|Year : 2019 | Volume
| Issue : 5 | Page : 1098-1104
Antiproliferative and apoptotic effects of the natural alkaline water (Zamzam) in breast cancer cell line MCF-7
Shiekhah M Al Zahrani1, Ulfat M Omar1, Sawsan A Rahimulddin1, Ayat B Al-Ghafari1, Alia M Aldahlawi2, Huda A Al Doghaither1
1 Department of Biochemistry, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia
2 Department of Biological Sciences, Faculty of Sciences, King Abdulaziz University; KFMRC, Immunology Unit, King Abdulaziz University, Jeddah, Saudi Arabia
|Date of Web Publication||4-Oct-2019|
Huda A Al Doghaither
Department of Biochemistry, King Abdulaziz University, P. O. Box 7775, Jeddah 21472
Source of Support: None, Conflict of Interest: None
Background: Zamzam water (ZW) is a natural alkaline water that contains several minerals that may represent a powerful tool for cancer therapy.
Objectives: In this research, in vitro antiproliferative and apoptotic effects of ZW were investigated in the human breast cancer cell line MCF-7.
Materials and Methods: This study was conducted between January 2015 and February 2016. The effects of ZW on the morphology and the cell viability of human breast cancer cell line MCF-7 were determined. The cell death type and cell cycle changes were investigated using flow cytometry. Finally, reactive oxygen species (ROS) were also measured by fluorometric technique.
Results: MCF-7 cells treated with either ZW with adjusted pH at 7.2 or unadjusted pH at 8 showed reduced cell viability of cancerous cells. The cell death occurred through the apoptosis pathway under both treatment conditions. The treated MCF-7 cells were arrested in the G2/M phase and decreased in the G1 phase. Only the unadjusted pH ZW sample demonstrated an increase in the production of both cytoplasmic and mitochondrial ROS in MCF-7 cells.
Conclusion: All the results in the present study indicated, for the first time, that ZW might have anticancer and apoptotic effects on breast cancer cell line.
Keywords: Antiproliferation, apoptosis, cell cycle, MCF-7, reactive oxygen species, Zamzam water
|How to cite this article:|
Al Zahrani SM, Omar UM, Rahimulddin SA, Al-Ghafari AB, Aldahlawi AM, Al Doghaither HA. Antiproliferative and apoptotic effects of the natural alkaline water (Zamzam) in breast cancer cell line MCF-7. J Can Res Ther 2019;15:1098-104
|How to cite this URL:|
Al Zahrani SM, Omar UM, Rahimulddin SA, Al-Ghafari AB, Aldahlawi AM, Al Doghaither HA. Antiproliferative and apoptotic effects of the natural alkaline water (Zamzam) in breast cancer cell line MCF-7. J Can Res Ther [serial online] 2019 [cited 2020 Feb 28];15:1098-104. Available from: http://www.cancerjournal.net/text.asp?2019/15/5/1098/244212
| > Introduction|| |
Cancer is considered to be one of the major causes of death worldwide, and it was responsible for approximately 13% of deaths in 2008. New cancer cases are expected to increase by about 40% by 2030. This disease is characterized by an uncontrolled increase in cell proliferation, which leads to tumor formation. Multiple signaling pathways such as irregularities in deoxyribonucleic acid (DNA) repair, cell cycle, apoptosis, and redox balance are affected simultaneously by tumor progression. Targeting these pathways for novel therapeutic strategies is being explored. Reactive oxygen species (ROS) are second messengers that participate in various physiological processes such as proliferation and apoptosis.,, Therefore, it was suggested that antioxidants might have an inhibitory effect on the promotion of tumors. Resistance to treatment with broad range of anticancer drugs and toxic side effects have resulted in a need for new anticancer therapies with diverse modes of action and little to no side effects. Cancer treatment strategies utilizing either chemotherapy or radiotherapy have undesirable and toxic side effects. Consequently, finding novel and effective therapies against cancer is highly necessary. Attention to the use natural compounds as new candidates that can replace the chemotherapy or radiotherapy has increased.,
Recently, scientists have aimed to develop oxygen-reduced water with high mineral combination and high power of hydrogen (pH), which is widely used as a fashionable type of drinking water. A novel example is electrolyzed-reduced water (ERW) which is characterized by a high pH, lower oxidation potential, and lower dissolved oxygen than any other forms of tap water or distilled water. This water has been shown to possess anticancer properties including inhibition of cell proliferation in lung A549 cancer cells, inhibition of tumor growth of B16 melanoma in tumor-injected mice, and enhancing the mitochondrial apoptosis pathway in human promyelocytic leukemia cells (HL-60) alongside glutathione (GSH).
Zamzam water (ZW) is a natural alkaline mineral water with unique natural characteristics that are different from any other water. It is named after the well that provides water to billions of people for thousands of years and has never dried up. This water has never been chemically treated or chlorinated. Chemical analysis of ZW shows that it contains many inorganic elements and ions such as sodium, calcium, magnesium, potassium, chromium, cobalt, copper, zinc, arsenic, lithium, selenium, strontium, cadmium, lead, chloride, fluoride, nitrate, sulfate, and bicarbonate. In addition, it has a higher pH value (7.9–8) compared to the pH of ordinary water (pH 6.5–7) as well as antimicrobial growth properties., Previous studies reported that ZW had antioxidant activity and improved the oxidative stress in type 2 diabetic patients  and in rats stressed with gentamicin  and carbon tetrachloride. Some studies suggested that the arsenic, selenium, and lithium in ZW might represent a powerful tool for therapy of several diseases, especially cancer.,,, Others found that several minerals such as arsenic trioxide, lithium chloride, and selenium could inhibit the proliferation of normal and cancerous cells.,, Moreover, it has been reported that cancer cells have died through the apoptosis pathway when they were exposed to selenium, arsenic trioxide, and cadmium chloride (CdCl2),, with a distinctive effect of arsenic trioxide on cell cycle. In addition, studies on breast cancer have shown that oral administration of sodium bicarbonate increases extracellular pH of tumors and reduces the formation of spontaneous metastases. It is suggested that due to the presence of certain elements and its high pH, ZW might have a powerful effect on the proliferation of cancer cells with potential antioxidant properties.
As far as we are aware, no research has been done to study the effects of ZW on breast cancer cells. Hence, the objectives of this research were to investigate the effects of ZW on morphological changes, cell viability, cell death mechanism, and cell cycle arrest as well as to estimate the level of cytoplasmic and mitochondrial ROS in MCF-7 cells.
| > Materials and Methods|| |
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), and L-glutamine were purchased from Sigma-Aldrich Company, Poole, UK. High-glucose Dulbecco's Modified Eagle's Medium (DMEM) powder, fetal bovine serum (FBS), and trypan blue 0.4% (w/v in phosphate-buffered saline [PBS]) were purchased from HyClone, Logan, UT, USA. Trypsin/ethylenediaminetetraacetic acid (EDTA) 0.25% (EDTA) (1x), antibiotics (penicillin/streptomycin), and 100x non-essential amino acids (NEAAs) were purchased from Gibco, Canada. PBS tablets were purchased from Oxoid, Hampshire, UK. Isopropyl alcohol and ethanol were purchased from BDH, Poole, England. Di-hydrodichlorofluorescein diacetate (CM-H2 DCFDA) and MitoSOX red were obtained from Invitrogen, Paisley, UK. The annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit and ribonuclease (RNase) were purchased from Abcam, UK.
Human breast cancer cell line MCF-7 was obtained from the Tissue Culture Bank at King Fahad Medical Research Center, King Abdulaziz University (KAU), Jeddah, Kingdom of Saudi Arabia (KSA).
Zamzam water source
ZW was obtained directly from the Zamzam well with the permission of the General Presidency for the Affairs of the Two Holy Mosques. The well is located in the Holy Mosque, Makkah, KSA, and is about 30 m (98 ft.) deep and 1.08–2.66 m (3 ft. 7 in to 8 ft. 9 in) in diameter. The Global Positioning System (GPS) coordinates are 21°25'19.2”N and 39°49'33.6”E. One large patch of ZW was stored in sterilized glass bottles at room temperature until it was used. Chemical analysis of ZW samples was carried out at the Zamzam Studies and Research Centre (ZSRC), The Saudi Geological Survey (SGS).
Preparation of culture media
The growth medium DMEM was supplemented with 10% FBS, 1% L-glutamine, 1% penicillin/streptomycin, and 1% NEAAs. The pH of the medium was adjusted to 7.2. The medium was sterilized by 0.22 μm filter and used to culture MCF-7 cells. After reaching the desired confluence at 70%, the growth medium was removed and ZW treatments were tested for 24 h.
The study was conducted at KAU, Jeddah, KSA, between January 2015 and February 2016. ZW dissolved in PBS was used as the treatment in this research. Two samples of ZW were prepared in PBS and designated as Z1 and Z2. In a sterilized glass bottle, a PBS tablet was dissolved in 800 ml of ZW and the pH was either adjusted to 7.2 (Z1) or left with its natural alkalinity, pH = 8 (Z2). MCF-7 cells cultured in PBS only were used as a negative control and designated as C, as previously described. The cytotoxic and apoptotic effects of the two treatments, Z1 and Z2, were studied in MCF-7.
MCF-7 cells were seeded in 25 cm2 flasks at the concentration of 8 × 105 cells/ml. After 24 h, the changes of cell morphology were photographed using a Nikon Eclipse microscope at 10x magnification, and Z1 and Z2 treatments were then compared to the control.
Cell viability assays
The cytotoxic effects of ZW treatment on MCF-7 were studied using MTT assay and trypan blue exclusion. MCF-7 cells were seeded in a 96-well tissue culture plate at a concentration of 1 × 104 cells/well. After 24 h of incubation at 37°C and 5% CO2, the culture medium was replaced with 200 μl Z1 or Z2 treatments or 200 μl PBS as a negative control. MTT assay was performed by adding 20 μl of MTT dye to the samples and then they were incubated for 2 h at 37°C. Treatments were discarded and the precipitates were dissolved with 200 μl of isopropyl alcohol. The developed color intensity was measured at 490 nm using a BioTek microplate reader. Three independent experiments were performed to ensure reproducibility. For trypan blue exclusion, MCF-7 cells were treated as in the morphology section with 60%-70% confluent reached after 24 h. Trypan blue dye (0.2%) was added to MCF-7 cells and counted using a hemocytometer.
Determination of cell death type by annexin-V/propidium iodide assay
To detect early and late apoptosis in the MCF-7 cell line, cells were seeded in 25 cm2 flasks at a concentration of 8 × 105 cells/ml. When cells were 70% confluent, they were incubated with Z1, Z2, and the control medium. After 24 h, the assay was carried out according to the manufacture's instructions using Beckman Coulter flow cytometry. Minimum 10,000 events were acquired in the gated regions. For cells labeled with annexin V-FITC, the emission was 520 nm, while for cells labeled with PI, an emission of 620 nm was used.
Cell cycle analysis
MCF-7 cells were seeded in 25 cm2 flasks at a concentration of 8 × 105 cells/ml. When cells were 60%–70% confluent, they were treated with Z1 and Z2 for 24 h followed by trypsinization. Pellets were washed twice with PBS and then cells were fixed with 1 ml of ice-cold fixing buffer (70% ethanol in PBS) and incubated at 4°C overnight. After 24 h, the fixation buffer was discarded and cells were re-suspended in 500 μl PBS followed by 5 μl of 5 mM RNase. Cells were incubated for 30 min at 37°C, dyed with 5 μl PI (1 mg/ml), and analyzed by BD FACSCalibur flow cytometry. At least 10,000 events were acquired in the list mode using an emission wavelength of 620 nm.
Measurement of cellular reactive oxygen species (ROS)
ROS level in MCF-7 was measured using the H2-DCFDA dye. MCF-7 cells were seeded in 25 cm2 flasks at a density of 8 × 105 cells/ml and treated with Z1 and Z2 culture medium for 24 h. Cells were incubated in the dark with 5 μl of DCFDA for 30 min at 37°C and 5% CO2. Cells were kept on ice and dark condition to reduce susceptibility to the photo-oxidation that could occur. The dichlorofluorescein (DCF) fluorescence was read at an excitation wavelength of 485 nm and at an emission wavelength of 528 nm using a BioTek fluorescence microplate reader.
Measurement of mitochondrial reactive oxygen species (mtROS)
Superoxide levels in the MCF-7 cell line incubated with Z1 and Z2 were measured by a BioTek fluorescence microplate reader using MitoSOX dye according to the manufacture's instructions. The red fluorescence was read at an excitation wavelength of 530 nm and at emission wavelength of 590 nm.
The data were analyzed using GraphPad Prism (version 6.0, GraphPad Software Inc., San Diego, CA). All experiments were performed at least three times to insure reproducibility. The significant differences were evaluated using P values by one-way analysis of variance followed by Bonferroni's test. The cutoff level for significance was <0.05.
| > Results|| |
Chemical analysis of ZW samples was carried out to identify and quantify the chemical composition of ZW samples. The concentrations of all elements were within the permissible limits of the WHO. The chemical analysis of the ZW showed that the pH of the water was 8, Total Dissolved Salts (TDS) was 413 mg/L, and total hardness was 161.41. The major components of ZW were Ca, Cl, HCO3, K, Mg, Na, NO3, SiO2, and SO4. Few traces of B, Ba, Br, F, Fe, I, Li, NH4, Ni, and Sr were also detected. The concentrations of Ag, Be, Bi, Cd, Cr, Cs, Cu, Hg, Mn, NO2, Pb, PO4, Sb, Sn, Ta, Tl, and Zn were <0.001 ppm. The concentrations of toxic elements including Al, As, Rb, and Se were <0.01 ppm.
The antiproliferative effects of the two treatments of Zamzam (Z1 and Z2) on MCF-7 are shown in [Figure 1]. The changes in the cell membrane of MCF-7 were more obvious in the high alkaline medium (Z2) than in the adjusted pH treatment (Z1).
|Figure 1: Effect of Zamzam water on MCF-7 cells compared to the control untreated cells. MCF-7 cells incubated for 24 h with Z1 and Z2 treatments compared to untreated control. Images were captured using light microscopy at 10x magnification|
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Determination of cell viability
The effects of ZW on cell viability were assessed by both MTT assay and cell count. The cancer cell viability was significantly decreased to 75.94% and 65.3% after treatment with Z1 and Z2, respectively [Figure 2]a. The reduction in cell viability of MCF-7 after ZW treatment was also confirmed by trypan blue cell count. As shown in [Figure 2]b, the cell viability was significantly reduced after treatment with Z1 and Z2 (82.96% and 47.23%, respectively).
|Figure 2: The effects of Zamzam water on the viability of MCF-7 by MTT and trypan blue. (a) MTT results of MCF-7 cells treated with Z1 and Z2 treatments compared to untreated cells. (b) Trypan blue cell count of MCF-7 cells treated with Z1 and Z2 treatments compared to untreated control cells. The data represent the mean of three independent experiments (n = 3 ± SEM). Comparisons of means were made using a one-way ANOVA followed by Bonferroni's test (***P ≤ 0.001, ****P ≤ 0.0001)|
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Apoptosis in MCF-7 cells
Apoptosis was significantly induced in MCF-7 treated cells compared with the untreated control in both early and late stages of apoptosis. The percentage of early apoptosis in the control was increased from 8.17% to 23.85% (P < 0.05) when treated with Z1 and further increased to 28.62% with the more alkaline medium Z2 (P ≤ 0.01). Similarly, the same pattern was followed for the late apoptotic stage, with a significant increase from 2.41% for the control to 10.69% and 13% for Z1 (P < 0.05) and Z2 (P ≤ 0.01), respectively (P ≤ 0.001). Cell necrosis did not change with the treatment. The percentages of necrosis in MCF-7 cells were 0.28%, 0.3%, and 0.42% (P > 0.05) in the untreated control, Z1, and Z2, respectively [Figure 3]a and [Figure 3]b.
|Figure 3: Detection of cell death of MCF-7 cells after treatment with Z1 and Z2 for 24 h by flow cytometry. (a) MCF-7 treated with Z1 and Z2 for 24 h. Flow cytometry analysis of annexin V in MCF-7 cells treated with Z1 and Z2 for 24 h. C1–C4 quadrants indicate: C1 = Cells stained with PI; C2 = Cells conjugated with annexin V and stained with PI: C3 = Healthy cells and C4 = Cells conjugated with annexin V. (b) Percentages of MCF-7 cells in early apoptosis, late apoptotic/necrotic cell death, and necrotic cell death pathways after treatment with Z1 and Z2 for 24 h (*P < 0.05, **P ≤ 0.01)|
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The effect of Zamzam treatments on cell cycle
[Figure 4]a and [Figure 4]b showed MCF-7 cell cycle phases after treatment with Z1 and Z2. Arrest in the G2/M phase was observed by noticing a significant increase in the cell population with an associated reduction in the G1 population in MCF-7 cells treated with both Z1 and Z2 treatments. Consequently, the S phase was not affected by the treatments. The percentages of MCF-7 cells in the G1 phase were 53.52%, 38.07%, and 41.59% for the control, Z1, and Z2, respectively (P ≤ 0.001). The percentages of cells in the S phase were 23.59%, 21.93%, and 20.54% (P > 0.05), while in the G2 phase, the percentages were 15.42%, 29.37%, and 31.29% for the control, Z1, and Z2 (P ≤ 0.001), respectively.
|Figure 4: Cell cycle arrest in MCF-7 cells treated with Z1 and Z2 treatments for 24 h by flow cytometry. Represented values are the means of three independent experiments (n = 3 ± SEM). Comparisons of means were made using one-way ANOVA followed by Bonferroni's test (***P ≤ 0.001). (a) MCF-7 cells treated with Z1 and Z2 compared to the untreated control. (b) The effects of Z1 and Z2 treatments on MCF-7 cell cycle distribution after 24 h. G1 phase, S phase, and G2 phase. In each phase, the percentages of cells were measured by flow cytometry|
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Cellular and mitochondrial reactive oxygen species measurement
The intensity of cytoplasmic ROS production was observed only with Z2 treatment. Cellular ROS production was not affected by Z1 treatment compared to the control [Figure 5]a. The intensity of ROS formation of Z2 was 79.5 RFU, while the ROS production of Z1 was 60 RFU, compared to control 58.5 RFU. The mitochondrial ROS (mtROS) production (superoxide anion) was significantly observed with only Z2 compared to the control. Moreover, mtROS production was not affected by Z1 treatment compared to the control. The fluorescent intensity of mtROS formation of Z2 was 16881.9 RFU, while the mtROS production of Z1 was 12430.7 RFU, compared to 13810.9 RFU [Figure 5]b.
|Figure 5: Measurement of cellular and mitochondrial reactive oxygen species in MCF-7 cells treated with Z1 and Z2 for 24 h. Comparisons of means were made using one-way analysis of variance followed by Bonferroni's test (*P < 0.05). (a) MCF-7 cells treated with Z1 and Z2 treatments for 24 h. Cells were loaded with CM-H2DCFDA, and the dichlorofluorescein fluorescence was measured using the BioTek fluorescence microplate reader to monitor reactive oxygen species formation. (b) MCF-7 cells treated with Z1 and Z2 treatments for 24 h|
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| > Discussion|| |
Zamzam is a natural alkaline water consumed by millions of people worldwide because of their religious belief in its healing power for several diseases. In this study, the antiproliferative and apoptotic effects of ZW on the MCF-7 cancer cell line were investigated for the first time.
The results of the current study showed that the incubation of MCF-7 cells with ZW inhibited growth and proliferation of cancer cells and changed the morphology of cells. In fact, there are two main characteristics of ZW that differs from any other water. The first is the presence of various concentrations of a number of inorganic minerals. Several studies have indicated that minerals such as arsenic trioxide, lithium chloride, selenium, cadmium–zinc combinations, and lead  can inhibit the proliferation of cancerous cells. The second characteristic of ZW is the high pH (7.9–8) compared to ordinary water, a property that has been demonstrated to reduce cancer cells proliferation in numerous studies. This was consistent with the current finding of Z2 treatment compared to the ZW with pH adjusted to 7.2 (Z1).
It has been reported in previous research that MCF-7 cells died through the apoptosis pathway when they were exposed to arsenic trioxide by reducing the Bcl-2/Bax ratio  or to CdCl2 by ROS production and oxidative damage. In addition, it was reported that selenium induced apoptosis in colorectal cancer cells. In another study, the treatment of ERW with GSH induced the mitochondrial apoptosis pathway in HL-60 cells. These findings are in agreement with our results in which apoptosis and not necrosis are induced. Moreover, the generation of ROS seems to be the mechanism that together with apoptosis resulted in reduced cancer cell viability. This result was observed only in Z2 treatment, which emphasizes the importance high pH level of ZW.
ROS level in the MCF-7 cell line was investigated to assess whether or not ROS played a significant role in the induction of apoptosis after exposure to Zamzam treatments. Our data demonstrated that both cytoplasmic and mitochondrial ROS formations were induced. This phenomenon was observed only with Z2 treatment. The unique combination of minerals in ZW might explain the increase of cytoplasmic and mitochondrial ROS. Arsenic and cadmium might be responsible for the induction of cytoplasmic ROS in MCF-7 cells. Arsenic trioxide can depolarize the mitochondrial membrane potential (MMP) and induce the opening of the mitochondrial transition pore. It also inhibits GSH peroxidase enzyme, increases cellular hydrogen peroxide content, and increases both ROS release from the mitochondria to the cytoplasm and intracellular ROS production., In addition, cadmium affects cells through very complex functions such as ROS production, disruption of mitochondrial function, and induction of apoptosis. Furthermore, the cytotoxic effects of copper chloride and CdCl2 on the hepatocyte occur as a result of mtROS formation due to their ability to decrease The mitochondrial membrane potential. Recently, the effects of ZW on colon cancer cell line HCT-116 and normal human skin fibroblast cell line HSF were investigated using the same treatments (Z1 and Z2) that were used in the current study. The study revealed that mitochondrial ROS production was induced only with Z2 treatment while Z1 treatment scavenged mtROS. These findings demonstrated that ZW might have different mechanisms in killing cancer cells that are dependent on the type of cancer cells.
Flow cytometry assays showed that cells were arrested in the G2/M phase and decreased in G1 phase. The percentages of MCF-7 cells in the G1 phase were 53.52%, 38.07%, and 41.59% for the control, Z1, and Z2, respectively. The percentages of cells in the G2/M phase were 15.42%, 29.37%, and 31.29% for the control, Z1, and Z2, respectively. It is well known that cyclin B is involved in the regulation of the G2/M phase and the M-phase transition. One study has reported that cells treated with arsenic trioxide were arrested at either the G1 or the G2/M phase. In addition, it has been found that the arresting of the G2/M phase in human tumor cell lines including MCF-7 by arsenic trioxide is due to an increase in the levels of cyclin B, which is a regulatory protein involved in mitosis.,, De Groot et al. observed a significant accumulation of renal principal cells in the G2 phase after lithium treatment compared to controls. Another study reported that inorganic selenium caused a slight arrest in the G2/M phase of the cell cycle in human T-cell acute lymphoblastic leukemia MT-4 cells.. In this study, it was assumed that the arresting effect of Z1 and Z2 on MCF-7 cells might be due to the increased cyclin B level through the effect of the alkaline pH and the fact that both lithium and selenium are found naturally in ZW. Furthermore, another study found that there is no significant effect on DNA fragmentation of normal peripheral blood mononuclear cells treated with ERW with GSH. However, it was reported in previous researches that alkaline-reduced water possesses potent antioxidant activity and an anticancer effect in normal mice. In addition, ERW protected DNA from oxidative damage. Al Doghaither et al. indicated that HCT-116 cells were arrested in the G2/M phase of the cell cycle and decreased in the G1 phase after 24 h of treatment with ZW.
| > Conclusion|| |
This study provides strong evidence that ZW has the ability to inhibit the growth and proliferation of breast cancer cells. This gives ZW a strong recommendation as a promising natural cancer treatment with no side effects. The unique combination of minerals and the alkalinity of ZW might be candidates for causing such effects. To confirm such findings, further researches are needed with different types of cancer cell lines in addition to in vivo studies on animals with induced tumors.
The authors are grateful to King Abdulaziz City for Science and Technology (KACST), Riyadh, Saudi Arabia, for their financial support (DS-36-166). The authors also would like to express their deepest gratitude to the General Presidency for the Affairs of the Two Holy Mosques for providing ZW samples. We are also grateful to the Zamzam Studies and Research Center, the Saudi Geological Survey, Jeddah, KSA, for the chemical analysis of the components of ZW samples.
Financial support and sponsorship
The research was funded from King Abdulaziz City for Science and Technology (KACST), Riyadh, Saudi Arabia (DS-36-166).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]