Effect of Exposure to Quran Recitation on Cell Viability , Cell Migration , and BCL 2 L 12 Gene Expression of Human Prostate Adenocarcinoma Cell Line in Culture

Introduction ne of the most frequent nondermatologic malignancies is prostate cancer (1). Different therapeutic methods, such as new treatment options, early diagnosis, and routine screening, have positive and important effects on the survival rate of prostate cancer patients (2). Based on the statistics, in 2012, about one million men were diagnosed with prostate cancer worldwide, 70% of whom were in developed countries. The incidence of prostate cancer in Iran is about 10 per 100,000 individuals (3). It is estimated that about 43% of male cancer survivors in the United States are survivors of prostate cancer, and the rate of 5-year survival across different stages is 99.7% (4). When sound travels through a medium, it can cause turbulence in it. Sound is defined as the compression of a waveform at a specific frequency that passes through air or materials (5). A number of studies have divided sound into three groups: 1) infrasound (below 20 Hz); 2) audible sound (20-20,000 Hz), and 3) ultrasound (above 20 kHz) (6). In the field of medicine, the use of ultrasound is well-known in the diagnosis and treatment of medical problems. Sound can also create mechanical stress and directly affect cells. The disturbance created by sound via the mechanism of particle interaction is probably the cause of such an effect (7). O Abstract

On the other hand, music is an integral part of many cultures worldwide; however, its various effects on human life are not widely known yet.Previous studies have shown that music may be useful in the alleviation of stress, medical care, and nociception in cancer and burn patients, as well as some patients undergoing surgical procedures.However, the main mechanisms through which these effects occur are still unidentified (8,9).Since the beginning of the 21st century, different studies have demonstrated that the response to sound, in general, and to music, in particular, is complex and might not be fully ascribed to emotion, given that cell types other than auditory hair cells can also respond directly to audible sound (6).In a study, sound was used to improve cell growth in plants (10).Other researchers have also confirmed the positive effects of sound on human beings.Human cells are well reacted to sound, and sound can resonate through human somatic and auditory cells (6,8).The approach of using sound for therapeutic purposes can introduce further non-invasive treatment options to patients.In Islam, Quran is the main source of principles that acts as a leader for Muslims and offers guidelines to solve social problems and improve human lives (11).Quran recitation has been already used as a treatment method in some Islamic countries (12,13).A previous study indicated that junior high school students who recited Quran regularly had less anxiety, depression, and stress as compared to other groups (14).Furthermore, other research demonstrated that those who listen to Quran regularly have a lower degree of anxiety, compared to those who do not listen to this holy book recitation (15).It appears that studies investigating the response of human body cells to Quran recitation are limited.Therefore, the aim of this study was to evaluate the effect of Quran recitation alone and along with cisplatin on the viability, motility, and BCL2L12 (BCL2-like 12) gene expression of PC-3 cells.This study was conducted to understand the effects of acoustic vibrations in the form of Quran recitation on human cells in culture.

Methods
Cell Line and Cell Culture: PC-3 cells were obtained from the National Cell Bank of Iran in Tehran.These cells were incubated in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 100 U/mL antibiotics (all from Gibco, Scotland) at 37°C with 95% O2 and 5% CO2 in an incubator.The experiments were done overnight after seeding in order to ensure adherence of the cells.Each experiment was repeated at least three times (16).
Treatment with Quran Recitation: The cells were exposed to Quran recitation using four speakers symmetrically surrounding the 96well plates for 2 h.To this end, the recitation of Surah Al-Fatiha by the recorded voice of Sheikh Abdul Basit was used for 70 times.A small platform was used so that the speakers were not in contact with the floor.As a control group, the cells had exposure to silent speakers to ensure the deletion of possible magnetic field or background noise produced by the speakers.Pressure levels of sound were maintained at 90 dB (6).MTT Assay: The effects of sounds on PC-3 cells were defined by an MTT assay (Sigma, USA).A total of l04 cells were exposed to Quran recitation for 2 h.However, l04 cells were treated with different concentrations of cisplatin for 48 h.Furthermore, l04 cells treated with IC50 concentration of cisplatin for 48 h were exposed to Quran recitation for 2 hours.Then, the cells were washed twice with culture medium, and MTT (0.5 mg/ml PBS) was incorporated to each well and incubated at 37°C for 4 h.Formazan was dissolved by isopropanol incorporation, and the absorbance was read at 540 nm using a microplate scanning spectrophotometer (ELISA Reader, OrganonTeknika, Netherlands) (17).
Cell Migration: A CytoSelect Cell Migration Assay Kit (Cell Biolabs, USA) was applied particularly to measure the effect of Quran recitation and cisplatin on the migration of PC-3 cells.The assay was conducted according to the manufacturer's guidelines.The 5×105 cells were suspended in 100 μl of RPMI without FBS and added to the upper chamber.In addition, 150 μl of culture medium with FBS was added to the bottom chamber and incubated at 37°C for 18 h.Migratory cells passed through the membrane pores in response to the chemoattractant.Furthermore, these migratory cells were separated from the membrane (18).
Power SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) was employed to perform quantitative PCR on the Rotor-Gene 6000 (Corbett Research, Australia).The thermal-cycling conditions of the PCR included 10 min at 95°C (one repeat) as primary denaturation and hot-start enzyme activation, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min.
Calculation of gene expression was performed using the comparative threshold cycle (Ct).The mean threshold cycle (mCt) was obtained from triplicate amplifications during the exponential phase.Then, mCt value of the reference gene (TBP) was subtracted from the mCt value of the target gene (BCL2L12) to obtain ∆Ct and ∆∆Ct values of each sample that were calculated from the corresponding Ct values as follows: ∆∆Ct=[Ct target -mCt reference] (treated sample) -[mCt target -mCt reference] (untreated sample) Finally, BCL2L12 gene expression/tbp gene expression ratio was computed by the ratio formula 2-∆∆Ct (20).
Statistical Analysis: The data were analyzed in SPSS software (version 16, SPSS Inc., USA).Each test was repeated at least three times.The data were expressed as mean and standard error, and analyzed using one-way ANOVA with Dunnett's post hoc test for comparison.In conclusion, the P-values and expression proportion related to the tested genes were calculated using the Relative Expression Software Tool (REST).P-value less than 0.05 was considered statistically significant.

Result
Cisplatin and Quran Recitation Cytotoxicity on PC-3 Cells : In this study, the cytotoxicity of cisplatin was determined by treating PC-3 cells with various concentrations of cisplatin (0-100 µM) for 48 h followed by an MTT assay.the total cell numbers were decreased by a lower dose of cisplatin at 2.5 µM (13.38%;P<0.05) and a higher dose of cisplatin at 100 µM (98.13%;P<0.001), compared to the control group (Figure 1a).Moreover, the cytotoxic activity of cisplatin increased with the elevation of cisplatin concentration.The half maximal inhibitory concentration (IC50) of cisplatin was computed at 7.4 µM.
The percentage of surviving PC-3 cells that were exposed to Quran recitation for 2 h was 91.8% (P<0.05; Figure 1b).Figure 1c illustrates that the percentage of live cells significantly diminished 48 h after cisplatin treatment at 7.4 µM concentrations and exposure to Quran recitation (43.8%;P<0.001).
The percentage of PC-3 cell migration exposed to Quran recitation for 2 h was 95.4% (P<0.05; Figure 1b).Figure 1c shows that Quran recitation significantly diminished the migration of PC-3 cells that were treated with 7.4 µM concentration of cisplatin (18.6%;P<0.001).

Mitigatory Effect of Cisplatin and Quran
Recitation on BCL2L12 mRNA Level: Exposure to cisplatin at IC50 concentrations for 48 h led to a significant decline in the mRNA level of BCL2L12 by a factor of 0.30, compared to the control group (P<0.001).Similarly, the PC-3 cells exposure to Quran recitation for 2 h significantly decreased the mRNA level of BCL2L12 by a factor of 0.48 (P<0.001).The ratio was calculated as 0.21 (P<0.001) for PC-3 cells that were treated with 7.4 µM concentration of cisplatin and also exposed to Quran recitation for 2 h.The Pvalues and expression ratio related to the genes were computed by the REST, and the investigated genes were normalized against TBP (Figure 2).

Discussion
The use of sound as an alternative and supplementary agent for therapeutic purposes has gained attention in recent years.This type of treatment is widely used as an alternative to clinical medicine in order to improve the health level of human life (21).Due to the fact that water (that acts as a good conductor for sound) forms about 70% of the body, sound can easily pass through the human body.The interaction between cells and sound has become a very challenging topic to be explored.Responses of human body cells to sound are a very complicated process (6).
Many studies stated that the response to sound not only may be limited to emotion, but can also affect the very small structures of living mechanisms like cells (8).This hypothesis relates the in vitro direct effects of sound on non-auditory cells to mechanical stress, which is reasonable since sound is a mechanical vibration that can cause mechanical stress (8).
Previous studies suggested that cellular responses to sound depend on different factors, such as the intrinsic characteristics of cellular type and nature of sound (22).Therefore, it seems that the use of appropriate sound can improve the human healing process.Meanwhile, Aggio et al. (2012) added that sound can reduce biomass production in the yeast cells (23).Another report showed that audible sound can alter the proliferation and protein activity of cells.It was also suggested that sound healing was able to alter the cell cycle, morphology, and functional parameter in non-auditory human cells (4).
The use of Quran recitation as a technique in sound healing approaches seems to be promising.Quran recitation as a natural sound can have unique qualities that are capable of healing the cancerous cells.The findings of the current study are in line with those of other authors who showed that Quran recitation can have positive effects on the cancerous cells.This idea is supported by a study, which showed that the recitation of Quran as an alternative therapy can lead to the improvement of chronic diseases (14).Tumiran et al. (2013) suggested that Quran recitation is a complementary sound healing technique that provides better outcomes as compared to routine sound healing (24).In another study, it was demonstrated that reciting or listening to Quran verses can have a promising effect on human cells (25).Quran recitation also has a fluent style, powerful expression, and relaxing sound.It has an inner tendency toward rhythm and harmony and can influence the audience (26).Babamohamadi et al. demonstrated that reciting the Holy Quran to a sick person will have considerable healing effects (12).Tumiran et al. (2013) confirmed several deep points regarding healing with Quran recitation.It seems that the sound of Quran recitation can provide a deep spiritual energy to both audience and reciter.The healing process using Quran recitation can affect listeners even if they do not understand the meaning of the verses (24).
To the best of our knowledge, this study was the first attempt investigating the restrictive effects of Quran recitation, and the viability and migration capacities of PC-3 prostate cancer cells through examining BCL2L12 mRNA levels.The significant inhibitory effects of cisplatin on the proliferation and migration of PC-3 cells exposed to Quran recitation was more than that on PC-3 cells that were treated with cisplatin alone.Furthermore, these results were observed with regard to the expression of BCL2L12.Therefore, there was a reduction in the expression of this gene in PC-3 cells that were exposed to Quran recitation.
However, downregulation in the BCL2L12 gene was greater in PC-3 cells that were treated with cisplatin.Several studies have demonstrated that audible sound waves can alter cell proliferation.Jones et al. showed that human gingival fibroblasts that have been exposed to 261 Hz sound changed the rate of cell proliferation according to the time and amplitude of exposition (27).Therefore, it is not surprising that in this study, we observed decreased cell proliferation induced by Quran recitation.
Cisplatin is an important antineoplastic drug that can display a significant clinical activity against a wide variety of solid tumors.Although cisplatin has a widespread clinical use as an anticancer medication, molecular mechanisms leading to cell death after cisplatin treatment are not well-understood.However, cisplatin-DNA crosslink may induce programmed cell death (19).
This study showed that cisplatin treatment resulted in the downregulation of BCL2L12 and was the main cellular event.BCL-2 family members, consisting of pro-apoptotic and antiapoptotic genes, are the key regulators of DNA damage-induced apoptosis.Although BCL2L12 is certainly involved in apoptosis, its antiapoptotic or pro-apoptotic role still remains controversial depending on the cellular context.
A high expression of BCL2L12 mRNA has been confirmed in many cancers.

Conclusion
Since the current study was the first report on the direct effects of Quran recitation on nonauditory cells in culture, more studies are needed to achieve further comprehension of these phenomena.Results of this investigation strongly suggested that Quran recitation could alter cell proliferation and migration in human PC-3 cells.Moreover, it was shown that BCL2L12 was downregulated in PC-3 prostate adenocarcinoma cell lines that were exposed to Quran recitation.Our results revealed that Quran recitation can also induce apoptosis in these tumor cells through the downregulation of BCL2L12 expression.Considering the type, frequency, range, and level of sound used during the cell culture, the effect of sound on human cells may be different.These results warrant further investigations in animal tumor models.

Figure 1 .
Figure 1.a) Comparison of dose-dependent effects of cisplatin on the viability and migration of the PC-3 cells (results are expressed as a percentage of inhibition rate, compared to control, and mean±standard deviation from at least three independent experiments (P<0.05),b) effects of exposure to Quran recitation for two hours on the viability and migration of the PC-3 cells (data are expressed as the mean±SD of at least three independent experiments), c) effects of exposure to Quran recitation for two hours on the viability and migration of PC-3 cells treated with 7.4 µM concentrations of cisplatin for 48 hours (data are expressed as the mean±SD of at least three independent experiments); asterisk indicates that treated group is statistically significant (*P<0.05,**P<0.01,*** P<0.001) from the control group.

Figure 2 .
Figure 2. Inhibitory effects of cisplatin (A), Quran recitation (B), and cisplatin with Quran recitation on the expression of BCL2L12 mRNA (all data were normalized against TBP, by REST software.Bars represent fold differences of the mean of normalized expression mean±SD.Significance was set at *P<0.05, **P<0.01,***P<0.001.)