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Alternative Cancer treatment by nigella sativa and alium atravilocum

Purpose

Figure (1)
Figure (1)

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

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

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.

,also we searched in vital

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

2.5gm Fresh flower of A. atroviolaceum (FAA). figure (2)

2,5gm nigella sativa . figure (3)

70% methanol of the doses

dimethyl sulfoxide (DMSO)

RPMI-1640 cell grown media

10% FBS

100 IU/ml penicillin streptomycin

Variables

Figure (2)

• 100 IU/ml penicillin streptomycin Variables Figure (2) Figure (3) Time : The time that the

Figure (3)

penicillin streptomycin Variables Figure (2) Figure (3) Time : The time that the dose spend in

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.

Eman Abdelshafy

Procedure

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 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 Scientific, NY, USA) the methanolic extract dissolved in in dimethyl sulfoxide (DMSO to obtain the stock solution (1000 μg/ml). 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 were maintained at 37 °C in a humidified atmosphere of 5% CO2. 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, 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 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. 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 incubation and photographed using a phase-contrast microscope. 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

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, 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 Leica fluorescence microscope DM 2500 with 100x magnification. Images were captured using an Alpha Imager. Each experiment was assayed three times (n=3)

Caspase-3 colorimetric assay: A caspase colorimetric assay kit was used to measure the caspase -3 activity in treated cell line according to manufacturers instructions. The cells (10^6/ml) were

placed in a six-well plate for 24 h before treatment with various concentrations of the compound. After 24, 48 and 72 h.

Data Figure (6)Induction of caspase-3/7 activity by thymoquinone in HepG2 cells and Figure (4 )Possible
Data
Figure (6)Induction of caspase-3/7 activity by thymoquinone in HepG2 cells and
Figure (4 )Possible mechanisms of thymoquinone (TQ) action. (1) TQ induces apoptotic cell death in cancerous tissues by
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.
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
0.025 mg/ml
0.25 mg/ml
1mg/ml
Control
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
Graphs
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
(d) observed under inverted light microscopy (40X). Live cells (L), cytoplasm condensation (CC), blebbing (B), shrinkage (S), apoptotic bodies (AB)
and debris (D). Similar cellular morphology was observed in three independent experiments (n = 3)
Fig (11) Effect of FAA on executioner caspases-3 activity in HepG2 cells. The cells were treated with IC25 = 42, IC50 = 26.67
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
independent experiments. The symbol * indicates significant difference from control (p < 0.05)
Fig. 10 Treated HepG2 stained with AO/PI observed under fluorescence microscope. Photographic documentation was carried out at 40x magnification. a
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
margination (NM), chromatin condensation (CC), nuclear fragmentation (NF), membrane blebbing (MB), and membrane loss (ML)
such as nuclear
Data analysis

Data analysis

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. 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

disassembly of cells, including DNA fragmentation, chromatin condensation, nuclear remodeling and membrane blebbing, as detected in the morphological study which suggested the caspase mediated 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 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 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 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 condensation (24 h), shrinkage and formation of apoptotic bodies (48 h) and the formation of debris (72 h) that are classic morphologies of apoptosis.

Results

MMT cytoxic assay Optimum dose of the compound to inhibit HepG2 cell proliferation The anti-proliferative activities of methanol extracts from FAA,NSE

illustrated an inhibition effect on the cell proliferation in a time and dose-dependent manner. HepG2 cells treated with the compound showed inhibited cell proliferation at

24 h with the IC50 value of 57.5±4.95 μg/ml, which markedly decreased to 44 μg/ml at 48 h and 26.67±3.5 μg/ml at 72 h (Fig.11). 3T3, was also considerably higher than FAA,NSE.while FAA,NSE showed selective cytotoxicity, as the CC50 of normal cell is>100 μl of FAA Microscopic examination THE Phase contrast microscopy of the cells revealed that Exposure of the cancer cells to the compound led to cytoplasm condensation (24 h), shrinkage and formation of apoptotic bodies (48 h) and the formation of debris (72 h) that are classic morphologies of apoptosis.There was a visible loss of contact and rounding of cell shape post treatment as compared to the tightly packed and distinctively epithelial monolayer formation in the untreated cells, indicative of apoptosis (Fig.9). 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 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. 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 operation and cytotoxic cells .

Applications

Application

operation and cytotoxic cells . Applications Application we believe our project the compound of (nigella sativa

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 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 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

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 and chemotherapy. The compound is available , in many country and it more cheaply than any treatment of cancer the doses for a period of not less than three months. 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

anticancer activity of Nigella

Research resources

Research resources

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cancer in Chhattisgarh state, India. Asian Pac J Trop Biomed. 2011;1:47150. 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;33:32537