Effect of a Japanese Energy Healing Method Known as Johrei on Viability and Proliferation of Cultured Cancer Cells In Vitro

Abstract

Objectives:

The objective was to explore the effect of a Japanese energy healing method known as Johrei on the viability and proliferation of cultured human cancer cells in vitro.

Design:

A randomly selected 96-well plate or a culture dish of various types of human cancer cell lines in culture were exposed to Johrei treatment. For comparison purpose, an equal number of untreated or volunteer-treated cultures were chosen as the control group. Johrei treatment was repeatedly performed at appropriate time intervals over the course of the experiments. Cell viability was examined by a colorimetric assay with a Cell Counting kit. Morphological changes were analyzed by phase-contrast and time-lapse microscopy. Cell proliferation and early and late stages of cell death were also determined with the use of a bromodeoxyuridine (BrdU) cell proliferation assay kit and an Annexin V-FLUOS Staining kit, respectively.

Outcome measures:

Quantitative data were presented as means±standard deviation. The outcome measures were the differences in viable cell numbers that remained under healing practice versus control conditions, and the statistical significance of differences in their mean values was assessed.

Results:

The viability loss of cultured human cancer cells in the Johrei group was significantly higher than that of either of the control groups, despite the fact that the responsiveness to Johrei varied with different cancer cell types. The proliferation rate of gastric cancer cells exposed to Johrei treatments for 72 hours was more significantly decreased compared with that of the untreated cells, whereas the extent of dying and/or dead cells in the Johrei group was more profound than that of the untreated cells.

Conclusions:

These results provide evidence that Johrei treatment induces the viability loss of various cancer cells in vitro, mainly due to the increased cell death and the decreased proliferation.

Introduction

A lternative energy healing methods including Reiki and Johrei are increasingly popular in the world as a potentially useful intervention to achieve good health.^{1

–13} The Japanese energy healing method known as Johrei was founded by Mokichi Okada (1882–1955) in 1935. According to Okada’ doctrine, Johrei is essentially a method of spiritual purification attained by the transmission of divine energies without touching the recipient through the hand of practitioners, thereby contributing to the achievement or sustenance of a state of health. While the majority of reports suggest beneficial effects of Johrei on various human psychologic and physical conditions,^8,9,13
–15 only a limited number of studies evidencing such beneficial effects by use of generally accepted scientific methods are available. In particular, it is not clear whether or to what extent nonspecific factors based on the healer–recipient relationship—including belief, expectation, and other psychologic aspects—affect the results obtained with human subjects. Accordingly, to minimize such psychologic and artificial effects and to verify beneficial effects of Johrei on the health of recipients, various approaches based on generally accepted scientific methods are needed. The in vitro studies with single cell lines have a great advantage over in vivo studies with human subjects and are highly effective for replications and verifications of these experiments, since such studies are able to be performed under tightly controlled experimental conditions that minimize psychologic and artificial aspects. In addition, researchers conducting the biologic assays and statistical analyses could be readily blinded to the experimental conditions.

Despite little scientific evidence showing the existence of Johrei energy, it was hypothesized that if its purported universal healing energy may be transmitted to the targets through the hand of the Johrei practitioner, it may directly interact with cells in culture, thereby leading to particular alterations of their structure or function. In this study, the culture system of various human cancer cell lines was utilized and the effect of Johrei treatment on the viability, proliferation, and death of the cultured cancer cells were investigated.

Materials and Methods

Participants

The Johrei healing method was performed by 6 trained Johrei practitioners who were qualified by the Japanese Johrei Society Sekai Kyusei Kyo Izunome. The mean age of these practitioners was 54.1 years, with a range of 40–63 years. All of the practitioners had more than 10 years experience treating people with Johrei. They concentrated on the transmission of Johrei healing energy to cultures by means of Ohikari, a symbolic tool given to the practitioner qualified to deliver this energy. Cancer cells seeded in 96-well plates or μ-dishes (ibidi, Martinsried, Germany) at appropriate densities were exposed to twice-daily Johrei healing treatments of 15 minutes (total 30 minutes per day) from approximately 15 cm away. The experiments took place over a period of 3 or 4 days. An equal number of unexposed plates and dishes were standing very far apart from the practitioners in the same laboratory and served as the control group. In some experiments, 6 volunteer operators who had no experience treating people with any energy healing methods and who did not wear Ohikari were chosen as another control group. During healing sessions, each member of the volunteer group was asked to treat cell cultures in the same way as the practitioners did. In experiments in Figure 1, each member in both groups was given separate plates.

FIG. 1.

Effect of healing treatments of each of the volunteer operators without switching with the team members on the viability of human gastric cancer AGS cells. The cultured cells seeded in 96-well plates at a density of 2000 cells/well (A) and 3500 cells/well (B) in a volume of 100 μL were subjected to twice-daily treatments of 15 minutes by each of the volunteer operators. Volunteer operators who participated in the experiments A and B were different from each other. Other details are the same as described in Fig. 2. A. Lane 1, control; lane 2, volunteer A; lane 3, volunteer B; lane 3, volunteer C. B. lane 1, control; lane 2, volunteer D; lane 3, volunteer E; lane 4, volunteer F. Data are the means±standard deviation of values for eight independent experiments in the respective experiments A and B, and 24 96-well plates were used in each experiment. *p<0.05, and ***p<0.001 versus the corresponding values for the untreated cells.

Culture conditions

A human gastric carcinoma cell line (AGS) was obtained from DS Pharma Biomedical Co. (Osaka, Japan). The human uterine cervix epithelioid carcinoma cell line HeLa, the human malignant lymphoma U937, and mouse B16 melanoma cells were from RIKEN BioResource Center (Tsukuba, Japan). The human prostate carcinoma cell line PC-3 was obtained from Human Science Research Resources Bank (Tokyo, Japan). Two (2) human prostate carcinoma cell lines (ALVA-41¹⁶ and PPC-1¹⁷) were kindly provided by J.Y. Bahk (Gyeogsang National University, Korea) with the permission of their original developers. Each of the cell lines was incubated at 37°C for 24 hours in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL) in a 5% CO₂ incubator. Then the cells were seeded in a 96-well plate in a volume of 100 μL at an appropriate density (AGS, 2000–3000 cells/well; HeLa and U937, 1000–1500 cells/well; ALVA-41, PPC-1 and PC-3, 500–1000 cells/well; B16 melanoma, 800–1500 cells/well) and cultured in the same medium at 37°C for ∼96 hours in a 5% CO₂ incubator. These plates were randomized to the Johrei and control groups. Each plate of the Johrei group was subjected to twice-daily Johrei application for 15 minutes. An equal number of untreated plates were allowed to stand far apart from the practitioners in the same laboratory and served as the control (Table 1, Figs. 2 –6). As another control group, each of the volunteer operators treated an equal number of plates in the same way as the practitioners did.

FIG. 2.

Effect of Johrei intention on the viability of human gastric cancer AGS cells. After incubation at 37°C for 24 hours in a 5% CO₂ incubator, the cells were seeded in 96-well plates at a density of 2500 cells/well in a volume of 100 μL and exposed to twice-daily Johrei treatments of 15 minutes by the practitioners. Each practitioner was switched every 24 hours with the other team member. An equal number of cultures that were not treated by either the practitioners or the volunteer operators were chosen as the control group (filled circles) and processed under the same conditions as the Johrei group (open circles). At the indicate time intervals, the cell viability was determined with a colorimetric assay using a Cell Counting kit-8. Data are the means±standard deviation of values for eight independent experiments, and 24 96-well plates were used in each experiment. *p<0.05, and **p<0.01, versus the corresponding values for the control group.

FIG. 3.

Effect of Johrei treatments by each of the practitioners without switching with the team members on the viability of human gastric cancer AGS cells. The cultured cells seeded in 96-well plates at a density of 2000 cells/well in a volume of 100 μL were exposed to twice-daily Johrei healing intention treatments of 15 minutes by each individual practitioner. An equal number of untreated cells in culture were chosen as the control group and processed under the same conditions as the Johrei group. Other details are the same as described in Figure 2. A. Lane 1, control; lane 2, Johrei practitioner A; lane 3, Johrei practitioner B; lane 3, Johrei practitioner C. NS, not significant. B. Lane 1, control; lane 2, Johrei practitioner D; lane 3, Johrei practitioner E; lane 4, Johrei practitioner F. Data are the means±standard deviation of values for eight independent experiments, and 24 96-well plates were used in each experiment. *p<0.05, **p<0.01, and ***p<0.001 versus the corresponding values for the control group.

FIG. 4.

Morphological assessment of Johrei-treated human gastric cancer AGS cells and the untreated cells by differential interference contrast microscopy. The cultured cells were seeded in a μ-dish in a volume of 2 mL at a density of 4500 (A) and 6000 cells/dish (B). The cells were exposed to twice-daily Johrei treatments of 15 minutes for 72 hours. A Johrei practitioner was switched every 24 hours with the other team member. An equal number of the untreated cells in culture were chosen as the control group and processed under the same conditions as the Johrei group. Photographs represent the representative results from experiments performed with five independent pairs of AGS cells. Magnification×100.

FIG. 5.

A cell proliferation assay of Johrei-treated and -untreated human gastric cancer AGS cells with the use of a BrdU assay kit. The cultured cells were seeded in a 96-well plate in a volume of 100 μL at a density of 3000 cells/well and then incubated at 37°C for 24 hours in a CO₂ incubator. Then, the cells were exposed to twice-daily 15-minute Johrei treatments. An equal number of untreated cells in culture were chosen as the control group and processed under the same conditions as the Johrei group. BrdU was added to the cultured cells at 24 and 72 hours after the initial treatment and further incubated at 37°C for 16 hours in a CO₂ incubator. After fixation, amounts of the BrdU incorporated into the cells were quantified with the use of anti-BrdU detection antibody. Lane 1, control; lane 2, Johrei practitioner A; lane 3, Johrei practitioner B; lane 4, Johrei practitioner C. Data are the means±standard deviation of values for three independent experiments, and 24 96-well plates were used in each experiment. *p<0.05, **p<0.01, and ***p<0.001, versus the corresponding values for the control group.

FIG. 6.

A quantitative cell death analysis of Johrei-treated and -untreated human gastric cancer AGS cells with the use of an Annexin V-FLUOS Staining kit. Cultured AGS cells exhibited higher rates of cell death with Johrei treatments. The cells cultured for 72 hours in a μ-dish were subjected to quantitative cell death analysis with the use of an Annexin V-FLUOS Staining kit for detecting early (yellow arrows) and late stages of cell death (red arrows). An equal number of untreated cells in culture were chosen as the control group and processed under the same conditions as for the Johrei group. Other details are the same as described in Fig. 4. The relative ratio of dying and/or dead cells to viable cells is significantly low in untreated cells (A), whereas that ratio is extremely high in Johrei-treated cells (B). The data represent the representative results from experiments with five independent pairs of AGS cells. Magnification×100.

Table 1.

Responsiveness of Various Types of Cancer Cells to Johrei Treatments

Source	Cell line	Cancer cell type	Susceptibility to Johrei
Human	AGS	Gastric cancer	+++
	U937	Malignant lymphoma	++
	HeLa	Uterine cervix epitheloid carcimona	+++
	PC-3	Prostate carcinoma	+
	ALVA-41	Prostate carcinoma	++
	PPC-1	Prostate carcinoma	+
Mouse	B16	Melanoma	++

Each cultured cell was seeded in a 96-well plate in a volume of 100 μL at optimal density (human gastric carcinoma cell line [AGS], 2000 cells/well; HeLa and U937, 1500 cells/well; ALVA-41, PPC-1 and PC-3, 500 cells/well; mouse B16 melanoma, 800 cells/well) and cultured in the same medium at 37°C for 72 hours in a 5% CO₂ incubator. During the incubation, each plate of cultured cells was subjected to twice-daily 15-minute Johrei healing-intention treatments. An equal number of unexposed cultures that were not treated by either the practitioners or the volunteer operators were chosen as controls and processed under the same conditions as the Johrei group. Based on the ratio of the viable cell number between the Johrei group and the control group, the relative responsiveness of each cell type to Johrei treatment was expressed as the ratio to that of AGS cells and classified into three groups:+, low responsiveness (<40% of that for AGS cells); ++, moderate responsiveness (40%–80%); +++, high responsiveness (more than 80%).

Measurement of cell viability and proliferation

At each time point, cultures treated by both groups were subjected to cell viability assay using the Cell Counting kit-8 reagent (Dojindo Molecular Technologies, Kumamoto, Japan). After the addition of the reagent (10 μL) to each well, cells were incubated for an additional 1 hour. Then the absorbance was determined on a microplate reader (Model 680, Bio Rad Laboratory, Tokyo, Japan) at the wavelength of 450 nm with a reference wavelength at 650 nm.¹⁸ The cell viability was also determined with the use of an Annexin V-FLUOS Staining kit (Enzo Life Sciences International, Inc., Plymouth Meeting, PA) for detecting early and late stages of cell death, according to the manufacturer's instruction. Cell proliferation was determined with the use of a bromodeoxyuridine (BrdU) cell proliferation assay kit (Exalpha Biologicals, Inc., Watertown, MA), according to the manufacturer's instruction.

Time-lapse microscopy

For a time-lapse analysis, AGS cells seeded in a μ-dish and cultured at 37°C for 24 hours in a 5% CO₂ incubator were transferred to a time-lapse microscope (Olympus IX81, Tokyo, Japan) equipped with a heated stage, a 5% CO₂ incubation chamber, and a Plexiglas® (acrylic plastic) environment chamber. Johrei treatments were administered 3 times a day for a total of 45 minutes (15 minutes for each) toward the incubation chamber from a short distance (Fig. 7B). The experiments took place over a period of 3 days. An untreated dish exposed to neither the practitioners nor the volunteers was incubated in the same way (Fig. 7A). Live cells were observed under the Olympus IX81 microscope with differential interference contrast objectives (10X) and the images were collected with a cooled CCD video camera DP30 (Olympus, Tokyo, Japan) at 10-minute intervals for 72 hours mounted on a time-lapse imaging system and saved as image stacks and then analyzed using MetaMorpho software (Molecular Devices).

FIG. 7.

Time-lapse microscopy analysis of human gastric cancer AGS cells. AGS cells were seeded in a μ-dish (35-mm diameter) in a volume of 2 mL at a density of 4500 and incubated at 37°C for 24 hours in a CO₂ incubator. Then the dish was transferred from the incubator to a time-lapse microscope equipped with a heat stage and incubation chamber. Johrei treatments were administered 3 times a day for a total of 45 minutes (15 minutes for each) toward the incubation chamber from a short distance (B). An equal number of untreated cells in culture were chosen as the control group and processed under the same conditions as the Johrei group (A). To monitor the progression of proliferation of AGS cells, time-lapse microscopy with differential interference contrast objectives (10×) was performed. Images were collected with a cooled CCD video camera at 10-minute intervals for 72 hours mounted on a time-lapse imaging system and saved as image stacks and then analyzed using MetaMorpho software. Photographs represent the representative results from experiments with five independent pairs of AGS cells and are shown as images of every 3.13 hours.

Statistical analysis

Quantitative data are presented as means±standard deviation. Results from cell viability experiments were compared between the Johrei group and either of the control groups. The statistical significance between mean values of either of the two groups was analyzed by the Welch's two-sample t-test. Values of p<0.05 were considered statistically significant.

Results

Effect of Johrei treatment on viability of human cancer cell lines

It is generally accepted that the viable cell number is determined by the balance between proliferating and dying cells. It was first found that the responsiveness of cultured cancer cells to Johrei treatment varied with different cell types. To determine whether Johrei treatment has a direct effect on the viability of cultures of various human cancer cell lines, the concentration of cultured cells seeded in 96-well plates were optimized. After several trials, the optimal density of each cancer cell type was determined as described in the Materials and Methods section. AGS cells seeded in 96-well plates at a density of 2500 cells/well were exposed to twice-daily Johrei healing intention treatments of 15 minutes by each practitioner who was then switched every 24 hours with the other team members. An equal number of untreated cultures chosen as the control group were standing for the same intervals as those treated by the practitioner. As shown in Fig. 2, while the number of viable AGS cells in both groups was time-dependently increased during the experimental period, the increasing rate of viable cells in the Johrei group was significantly lower than that in the control group.

To investigate whether the decrease in viable AGS cells by the practitioners could also be induced by Johrei treatments of each of the practitioners without switching with the team members, AGS cells were exposed to twice-daily 15-minute Johrei treatments by each practitioner. Although the survival rate of the cells was to an appreciable extent different among the practitioners, the number of viable AGS cells treated by all of the practitioners was significantly lower than that of the untreated cells (Fig. 3A and B). Moreover, when AGS cells were exposed to twice-daily 15-minute healing intention treatments by 6 volunteer operators chosen as another control group, the number of viable AGS cells was not significantly changed by the treatment of each of the volunteer operators (Fig. 1A, B). However, as exceptional instances, the number of viable cells was significantly decreased at 1 day after the treatment by the volunteer operator A and inversely increased at 2 days after the treatment by the volunteer operators C and F and at 3 days after the treatment by the volunteer operator E.

Morphological assessment

Morphological analysis revealed that the number of AGS cells in the Johrei group at 72 hours after incubation was apparently lower than that in the untreated group (Fig. 4). To determine whether the observed difference between the Johrei group and either of the two control groups was actually related to altered cell viability, movies of actively proliferating AGS cells were generated using time-lapse microscopy. Johrei treatments were administered by the Johrei group members in 3 times daily treatments of 15 minutes toward the incubation chamber from a short distance. Figure 7 shows the same time picture frames selected every 80 minutes from images of cultured AGS cells that were collected with a cooled CCD video camera at 10-minute intervals for 72 hours. The untreated cells were time-dependently increased and actively fused and consolidated into larger masses during this period of time (Fig. 7A). By contrast, the Johrei-treated cells were more slowly increased compared with those in the control group over the same time intervals (Fig. 7B). Strikingly, most of the proliferating AGS cells in the Johrei group appeared to be restricted in their movement at the early stage and to be unable to progress further morphologically. It is also noteworthy that significant amounts of Johrei-treated AGS cells rotated freely at the early stage and ultimately died.

Measurement of proliferation and early and late stages of cell death

To determine whether the decreased viability of AGS cells applied to Johrei treatments was due to the decrease in the proliferation extent and/or the increase in the extent of cell death, cell proliferation and death assays were performed with the use of a BrdU assay kit and an Annexin V-FLUOS Staining kit for detecting cell death, respectively. BrdU is known to be incorporated into newly synthesized DNA strands of actively proliferating cells. As shown in Figure 5, the incorporated BrdU levels in the cells treated by each of the Johrei practitioners at 24 hours were significantly higher than those in untreated cells. However, the BrdU levels in Johrei-treated cells at 72 hours were apparently lower than those in untreated cells. The results strongly suggest that Johrei application to the cells increases their proliferation at early stages of the treatments but decreases it at late stages of the repeated treatments. To further determine the induction of cell death by Johrei treatments, AGS cells cultured for 72 hours in a μ-dish were evaluated with the use of an Annexin V-FLUOS Staining kit for detecting early and late stages of cell death. The data in Fig. 6 demonstrate that the relative ratio of dying and/or dead cells to viable cells is significantly low in untreated cells, whereas it is extremely high in Johrei-treated cells. The results clearly indicate that Johrei treatments induce not only the reduction of the number of viable cells but also the increase of dying and/or dead cells.

Susceptibility of various cancer cell types to Johrei treatments

It is generally considered that the number of viable cancer cells depends on a balance between the Johrei energy to induce the viability loss of cancer cells and their proliferation activity. Therefore, if the Johrei energy exceeds the proliferation potency of cancer cells, the number of viable cells could be decreased. If the latter is superior to the former, on the contrary, the viable cell number could be increased. It was thus determined whether the viability loss of AGS cells induced by repeated Johrei treatments was common to other cancer cell types and whether and to what extent various cancer cell types were susceptible to Johrei treatments. Each cancer cell type was seeded in 96-well plates at an optimal density in a volume of 100 μL and was exposed to twice-daily 15-minute Johrei treatments by each of the practitioners, who were switched every 24 hours with the other team members. An equal number of untreated cultures were chosen as controls. Of human cancer cell types, AGS and HeLa cells showed the highest susceptibility to Johrei treatments (Table 1). Among three human prostate carcinoma cell lines, ALVA-41, which was androgen dependent, and well-differentiated carcinoma cells originated from bone metastasis had relatively high responsiveness to Johrei treatments. By contrast, PC-3 and PPC-1, which were androgen independent and poorly differentiated carcinoma cells originated from bone metastasis and primary prostate carcinoma, respectively, showed the most resistance to Johrei treatments, thus resulting in only a little viability loss. Mouse B16 melanoma cells showing resistance to cathepsin E–mediated TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-dependent apoptosis¹⁹ had a relatively high responsiveness to Johrei treatments. The results thus suggest that Johrei treatments can induce the viability loss of all the cultured cancer cell types tested, although the responsiveness to Johrei treatments varies with cancer cell types.

Discussion

Given the difficulty in replications and verifications of in vivo studies, especially with human subjects, various approaches based on generally accepted scientific methods are necessary to verify the suggestive value of Johrei practice. In this study, the culture system of various cancer cell lines was used and the first evidence of the positive effect of Johrei treatments on the viability loss of various cancer cells in vitro were provided. In pilot experiments, it was noticed that the effect of Johrei treatment on the viability of various cancer cell types is variable, depending on experimental conditions employed (e.g., the cell density seeded in wells of plates or μ-dishes, and the frequency and duration of Johrei treatment). Therefore, the optimal conditions of Johrei experiments for each type of cancer cells had to be determined. The present results clearly demonstrated that the viability of all the cancer cell types tested was significantly decreased by Johrei treatments performed under such optimal conditions, although their responsiveness to Johrei practice appeared to vary with different cancer cell types. AGS and HeLa cells showed the highest susceptibility to Johrei treatments, and ALVA-41 had a relatively high responsiveness to Johrei treatments. By contrast, PC-3 and PPC-1 showed the most resistance to Johrei treatments. A previous scientific study with computerized time-lapse microscopy revealed that the cell death and proliferation rates of cultured human brain cancer cells (SF188GBM) were not significantly changed by Johrei treatment and suggested that the failure to observe evidence of a reproducible cellular response to Johrei treatment was due to a significant difference in cell division in the baseline period between control and Johrei treated samples.¹⁰ The difference in the efficacy of Johrei practices on cancer cells between the previous and present studies is most likely to arise from the differences in experimental conditions.

Meanwhile, it was revealed that the viability loss of AGS cells induced by the alternate intention of the practitioners was also induced by each of the practitioners without switching with the team members. Interestingly, the extent of the viability loss of AGS cells appeared to vary with each of the Johrei practitioners. The viability loss of AGS cells by Johrei treatments was further substantiated by morphological analysis with the use of differential interference contrast microscopy and time-lapse microscopy. Strikingly, the results with time-lapse microscopy clearly demonstrated that the viability loss of the cells by Johrei treatments was apparent after 24 hours of incubation.

Next, the possible mechanism for the viability loss of AGS cells by repeated Johrei treatments was investigated by both cell proliferation and death assays with the use of a BrdU assay kit and an Annexin V-FLUOS Staining kit, respectively. The extent of proliferation was apparently decreased at 72 hours after Johrei treatments compared with untreated cells. By contrast, the number of dying and/or dead cells was markedly increased in Johrei-treated cells compared with untreated cells. The results strongly suggest that the decrease in viable AGS cells in the Johrei group is mainly due to the reduced proliferation and the increased cell death. Considering that the practitioners applied Johrei to cancer cells only with the intention of doing transmission of its healing energy and that they were not informed about the type (either normal or abnormal cells) and nature (malignancy) of cells used until the end of the said experiment, they are unlikely intending either “healing” or “killing” to have occurred. It is also assumed that the pleiotropic, beneficial influences of Johrei healing energy might be complex because it dynamically interacts with various cellular molecules through regulatory, buffering, and feedback mechanisms. At present, it is not clear how Johrei treatment induces the viability loss of cancer cells, but the authors believe that the underlying mechanism will be addressed in a subsequent work.

Conclusions

In vitro studies using various human cancer cell lines indicate that Johrei treatment induces the viability loss of these cells, which is associated with increased cell death and decreased proliferation. The results may support the suggestive value of Johrei practice for maintaining good health. To better understand the beneficial efficacy of Johrei in human health, further studies are needed to elucidate whether and how Johrei treatment affects the viability of normal human cells in culture.

Footnotes

Disclosure Statement

No competing financial interests exist.

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