The Anti-Cancer Property of Mumie as Natural Product on Human Cervical Cancer Cell Line (HeLa)

Journal of Environmental Treatment Techniques

2021, Volume 9, Issue 1, Pages: 196-202

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

https://doi.org/10.47277/JETT/9(1)202

The Anti-Cancer Property of Mumie as Natural

Product on Human Cervical Cancer Cell Line

(

HeLa)

Azin Tavassoli, Malihezaman Monsefi

Biology Department, College of Sciences, Shiraz University, Shiraz, Iran

Received: 15/08/2020

Accepted: 25/10/2020

Published: 20/06/2021

Abstract

Mumie is a natural component found in some mountains, such as the Himalayas, as well as in some mountainous of Iran. It contains

of humic and phenolic compounds that have antioxidant and anti-cancer properties. Therefore, in this study, anti-cancer and antioxidant

properties of mumie were examined on Human Cervical Cancer Cell Line (HeLa). HeLa cells and normal fibroblasts (NIH) were cultured

in DMEM/F12 with mumie at concentrations of 0, 100, 200, 300, 400, 500 and 1000 µg/ml for 24 and 48 h. The bioviability of these

cells were evaluated using MTT assay. Chromatin condensation and apoptosis of these cells were examined using acridine orange and

2 2

aniline blue staining respectively. Antioxidant property of mumie on NIH cells was evaluated by 10 mM H O and neutral red test. MTT

assay revealed bioviability of HeLa cells decreased but chromatin condensation increased in concentration of 100 μg/ml mumie treated

culture. Apoptosis of the HeLa cells were observed in 100 μg/ml mumie treated culture. Mumie did not affect the bioviability, chromatin

condensation and apoptosis of NIH cells but 500 and 1000 μg/ml concentrations were toxic and induced cell death. The cell cultures in

different concentrations of mumie after 24and 48 h showed the similar results. NIH cells bioviability increased in 500 and 1000 μg/ml

concentrations of co-culture of H O and mumie that confirmed the antioxidant property. It concluded that even low concentrations of

2 2

mumie could destroy HeLa cells without any side effect on normal cells. Therefore, it can be used for cervical cancer treatment but

further research is needed.

Keywords: Apoptosis, Cervical cancer, Mumie

the natural substances that individuals have utilized as a healing

Introduction

compound for a long time. It is a brownish, rocky exudate

found in some mountainous areas. The chemical content of the

mumie is mostly originated from organic humic substances and

plants (7). Other mumie components include phenolic

compounds and some minerals such as fatty acids, albumin,

sterols, amino acids, 3,4 benzocoumarin, aromatic carboxylic

acids and polyphenols. The efficacy of mumie has been proven

for treating diseases such as anemia, diabetes, high cholesterol,

joint and bone disorders, and chronic pain (8). Also, it mainly

used to treat people with weakness, inflammation, bone

fracture, bleeding and wound healing (9). Based on (10)

administration of 200–600 mg/dose of shilajit in mice results in

morphological and phagocytotic changes in peritoneal

macrophages. (11) showed that shilajit can diminish gastric

ulcer record by increasing mucus barrier. They also proved that

shilajit has anti-inflammatory effect in carrageenan-induced

acute pedal edema, granuloma pouch and adjuvant-induced

arthritis in rats.

Cancer burden rise to 18.1 million infection and 9.6 million

deaths worldwide in 2018 (1). Common cancer treatments such

as chemotherapy, radiation therapy, and hormone therapy have

numerous side effects. Some typical side effects of

chemotherapy are vomiting, constipation or diarrhea and hair

loss (2). In recent years, increasing the new cases of cervical

cancer among all types of cancer has become a major medical

concern. However, the treatment of intermittent cervical cancer

disease remains ineffectual to a great extent (3). Consequently,

researchers are attempting to find more effective remedies to

cure this type of cancer. One of the approaches that has been

taken into consideration of numerous scientists today, is the

utilization of natural compounds like herbs. The use of herbs in

disease treatment has existed for thousands of years in many

parts of the world. Plants are one of the most important natural

sources for cancer treatment because many of them are capable

to inducing apoptosis pathway and blocked in cancer cells (4).

Approximately, 50ꢀ% of the modern anticancer drugs have

been originated from the natural products. Therefore, the use of

natural products in the development of new drugs has been a

great interest for researchers. For the cancer treatment,

Traditional Chinese Medicine has been used natural products

as adjuvant therapy in conjunction with chemotherapy and

radiotherapy for inhibiting the toxic effects of the treatments,

as well as improving overall efficacy (5, 6). Mumie is one of

There are many researches for healing effects of mumie in bone

fracture. Also, some researcher has been reported anti-cancer

property of mumie but we did not found its effect on cervical

cancer. Considering the potency of mumie in curing all above

mentioned medical complications, the aim of the present study

was to investigate the anti-cancer properties of mumie on

human cervical cancer cells (HeLa).

Correspondence author: Monsefi Malihezaman, Prof. of Anatomy, Biology Department, College of Sciences, Shiraz University

Shiraz-Iran. Tel: 0098-713-3167357, Fax: 0098-713-2280916, Email address: monsefi.g@gmail.com and

monsefi@susc.ac.ir.

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2021, Volume 9, Issue 1, Pages: 196-202

method score 1 to 4 represents light blue nuclei or normal cells,

semi-dense nuclei, dens nuclei and dark blue nuclei or

compressed cells respectively.

Materials and methods

.2 Extract preparation

Black mumie were purchased from a commercial source in

Kerman, Iran. Mumie were powdered and 0.01 g were solved

in 12.5 ml culture media in a water bath for 10 min. Mumie

extract were vortexed for 10 minutes and sterilized with 0.45

µm syringe filter. The concentrations of 50, 100. 150, 200, 250,

5.2 Acridine orange/Ethidium bromide (AO/EB) staining

AO/EB staining is used for evaluation of nuclear

morphology in apoptotic cells. The number of 5×10 cells were

cultured in each well of 24 well plate and then were treated with

different doses of mumie for 24 and 48 h. After the treatment

period, HeLa and NIH cells were harvested and rinsed with

PBS. The pellets were resuspended in AO/EB solution

including 5 μl of AO (Merck, Germany) and 5 μl of EB

00, 400, 500, 1000, 2000 and 3000 µg/ml were prepared. After

pilot study, the concentrations of 100. 200, 300, 400, 500 and

000 µg/ml were selected.

.2 Cell culture

(

SinaClon, Iran). After 10 min, the cells were observed using a

The effects of mumie on the human cervical cancer cell line

fluorescence microscope (Nikon Eclipse-E600) and

photographs were taken at ×100 magnification using a digital

camera (Nikon, Japan). Acridine orange is a vital dye and stains

both live and dead cells. Ethidium bromide stains only cells that

have lost membrane integrity. Live cells will appear uniformly

green. Early apoptotic cells stain green and contain bright green

dots in the nuclei as a consequence of chromatin condensation

and nuclear fragmentation. Late apoptotic cells also incorporate

ethidium bromide and therefore stain orange, but, in contrast to

necrotic cells, the late apoptotic cells show condensed and often

fragmented nuclei. Necrotic cells stain orange, but have a

nuclear morphology resembling that of viable cells, with no

condensed chromatin. The nuclei of 100 cells in each

concentration of each well were scored based on the percentage

of cells nuclei color as normal cells, early apoptotic cells and

apoptotic cells.

(

HeLa) were evaluated. A NIH cell line from the fibroblast of

Swiss albino mouse embryo tissue as a normal cell was cultured

with the same method to compare with the cancer cell line.

These cells were cultured in DMEM/F12 (Dulbecco's Modified

Eagle Medium, Gibco, USA) containing 10% fetal bovine

serum (FBS, Gibco, USA) and 5% Penicillin/Streptomycin

(

Gibco, USA). The cells were incubated at 37°C and 5% CO2.

The media were changed every 2 days. After 80-90%

confluency on the flask, the cells were passaged using Trypsin

.25% (Bio-IDEA Iran).

Cells number and viability were estimated using a

haemocytometer under a light microscope after vital staining

with trypan blue (Sigma-Aldrich, USA). Number of 1×10 cells

were grown and maintained in 100 µl per each well of 96 well

plate and 5×10 cells in 200 µl per each well of 24 well plate.

All cells were incubated at 37°C with 5% CO for 24 and 48 h.

Each concentration was repeated in 3 wells of well plate and in

three well plates separately.

.2 H

To investigate the antioxidant properties of mumie, H

was added to various concentrations of mumie. H causes

cell death by oxidation stress and DNA damage. Antioxidant

substances were reduced H activity in the cells. The number

2 2

O assay and Neutral red staining

2 2

.2 MTT assay

To evaluate the cytotoxicity effects of mumie on the HeLa

_c_e_l_l_s_,_M_T_T_c_o_l_o_r_i_m_e_t_r_i_c_a_s_s_a_y_w_a_s_a_p_p_l_i_e_d_._B_r_i_e_f_l_y_,₁_₁₀4

2 2

of 5×10 cells were cultured in each well of 24 well plate and

then were treated with different doses of mumie for 24 and 48

h. Negative control wells contained culture media and in

cells/well were transferred into 96-well culture plates

containing 100 µl of DMEM/F12 and incubated for 24 h. NIH

cell line was cultured with the same method to compare with

the cancer cell lines. The culture medium was replaced by fresh

medium containing mumie extract at concentration of 100. 200,

2 2

positive control wells H O were added to culture media.

Neutral red staining was then performed to examine living and

dead cells. Neutral red is a vital dye that across the

mitochondria of living cells but cannot enter in the

00, 400, 500 and 1000 µg/ml after 24 h and incubated for 24

and 48 h. MTT solution was prepared at 1mg/ml in PBS and

was filtered through a 0.2 µm syringe filter. Then MTT solution

at 1:10 of total volume was added into each well. Cells were

mitochondria of dead cells. The number of 5×10 NIH cells

were cultured in each well of 24 well plate and then were

treated with different doses of mumie for 24 and 48 h. 150 μl

of neutral red dye was added to each well then fixed for 1 min

by formal calcium. 200 µl of acid alcohol (3%

hydrochloric acid in 95% ethanol) was added to each well for

incubated for 4 hours at 37°C with 5% CO and complete

humidity. After 4 hours, the MTT solution was removed and

replaced with 200 µl of DMSO. The plate was further incubated

for 2 hours at room temperature, and the optical density (OD)

of the wells was determined using a plate reader at a test

wavelength of 570 and 690 nm. Percent of the cell viability was

calculated using the equation: (mean OD of treated cells/mean

OD of control cells)  100.

h. Finally, the supernatants were transferred to the new plate

and the light absorbance was measured at a wavelength of 540

nm by a microplate reader. Normal cells revealed higher color

intensity than to dead cells.

.2 Statistical analysis

The data were analyzed with One-Way ANOVA using

.2 Chromatin condensation assay

The number of 5×10 cells were cultured in each well of 24

SPSS version 17.5. The p<0.05 were considered as significant

level. The Tukey post hoc test was used to obtain mean and

standard deviation for each parameter and the graphs were

plotted using EXCLE software.

well plate and then were treated with different doses of mumie

for 24 and 48 h. HeLa and NIH cells were fixed in 3%

glutaraldehyde in PBS 0.2 M 30 min then stained with 5%

aniline blue (Acros Organics, USA) in 4% acetic acid 10 min

at pH=3.5. Each well was examined by invert microscope and

their photographs were taken by digital camera (Nikon, Japan).

Condensed chromatin was stained as dark blue. Intensity of

reactions of 100 nuclei in each concentration of each well were

scored based on the percentage of cells nuclei color. In this

Results

The human cervical cancer cell line (HeLa) of mumie

treated cultures showed a significant reduction in cell viability

in a concentration and time dependent manner.

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2021, Volume 9, Issue 1, Pages: 196-202

Figure 1: The bio-viability effects of mumie on HeLa and NIH cells cultures at different concentrations after 24 and 48h using MTT assay.*Significant

difference (P<0.05) compared to the control culture

Figure 2: Chromatin condensation of HeLa and NIH cells at different concentrations of mumie treated culture after 24 and 48 h.

*Significant difference (P<0.05) compared to the control culture

The viability of Hela cells reduced from 100 µg/ml

concentration significantly (P<0.05). IC50 of HeLa cells after

cells viability decreased at 300 µg/ml concentration. IC50 of

HeLa cells after 48 h of mumie treatment were 310.4 µg/ml.

NIH cells had an IC50 of 998.8 and 807.8 µg/ml after 24 h and

4 h of mumie treatment were 379.8, whereas after 48 h these

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2021, Volume 9, Issue 1, Pages: 196-202

8 h cultures respectively. The results confirmed the sever

cytotoxic effect of mumie on cervical cancer c:ell lines (Fig. 1).

The aniline blue staining revealed the round and euchromatin

nuclei of the HeLa cells in the control culture changed to the

condensed and dark chromatin in the different concentrations

of mumie treated cultures. Chromatin condensation appears in

treated culture (Fig. 4). The apoptotic changes such yellowish

nuclei and well condensed and small cytoplasm saw in 300

μg/ml and higher concentration of mumie treated culture. In

400 μg/ml concentration of mumie treated culture near 40%

cells showed apoptotic appearance. HeLa cells of 1000 μg/ml

concentration of mumie treatment showed yellow to orange

color that referred to their apoptosis (Fig 5D). The results of

apoptosis of HeLa cells of mumie treated cultures after 48 h

were similar to it in 24 h culture (Fig. 4 and 5E-H). NIH cells

mumie treated cultures after 24 h did not show any apoptotic

changes except in the highest concentration of 1000 μg/ml (Fig.

4) but after 48 h they revealed some early apoptotic changes in

200 μg/ml concentration of mumie treatment (Fig. 4) but the

more than 80% of NIH cells were normal (Fig. 4 and 5I-L).

00 μg/ml of mumie concentration and nuclei compression saw

at 300 μg/ml of mumie concentration treated culture (Fig 2).

Higher HeLa cells of the control culture (almost 80%) showed

dark green nuclei and spindle like cytoplasm using AO/EB

staining (Figure 5A). HeLa cells in low concentration (100

μg/ml) of mumie treated culture showed the early apoptotic

changes such as light green nuclei and round and compressed

cytoplasm (Fig. 5B). The population of early pre-apoptotic

cells increased in 200 μg/ml and higher concentration of mumie

Figure 3– The effects of mumie on HeLa and NIH cells cultures at different concentrations of control, 300 and 1000 µg/ml respectively. (A-C) showed

HeLa cells cultured after 24 h, (D-F) showed HeLa cell cultures after 48 h. (G and H) showed NIH cell cultures after 24 h and (I) after 48h. Aniline blue

staining.

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2021, Volume 9, Issue 1, Pages: 196-202

Figure 4: apoptosis of HeLa and NIH cells at different concentrations of mumei treated culture after 24 and 48 h.*Significant difference (P<0.05)

compared to the control culture

Figure 5: The effects of mumei on HeLa and NIH cells cultures at different concentrations of control, 200, 400 and 1000 µg/ml respectively. (A-D)

showed HeLa cells cultured after 24 h, (E-H) showed HeLa cell cultures after 48 h. (I-K) showed NIH cell cultures after 24 h and (L) after 48h. Acridine

orang/Ethidium bromide staining.

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2021, Volume 9, Issue 1, Pages: 196-202

an effective substance for inhibiting proliferation in cancer

cells such as Hep3B, HT29 and PC3. It upregulates the mRNA

levels of apoptotic genes of PC3 cells (18). Large population of

HeLa cells showed either condensed chromatin after aniline

blue staining or apoptotic signs after Ao/Eb staining (Figures

-4). Mumie has induced chromatin condensation and

apoptosis in all concentration of HeLa treated culture in both

4 and 48 h. Apoptosis and chromatin condensation was

increased with increasing concentrations of mumie. Chromatin

condensation referred to compaction and hyperchromic nuclei

of HeLa cells before they died. During cell death, chromatin

undergoes the changes from a heterogeneous, genetically active

network to an inert, highly condensed form. According to our

results, different condensation of mumie did not show any side

effects on NIH nuclei except for the highest dose of 1000 µg/ml

concentration that proved toxic, leaving the highest possible

number of cells dead. Humic acid (the major component of

mumie) induced apoptosis in human premyelocytic leukemia

HL-60 cells and showed loss of cell viability, chromatin

condensation, and internucleosomal DNA fragmentation of

these cells. Furthermore, caspase-3 and the specific proteolytic

cleavage of poly (ADP-ribose) polymerase activated, and Bax

protein levels increased while Bcl-2 reduced (14). (19) has been

reported cytotoxic effects of humic acid on human breast

cancer cells.

polyphenols can repress the tumor generation and inhibit

cancer cells growth. Tea decreases the danger of breast cancer

(20). consumption of green tea and tomato resulted in reduction

of prostate cancer in mouse models (21) (22). Our results

revealed antioxidant activity of mumie on NIH cells.

Mitochondria efficiency in generating ATP decrease in animals

and in humans is associated with aging and oxidative stress.

Figure 6: Anti-oxidant property of HeLa and NIH cells at different

concentrations of mumie treated culture after 24 and 48 h. *Significant

difference (P<0.05) compared to the control culture

H O

2 2

assay of NIH cell mumie treated culture after 24h

revealed anti-oxidant property of high concentrations (500 and

000 μg/ml) of this natural material. The NIH cell viability

increased 7 and 12 folds in concentration of 500 and 1000

μg/ml mumie treated culture respectively when they compared

to positive control culture but there is no anti-oxidant property

in 48 h culture (Fig. 6).

Discussion

The cell proliferation measurement using MTT assay

revealed mumie decreased HeLa cell viability even in low

concentration and short time (24h) significantly (Figure 1).

There was a 19, 31, 38, 43, 48 and 68 % (pꢁ<ꢁ0.05) reduction in

the cell proliferation when the cells were incubated with 100,

(

23)treated mice with 20 mg of a mixture of the 3-

hydroxydibenzo-α-pyrone (3- OH-DBP) and 3,8-

dihydroxydibenzo-α-pyrone [3,8-(OH)2-DBP isolated from

shilajit. They revealed shilajit DBPs and their redox products

upholds creation of ATP by hepatic mitochondria ((23)2009).

Free radical scavenging activity of shilajit by 2,2-Diphenyl-1-

picrylhydrazyl (DPPH) assay has been reported (Rege et al.,

00, 300, 400, 500 and 1000 μg/ml concentration of mumie

after 24h respectively. The mumie treated culture after 48 h

showed 35, 45, 65 and 92% reduction in cell viability in 300,

00, 500 and 1000 μg/ml concentration of mumie after 48h

012). The effect of shilajit against some biogenic free radicals

respectively. Mumie is not significant effects on NIH cell

viability in similar condition to HeLa cells except in higher

concentration of 1000 μg/ml mumie treated culture in both 24

and 48 h that revealed 49 and 70 % reduction in viability

respectively (Figure 1). MTT is a NADPH-dependent cellular

oxidoreductase enzyme assay. It resulted insoluble purple

color formazan, which represents the numbers of the viable

cells (12). MTT assay indicates mumie anti-proliferative

property in HeLa cells.

Mumie is a natural compound that mostly contains of

humic substances. Many people around the world have used

Mumie (shilajit) as a treatment for a variety of ailments.

Different doses of shilajit from 200 to 1000 mg/kg on rats

showed it has no side effects on vital organs including heart,

liver, and kidneys (13). Many studies have suggested that

mumie have immunomodulatory and anti-inflammatory

activity. These examinations recommend that the lytic potential

of polymorphonuclear leukocytes could be improved by

shilajit. Mumie contains fulvic acid and humic acid, which are

responsible for its biochemical activities. Humic matter has

been reported to be anti-cancer agent as it inhibited the cancer

cell growth and induced the apoptosis (14). The cytotoxic

properties of humic acid was accompanied the ROS production

-•

including (O ) , (•OH), and (NO) revealed it provided

complete protection to methyl methacrylate (MMA) against

hydroxyl radical-induced polymerization and acted as a

reversible NO-captodative agent (24).

5 Conclusion

Mumie vastly used in southern Iran for a number of health

complaints. In our study, Mumie had shown as potential anti-

proliferative and apoptotic properties. The evidence from the

present study suggests that Mumie may be a factor in diet that

may lower the risk of cancer and may inhibit the tumor growth

and proliferation, apoptosis and oxidative stress.

Ethical issue

Authors are aware of, and comply with, best practice in

publication ethics specifically with regard to authorship

(

avoidance of guest authorship), dual submission, manipulation

of figures, competing interests and compliance with policies on

research ethics. Authors adhere to publication requirements

that submitted work is original and has not been published

elsewhere in any language.

(

15) and NO synthesis (16).

Competing interests

The authors declare that there is no conflict of interest that

would prejudice the impartiality of this scientific work.

Fulvic acid can increase extracellular anti-cancer mediators

from RAW 264.7 cells that caused MCA-102 fibrosarcoma

cells apoptosis. It also induced NF-ĸB activity which

modulated the expression of NO and iNOS (17). Fulvic acid is

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2021, Volume 9, Issue 1, Pages: 196-202

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Authors’ contribution

All authors of this study have a complete contribution for

data collection, data analyses and manuscript writing.

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