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Abdel-Hamid et al. Antitumor and chemosesitizing efficacy of Kochia indica on human HCC

N.V [half maximal inhibitory concentration (IC ) for 125 g for 5-7 min. The cell pellet was suspended again
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agent separately] = N .V (in combination) in the medium and dispense into a 25 cm culture
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2
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N= IC for the agent, V = volume of the agent flask. The culture was incubated at 37 °C in a 5% CO 2
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separately = 1 incubator and thereafter, was ready for sub culturing
N = the concentration of the agent in the combination, into working groups.
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V = the volume of the agent in the combination
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Experimental cell groups
Cell culture media, including DMEM and fetal HepG cells were divided into 4 groups described as
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bovine serum (FBS) were purchased from GIBCO ® follow: (1) first group contained HepG cells cultured
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(Invitrogen). Penicillin and streptomycin, the culture in a media and left without any treatments to serve as
antibiotics, were purchased from (Aldrich-Sigma a negative control; (2) second group contained HepG
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Company, CA, USA). DMSO, fluorescence dye, Sodium cells cultured in a media treated with the following
bicarbonate (NaHCO ), neutral red, 4-(4’-nitrobenzyl) doses (15.625, 31.2, 62.5, 125, 250 µg/mL) of 5-FU
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pyridine (NBP) and 5-FU were also purchased from as a standard chemotherapy for HCC and left to serve
Sigma-Aldrich. All chemicals and reagents used in the as a positive control; (3) third group contained HepG
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experiments are of highly purified grades. cells cultured in a media and treated with the following
doses (12.5, 25, 50, 100, 200 µg/mL) concentrations
Hepatoma cell line of Kochia indica ethanol extract alone and left as an
Hepatoma cell line (HepG ) was obtained from Holding experimental group; (4) fourth group contained HepG
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Company for Biological and Vaccine Production, cells cultured in a media treated with a combination of
Cairo, Egypt. HepG was maintained in the cell culture 5-FU and Kochia indica extract in different ratios (1:1,
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laboratory, Medical Research Institute, Smouha, Alex, 2:1, 1:2, 1:9) and left as an experimental group.
Egypt. HepG is a perpetual cell line consisting of
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human liver carcinoma cells, derived from the liver Cell viability determination
tissue of a 15-year-old Caucasian male patient who The HepG culture was sub-cultured at 37 °C
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had a well-differentiated HCC. under a humidified environment containing 5%
CO incubator. Cytotoxic effect of the tested agents
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Natural agents on tumor cells was tested by the neutral red (NR)
Kochia indica leaf plant was collected nearby Tanta method described by Fotakis and Timbrell. [14] In brief,
city, Al-Gharbiya Governorate, Egypt. The plant tumor cells were seeded in 96-well plates (100 μL/
was authenticated visually in taxonomy laboratory well at a density of 3 × 10 cells/mL) and treated with
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at Botany Department, Faculty of Science, Tanta various concentrations of the used agents for 24 h.
University, Egypt. The collected plants were washed Then, cells were washed twice with 1× phosphate
under running tap water and blotted where they were buffer saline and the supernatant was discarded.
cut into small pieces and kept for drying in oven at A total of 100 μL of NR solutions (50 μg/mL)
temperature 40 ± 2 ºC for 5 days. The dried plant was added to each well and incubated at 37 °C for
material was ground into powder and stored in air tight another hour. The NR dye was then dissolved in
container as described by Prayong et al. [12] The crude 100 μL of 0.33% HCl. Absorbance of NR dye was
ethanolic extracts were dissolved in DMSO at 20 mg/mL detected by a dual-wavelength UV spectrometer
as stock solutions which were then diluted with DMEM (Anthos 2010; Biochrom, UK) at 450 nm wavelenth.
to desired working concentrations ranging from 10 to The cytotoxicity was determined against untreated
250 μg/mL. The final concentration of DMSO in each cells by the following equation of Machana et al.: [15]
sample did not exceed 1% v/v, to keep the cytotoxicity cytotoxicity (%) = [100 × (absorbance of untreated
of DMSO at less than 10%. group - absorbance of treated group)]/absorbance of
untreated group. Therefore, the cell viability (%) = 100
Preparation of HepG culture media - cytotoxicity.
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The HepG culture media was prepared according to
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Van der Bliek et al. [13] Briefly, the vial containing HepG To calculate the IC values of the agents under
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was placed in a 37 °C water bath until the contents were investigation, cytotoxicity (%) was plotted against
thawed and decontaminated immediately by dipping in agent’s working concentrations [14] to give linear equation
or spraying with 70% ethanol. The vial contents were from which IC was calculated. Also, the quantitative
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transferred to a centrifuge tube containing 9.0 mL of efficacy of the combination exposure between 5-FU
absolute DMEM. To prepare DMEM growth medium, and Kochia indica extract was determined as a
FBS was added to reach 10% final concentration. combination index (CI) according to the following
Prepared cell culture media were spin at approximately equation: [16] CI = (5-FU) in combination/(5-FU) alone +
Hepatoma Research ¦ Volume 3 ¦ July 21, 2017 151