Original researchOpen Access

AbstractTop

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for 84% of all lung cancer diagnoses. In advanced NSCLC, including adenocarcinoma and squamous cell carcinoma, median survival time (MST) rarely exceeds 10-12 months. Reduced deuterium (D) concentration in water of tissue culture media and in drinking water for humans has shown a strong anticancer effect in previous investigations. In the present study, 1 parts per million (ppm) decrease of D-concentration every 8 hours resulted in reduced growth rate of the A459 lung cancer cell line in vitro, and the cell migration was also dose-dependently reduced. Retrospective study of 183 NSCLC patients consuming commercially available deuterium-depleted water (DDW) revealed a severalfold increase of MST, which was 149 months for 19 patients and 40 months for 110 patients, who started DDW-consumption at early or advanced stage, respectively. Interestingly, MST showed a significant difference by gender (107 months in females and 41.2 months in males). Application of DDW in combination with surgery plus other conventional therapies (68 patients) gave 149 months MST, while for DDW combined with chemotherapy only (48 patients) MST was 43.7 months. The present results support earlier data that integration of D-depletion to conventional therapies increases the efficacy of therapy, reduces relapse rate and increases MST.

Keywords: deuterium depletion; deuterium-depleted water; lung cancer; non-small cell lung cancer; NSCLC; A459 cell line

IntroductionTop

In the last century, carcinoma of the lung has progressed from an uncommon and obscure disease to the most common malignancy in the world and the most common cause of death from cancer. In 2018, there were 121,680 new cases of lung cancer among men and 112,350 among women in the US, equivalent of 641 lung cancers diagnosed per day [1]. Despite all efforts at management, and recently registered more efficient new drugs [2], the prognosis of advanced lung cancer in stage IV, including both small-cell lung cancer (SCLC) and non-small-cell lung cancer patients (NSCLC) is extremely poor, with a median survival time (MST) of 8-12 months [3-5]. NSCLC is the most common type of lung cancer, accounting for 84% of all lung cancer diagnoses (Cancer. Net). In advanced NSCLC, such as adenocarcinoma and squamous cell carcinoma, the MST rarely exceeds 10-12 months; and adjuvant therapies can extend the survival of these patients by a few months only [6]. In special cases however, a significant increase (15.4 months) in survival could be achieved in NSCLC patients who were diagnosed with epidermal growth factor receptor (EGFR) gene mutation, thanks to targeted drug development [7, 8].

A novel, submolecular approach to tumor cell physiology can, however, provide a better alternative form of cancer treatment. Studies investigating the possible role of naturally occurring deuterium (16.8 mmol/L in natural waters) revealed that deuterium-depleted water (DDW) inhibited tumor cell growth in vitro and caused tumor regression in vivo [9]. The data suggest that the significantly different chemical and physical properties of hydrogen (H) and deuterium (D) [10-12] and the changing D/H ratio have a high impact on cell physiology [13, 14]. In order to investigate the possible role of D in cell cycle regulation, numerous studies have been conducted on different cell lines in culture media containing DDW, which verified the determinative role of D in cell signaling and tumor growth [9, 15-18]. The apoptosis-triggering effect of DDW has been demonstrated in vitro [15] and in vivo [19]. D-depletion also exerts influences on protooncogenes and tumor suppressor genes. Induction of the expression of c-Myc, Ha-ras, and p53 genes by carcinogen exposure was significantly inhibited when experimental animals were given DDW to drink [20]. Cancer cells are extremely sensitive to D-depletion, which can cause tumor necrosis induced by ROS (reactive oxygen species) [21]. Recent studies confirmed that D is a natural cell growth regulator that controls mitochondrial oxidation-reduction balance [21-23]. The clinical outcome of D-depletion was investigated in prospective [24] and retrospective studies [24-26]. In four lung cancer cases with brain metastases, a remarkably long survival time was observed, which was an exceptional response of brain metastases from lung cancer [27].

Here we confirm previously published in vitro data on cell growth inhibition by deuterium depletion, applying the most advanced technology (xCELLigence RTCA system [Roche Applied Sciences]) and present a retrospective, follow-up study to show the impact of DDW-consumption on the outcome of NSCLC patients. The daily amount of fluid intake of the 183 lung cancer patients was replaced with DDW for at least 91 days, without altering the conventional treatment regimens. The primary end point of the human study was the median survival time (MST). Different subgroups were created to compare the data to historical controls.

The data indicate that the combination of D-depletion with existing conventional therapies results in a severalfold increases of MST.

Materials and methodsTop

Production of deuterium-depleted drinking water
DDW was produced from ordinary drinking water of natural D-content (150 ppm equivalent to 16.8 mmol/L) by fractional distillation [25, 28] using a GMP-conform technology. Final D-level was in the range of 38 to 25 ppm, verified by a Liquid-Water Isotope Analyser-24d (manufactured by Los Gatos Research Inc., USA) with ±1 ppm precision. For the marketed product intended for human consumption (Preventa®), DDW was mixed with natural spring water in order to adjust the exact D concentration, and to replace minerals. In the present study, drinking water with 105, 85, 65, 45, and 25 ppm D concentration were used.

Culture media and cell lines
Two stock solutions were made up, each containing 6.68 g DMEM, 0.11 g NaHCO₃, 0.01 g Streptomycin and 500 µl Penicillin, dissolved in either distilled water or DDW that contained 24 ppm D.

These solutions were sterile filtered (Acrodisc Premium 25 mm Syringe Filter with 0.2 µm GHP Membrane, Pall Life Sciences, cat. P NAP-4564T) and mixed to obtain culture media with the necessary D concentrations in each experiment. A549 lung cancer cells were cultivated at 37°C, 5% CO₂.

Measurement of the inhibitory effect of gradual D-depletion on the growth of A549 cells
The inhibitory effect of DDW on cell growth was studied using the label-free, real-time detection technology, xCELLigence RTCA system. The system is a microelectronic cell sensor method based on microelectrodes integrated in the bottom of the wells of a microtiter plate (96-well E-plate). Presence of the cells on top of the electrodes affects the local ionic environment, leading to an increase in the electrode impedance, which enables continuous monitoring of adhesion, proliferation, and viability of the observed cells (the more cells are attached to the electrodes, the larger is the increase in electrode impedance). The system was operated according to the instructions in the Users’ Manual. The cells were seeded into wells of the 96-well E-plate at 4,000 cells per well, cultured at 37°C in 5% CO₂ atmosphere, and continuously monitored. A dimensionless parameter termed Cell Index (CI) can be derived from the relative change in measured electrical impedance [29]. Therefore, CI is suitable for describing cell count and viability. Decrease in CI after treating the cells with an agent in the medium indicates inhibitory effect.

Before being seeded into E-plates, the cells were starved overnight without fetal calf serum (FCS). At 24 hours after seeding, the medium was changed to test media (D at different concentrations; 150 ppm, 135 ppm, 125ppm, 115 ppm, 105 ppm, 85 ppm, 65 ppm and 40 ppm) and the cells were observed between 23 and 70 hours. The experiment was carried out in four replicates.

Measurement of the effect of gradual D-depletion in the media of A549 lung cancer cells
In this test, the A549 lung cancer cells were treated with media containing D at gradually decreasing concentrations, starting at 150 ppm. The medium was replaced every 8 hours with a new one of D concentration reduced in the first series by 0 ppm (control) or by 5, 7.5, or 10 ppm, in altogether 8 consecutive steps. In the second series, D-level reductions were 0 ppm (control), 1 ppm, 2.5 ppm or 5 ppm. Cells were monitored real time from 30 hours to 100 hours, 24 hours after seeding them in the E-plates, CI was measured and plotted for each cell line and for each D concentration in cell media.

Measurement of the effect of D-depletion on the migration of A549 lung cancer cells
Migration of the cancer cells was measured using the cellular invasion/migration-plate (CIM Plate) in the Real-Time Cell Analyzer (RTCA) system. The CIM plate is composed of an upper and a lower chamber. The upper chamber has 16 wells that are sealed at the bottom with a microporous polycarbonate membrane containing microelectrode sensor arrays integrated on its bottom surface. Cells during migration through the electrode sensor arrays affect the impedance of the electrodes that leads to a CI increase. The driving force of the migration is the chemo attractant imbalance between the two chambers, namely that the medium in the upper chambers does not contain FCS while that in the lower chamber is supplemented with FCS.

All assays were conducted on freshly seeded cells. Cells were suspended in serum-free medium containing D at a concentration of 150 ppm, 135 ppm, 120 ppm or 105 ppm and then 2×104 cells in 150 µL of the medium were seeded into each upper well of the CIM plate. CI was determined in real time through 80 hours.

Administration of DDW
The aim of the treatment was to reduce D concentration in the patients’ body by replacing the normal fluid intake with DDW, consuming 1.5-2 litres per day. The patients used it as a supplement (and not replacement) of the conventional therapies. DDW-consumption typically started with 105 ppm D and was changed to 85 ppm, 65 ppm, 45 ppm and 25 ppm every 1 to 3 months. Depending on stage of disease and response to deuterium depletion, DDW-consumption was suspended after long progression-free interval or complete remission for 3 to 6 months, and started again for repeated 4 to 6-months’ durations. Over the time, the treatment periods were shortened, although each had to be at least 3-4 months long and later the 3-6 months long breaks were prolonged (Figure 1). The duration of DDW-consumption in the follow-up population was between 91 and 3,793 days.

Figure 1 Consort flow diagram of the proposed application of D-depletion according to staging at diagnosis.

The patients were continuously updated with, aware of, consented and provided access to all available information and publications regarding deuterium depletion.

Data collection
Our study had access to the data of 377 lung cancer patients consuming DDW between April 1993 and October 2019. The criteria of eligibility were histologically confirmed NSCLC tumor and duration of DDW-consumption longer than 90 days. Based on these two criteria, 183 patients (85 males and 98 females) were enrolled in the retrospective study.

The aim of the study was to investigate the effect of DDW-consumption on the outcome of NSCLC in combination with conventional therapies. The patients received the conventional forms of therapy, and underwent follow-up examinations, in different hospitals in Hungary. Data collection was closed in March 2020. Patient documentation was evaluated retrospectively, with survival as the only endpoint of the study.

Statistical evaluation
The primary endpoint of the study was survival, and the Kaplan-Meier method was applied to calculate survival curves. Statistical analysis was performed using MedCalc Statistical Software, Version 12.3.0. Because the study was performed retrospectively and patients started DDW-consumption at different time points after diagnosis, MST was computed both from the time of diagnosis of lung cancer and from the start of DDW-consumption. The mean difference is significant at 0.05 level.

For correlation analysis, the Pearson method was used. Variables were duration of DDW-consumption in days and survival time in days. The calculations were performed by Adware Research Ltd (Balatonfüred, Hungary).

ResultsTop

Retrospective study with 183 NSCLC lung cancer patients receiving conventional therapies and consuming DDW

Disease characteristics of 183 DDW-consuming lung cancer patients
The 183 evaluated patients were diagnosed with NSCLC before they started to consume DDW. Each patient had tumor at the start of DDW-consumption, except for 10 patients, who were operated earlier, and the tumor had been removed. Patients were in different stages at the start of DDW-consumption, and were divided to three groups (Table 1) based on TNM classification: group A, (stage IA, IB and stage IIA) group B (stage IIB, stage IIIA) and group C (stage IIIB and stage IV) [30]. Of these 183 patients, 113 (61.7%) had adenocarcinoma, 47 (25.7%) had squamous cell carcinoma, and 23 (12.6%) had mixed or uncharacterized NSCLC (Table 1). All patients underwent conventional treatments with chemotherapeutic agents, radiotherapy, surgery, targeted therapy, or their combinations.

The median age in the study population was 60 years (mean was 59.3±9.55 years). The patients started DDW-consumption at different times after diagnosis. The median length of time between diagnosis and start of DDW-consumption was 2.9 months, the mean was 9.9±18.4 months (mean±SD). The broad standard deviation reflected that in some cases a couple of years elapsed until inclusion in the study, whereas other patients were enrolled after only a few months or close to diagnosis. The median duration of DDW-consumption was 20.4 months, and its cumulative duration, 2,480.5 months (206.7 years). During the 5,740.8 months (478.4 years) cumulative length of total follow-up (from diagnosis to the end of follow-up) 59 patients died (32.2%). The median follow-up period from the diagnosis was 21.1 months (1.75 years), although the SD (33.8 months) of the 32.7 months average follow-up period indicated a wide range of values. Seven patients (3.8%) were followed for a period longer than 10 years, and 77% of the patients were alive over a year after the diagnosis.

Calculations of MSTs of the cancer patients
The 183 patients consuming DDW in addition to conventional treatments showed increased MST compared to historical control (8-12 months). Two different MSTs were calculated, one from the date of diagnosis and one from the start of DDW-consumption. MST from the diagnosis was 61.9 months (95% CI: 40.3 – 83.5), and from the start of DDW-consumption it was 48.4 months (95% CI: 20.9 – 75.7).

Differences of MSTs in the whole cohort by gender
There were striking differences between the two genders in the calculated MST values. Calculating from the date of diagnosis, MST was 41.2 months (95% CI: 29.4 – 53.0) in the male and 107.0 months (95% CI: 61.3 – 152.8) in the female subgroup (significant difference, p=0.030). Calculating MSTs from the date of DDW-consumption, the males’ MST was 33.2 months (95% CI: 16.0 – 50.4), and the females’ MST was 82.0 months (95% CI: 35.1 – 128.9); the difference was significant (p=0.009) (Figure 2).

Figure 2 Kaplan-Meier graphs of survival in male and female subgroups, (a) shows survival from the diagnosis, (b) shows survival from the start of DDW-consumption.

Differences of MST in the whole cohort by histology
The data indicated that survival was strongly influenced by histology of NSCLC. Table 2 shows the MST of patients by different histology and gender.

It is important that 38 (34%) male and 75 (66%) female patients had adenocarcinoma; whereas 36 (77%) male and 11 (23%) female patients had squamous cell cancer at the start of DDW-consumption, which may explain the striking differences of MST in the cohort by gender (Table 2).

MST in the whole cohort and in the subgroups created by stages of cancer at the start of DDW-consumption
Difference between subgroups was statistically significant when calculating the MST from the diagnosis (p=0.037) or from the start of DDW-consumption (p=0.015) which clearly confirmed the importance of tumor diagnosis at as early stage as possible. The data also show (Table 3) that even in the worst-case scenario, in group C, the MST was severalfold longer compared to historical control [2-6].