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

Abstract

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.

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Metadichol ; An agonist that expresses anti-aging
gene Klotho in various cell lines
Palayakotai R Raghavan (  raghavan@nanorxinc.com )
nanorx inc
Research Article
Keywords: Klotho, Metadichol, anti-aging, anti-tumor, primary cancer cells, broblasts
Posted Date: February 28th, 2023
DOI: https://doi.org/10.21203/rs.3.rs-2635049/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License

Metadichol promotes the expression of the anti-aging gene Klotho in various cell lines
Palayakotai R. Raghavan
Nanorx Inc., PO Box 131, Chappaqua, NY 10514, USA
Email: raghavan@nanorxinc.com
Abstract
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.
Keywords: Klotho, Metadichol, anti-aging, anti-tumor, primary cancer cells, fibroblasts

Introduction
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.
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. (Figure1)
Methods
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.
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).
Results and Discussion
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) ( Figure 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.
Abbreviated Cell Lines Description
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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Tables
Table 2. Primers used for qPCR
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
Gene
Primer pair
Sequence
Tm
P ro d u c t s i z e
(base pairs)
β–Actin
FP
TCCTCCTGAGCGCAAGTACTCT
62.1
153
RP
GCTCAGTAACAGTCCGCCTAGAA
62.4
KLOTHO
FP
GGGAGGTCAGGTGTCCATTG
55.88
152
RP
TGCTCTCGGGATAGTCACCA
53.83

Figure 1. Klotho fold expression changes in various cell lines

KLOTHO-WESTERN BLOT DATA IN HESC BG01V AND
PANC1,MIAPACA AND COLO205 CELL LINES

hESC BG01V cell line culture and treatment
!Cell line:
!hESC BG01V (ATCC® SCRC-2002™), Homo sapiens, embryonic stem cells
!Cell Culture:
!hESC BG01V cells were cultured in 1:1 Mixture of Dulbecco's Modified Eagles Medium and Ham's F-12 medium containing 1.2g/L sodium bicarbonate,
2.5mM L-glutamine, 15mM HEPES and 0.5mM sodium pyruvate, supplemented with 2mM L-Alanyl-L-Glutamine, 0.1mM Non-essential amino acids,
0.1mM 2-mercaptoethanol; 4ng/ml bFGF (80%), Knockout serum replacement (5%), fetal bovine serum (15%), 100units/ml penicillin G, and 100µg/ml
streptomycin at 37°C, 5% CO2 incubator. hESC BG01V cells were treated with various concentrations of test sample Metadichol (1pg/ml, 100pg/ml, 1ng/
ml, and 100ng/ml incubated for 24hrs. Post incubation, the cells were harvested for isolation of protein using RIPA buffer (Sigma; R0-278)

Isolation of protein using RIPA buffer
1. The cells, post harvesting, were washed twice using 1XPBS.
2. The cell pellets were gently suspended in 500µl of RIPA buffer with 1X Protease Inhibitor (Sigma; P-8340)
3. The cells were incubated for 30mins by gentle mixing every 5mins.
4. Post incubation, the cells were centrifuged at 10,000rpm for 12-15 minutes.
5. The protein lysates in the supernatant was transferred to fresh sterile tubes and stored in -20˚C until further use.

SDS-PAGE and Western Blot procedure
1. 100ug protein sample from each cell lysate was mixed with 5X loading dye and heated for 6 min at 980C.
2. Protein samples were loaded and separated on 12% SDS-PAGE gel using Mini protean Tetra cell (Bio-Rad).
3. Methanol activated 0.45uM PVDF membrane was pre-wet in transfer buffer for 10 min @ RT.
4. Protein transfer was done for 10 min in Turbo Transblot (Bio-Rad) apparatus.
5. Blot was blocked in 5% BSA + TBST for 1 hr @RT.
6. Blot was incubated with 10 Ab @ dilution: 1: 1000 for overnight @ 40C on shaker.
7. Washed 3 times with TBST for 5min @ RT
8. Blot was incubated with 20 Ab (Anti-Rabbit HRP- IgG) @ dilution 1: 1000 for 1hr @ RT.
9. Washed 3 times with TBST for 5min @ RT
10. Blot was rinsed with ECL reagent (two component system) for 1 min in dark and image was captured with 40 sec
exposure in Chemidoc MP imaging system (Bio-Rad).

Weste rn Blo t f or the ex pre ssi on of G APD H, KLO THO in hE SC BG 01V
cells.
Metadicho
l
Band!Intensity!Of!
Protein
Normalised!
Protein
expression
Relative!Protein!!
Expression
GAPDH
KLOTHO
Control
32916.56
15318.803
0.47
1.00
1pg!
28754.782
16261.953
0.57
1.22
100pg!
27463.368
14343.711
0.52
1.12
1ng
22421.296
6319.468
0.28
0.61
100ng!
28068.631
4156.054
0.15
0.32
KLOTHO Protein expression in hESC BG01Vtreated with
sample
Fold regulation
0.00
0.33
0.65
0.98
1.30
Treament
Control
1pg
100pg
1ng
100ng
0.3182
0.6056
1.1223
1.2152
1
36KDa!
65!KDa

Western Blot of Klotho in Panc-1 cells
Panc 1
Klotho expression
Metadichol (Conc.)
Lane
Relative Gene
Expression
Control
1
1.00
1µg/ml
2
0.65
1ng/ml
3
0.58
1pg/ml
4
1.23
β-actin
Klotho

Western Blot of Klotho in Panc 1 cells
Expression of Klotho Expression of β-Actin

Weste rn blo t d ata on an aly sis of ex pre ssi on of Kl oth o p rot ei n
Panc 1
Metadichol
(Conc.)
Band Intensity Proteins
Normalised
Relative Gene
Expression
β-actin
Klotho
Control
18858.602
18953.894
1.01
1.00
1µg/ml
16428.522
10724.974
0.65
0.65
1ng/ml
16059.894
9369.045
0.58
0.58
1pg/ml
22344.338
27602.016
1.24
1.23
Klotho Protein Expression in Panc 1 cells treated with Metadichol

Western Blot of Klotho in Panc-1 cells
β-actin
Klotho
Colo 205
Klotho expression
Metadichol (Conc.)
Lane
Relative Gene
Expression
Control
1
1.00
1µg/ml
2
0.93
1ng/ml
3
0.87
1pg/ml
4
0.48

Western Blot of Klotho in Colo 205 cells
Expression of Klotho Expression of β-Actin

Weste rn blo t d ata on an aly sis of ex pre ssi on of Kl oth o p rot ei n
Colo 205
Metadichol
(Conc.)
Band Intensity Proteins
Normalised
Relative Gene
Expression
β-actin
Klotho
Control
25292.128
26710.68
1.06
1.00
1µg/ml
20753.811
20309.42
0.98
0.93
1ng/ml
18488.711
16999.69
0.92
0.87
1pg/ml
32729.957
16477.58
0.50
0.48
Klotho Protein Expression in Colo 205 cells treated with Metadichol

Western Blot of Klotho in Panc-1 cells
Panc 1
Klotho expression
Metadichol (Conc.)
Lane
Relative Gene
Expression
Control
1
1.00
1µg/ml
2
0.65
1ng/ml
3
0.58
1pg/ml
4
1.23
β-actin
Klotho

Western Blot of Klotho in Panc 1 cells
Expression of Klotho Expression of β-Actin

Weste rn blo t d ata on an aly sis of ex pre ssi on of Kl oth o p rot ei n
Panc 1
Metadichol
(Conc.)
Band Intensity Proteins
Normalised
Relative Gene
Expression
β-actin
Klotho
Control
18858.602
18953.894
1.01
1.00
1µg/ml
16428.522
10724.974
0.65
0.65
1ng/ml
16059.894
9369.045
0.58
0.58
1pg/ml
22344.338
27602.016
1.24
1.23
Klotho Protein Expression in Panc 1 cells treated with Metadichol

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Disease-Connect.org
Gene KL
7
>
Gene View: The network shows the diseases associated with KL based on the GWAS/OMIM/DEG records (P < 1e-8).
Legend Label Layout P-value Edge Option Export Table View Hierarchical View
Neoplasms, Complex and Mixed
P-value < 1.0e-12 Node: 40
(<0
► ,- I••
+ J
Edge: 339