Evaluation of Lomustine-Loaded Iron Nanoparticles on Caspase-6 Gene Expression and Cell Viability in U87Mg Cell Line

http Introduction Cancer is recognized as one principal cause of mortality worldwide. A large number of people around the world die from cancers every year. In 2012, about 8.2 million deaths from cancer were reported in the world (1). cancer is considered as the third cause of death after cardiovascular diseases and accidents in Iran (2). Among all types of cancers, brain cancer has been identified as one of the most deadly malignancies due to the late detection and limitations of current therapies (3, 4). Researchers have shown that approximately 60% of these tumors are of glioblastoma type (5). Glioblastoma occurs in different parts of the central nervous system and is considered as the main leading cause of cancer death in people under 35 years of age (6, 7). Currently, standard treatments for this type of cancer include surgery and radiation therapy plus chemotherapy (8). After the surgery, the patient undergoes chemotherapy to cure residual cells and metastases (9). Evaluation of Lomustine-Loaded Iron Nanoparticles on Caspase-6 Gene Expression and Cell Viability in U87Mg Cell Line


Introduction
Cancer is recognized as one principal cause of mortality worldwide. A large number of people around the world die from cancers every year. In 2012, about 8.2 million deaths from cancer were reported in the world (1). cancer is considered as the third cause of death after cardiovascular diseases and accidents in Iran (2). Among all types of cancers, brain cancer has been identified as one of the most deadly malignancies due to the late detection and limitations of current therapies (3,4). Researchers have shown that approximately 60% of these tumors are of glioblastoma type (5). Glioblastoma occurs in different parts of the central nervous system and is considered as the main leading cause of cancer death in people under 35 years of age (6,7). Currently, standard treatments for this type of cancer include surgery and radiation therapy plus chemotherapy (8). After the surgery, the patient undergoes chemotherapy to cure residual cells and metastases (9). Int Electron J Med. Vol  Although this may increase the survival of patients after surgery, the prognosis of this cancer is still not convincing. One of the limiting factors is the resistance of cancer cells to chemotherapy drugs and the low doses of these drugs next to cancerous cells because of the side effects, early renal and liver excretion, as well as the presence of a blood-brain barrier (10,11). Accordingly, these limiting factors should be eliminated to achieve the desired result. Various methods have been proposed in this regard. The use of nanoparticles (NPs) is an effective and attractive method that has received special attention due to its nontoxicity, the presence of a modifiable surface for coating with biocompatible materials and targeting materials, and the ability to bind to therapeutic molecules (12,13). NPs can play an important role in the treatment of brain cancer and increase the life span of patients because of the portability of chemotherapeutic drugs, increased drug concentration at the tumor site, and improved drug uptake by cancer cells (14). Chemotherapy drugs can be loaded onto iron oxide (Fe 3 O 4 ) NPs in various ways. The best method is to use polymers with good biocompatibility and biodegradability properties (15). Targeted molecules can also be attached to this polymer and can enhance the specificity and uptake of the complex of nano plus chemotherapeutic drugs. Folic acid (FA) is one of the molecules that is used for targeting therapy with the NP, and its receptors are expressed on many malignant cells, including brain cancer cells (16). In addition, lomustine (LUM) is one of the powerful chemotherapy drugs that can be used to treat these cells. Further, it is a lipophilic drug that has the ability to cross the blood-brain barrier although side effects and therapeutic benefits are not satisfactory. The targeted NPs can increase the uptake of this drug into brain cancer cells and decrease the side effect (17). Given the above-mentioned explanations, the present study evaluated and compared the effect of nano, nanofolic, nano-LUM, LUM, and complex on cell viability and apoptosis in U87MG brain tumor cells.

Nanoparticle Synthesis and Modifications
Fe 3 O 4 -NPs were synthesized through the standard method. For surface modification, in a bottom of round flask equipped with a condenser and a stirrer, a mixture of APTES (5 mL, Sigma, 440140) and Fe 3 O 4 (4.03 g) was refluxed in a dry toluene (100 mL, Sigma, 244511) for 24 hours at 70°C. To separate NPs from the reaction mixture, an external permanent magnet was used, washed with milli-Q water (Sigma) and ethanol for several times, and then dried at 60°C under the vacuum. Next, cyanuric chloride (0.25 g, Sigma, C95501) was added to the mixture (0.50 g) in the dry tetrahydrofuran (40 mL, Sigma, 401756), subsequently, the mixture was stirred for 2 hours at 0˚C. The triazine functionalized MNPs (CC-Fe3-O4) were separated from the NP mixture by the magnetic field. Finally, the NPs were washed with tetrahydrofuran several times and dried at 40˚C under the vacuum.

Characterization
The size and shape of NPs were determined by TEM (Philips CM10 HT-100 kV). In addition, photoelectron spectroscopy with the X-ray was applied to assess the composition of synthesized NPs. Further, dynamic light scattering was used for sizing NPs and analyzing the hydrodynamic diameter of IONPs (typically ranging between 30 and 190 nm) through the Stokes-Einstein equation. Additionally, IONPs were electrostatically characterized by using the potential (Zeta potential) measurement to determine their surface charge. In general, surface charge, and in turn, the Zeta potential can be modified by employing a polymer coating. Furthermore, 30 mg of nano-LUM was dissolved in phosphate-buffered saline with a pH of 7.4 and digested with ethanol (95% v/v) in order to determine the encapsulation efficiency (EE). After centrifuging at 19400 ×g for 30 minutes, the supernatant was used for analysis. The total mass of LUM in NPs divided by the mass of LUM used in the formulation was considered as EE.

Cell Culture and Group Treatment
The U87-MG cell line was obtained from the Animal Cell Culture Division of Pasteur Institute (Iran, Tehran). The cells were then cultured in DMEM/F12 (Dulbecco's Modified Eagle Medium, Bioidea, Iran) with a 10% FBS (fetal bovine serum, Gibco, Germany), 100 μg/mL of streptomycin (Bioidea, Iran), and 100 U/mL of penicillin (Bioidea, Iran), and kept in a humidified atmosphere at 37°C with 5% CO 2 . Nano, nano-folic, nano-LUM, LUM, and complex (nano-folic-LUM) were dissolved in dimethyl sulfoxide (DMSO, Sigma, USA). After the U87-MG cells were more than 80% confluent and growing exponentially, they were counted and plated in a T75 culture flask and incubated with certain concentrations of nano, nano-flic, nano-LUM, LUM, and complex according to the 50% inhibitory concentration (IC50) and the tests were performed at certain times (i.e., 1, 3, and 5 days).

IC50 Assay
This evaluation in U87-MG cells was acquired after 1 day of treatment. Briefly, 10 4 cells were placed into a 12well plate and treated for 24 hours with various drug concentrations (i.e., 4.25, 7.5, 24.37, and 41.25 µg/mL), and then 3(4, 5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT, Sigma, the USA) survival assay was carried out to evaluate the cell viability in each group. Next, a percentage of cell viability versus drug concentration was used to obtain the IC50 value.

MTT assay
The effect of control, nano, nano-follicle, nano-LUM, iejm.hums.ac.ir http LUM, and complex on cell viability was determined by the MTT assay. To this end, the U87-MG cell line was cultured in 96-well plates at a density of 10×10 3 cell/well in a culture medium containing 10% FBS and incubated overnight. To evaluate the effect of drugs on cell growth, the cells were treated according to the IC50 value for 1, 3, and 5 day(s). After certain times of incubation in each group, 50 μL of 1 mg/mL MTT tetrazolium salt (Sigma) was added to each well and then the cells were reincubated at 37 º C for 4 hours. Next, the culture medium containing MTT was removed and replaced with 100 μL DMSO (Sigma) to dissolve the produced cell membrane and formazan crystals in lively cells for 2 hours. Finally, absorbance values were read at 570 nm and the percentage of cell viability was estimated using the following formula: Mean optical density (OD) of the treated group/mean OD of the non-treated group ×100

Real-Time Polymerase Chain Reaction for Caspase-6 Expression
U87-MG cells were plated at a density of 5 × 10 5 in the T27 culture flask overnight and incubated with control, nano, nano-folic, nano-LUM, LUM, and complex for 1, 3, and 7 days according to the IC50 value. After treatment time, the RT-PCR was carried out to determine the caspase-6 expression rate in each group vs. the control group.

Statistical Analysis
One-way analysis of variance with the post-hoc Tukey's test was performed to determine statistical significance among different groups using SPSS software. All quantitative data were presented as the mean ± standard deviation, and acceptable significances were at a level of P ≤ 0.05. Figure 1 displays the transmission electron microscopy analysis of LUM-loaded NPs and LUM-loaded NPs modified with FA. It mainly proves that NPs had a spherical shape, and zeta potential values were in the delicate range of -52.4±0.06 to -22.85±0.47 mV. In addition, the poly-dispersity index (particle size distributions), EE, and drug loading content are listed in Table 1. Dynamic light scattering results showed that all synthesized NPs had a hydrodynamic dimension of 19-52 nm. Further, the average size of the hydrodynamic particle was 19.3±0.72, 28.52±0.46, 46.24±1.01, and 52.61±1.12 for magnetic triblock copolymers, magnetic triblock copolymers modified with FA, LUM-loaded magnetic triblock copolymers, and LUM-loaded magnetic triblock copolymers modified with FA, respectively.

Cytotoxicity Assays
The viability of U87Mg cells was determined by the MTT assay for nano, nano-folic, nano-LUM, LUM, and complex drugs. In nano-LUM, LUM, and complex groups, the cell viability decreased by increasing the incubation time ( Figure 3).

Caspase-6 Expression After the Treatment of the U87-MG Cell Line
Caspase-6 expression analysis was used as an executive apoptotic gene to study the gene expression modified effects of each material in U87MG cell lines. As shown in Figure 5, U87Mg cell treatment with LUM significantly increased in the caspase-6 expression after 3 and 5 days. In nano-LUM and complex, the caspase-6 expression increased compared to the control group as well. However, caspase-6 expression decreased on the fifth day compared to the third day.

Discussion
Attention has focused on the cytotoxicity of NPs by the   (18,19). NPs are attractive for utilization in various biological fields and have shown commitment to deliver biologic agents to cells (20). Nevertheless, researches demonstrated that NPs have meaningful unacceptable effects on normal cells (21). The characteristics of NPs induce cytotoxicity in cells (22). The manipulation of NPs by biocompatible polymers is required to support the safe utilization in medicine fields (23,24). Consequently, in our investigation, new modified NP surface by LUM and FA was applied to make a proper nano-size vehicle for medical aims. The results of our study showed that synthesized NPs had a proper size and shape with a super-paramagnetic function. The findings of another study represented that covering the surface of NPs with other drugs makes a decrease in magnetization NPs (25). Therefore, the coating of NPs with drugs can reduce drug concentration (26 In another study, NPs influenced the cell apoptosis rate through the stimulation of reactive oxygen species production and then DNA damage, and finally, P53 and caspase-6 overexpression (29). NPs in the cells degenerated into free mineral ions by lysosomal hydrolyzing enzymes (30). Cell organelle such as nucleus or mitochondria can be discharged from free iron ions. In addition, the hydroxyl free radicals effectively react with the DNA chain and some important proteins result in DNA breaks and protein damages and the stimulation of cell apoptosis and death (31). The present study explored the effects of both NPs and LUM-NPs on cell apoptosis and the findings showed that LUM-NPs significantly induced apoptosis in cancerous cells after 3 days.
The caspase-6 activity as apoptosis markers was analyzed to confirm the inhibitory effect of LUM-NP -induced apoptosis. Our results demonstrated that LUM-NPs significantly increased the expression of caspase-6 activity.
Other studies also confirmed that caspase has a key role in apoptosis (32,33). The results of this study further approved that caspase-6 was stimulated by LUM-NPs following 3 days whereas folic NPs and LUM-folic-NPs

Caspase-6 Expression After the Treatment of the U87-MG Cell Line
Caspase-6 expression analysis was used as an executive apoptotic gene to study the gene expression modified effects of each material in U87MG cell lines. As shown in Figure 5, U87Mg cell treatment with LUM significantly increased in the caspase-6 expression after 3 and 5 days. In nano-LUM and complex, the caspase-6 expression increased compared to the control group as well. However, caspase-6 expression decreased on the fifth day compared to the third day.    iejm.hums.ac.ir http represented no significant effects on caspase-6 activity. Finally, our results revealed that LUM-NPs induce apoptosis in U87-MG cell lines while NPs and LUM-folic-NPs had no effects on cell programmed death.

Conclusion
In general, our results demonstrated that NPs increased the cytotoxic effects after 5 days when compared with the control group although it was not significant.