沙漠研究 22-1, 191 -194 (2012 )"ICAL 1 / DT X"

Journal of Arid Land Studies－Refereed Paper－

Cytotoxic Profiling of Some Compounds of Natural Origin against HepG2
Liver Cancer Cell Line in-vitro
Ali M. MAHMOUD1) and Hany A. EL-SHEMY*1)
Abstract: Cancer is so far a national and international health problem. WHO reports showed constant rates of mortality caused byvarious types of cancer through the years 1950-2000. There is a wide gap between the disease incidence (0.16%), disease burden(5.6%) and the mortality caused by cancer (12.5%) worldwide. These gaps denote clearly the lack of effective treatment for cancer
compared to other mortality causes. Despite the availability of several anticancer agents, the treatment of cancer remains medical
hurdle in the developed and developing countries. Discovery of natural products with potential anti cancer activity is very initiative
trend in countries with rich botanical flora. Liver cancer is very serious solid tumor which is highly abundant in areas endemic with
hepatitis viruses such as middle and Far East. Herein, we have assessed the cytotoxic characteristics of several natural molecules(cerulinin, chrysin, honikiol, limonin, mevinolin, resveratrol, salicin, retinol, ascorbic acid and calciferol) against HepG2 liver solidtumor cell line. After exposure to serial concentrations of the test compounds for 72 h, SRB-u assay was undertaken and viability
assessment was performed via fitting to Emax model to identify the cytotoxicity parameters such as, IC50 and resistant fraction(R-value). Most of tested compound showed sort of cytotoxic effects against HepG2 cell line with IC50's ranged from 1.1 to 33.1µg/ml. Cerulinin and mevinolin showed the highest potency with IC50 less than 5 µg/ml. Chrysin, honikiol and resveratrol showed
moderate potency with IC50 ranging from 5-10 µg/ml. Ascorbic acid was weak cytotoxic agent against HepG2 cell line. Limonine,
salicin and retinol failed to exert any cytotoxicity against liver cancer cell line in-vitro. Impressively, molecules of potent andmoderate potency (except chrysin) showed low resistant fraction in HepG2 liver cancer cell line. In conclusion, our data showedwide range of variable efficacy of several molecules of natural origin against liver solid tumor.
Key Words: Anti-cancer screening, L cytotoxicity, Liver cancer, Natural products

1. Introduction
Natural products from plants have been valuable sources
for anticancer drug discovery (Schwartsmann et al., 2002). A
screening program was initiated by Leven et al. (1979) that
identified many antibacterial antifungal, antiviral, antiparasitic,
and other pharmacologically active substance activities inhigher plants (Jang et al., 1997).
Traditionally, resveratrol of red grape wine was considered
as general anti-aging material with classical antioxidant
properties due to its polyphenolic composition (Yao et al.,2009). Further studies revealed anti-inflammatory, vascularendothelial protective and lipid metabolism enhancement
effects for Resveratrol. Resveratrol has been found tointerfere with the expression/activity of several
inflammatory/immunoregulatory cytokines and enzymes suchas cycloxegenase, TNF-α, NF-κB, INF-γ and IL-1, IL-4 andIL-6 (Han et al., 2008). Cancer preventive property ofresveratrol has been attributed mainly due to its antioxidant
anti-inflammatory/immunoregulatory profile (Yang et al.,2009). In addition, several reports have indicated potential
anti-proliferative or anti-cancer effect of resveratrol per seagainst many types of human cancers (Alfaras et al., 2009; Cuiet al., 2010 ; Cui et al., 2010 ; Gagliano et al., 2010).
Resistance of solid tumor to anticancer treatment has been
attributed currently to pharmacokinetic reasons rather thanresistance at the cellular level. Anticancer agent need to be
introduced to each single tumor cell in a cytotoxic
concentration to generate tumor killing effect (Tredan et al.,2007; Hicks et al., 2003). This study aim to measure theactivities of some natural compounds as anticancer (liver).

2. Materials and Methods
2.1. Chemicals and drugs
Pure compounds were purchased from Sigma Chemical
Co. (St. Louis, MO). RPMI-164 media, fetal bovine serum
and other cell culture materials were purchased from AATC
(Houston, TX). Other reagents were of the highest analyticalgrade.

* Corresponding Author: helshemy@hotmail.com
1) Faculty of Agriculture Research Park (FARP) and Biochemistry Department, Faculty of Agriculture, Cairo University, 12 613 Giza, Egypt

Fig.1. HepG2 monolayer culture at 70-80% confluent ratio.

D-R curve of resveratrol in HepG2 cell line
D-R curve of Cerulinine in HepG2 cell line
120120100
100

Survival (%)

80

Survival (%)

80

60

60

40

40

20

2000.01

00.01

0.1

1

10100

0.1

1101001000

Conc. (ug/mlM)

Conc. (ug/ml)

D-R curve of mevalionin in HepG2 cell line
D-R curve of Honikiol in HepG2 cell line
10012080

Survival (%)

100

Survival (%)

80

6060

40

40

2020000.01

0.11010.11001101001000

Conc. (ug/ml)

Conc. (ng/ml)

Fig.2. The effect of natural compounds on the dose response curve (D-R) of HepG2 liver cancer cells.
Table 1. Effect of natural compounds on the cytotoxicity parameters of Hep G2 cell line.
Compounds

Resveratrol

Cerulinine

chrysin

Honikiol8.93±0.12

2.97±0.15

8.70±2.65

4.60±0.35

µg/ml

µg/ml

µg/ml

µg/ml

4.70±0.23%

0.00±0.00 %16.93±16.93%

0.00±0.00 %Limonene

Mevinolin

Salicin

VIT A-----l>100%>100%

Vit-C

Vit D

33.10±2.36

10.04±1.83µg/mlµg/ml

0.27±0.2 %0.00±0.00 %ParameterIC50--

1.06±0.12µg/ml

ResistanceFraction> 100%

3.74±1.67 %2.2. Cell culture
Human hepatocellular cancer cell line (HepG-2) was
obtained from the Vaccera (Giza, Egypt). Cells weremaintained in RPMI-1640 supplemented with 100 µg/mL
streptomycin, 100 units/mL penicillin and 10% heat
-inactivated fetal bovine serum in a humidified, 5% (v/v) CO2
atmosphere at 37ºC (Fig. 1).
2.3. Cytotoxicity assays
The cytotoxicity of crude extract and the fractionatedcompounds was tested against HepG-2 cells by SRB assay aspreviously described (Skehan et al., 1990). Exponentiallygrowing cells were collected using 0.25% Trypsin-EDTA andplated in 96-well plates at 1000-2000 cells/well. Cells wereexposed to each test compound for 72 h and subsequently
fixed with TCA (10%) for 1 h at 4ºC. After several washings,
cells were exposed to 0.4% SRB solution for 10 min in darkplace and subsequently washed with 1% glacial acetic acid.
After drying overnight, Tris-HCl was used to dissolve theSRB-stained cells and color intensity was measured at 540 nm(Skehan et al., 1990).
2.4. Data analysis
The dose response curve of compounds was analyzed
using Emax model (Eq. 1).
[D]m  + R% Cell viability = (100 − R ) × 1 −mm

K d + [D] … (Eq. 1)
Where R is the residual unaffected fraction (the resistancefraction), [D] is the drug concentration used, Kd is the drugconcentration that produces a 50% reduction of the maximum
inhibition rate and m is a Hill-type coefficient. IC50 wasdefined as the drug concentration required to reduce
fluorescence to 50% of that of the control (i.e., Kd = IC50 when
R=0 and Emax =100-R) (Al-Abd et al., 2008).

3. Results and Discussion
Cerulinine, Vit D and Honkiol were showed moderately
active as single agent for the liver cancer.
Mevenoline was moderately active as single agent for theliver cancer (Table 1, Fig. 2).
Vitamin A and C were showed weak activities for livercancer.
Additionally, to study the effect of RES on the cytotoxic
profile of two corner stone chemotherapeutic agents (Table 1)(Fig. 2) for 72 h incubation peroid.
Several studies have demonstrated that mixtures in extracts
from herbal medicines had an anticancer potential in vitro or invivo (Bonham et al., 2002; Hu et al., 2002; Chen et al., 2005;
El-Shemy, et al., 2007). Aqueous extracts from willow (Salixsp.; Saliceae) leaves prevented proliferation of three cancer celltypes acute myeloid leukemia (AML), acute lymphoblastic
leukemia (ALL) and Ehrlich ascites carcinoma cells (EACC;
El-Shemy et al., 2007). Alcohol extractsof Ganodermalucidum (Hu et al., 2002) induced apoptosis in MCF-7 humanbreast cancer cells. Three of ten sesquiterpene lactone
compounds from Vernonia chinenesis were active against
P-388 and A-549 tumor cell lines (Chen et al., 2005).
Therefore, the complex mixtures in crude extracts may be
more effective than single purified compounds.
The safety profile of Mevinolin (MVN) both inexperimental and clinical stages are encouraging further
clinical trials for the treatment of various types of tumors.
The dose of MVN suggested for anti-cancer treatment isbelieved to be clinically safe 6,7. Therefore, very high dosesof MVN administered every four hours to patients have beenfound very tolerable (Holstein et al., 2006). MVN and othernatural statins are believed to be better treatment option forcancer than synthetic statins (Ahn et al., 2008). MVN was
shown to be a clinically safe drug of natural origin that is
known to inhibit the HMGCo-A reductase activity andinterfere with steroidogenesis (Kumari et al., 2009). In future,gene silencing studies would be recommended to understandthe exact mechanism of different compounds on HepG-2 cellline.

Acknowledgement
This research was fully funded by grants from the National
CFIDS Foundations Inc, Needham, MA 02492-3931, USA.
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