |
Secretome derived from reovirus-infected mesenchymal stem cells | 27.9 | 90.1 | 56.4 |
Met+secretome derived from reovirus-infected mesenchymal stem cells | 42 | 92.1 | 69.3 |
I+secretome derived from reovirus-infected mesenchymal stem cells | 62.7 | 95.8 | 79.3 |
I+Met+secretome derived from reovirus-infected mesenchymal stem cells | 64.8 | 97.4 | 85.7 |
Secretome derived from mesenchymal stem cells | 16.4 | 40.1 | 21.4 |
Met+secretome derived from mesenchymal stem cells | 22.4 | 66.6 | 28.2 |
I+secretome derived from mesenchymal stem cells | 23.6 | 79.9 | 53.3 |
I+Met+secretome derived from mesenchymal stem cells | 25.9 | 85.8 | 73.2 |
Treatment regimen . | Apoptosis rate . | ||
---|---|---|---|
after 48 h for L929, % . | after 72 h for L929, % . | after 72 h for CT26, % . | |
Control | 1.1 | 1.1 | 3.6 |
Virus (MOI = 1) | 38.8 | 91.3 | 60.8 |
Secretome derived from reovirus-infected mesenchymal stem cells | 27.9 | 90.1 | 56.4 |
Met+secretome derived from reovirus-infected mesenchymal stem cells | 42 | 92.1 | 69.3 |
I+secretome derived from reovirus-infected mesenchymal stem cells | 62.7 | 95.8 | 79.3 |
I+Met+secretome derived from reovirus-infected mesenchymal stem cells | 64.8 | 97.4 | 85.7 |
Secretome derived from mesenchymal stem cells | 16.4 | 40.1 | 21.4 |
Met+secretome derived from mesenchymal stem cells | 22.4 | 66.6 | 28.2 |
I+secretome derived from mesenchymal stem cells | 23.6 | 79.9 | 53.3 |
I+Met+secretome derived from mesenchymal stem cells | 25.9 | 85.8 | 73.2 |
I, irinotecan (40 μg/mL); Met, metformin (5 mmol); Sec, secretome derived from AD-MSCs; V, reovirus T3D (MOI 1);
AD-MSCs, adipose tissue mesenchymal stem cells.
Overall comparison of apoptosis results from flow cytometry test on L929 and CT26 cells at 48 and 72 h.
Overall comparison of apoptosis results from flow cytometry test on L929 and CT26 cells at 48 and 72 h.
a Apoptosis assay for L929 cells after 72 h of treatment. b Apoptosis assay for CT26 cells after 72 h of treatment. c Apoptosis assay for L929 cells after 48 h of treatment. The vertical axis is annexin V, and the horizontal axis is PI: Q1: early apoptosis, Q2: secondary apoptosis, Q3: live cells, and Q4: necrosis.
a Apoptosis assay for L929 cells after 72 h of treatment. b Apoptosis assay for CT26 cells after 72 h of treatment. c Apoptosis assay for L929 cells after 48 h of treatment. The vertical axis is annexin V, and the horizontal axis is PI: Q1: early apoptosis, Q2: secondary apoptosis, Q3: live cells, and Q4: necrosis.
Discussion
In this in vitro study, inoculation with reovirus reduced cell viability of both the L929 cell line and the colorectal cancer cell line, CT26. combination therapy comprised reovirus, irinotecan, and metformin showed an additional cytotoxicity effect. We also found that combining secretome derived from AD-MSCs with irinotecan and metformin increased inhibitory effects of these two drugs. In all cases, regimens containing the secretome derived from reovirus-infected AD-MSCs had higher inhibitory effects compared to the regimens containing secretomes from intact AD-MSCs. Similarly, the combination of irinotecan, metformin, and secretomes derived from AD-MSCs induced higher rates of apoptosis in both cell lines compared to monotherapy regimens. This additional apoptosis induction was more pronounced when using secretome derived from reovirus-infected AD-MSCs.
Previous studies have reported antineoplastic effects of irinotecan and metformin. Keyvani-Ghamsari showed an increased rate of apoptosis in MCF-7 breast cancer cells by increasing the concentration of irinotecan [40]. Zhu et al. [41] studied the simultaneous effect of irinotecan and the TRAIL factor on TRAIL-resistant HT-29 colon cancer cells. In a synergistic manner, low dose irinotecan increased the antitumor effect of the TRAIL factor on cancer cells [41]. Furthermore, a recent study by Shen et al. [42] about metformin showed that it can cease the G1 phase of the cell cycle and reduce the translation of the Myc oncogene precursor protein in colorectal cancer cells. Similarly, we observed antineoplastic effects of both drugs in our study.
Promising results of in vitro and in vivo studies have raised the hopes for using MSCs and oncolytic viruses along with the conventional methods to fight cancer. Some clinical trials are currently examining oncolytic virus-loaded secretomes of MSCs. T-VEC (engineered herpes virus) was the first oncolytic virus confirmed by Food and Drug Administration and the European Union for treatment of melanoma [6, 43]. The type 3 reovirus (Reolysin) has been evaluated extensively in preclinical and clinical investigations [44, 45]. Hirasawa et al. [46] investigated the effect of oncolytic reovirus (MOI = 20) on Ras protein-mutated and non-Ras protein-mutated ovarian and colon cancer cells in vivo and reported about 95% cell death rate in the Ras protein-mutated group after 72 h of virus inoculation [46]. Desirable results like this led to the first study to examine the combination of chemotherapy and Reolysin on HCT116 in 2004. This study reported synergistic effects of Reolysin and gemcitabine on colorectal cancer cells [47]. Another study by Maitra et al. [17] examined the synergistic effect of irinotecan, reovirus on KRAS-mutated HCT116, and non-KRAS-mutated WT Hke3 cells. The authors found that reovirus in combination with irinotecan exerted higher growth inhibitory effects on KRAS-mutated cells compared to the virus alone [17]. Similarly, we found that reovirus combined with irinotecan had higher growth inhibition of colorectal cells compared to reovirus alone after 48 and 72 h. The additive effect of metformin together with irinotecan and the virus was also observed after 48 and 72 h.
Considering potential side effects of using adipose-derived MSCs, such as embolism and transmission of infection, researchers have used endocrine secretions of these cells called secretome [28, 32‒34]. A recent study by Serhal et al. [48] showed that co-culturing human liver cancer cells with AD-MSCs and also exposure to the conditioned media derived from MSC considerably decreased the cell proliferation and increased the apoptosis rate of cancer cells. However, the authors did not expose the cells to isolated secretomes as performed in our study. In further support, in vitro and in vivo treatment of neuroblastoma cells with secretomes derived from MSCs infected with adenoviral oncolytic virus showed higher antitumor effects compared to treatment with the virus alone; this treatment also increased chemotaxis of T cells into the tumor site [49]. In a clinical study, children with malignant neuroblastoma underwent weekly treatments with Celyvir – autologous bone marrow-derived MSCs. This resulted in a significantly better response to the conventional treatment compared to the control group [50].
In contrast to our findings and the studies mentioned above, Hendijani et al. [51] showed that combination of doxorubicin, an anticancer drug, with secretomes of Wharton’s jelly-derived MSCs did not alter cell proliferation, apoptosis, or drug resistance in lung cancer cells. In another study, MSC secretomes increased the rate of proliferation and migration of cancer cells [28]. Furthermore, glioblastoma cancer cells did not show sensitivity to a combination of secretome and the anticancer drug temozolomide [52]. It should be noted that depending on factors such as the source of MSCs, the number of their passages, the amount of serum, and the type of proteins involved, secretions of MSCs show varied effects on cancer cells and the immune system (stimulatory or inhibitory). This could explain some of the inconsistencies observed among the studies discussed.
As suggested by Maitra et al. [17], synergistic effects of irinotecan and the oncolytic reovirus could be through the mediation of the P21 protein. Collisions of irinotecan and the DNA-I Topo with the replication fork leads to DNA damage at the S-phase checkpoint with subsequent expression of P53. Increased P21 expression inhibits phosphorylation of the retinoblastoma protein (pRb) and results in G1/S cell cycle arrest [17]. On the other hand, reovirus can cause cell death by autophagy and altered mTOR expression. Similarly, metformin inhibits mTOR activity, which could be one of the explanations for the synergy between reovirus and metformin [24‒26]. Moreover, by relying on a series of studies that have examined the effects of MSC-driven secretome on cancer cells and having observed a significant increase in apoptosis rate due to an upregulation of caspase 3 and 9 genes and a downregulation of anti-apoptosis genes in cancer cells, an explanation for the synergy between reovirus and these secretomes could be the ability of reovirus to inactivate the internal pathway of apoptosis and prevent the expression of antiapoptotic genes [53, 54].
Extensive dimensions of the interactions between MSCs, oncolytic reovirus, and antineoplastic drugs remain unclear and leave many questions unanswered. In order to unravel the mechanisms underlying these interactions, future studies could examine the RNA and protein expression levels of dominant genes involved in internal and external apoptosis, the cell cycle, autophagy, and necrosis in treated cancer cells. Evaluation of the signaling pathways and some pre-inflammatory and anti-inflammatory interleukins could also be helpful. It is also possible to place reovirus, metformin, and irinotecan drugs in the vicinity of the secretome derived from other tissues and obtain a series of data based on the time and number of different passages and the amount of FBS. Finally, the results of present in vitro study suggest that this combination therapy could merit potential considerations in management of colorectal cancer patients. However, extensive in vivo and clinical studies are required to provide further confirmatory evidences.
As far as we know, in vitro anticancer effect of combination regime of ReoT3D, irinotecan, and metformin against CT26 cells has not yet been described. Therefore, we explored the potential anticancer effects of ReoT3D in KRAS-activated pathway in CT26 cells when supplemented with irinotecan and metformin for first time.
The major limitation of oncolytic viruses is the viral delivery to metastatic cancer sites and its therapeutic effects relatively poor intra-tumoral penetration. Also, we used combination of metformin with irinotecan as an effective treatment approach for targeting colorectal cancer cells that are resistant to irinotecan monotherapy, due to its decreasing intracellular level and uptake as well as mutations occurred in topoisomerase I.
In the near future, collecting the secretions of MSCs in different passages and infecting these secretions with different MOIs of the virus and examining their effect by flow cytometry for improving this study are needed, and expanding the result through in vivo assay for investigating their synergism effect as an anticancer strategy. Also, future studies should focus on identifying and characterizing different kind of colorectal cancer based on molecular organization, and it is important to consider potential confusing factors in cancer combination therapy which can be target the genomic, metagenomic-induced events.
Conclusion
In this in vitro study, we found that combination of T3D reovirus (oncolytic virus) and metformin with anticancer drug irinotecan resulted in higher rates of growth inhibition and apoptosis induction in colorectal cancer cell line. This synergistic effect was even more pronounced when using combination of secretome derived from reovirus-infected AD-MSCs, metformin, and irinotecan. Regimens containing secretome derived from noninfected AD-MSCs showed less cytotoxic effects. The apoptosis rate increased intensely from 48 h to 72 h in all cases. Future studies should explore mechanisms underlying these synergistic effects. Some candidate mechanisms could be upregulation of autophagy, P21, P53, and proapoptotic factors as well as downregulation of mTOR pathway-related and other pro-proliferative factors. Furthermore, in vivo and clinical studies could shed further lights on clinical applicability of these findings.
Acknowledgments
We thank the Deputy of Research at Tarbiat Modares University for their financial support and providing assistance. We also thank Dr. Seyed-Khorrami SM for his kind attempt to improve manuscript Figures quality.
Statement of Ethics
Ethical approval for this study was obtained from the Ethics Committee of Tarbiat Modares University (code No. IR.TMU.REC.1396.684).
Conflict of Interest Statement
The authors declare no conflicts of interests.
Funding Sources
The results described in this paper were part of student thesis, which was supported by the Grant No. Med-75118 from the Research Deputy of Tarbiat Modares University, Faculty of Medical Sciences.
Author Contributions
Maliheh Elhamipour carried out the experience and wrote the manuscript draft. Hoorieh Soleimanjahi designed and supervised the research and revised the draft. Asghar Abdoli was an adviser of project, Negar Sharifi performed the experience. Hesam Karimi analyzed the data, and Saeed Soleyman Jahi and Ruth Kvistad revised and edited the manuscript as a native English scientist.
Data Availability Statement
All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.