Alternative Cancer treatment by nigella sativa and alium atravilocum
Eman Abdelshafy
Purpose
Cancer is the most serious diseases facing humans
around the world , in spite of multiple attempts to
control it, but Cancer is cause of death worldwide
and accounted for 8.2 million deaths (around 13%of
Figure (1)
all deaths). Liver cancer one of the most dangerous type of cancer the number of people carry it
increase annually as shown in figure (1). Primary liver cancer is cancer that begins in the liver.
About 80% of primary liver cancer is hepatocellular carcinoma (HCC). Other subtypes of primary
liver cancer include bile duct cancer and angiosarcoma, a cancer of the blood vessels in the liver.
Primary liver cancer is globally the sixth most frequent cancer (6%) and the second leading cause
of death from cancer (9%). In 2012 it occurred in 782,000 people and resulted in 746,000 deaths.
In 2013, 300,000 deaths from liver cancer were due to hepatitis B, 343,000 to hepatitis C, and
92,000 to alcohol. Higher rates of liver cancer occur where hepatitis B and C are common. Males
are more often affected with HCC than females. Unfortunately, common treatment for this
disease is chemotherapy treatment and radiation those are two of the most dangerous types of
treatments as they have severe side effects, such (baldness-Digestive and nervous system
disorders-anemia-Anorexia) and many others, so, we had must study other alternative plants that
owns certain chemical properties can handle some types of cancer and relieve chemotherapy in
other types. the best of these plants ‘after long studying are the combination of (Nigella sativa
and Allium atroviolaceum) because they have a many anticancer activity that can treat cancer
without any side effect
Back ground research
first we understand what is cancer and major kind of it and causes and effect of cancer on
human http://www.wcrf.org (worldwide cancer research) and http://www.who.int/en/body to
know specially liver cancer and ordinary ways to treat it
www.medicinenet.com/liver_cancer_hepatocellular.../article.html ,also we searched in vital
process and protein in human which had effect in death of cancer cell such as apoptosis and ,we
searched about prior solution to treat cancer and most researches in this field also we know
about traditional medicinal of herbs and Spices have been used to prevent or treat medical
conditions As piper from https://www.ncbi.nlm.nih.gov/pubmed/.also we make wide search
about nigella sativa and TQ https://www.ncbi.nlm.nih.gov/pubmed/ and know all chemical
composition of it and how it contain many material that can treat many
https://www.ncbi.nlm.nih.gov/pubmed/ diseases include cancer line from.
www.ncbi.nlm.nih.gov/pubmed/https:// http://www.tbyil.com/ also we made wide search on
Allium atroviolaceum Curative properties https://www.ncbi.nlm.nih.gov/pubmed/16676298 we
recognize apoptosis and cytotoxic effect on the cell destroy on the body patient. Also made
search on MTT and NRU are assays to determine the level of cytotoxic on the cells
https://www.ncbi.nlm.nih.gov https://www.ncbi.nlm.nih.gov/pubmed/18600217.
Hypothesis
Increase the efficiency of TQ major extract from N.S by adding A. atroviolaceum that
increase the ratio of died cells by inducing apoptosis and cytotoxic effect on live cancer cell
specially hepatocellular carcinoma (HCC) , also proved the anti-cancer activities of A.
atroviolaceum.
Material
Figure (2)
• 2.5gm Fresh flower of A. atroviolaceum (FAA). figure (2)
• 2,5gm nigella sativa . figure (3)
• 70% methanol of the doses
• dimethyl sulfoxide (DMSO) Figure (3)
• RPMI-1640 cell grown media
• 10% FBS
• 100 IU/ml penicillin streptomycin
Variables
Time: The time that the dose spend in the cell affect the ratio of inducing cellular operations
and exert more cytotoxic effect on cancer cell line.
Concentration: different concentration of dose also affect the inhibition of cell Variability.
Cancer staging: The range of the stage of that cancer cell cancer affect the Possibility of
treatment type of kits used: the result may differ from kit to another.
Abstract
Phytochemical compounds are emerging as a new generation of anticancer agents with limited
toxicity in cancer patients. The purpose of this study was to increase the efficiency of the
anticancer activities of TQ the major compound of nigella sativa including exhibits
antiproliferative effect, cytotoxic effect, induces apoptosis. By adding A. atroviolaceum a plant of
the genus Allium has been used in folk medicine to protect against several diseases, such as
cancer. To evaluate in vitro cytotoxicity of combination of TQ and A. atroviolaceum, methanol
extract of the compound at a doses range from 100 to 3.12 μg/ml was assessed against the
HepG2 hepatocarcinoma cell line, and also on normal 3T3 cells, by monitoring proliferation
using the MTT assay method. A microscopy study was undertaken to observe morphological
changes of HepG2 cells after treatment and apoptosis were studied using flow cytometry. The
apoptosis mechanism of action was assessed by the level of caspase-3 activity and expression of
apoptosis related genes, Bcl-2, Cdk1 and p53 The results demonstrated growth inhibition of
cells in combined doses - and time-dependent manners, while no cytotoxic effect on normal cell
3T3 was found. The results revealed the highly occurrence of apoptosis.
Procedure Results
Plant material: The dried material (N.S and (FAA)) was homogenized to obtain a coarse powder and stored in airtight bottles. Approximately 5 gm of the powdered material was subjected to
MMT cytoxic assay Optimum dose of the compound to inhibit HepG2 cell
soxhlet (Electrothermal Eng.,sabry Mahfouz) extraction using 150 ml 70% methanol. The extract was concentrated under reduced pressure by rotary evaporator and solidified by freeze drier (SP
proliferation The anti-proliferative activities of methanol extracts from FAA,NSE
Scientific, NY, USA) the methanolic extract dissolved in in dimethyl sulfoxide (DMSO to obtain the stock solution (1000 μg/ml).
illustrated an inhibition effect on the cell proliferation in a time and dose-dependent
Cell culture: Human hepatoma HepG2 cells and mouse normal embryo cells (3T3) were grown in RPMI-1640 supplemented with 10% FBS and 100 IU/ml penicillin streptomycin. The cultures
manner. HepG2 cells treated with the compound showed inhibited cell proliferation at
were maintained at 37 °C in a humidified atmosphere of 5% CO2.
24 h with the IC50 value of 57.5±4.95 μg/ml, which markedly decreased to 44 μg/ml
MTT Cytotoxicity assay: HepG2 and normal 3 T3 cells were seeded at a density of 1×10^6/well into 96-well culture plates then exposed to various concentrations of FAA and N.S extractes (100, 50,
at 48 h and 26.67±3.5 μg/ml at 72 h (Fig.11). 3T3, was also considerably higher than
25, 12.5, 6.25 and 3.12 μg/ml). After 24, 48 and 72 h, 20 ug/ml of MTT solution was added to each well and incubated for 4 h. After addition of 100 μl of DMSO, the absorbance was measured with
FAA,NSE.while FAA,NSE showed selective cytotoxicity, as the CC50 of normal cell
an ELISA reader at a test wavelength of 540 nm and a reference wavelength of 690 nm. Cytotoxicity (%) = Absorbance of treated cells/absorbance of negative control × 100.
is>100 μl of FAA
Microscopic examination: after HepG2 cells cultured and being treated with IC50 concentration of the combound, morphological apoptotic changes were examined after 24, 48 and 72 h
Microscopic examination
incubation and photographed using a phase-contrast microscope.
THE Phase contrast microscopy of the cells revealed that Exposure of the cancer cells
Acridine orange/propidium iodide (AO/PI) double staining: Acridine orange/propidium iodide (AO/PI) double staining was used to observe the changes of apoptotic cell nuclei. The cells were
to the compound led to cytoplasm condensation (24 h), shrinkage and formation of
seeded at a density of 1×10^6 cells per well of six-well plate and after incubation for 24 h, the old media were replaced with the media treated with IC50 of the compound . After 24, 48 and 72 h,
apoptotic bodies (48 h) and the formation of debris (72 h) that are classic
the cells were washed with PBS. The mixture of 10 μg/ml acridine orange and 10 μg/ml propidium iodide (dissolved in PBS) was added to HepG2treated cells and then immediately observed under
morphologies of apoptosis.There was a visible loss of contact and rounding of cell
Leica fluorescence microscope DM 2500 with 100x magnification. Images were captured using an Alpha Imager. Each experiment was assayed three times (n=3)
shape post treatment as compared to the tightly packed and distinctively epithelial
Caspase-3 colorimetric assay: A caspase colorimetric assay kit was used to measure the caspase -3 activity in treated cell line according to manufacturer’s instructions. The cells (10^6/ml) were
monolayer formation in the untreated cells, indicative of apoptosis (Fig.9).
placed in a six-well plate for 24 h before treatment with various concentrations of the compound. After 24, 48 and 72 h.
Acridine orange/propidium iodide (AO/PI) double staining
cells were stained with AO/PI mixture and nuclear morphology changes were
observed under the fluorescence microscope. The cells treated with the compound
Data showed nuclear margination and chromatin condensation (24 h), membrane blebbing
(48 h), nuclear fragmentation and membrane loss (72 h). Cells stained with orange
colour indicated loss of cell membrane integrity. Morphological damage was seen in
cell lines when treated with the compound as compared to undamaged nuclei in
untreated cells. The untreated cells were live and stained bright green (Fig.10).
Conclusion
Conclusion
After making the experiment and record the result our finding support the hypothesis.
Figure (6)Induction of caspase-3/7 activity by thymoquinone in HepG2 cells and that the Allium atroviolaceum cases anti-proliferative properties in human
heptaocarcinoma (HeoG2) and pro-optotic also cytotoxic effect which increase the
efficiency of the anticancer activity of (TQ) major extract fro nigella sativa by 16% ant
the total efficiency of the compound of the dosage on cell viability is 88% .this
compound can be drug witch help in removing cancer cells by enhancing apoptosis
Figure (4 )Possible mechanisms of thymoquinone (TQ) action. (1) TQ induces apoptotic cell death in cancerous tissues by
operation and cytotoxic cells .
upregulating expression of apoptotic genes (caspases and bax) and down-regulating expression of anti-apoptotic genes (e.g.,
bcl 2); (2) TQ suppresses Akt activation by dephosphorylation and thus blocks cancer cell survival; (3) TQ deactivates
NFkappa B pathway by inducing cytokine production, and thus control oncogenic expression; (4) TQ increases the activities
of antioxidant enzymes and protects cell against cancer; (5) TQ protects normal cells’ injury caused by ionizing radiation in
the treatment of cancer; (6) TQ prevents CYP450 enzymes from damage. ‘+’ indicates increasing effect and ‘-’ indicates
decreasing effect.
Control
0.025 mg/ml
Figure 5. Cytotoxicity Assessments by MTT and NRU Assay in A-549 Cells Following the Exposure of Various Concentrations of Seed Oil of Nigella Sativa
0.25 mg/ml 1mg/ml Applications
Application
we believe our project the compound of (nigella sativa and Allium atroviolaceum) can
make a difference in the fight against cancer It’s crucial to remember that cancer is not
one disease – it’s more than 200. All different, unique diseases, which require different
Figure (7). Morphological Changes Hepg2 cells Exposed to Various Concentrations of the N.S . Images were taken using an inverted phase contrast microscope (OLYMPUS CKX 41) at 205 magnification
approaches for treatment. Treatments that work for some cancers don’t work for others
and sometimes those treatments simply stop working. In 2013 the global cancer burden
was estimated to be at least 14.1 million new cases and 8.2 million cancer deaths. More
Graphs than half of all cancers and cancer deaths in 2012 occurred in less developed regions of
the world, and these proportions will increase further by 2025. In times of diminishing
resources for health budgets the expensive drugs and technologies which we may have
access to in developed countries will be beyond the reach of many millions of people
globally. Our project about anti-cancer activity of compound herb Nigella sativa and
Allium atroviolaceum , it can treat more than 20 types of cancer the most important
Liver, brain, breast, skin, leukemia, Prostate cancer etc... anticancer activity of Nigella
sativa includes exert anti-proliferative, pro-apoptotic, anti-oxidant, cytotoxic, anti-
mutagenic, ant metastatic, and NK cytotoxic activity enhancing effects against various
primary cancer cells and cancer cell lines. Also chemical composition of Nigella sativa
play a vital part in forming Prostaglandin (PG) E1 which balances and strengthens the
immune system also black seed oil offers a powerful protective effect against radiation
figure (8) inhibition effect on the cell proliferation in a time and dose-dependent manner.
Fig. 9 Representative images to show morphological observation of HepG2 No treatment (a), treatment with FAA for 24 h (b), 48 h (c), and 72 h
and chemotherapy.
The compound is available , in many country and it more cheaply than any treatment of
cancer
(d) observed under inverted light microscopy (40X). Live cells (L), cytoplasm condensation (CC), blebbing (B), shrinkage (S), apoptotic bodies (AB) the doses for a period of not less than three months.
and debris (D). Similar cellular morphology was observed in three independent experiments (n = 3)
The compound TQ and F.A.A safe and effective herb that can be used by almost anyone.
In general, is not associated with serious side effects. No irritations or side effects are
caused when the right dose is correctly applied recommended dosage for cancer
therapy.
For the future plane we try now to increase the efficiency of the compound by adding
addition extract from other anticancer harp and turn it to drug for cancer patient
Research resources
resources
Fig (11) Effect of FAA on executioner caspases-3 activity in HepG2 cells. The cells were treated with IC25 = 42, IC50 = 26.67 Fig. 10 Treated HepG2 stained with AO/PI observed under fluorescence microscope. Photographic documentation was carried out at 40x magnification. a
and IC75 = 11.67 of FAA 24, 48 and 72 h treatment. Results are expressed as the mean optical density (405 nm) ± SD of three Control (untreated) cells (b) Cell treated with FAA at 24 h (c) 48 h (d) 72 h. Treated cells showed the typical characteristic of apoptosis such as nuclear
independent experiments. The symbol * indicates significant difference from control (p < 0.05) margination (NM), chromatin condensation (CC), nuclear fragmentation (NF), membrane blebbing (MB), and membrane loss (ML)
1.Attoub, S., Sperandio, O., Raza, H., Arafat, K., Al‐Salam, S., Al Sultan, M. A., ... & Adem, A. (2013).
Data analysis Thymoquinone as an anticancer agent: evidence from inhibition of cancer cells viability and invasion in vitro
and tumor growth in vivo. Fundamental & clinical pharmacology, 27(5), 557-569.
2.Al-Sheddi, E. S., Farshori, N. N., Al-Oqail, M. M., Musarrat, J., Al-Khedhairy, A. A., & Siddiqui, M. A. (2014).
Cytotoxicity of Nigella sativa seed oil and extract against human lung cancer cell line. Asian Pac J Cancer Prev,
15(2), 983-987.
A comparison of nuclear morphology at these various stages by AO/PI stain using fluorescent microscopy suggested that the FAA and TQ treated HepG2 cells displayed nuclear morphological changes. 3.Arıcan GO, Çakır O, Arıcan E, Kara T, Dağdeviren O, Arı S. Effects of Geven root extract on proliferation of HeLa
Untreated cells were observed with a green intact nuclear structure whereby, early apoptosis is obvious by intercalated AO within the fragmented DNA. Moreover, the cleavage of caspase-3 accelerates cells and BCL2 gene expressions. Afr J Biotechnol. 2014;11:4296–304.
4. Papadopoulos EI, Yousef GM, Scorilas A. Cytotoxic activity of sunitinib and
disassembly of cells, including DNA fragmentation, chromatin condensation, nuclear remodeling and membrane blebbing, as detected in the morphological study which suggested the caspase mediated everolimus in Caki-1 renal cancer cells is accompanied by modulations in the expression of apoptosis-related
apoptosis in HepG2 cells .It has been reported that caspase-3 is essential for cleavage of multiple protein substrates, including Bcl-2 The activity of caspase-3, the terminal effector in the apoptotic microRNA clusters and BCL2 family genes. Biomed Pharmacother. 2015;70:33–40.
cascade and the enzymatic major marker of apoptosis, was assessed in a time- and dose-course manner. Morphological changes in the cells and the nuclei were observed under 40X magnification of the 5. Jain R, Jain SK. Screening of in vitro cytotoxic activity of some medicinal plants used traditionally to treat
inverted phase contrast microscope, aided by AO/PI staining after treatment for 24, 48 and 72 h. Phase contrast microscopy of the cells revealed the original morphological form of control cells, most of cancer in Chhattisgarh state, India. Asian Pac J Trop Biomed. 2011;1:47–150.
which were adherent to the surface,but the presence of floating or detachment of nonviable cells in a dose- and time-dependent manner. Exposurenof the cancer cells to FAAand TQ led to cytoplasm 6.Bhadury P, Mohammad BT, Wright PC. The current status of natural products from fungi and their potential
as anti-effective agents. J India Microb Biotechnol. 2006;33:325–37
condensation (24 h), shrinkage and formation of apoptotic bodies (48 h) and the formation of debris (72 h) that are classic morphologies of apoptosis.