Metadichol ; An agonist that expresses  anti-aging gene Klotho in various cell lines

doi:10.21203/rs.3.rs-2635049/v1

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Metadichol ; An agonist that expresses anti-aging gene Klotho in various cell lines

https://doi.org/10.21203/rs.3.rs-2635049/v1

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10 October, 2023 Editorial note (correction). This submission initially contained a declaration of no competing interests for this submitted work; however, the author has now provided a corrected competing interest declaration as follows: The author, PR Raghavan, is the CEO of Nanorx Inc., the manufacturer of the Metadichol® supplement mentioned in this work, and, as such, has professional and financial interests related to research outcomes of this supplement.

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Klotho is a well-known tumor suppressor hormone that exhibits anti-cancer and anti-aging properties. Klotho levels are low or non-existent in cancer patients. Klotho protein levels decrease with aging; maintaining consistent levels may prevent disease and promote healthier aging. Metadichol is a nano emulsion of long-chain alcohols C26, C28, and C30, of which C-28 constitutes over 85%. Any small molecule that can elevate Klotho can, in principle, help reverse many diseases in which Klotho levels are low. Previously, we showed that treatment of the pancreatic cancer cell lines PANC1, MIA-PACA, and COLO-205, combined with Metadichol, a lipid emulsion consisting of long-chain alcohols at 1-100 pg/mL concentrations, resulted in a 4- to 10-fold increase in Klotho expression as determined by qRT-PCR, This study aimed to demonstrate that Metadichol promotes Klotho expression in a wide variety of cell lines, such as primary cancer, stem, and somatic cell lines. Cells were treated with various concentrations of Metadichol ranging from 1 pg to 1 µg. Three to fifteen fold increase in Klotho expression was observed compared with baseline, as measured by qRT-PCR and qualified by western blot analysis. Metadichol is a natural agonist of Klotho expression and is non-toxic at levels up to 5000 mg/kg in rats. and has a potential therapeutic role in cancer and reversing aging.

Klotho

Metadichol

anti-aging

anti-tumor

primary cancer cells

fibroblasts

In Greek methodology, Klotho was known as the goddess of lifespan regulation [1]. Following its initial discovery in the kidneys in 1997 [2], Klotho has been shown to block tumor growth and metastasis, modulate chemotherapeutic drug resistance and improve overall survival [3]. The injection of Klotho into animals decreased breast cancer growth regarding tumor size, weight, and visual appearance and resulted in no significant side effects. Low levels of Klotho are associated with cancer and many other diseases [4–6], as shown in Table 1. An increasing number of studies [4] have focused on the use of Klotho in extending longevity and counteracting the effects of aging on physical function. Recently, significant progress has been made in identifying factors (7) contributing to the aging process, which has contributed to developing therapeutic strategies to prevent, delay, or reverse the age-related decline. Many studies have attempted to induce Klotho gene expression, emphasizing its potential benefit when injected into animals.

Table 1

List of diseases associated with low levels of Klotho
Acetylcholine and Nitric Oxide Dysregulation	Hypertension
Aging (highly accelerated)	Impaired Cognition (such as Alzheimer's Disease)
All-Cause Mortality	Hyperphosphatemia
Anemia	Lung Damage
Anorexia	Multiple System Atrophy
Atherosclerosis (as well as calcification of the arteries)	Pseudoexfoliation Syndrome
Bone Loss (such as osteoporosis and low bone mass)	Rheumatoid Arthritis
Cancers · Bone · Brain · Breast · Colon · Stomach · Kidney · Liver	Sarcopenia
	Skin Atrophy (such as scleroderma)
	Stroke
	Vascular Disease (such as coronary artery disease)
	Hyperparathyroidism
	Inflammatory Bowel Disease

Clinical and preclinical studies have demonstrated that Klotho is an essential anti-aging molecule [8] that affects lifespan, health, and cognitive function [9]. Laboratory animals that do not express Klotho exhibit a shorter lifespan and cognitive impairment. In contrast, mice that overexpress Klotho have a longer lifespan and enhanced cognition and memory [10]. Klotho significantly inhibits the growth of lung cancer [11], pancreatic carcinoma [12], colorectal carcinoma [13], breast cancer [14], hepatocellular carcinoma [15], ovarian carcinoma [16], melanoma [17], diffuse large B cell lymphoma [18].

Several molecules, such as PPARγ agonists [19], testosterone [20], and resveratrol [21], either directly promote Klotho over expression in vitro or inhibit Klotho down regulation in vivo. Jung et al reported a novel molecular mechanism by which a small molecule [N-(2-chlorophenyl)-1H-indole-3-caboxamid] induces Klotho expression [22]. Other studies [23] have used CRISPR methodology to upregulate Klotho transcription and production in two different cell lines, one of which was a neuron-like cell line.

Pharmaceutical companies have been developing Klotho agonists that up regulate Klotho expression are of significant interest for treating diseases. For example, Klotho Therapeutics has developed a patent-pending treatment based on Klotho that affects aging [24]. Klogenix has been working on a two-pronged approach to target endogenous Klotho's natural production and regulation and deliver Klotho genetic material directly to patient cells, thus enabling them to produce the protein [25].

Previously, we showed that treatment of the pancreatic cancer cell lines PANC1, MIA-PACA, and COLO-205, combined with Metadichol, a lipid emulsion consisting of long-chain alcohols at 1-100 pg/mL concentrations, resulted in a 4- to 15-fold increase in Klotho expression as determined by qRT-PCR [26]. In the present study, we have extended our original work to determine the effects of Metadichol in many other cell lines, including primary cancer cell lines, stem cell lines, and fibroblasts. (Fig. 1)

Experimental procedures

This work was carried out by a service provider: Skanda Life Sciences, Bangalore, India.

Chemicals and reagents

The A549, Colo-205, PANC1, MDAMB31, HeLA, HepG2, and human cardiac fibroblast cells were purchased from the ATCC (USA). Primary breast cancer cells were obtained from BIOIVT (Detroit, Michigan, USA). Primary antibodies were purchased from ABclonal (Woburn, Massachusetts, USA) and E-lab Science (Maryland, USA). Primers were obtained from SahaGene, Hyderabad, India (Table 2). All other molecular biology reagents were purchased from Sigma Aldrich.

Table 2

Primers used for qPCR
Gene	Primer pair	Sequence	Tm	Product size (base pairs)
β–Actin	FP	TCCTCCTGAGCGCAAGTACTCT	62.1	153
β–Actin	RP	GCTCAGTAACAGTCCGCCTAGAA	62.4	153
KLOTHO	FP	GGGAGGTCAGGTGTCCATTG	55.88	152
KLOTHO	RP	TGCTCTCGGGATAGTCACCA	53.83	152

Cell line maintenance and seeding

The cells were cultured in a suitable medium with or without supplements in the presence of 1% antibiotics in a humidified atmosphere of 5% CO₂ at 37°C. The medium was changed periodically until the cells reached confluency. Cell survival was determined using a hemocytometer. When the cells reached 70–80% confluency, single-cell suspensions were seeded into 6-well plates at a density of 10⁶ cells per well and incubated for 24 h at 37°C in 5% CO₂. Afterward, we rinsed the cell monolayer with a serum-free medium and added Metadichol at predefined concentrations.

Cell treatment

Metadichol was prepared at various concentrations (1 pg/mL, 100 pg/mL, one ng/mL, and 100 ng/mL) in serum-free media, and the mixture was added to predesignated wells. The control cells received media without drugs. The cells were incubated, then gently rinsed with sterile phosphate-buffered saline (PBS) solution. Quantitative RT-PCR (q RT-PCR) and western blot analysis were done as described below.

RNA isolation

RNA was isolated from each treatment group using TRIzol reagent (Invitrogen). Cells (10⁶) were collected into 1.5-mL microcentrifuge tubes and centrifuged at 5,000 rpm for 5 min at 4°C. Then, 650 µL of TRIzol was added to the pellet, mixed, and incubated on ice for 20 min. Subsequently, we added 300 µL of chloroform, mixed the samples with gentle inversion for 1–2 min, and set them on ice for 10 min. The samples were centrifuged at 12,000 rpm for 15 min at 4°C. The upper aqueous layer was transferred to a new sterile 1.5 mL centrifuge tube, and an equal amount of pre-chilled isopropanol was added. The samples were incubated at − 20°C for 60 min, then centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was carefully removed, and the RNA pellet was washed with 1.0 mL of 100% ethanol and 700 µL of 70% ethanol with centrifugation as previously described. The RNA pellet was air-dried at room temperature for approximately 15–20 min and resuspended in 30 µL of DEPC-treated water. RNA concentration was measured using a Spectradrop (Spectramax i3x, USA) spectrophotometer (Molecular Devices).

cDNA synthesis

Complementary DNA (cDNA) was synthesized from 2 µg of total RNA using the Prime Script cDNA synthesis kit (Takara) with oligo dT primers following the manufacturer’s instructions. The reaction volume was 20 µL, and cDNA synthesis was performed at 50°C for 30 min, then incubating at 85°C for 5 min using an Applied Biosystems instrument (Veritii). The resulting cDNA was used as a template for qPCR.

Primers and qPCR

A final reaction volume of 20 µL of PCR mixture was prepared, consisting of 1 µL of cDNA, 10 µL of SYBR green Master Mix, and one µM complementary forward and reverse gene-specific primers. The samples were run under the following conditions: initial denaturation at 95°C for 5 min followed by 30 cycles of secondary denaturation at 95°C for 30 seconds, annealing at the optimized temperature for 30 seconds, and extension at 72°C for 1 min. We defined the number of cycles that allowed amplification in the exponential range without reaching a plateau as the optimal number of cycles. The results were obtained using CFX Maestro software. We calculated fold-change using the comparative CT method and determined the relative expression of each target gene relative to a housekeeping gene (β-actin) and untreated control cells. ΔCT was calculated for each treatment using the following formulas: ΔCt = Ct (target gene)–Ct (reference gene), ΔΔ Ct = ΔCt (treatment group)–ΔCt (control group). The fold-change was calculated for target gene expression for each treatment using the formula: Fold change = 2^ (−ΔΔCt).

Protein isolation and western blot analysis

Total protein was isolated from 10⁶ cells using radio immuno precipitation assay buffer supplemented with the protease inhibitor phenylmethyl sulfonyl fluoride. The cells were lysed for 30 min at four °C with gentle inversion, centrifuged at 10,000 rpm for 15 min, and the supernatant was transferred to a new sterile tube. The Bradford method ( BIO-RAD USA) was used to measure the protein concentration. Protein (25 µg) was mixed with 1X sample loading dye containing SDS and loaded onto a polyacrylamide gel. The proteins were separated under denaturing conditions using a Tris-glycine running buffer. The proteins were transferred to PVDF membranes (Invitrogen) using a Turbo transblot system (Bio-Rad, USA), blocked with 5% BSA for one h, incubated with the respective primary antibody overnight at four °C, followed by a species-specific secondary antibody for one h at RT. After washing, the membranes were incubated with ECL substrate (Merck) for 1 min in the dark. The images at suitable exposure settings were captured using the ChemiDoc XRS system (Bio–Rad, USA).

The highest Klotho expression was observed in MDAMB-231 ( Triple-Negative Breast Cancer cells developed at MD Anderson Institute Houston), followed by stem cells, then cardiac fibroblasts (NHCFs) ( Fig. 1). Although the expression of Klotho induced by small molecules is elevated in kidney cells [27], we found that Metadichol increased Klotho expression in many different cell types and thus may represent a universal Klotho agonist. Because Klotho exhibits anticancer activity, its level of expression in cancer cells is significant. Down regulation of Klotho has been observed in several cancers, such as pancreatic cancer, HCC, and others (28–29).. Epigenetic modulation, such as promoter methylation and histone deacetylation, also contributes to the dysregulation of Klotho in cancer.

Forester et al. [30–31] suggested that the liganded vitamin D receptor (VDR) upregulates Klotho via vitamin D response elements (VDRE). Metadichol is a VDR inverse/protein agonist [32–33]. Among the consequences of the enhanced expression of Klotho is an increase in telomerase activity. Similarly, Metadichol can up regulate telomerase [34] and thus potentially prevent stem cell aging [35].

Consequently, the down regulation of Klotho enhances proliferation and reduces apoptosis in cancer cells. Conversely, the over expression of Klotho results in cancer cell inhibition [36–38]. Similarly, our results suggest that Metadichol could be useful in inducing apoptosis in cancer cells by increasing Klotho expression and this also has benefits in other diseases. The current study and previously published results (39–42) suggest that the observed results could have been been due to increased Klotho expression (43). The potential therapeutic utility of Metadichol in elevating Klotho levels warrants further study in vitro and in vivo.

PANC1: human pancreatic cancer cell line isolated from a pancreatic carcinoma of ductal cell origin

HCAF-PPCC: (Human Cancer associated Fibroblast primary prostate cancer cell.

MIA-PACA: Human pancreatic cancer cell line

HCAF-TNBCC; Human Cancer associated Fibroblasts-Triple Negative Breast Cancer Panel

COLO-205: Cell line is made up of epithelial cells isolated from ascitic fluid derived from a 70-year-old, male with colon cancer

A-549: Adenocarcinomic human alveolar basal epithelial cell

HEPG2 : human liver cancer cell line

HELA; Immortalized cell line used in scientific research. It is the oldest and most commonly used human cell line. The line is derived from cervical cancer cells

hESC BGO1V; cells are pluripotent and can differentiate to representatives of the three primary germ layers.

MDAMB 231; isolated at M D Anderson Institute, Houston, USA from a pleural effusion of a patient with invasive ductal carcinoma) is commonly used to model late-stage breast cancer

Author Contributions

All work was planned and supervised by the author (PPR), who is solely responsible for its content.

Acknowledgments; We thank Dr Michelle Muller of Sferlap S.A Switzerland for helpful discussion.

Conflicts of Interest; None

Funding; Nanorx, Inc. R&D Budget provided funding.

Competing Interests: None

Availability of data and material

All data are in the manuscript and the supplementary material provided.

Supplementary material : Western Blot data and Klotho Diseases Network

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Download PDF

Journal Publication

published 28 Sep, 2023

Read the published version in Fortune Journal of Health Sciences →

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You are reading this latest preprint version

Metadichol ; An agonist that expresses anti-aging gene Klotho in various cell lines

Status:

Journal Publication

Version 1

Editorial Note

Abstract

Figures

Introduction

Methods

Experimental procedures

Chemicals and reagents

Cell line maintenance and seeding

Cell treatment

RNA isolation

cDNA synthesis

Primers and qPCR

Protein isolation and western blot analysis

Results And Discussion

Abbreviated Cell Lines Description

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 1