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.

196

Journal of Environmental Treatment Techniques

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