Abstract

We demonstrate for the first time that proprietary ultra-high diluted (D30) extracts of Viscum album L. sub-species mali, populus, salix and tilia can inhibit proliferation of adrenal cancer Y-1 cells. Adrenal carcinoma is a rare but severe form of cancer. Adrenal overactivity can have devastatingly negative health effects like worse outcome cancer progression, resistant hypertension, type 2 diabetes (T2DM), stress and more. The conventional treatment of both adrenal cancer and the overactive adrenal function is often risky (surgery) or associated with a high level of undesirable side effects. Thus, the potential ability of ultra-high diluted mistletoe extracts as safe, inexpensive agents to aid as standalone or adjuvant treatments to dampen pathological adrenal functions would be of considerable interest.

Keywords:Adrenocortical carcinoma; Adrenal cortex; Viscum album L; mistletoe; Y-1 cells; Ultra-high dilutions

Introduction

Beginning with the work of Rudolf Steiner in the 1920’s [1] pharmaceutically prepared mistletoe extracts enjoyed increasing worldwide, a prominent place among the plant-based adjuvant treatments for cancer. The specific anti-tumoral activity was shown to be enhanced by defined harvest times, understanding the importance of the host tree that carries the semi-parasitic mistletoe and finally by a manufacturing process involving centrifugation [2-4]. Clinical studies have shown the mistletoe therapies to be safe and effective in many aspects [5] and have shown the mistletoe therapy to have a positive effect on tumor regression [6, 7], increase in health related qualitive of life [8-10], diminishing fatigue [11], limited improvement in overall survival length [12], and minimizing side effects to aggressive conventional treatments [13]. In vitro studies found that mistletoe extracts (ME) caused apoptosis in a variety of cell lines, such as in Ewing Sarcoma cells [14], 562 leukemia cells [15], human A253 cancer cells [16], LNCaP prostate cells [17], endothelial cells [18] and more. Further studies have begun to elucidate the mechanism of action of the ME’s. These include, among others, effects on membrane receptors, enzymes, ion channels, transporter proteins and transcriptional targets [19]. It was also found that mistletoe lectins act as cytokine inducers [20] and have an effect on immune escape proteins PD-L1, PDL-2 and MCH-I in multiple cancer cell lines [21]. Recently the importance of high and ultra-high dilutions of ME’s produced in “homeopathic” step wise fashion have gained increasing attention. Vallance, Quaresma, Nonato and Batista [22-25] discussed the phenomena that high dilution can maintain biological activity. Specifically, high and ultra-high dilutions of ME’s have been shown in vitro to have cytotoxic effects [26], even to the CH200 dilution [27], inhibit the growth of osteosarcoma cells [28], inhibit proliferation of 562 leukemia cells [29], inhibit the proliferation of prostate cancer cells while leaving normal prostate cells unaffected [30], and inhibit murine melanoma cells [31,32].While considerable effort has gone into elucidating the effect of ME’s, both concentrated and in high dilutions, on other cell lines, no experiments have been previously done on Y-1 adrenal cancer cells. A search of PubMed [33] listed a little over 100 citations of studies that have been done using theY-1 adrenal cancer cell line but none with any homeopathically diluted (potentized) substances.

Materials and Methods

Mistletoe Extraction and Potentization

The mother tincture (Ø) of each mistletoe was obtained through a proprietary method of extraction. The sub species of mistletoe was harvested from each host tree at two different opposing seasons (e.g. summer and winter). After the separate extraction of each seasonal harvest, the summer harvested extraction was allowed to drip by gravity downwards into the centrifugally rotating extract from the winter season. The resultant final mother tincture was designated “Viscum host tree name”, ex. Viscum mali. This final mother tincture contained the extracted contents of 200mg dry mistletoe per ml. We used Viscum album L. of four sub-species: V. mali, populus, salix, tilia (Table 1). The first dilution, prepared in “homeopathic” style, was designated as D1. It was made by 1:10 proportion i.e. using 1 ml of the mother tincture and 9 ml sterile diH₂O, succussed for two minutes, followed by one minute of rest. The D2 dilution was made again by 1:10 proportion i.e dilution of 1 ml D1 with 9ml diH2O as before and this method was repeated to D30.

Cell Culture

The Y-1, a mouse adrenal cell line (ATCC, CCL-79), was described by the supplier as exhibiting epithelial morphology that was isolated from the adrenal gland of a male mouse. The mouse was tested and found negative for ectromelia virus (mousepox). Rainey et al. [34] have reviewed the Y-1 characteristics in depth and gave details supporting its origination from an adrenal carcinoma of the zona fasciculata of the adrenal cortex. The Y-1 cells were typically seeded at 20% confluency in 75cm2 flasks and grown to approximately 80% confluency at 37˚C, 5% CO2, which typically took four days. The medium used was ATCC formulated F-12K (ATCC, 30-2004) supplemented with 2.5% Fetal Bovine Serum (FBS; ATCC, 30-2020), 15% horse serum (Gibco, 26050070) and 1% penicillin streptomycin (ATCC, 30-2300). Once confluent, the medium was removed, and the cells rinsed with 3 mL 0.25% Trypsin, 0.53 mM EDTA (TE; ATCC, 30-2101) and the cell monolayer was then harvested by adding 3 mL TE to the flask and incubated at room temperature for 5 minutes, followed by 6 mL fresh medium. The suspension was transferred to a 15- ml centrifuge tube, and sedimented at 130 x g for 7 minutes. The supernatant was removed, and the pellet resuspended in fresh media. The countess 3 (Invitrogen, AMQAX2000) was used to count cell inoculum and to check viability.

Cell Viability Assay

Cells were plated in white, 96-well plates (Costar, CLS3917) at a density of 1 x 104 cells per well, and a volume of 90 μl per well. The controls contained 10 μl diH2O (0% and 100% controls) and the experimental wells contained 10 μl of the appropriate potency (D2 and D30), with four replicates per treatment or control. Plates were left for 1 hour at room temperature, before subsequent incubation at 37˚C and 5% CO2, to produce uniform cell distribution on the growth surface and reduce edge effect. Cell growth was assessed using the Cell Titer-Glo luminescent cell growth assay (Promega, G9243), using 100 μl of reagent per well according to the manufacturer’s instructions. The luminescence was measured in a BioTek, Synergy LX plate reader. Cell growth was assessed for 0% control wells after an initial 18 hours of incubation, while sample and 100% control wells were assessed after an additional 48 hours of growth.

Statistical calculations

Cell count analysis was performed on four measurements: 16 wells in total (two sets of quadruplicates on two different plates). All samples were included in the analysis. Data are expressed as mean ± standard deviation. Significance (p < 0.05) and STDEV calculations were done with the Microsoft Excel software.

The percentage growth was measured according to the following formula:

% Growth = 100 x (Sample – 0% Control)/ (100% Control– 0% Control)

Results and Discussion

All four Viscums: mali, populus, salix and tilia showed a statistically significant inhibiting effect on the proliferation of the Y-1 cells at the ultra-high dilution of D30 (Table 2 & Graph 1). Incidentally, it needs to be emphasized that demonstrating a biological activity of a substance at that dilutional level is in itself significant since a 1:10 dilution of a substance in water, repeated 30 times, presumably eliminates all molecules of the original substance and according to current thinking only “water remains”. The results at the D2 concentration are difficult to interpret. The seemingly stimulatory effect of V. mali, salix and tilia (populus not high then control) could be taken as a real sign of stimulation or it could be interpreted as simply as artifact since the intense color of the Viscum mother tincture up to the D2 dilution could be interpreted by the counting device as a higher number of cells (higher RLU values). The inhibition of adrenocortical cells by these ultra-high diluted, proprietarily produced mistletoe extracts, opens important new avenues in the pursuit of safe, inexpensive, well tolerated treatments in several areas of human suffering either as standalone or at least adjuvant treatment possibilities. Below are several listed conditions where a reduction in very side effect laden therapies might be welcomed. Currently, the rare but devastating adrenal cancer in children has only extremely intrusive management options like etoposide, doxorubicin, and cisplatin (EDP) with mitotane. Limited research has been reported on other treatment modalities for pediatric ACC, including mitotane, pembrolizumab, cabozantinib, and chimeric antigen receptor autologous cell (CAR-T) therapy [35]. On the other hand, adrenal cortical hyperactivity of androgens, cortisol and aldosterone are of interest here. Only a few examples of some of the clinical consequences of these overactive hormone levels will be mentioned.

An overproduction of androgenic steroids leads to exaggerated male characteristics in both men and women, such as hairiness of the face and body, baldness, acne, deeper voice, etc. [36]. An overproduction of the aldosterone hormone can lead to high blood pressure and to those symptoms associated with low levels of potassium, such as weakness, muscle aches, spasms, and sometimes paralysis [36]. Furthermore, multiple studies demonstrate that the adrenal gland, through its release of cortisol can worsen the progression of the tumorous process. Reviewing multi center studies Herrera-Martinez et al conclude that higher cortisol secretion while not increasing the risk of malignant neoplasm, can affect its clinical course, treatment requirements and mortality, leading to a worst prognosis [37]. Schrepf et al found that high nightly cortisol level predicted a halving of the survival rate and 46% increase in death rate [38]. Zeitzer et al describe an aberrant spike in cortisol level at night associated with progression of breast cancer in women [39]. Finally, Sephton et all point out even a connection between the high cortisol and the circulating natural killer (NK) cells leading to a worse outcome in breast cancer survival [40]. In terms of treatment, aldosteronism calls for spironolactone, eplerenone or calcium channel blockers [41]; hypercortisolism syndrome, depending on the exact cause, may need mifepristone, levoketoconazole [42] and more. (Hypercortisolism, well established as a “stress marker”, will be addressed in a subsequent paper). Considering the benefits of controlling adrenal overactivity with ultra-high diluted mistletoe extracts further in-depth investigations are warranted.

Authors Participation

i. R Rentea MD - designed the experimental project, wrote the article; and participated in the extraction of the mistletoe material.

ii. M Mueller M. Sc – executed, calculated the experimental data, and wrote the Materials & Methods section.

iii. M Kamsler MD - participated in the extraction of mistletoe material.

Conflict of Interest

The authors report no conflict of interest.

Acknowledgement

We thank Cosmin Borcea, Romania, for harvesting the mistletoe plants.

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