healthmedicinet

Cell line and culture conditions

The Burkitt lymphoma cell line Raji was purchased from the Shanghai Institute Cell
Resources Bank. Raji cells were maintained in RPMI 1640 (Hyclone, Logan, USA) supplemented
with 10 % fetal bovine serum, 2 mM L-glutamine (Hyclone) and antibiotics (100 U/ml
penicillin and 50 ?g/ml streptomycin) at 37 °C in a humidified 5 % CO
₂
in-air atmosphere.

Reagents and antibodies

As described previously 2], Rap (Calbiochem, San Diego, CA, USA) was dissolved in dimethyl sulfoxide (DMSO,
Sigma, St. Louis, MO, USA) and used at a concentration of 10 nM. Dex (Sigma) was dissolved
in ethanol and used at a concentration of 1 ?M. The final concentrations of DMSO and
ethanol in the medium were 0.05 % and 0.01 %, respectively, at which cell proliferation
or viability was not obviously altered. Propidium iodide (PI), 3-methyladenine (3-MA),
2-deoxyglucose (2-DG) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) were purchased from Sigma. The pan-caspase inhibitor z-VAD-fmk was purchased
from RD Systems (Minneapolis, MN, USA). The Annexin V-PI Kit was purchased from Roche
(Mannheim, Germany). Antibodies to phospho-glucocorticoid receptor (p-GR) (Ser211),
p70S6K, p-p70S6K (Thr421/Ser424), 4E-BP1, p-4E-BP1 (Thr37/46), AMP-activated protein
kinase (AMPK), phospho-AMPK (p-AMPK) (Thr172), Cyclin D, p27, Bax, Mcl-1, and Bcl-2
were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody for
p21 was purchased from BD Bioscience (San Jose, CA, USA). Antibodies to extracellular
signal-regulated kinase (ERK) and phospho-ERK (p-ERK) were purchased from Upstate/Millipore
(Billerica, MA, USA). Antibody to LC3 was purchased from Sigma. Antibodies to GR,
Bim, Cyclin A, horseradish peroxidase (HRP)–conjugated donkey anti-rabbit antibody
and HRP-conjugated sheep anti-mouse antibodies were obtained from Santa Cruz Biotech
(Santa Cruz, CA, USA). The actin antibody was obtained from Kangchen Bio-Tech (Shanghai,
China).

Cell treatment

Logarithmically growing cells were harvested and plated in 96-well sterile plastic
culture plates and 25-cm
²
flasks (Corning Inc.), to which various concentrations of Rap or Dex, specifically
10 nM Rap (Rap group), 1 ?M Dex (Dex group), 10 nM Rap plus 1 ?M Dex (Rap?+?Dex group)
and 0.05 % DMSO plus 0.01 % ethanol (Control group), were added. At the end of the
incubation period, cells were transferred to sterile centrifuge tubes, pelleted by
centrifugation at 400?g at room temperature for 5 min, and prepared for analysis as described below.

Cell viability assay

MTT assays were performed as described previously. Briefly, cells were seeded in 96-well
plates (100,000/ml) and incubated for 24 or 48 h. Next, 0.5 mg/ml MTT (final concentration)
was added to each well for 4 h at 37 °C. Then, solubilization buffer (10 % SDS in
0.01 M HCl) was added to each well, and the plates were further incubated for 24 h
at 37 °C. The spectrophotometric absorbance was measured at 570 nm (reference 690 nm)
using a multi-plate reader (Multiskan Spectrum, Thermo Electron Co., Waltham, MA,
USA). Values were obtained by comparing the experimental cells with their respective
controls. Mean values were calculated from triplicate cultures. Coefficient of drug
interaction (CDI) was used to analyze the effects of drug combinations. The CDI is
calculated as follows: CDI?=?AB/(A?×?B). According to the absorbance of each group,
AB is the ratio of the combination groups to control group; A or B is the ratio of
the single agent group to control group. Thus, a CDI value 1, =1 or 1 indicates
that the drugs are synergistic, additive or antagonistic, respectively.

Cell cycle analysis

For each analysis, 10
⁶
cells were harvested 48 h after treatment and fixed overnight in 70 % ethanol at 4 °C.
Cells were then washed and stained with 5 ?g/ml PI in the presence of DNAse-free RNAse
(Sigma). After 30 min at room temperature, the cells were analyzed via flow cytometry
(Beckman Coulter Inc., Miami, FL, USA), acquiring 30,000 events.

Apoptosis assay

The samples were washed with phosphate-buffered saline (PBS) twice and stained with
annexin V-FLUOS and PI using Annexin-V-FLUOS staining kit (Roche) according to the
manufacturer protocol. The percentages of annexin-V single positive cells were determined
by flow cytometry (Beckman Coulter), as the percentages of cells in the early stages
of apoptosis.

Glucose consumption assay

Glucose consumption was measured with a Glucose (HK) Assay Kit (Sigma). Briefly, 1?×?10
⁶
cells were grown in 10 ml RPMI containing 2 g/l glucose. After 48 h, the medium was
collected by centrifugation to remove the cells. Medium from each condition was incubated
for 30 min with the glucose assay reagent. Spectrophotometric absorbance was measured
at 340 nm using a multi-plate reader (Multiskan Spectrum). Values were obtained by
comparing with a glucose standard solution.

Lactic acid assay

Lactic acid production was measured with a Lactic Acid Assay Kit (Jiancheng, Nanjing,
China). Briefly, 1?×?10
⁶
cells were grown in 10 ml RPMI. After 48 h, the medium was collected by centrifugation
to remove the cells. Medium from each condition was incubated with the lactic acid
assay reagent according to the manufacturer protocol. Spectrophotometric absorbance
was measured at 530 nm using a multi-plate reader (Multiskan Spectrum). Values were
obtained by comparing with a lactic acid standard solution.

In vivo studies

All animal studies were conducted in accordance with the guidelines established by
the internal Institutional Animal Care and Use Committee and Ethics Committee guidelines
of Sichuan University. All animals were kept under specific pathogen-free conditions
in Laboratory Center of West China Second Hospital, Sichuan University. Female Balb/c
(nu/nu) mice (Laboratory Animal Center of Sichuan University, Chengdu, China), 5–6
weeks of age, 16-18 g of weight, were inoculated with 3?×?10
⁶
Raji cells subcutaneously (s.c.) in the right flank with an inoculation volume of
0.2 ml. Tumor size was measured by calipers every 2 days. The approximate tumor volume
was calculated using the equation V?=?(length?×?width?×?depth)/2. Once palpable tumors
were established (tumor volume reaching 30–40 mm
³
), animals were randomized into 4 groups, each containing 6 mice. Mice were injected
intraperitoneally daily with 3 mg/kg/d Rap (Rap group), 15 mg/kg/d Dex (Dex group),
3 mg/kg/d Rap plus 15 mg/kg/d Dex (Rap?+?Dex group) or PBS (Control group). All animals
were ear-tagged and monitored individually throughout the experiment.

Mitochondrial membrane potential detection

The mitochondrial membrane potential (??m) was measured using Rhodamine 123 (Rh123)
staining. In brief, Rh123 (10 ?M) was loaded into cells for 20 min at 37 °C. The fluorescence
intensity of cells was analyzed by flow cytometry (Beckman Coulter) with an excitation
wavelength at 488 nm and an emission wavelength at 525 nm.

Analysis of autophagy with MDC staining

The cells were suspended in 0.05 mM Monodansylcadaverine (MDC, Sigma) and incubated
at 37 °C for 40 min. Then, the fluorescent changes were observed by fluorescence microscopy
(Olympus, Tokyo, Japan) with the emission wavelength at 525 nm.

Transmission electron microscopy

Cells were harvested after treatment. Following fixation in 2 % paraformaldehyde/2.5 %
glutaraldehyde, pellets were rinsed and post-fixed in 1 % osmium tetroxide/1.25 %
potassium ferrocyanide. Samples were dehydrated in a graded series of ethanol, followed
by propylene oxide and infiltrated and embedded in Polybed 812 resin. Ultrathin 70-nm-thick
sections were taken from areas selected by light microscopy, mounted on 200 mesh copper
grids, and stained with uranyl acetate and lead citrate. These were observed and photographed
using a Jeol J EM-1200EX transmission electron microscope (Jeol Ltd., Tokyo, Japan).

Western blotting analysis

Cells (10
⁶
) were washed twice in cold PBS and then lysed by Laemmli sample buffer (Bio-Rad).
Samples were boiled for 5 min at 100 °C. Proteins were separated by 10 % or 15 % SDS–polyacrylamide
gel electrophoresis and transferred onto nitrocellulose membranes (0.22 ?m or 0.45 ?m,
Millipore). Non-specific binding sites were blocked with 5 % non-fat dry milk dissolved
in TBS (10 mM Tris–HCl, pH 7.6, 137 mM NaCl) with 0.1 % Tween 20 (TTBS) for 2 h at
room temperature, followed by incubation with primary antibody for 2 h at room temperature
or at 4 °C overnight. The membranes were then washed 3 times in TTBS and incubated
for 2 h at room temperature with secondary HRP–conjugated donkey anti-rabbit antibody
or HRP-conjugated sheep anti-mouse antibody (Santa Cruz) diluted 1:5000 in TTBS with
5 % non-fat milk. Proteins were visualized by incubation with ECL plus (Millipore).
All experiments were carried out independently at least 3 times. The level of Actin
protein was used as a control for the amount of protein loaded into each lane.

Statistical analysis

All assays were performed in triplicate, and data are expressed as mean values?±?SD.
One-way ANOVA was used to compare two groups. A p-value??0.05 was considered to be significant.