Contibuted equally
Curcumin is an inexpensive, natural plant ingredient with protease inhibitor effects. The present study aimed to analyze the inhibitory effects of curcumin on the multiple myeloma (MM) RPMI 8226 cell line, and examine the underlying mechanism that promotes the apoptosis of RPMI 8226 cells. A growth curve was constructed in order to observe the relative growth velocity, and MTT was used to analyze the effect of different concentrations of curcumin on inhibiting the proliferation of the RPMI 8226 cells. The mRNA expression of the
Multiple myeloma (MM) is a type of plasma cell-derived malignancy, which leads to the formation of multiple bone lesions and to disruption in the production of normal blood cells (
Curcumin is a primary active ingredient derived from the spice, turmeric. Curcumin suppresses tumor growth and inhibits cellular proliferation, invasion, angiogenesis, metastasis and osteoclastogenesis, which are processes that involve multiple cellular targets, such as nuclear factor (
The tumor suppressor protein, p53 (
In the present study, the proliferation rate of the MM RPMI 8226 cell line was analyzed following treatment with curcumin. In addition, changes in the expression of the
In total, RPMI 8226 cells (105/ml; School of Life Sciences, Zhengzhou University, Zhengzhou, China) were seeded into six-well plates and cultured in triplicate in RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (Invitrogen Life Technologies), with or without curcumin. The final concentrations of curcumin were 0, 1, 2.5, 5, 7.5, 10, 15, 20 and 40 μmol/l. The final volume of medium in each well following the addition of curcumin was 1 ml. The number of cells in each well was counted every 24 h, and the cells were cultured as usual with the same medium until 96 h.
First, 105/ml RPMI-8226 cells were seeded into 96-well plates and cultured in six repeated wells. The experimental groups contained the cells and RPMI-1640 medium with 10% fetal bovine serum and curcumin. The positive control groups contained the cells and RPMI-1640 medium with 10% fetal bovine serum. The negative control groups contained RPMI-1640 medium alone with 10% fetal bovine serum. The final concentrations of curcumin were 1, 2.5, 5, 7.5, 10, 15, 20 and 40 μmol/l. The final volume of medium in each well following the addition of curcumin was 200 μl. Next, the surrounding wells were covered with 200 μl phosphate-buffered saline (PBS). The plates were then incubated at 37°C with 5% CO2 for 24, 48 and 72 h. Following this, 20 μl MTT solution (5 mg/ml in PBS, Sigma-Aldrich, Santa Clara, CA, USA) was added to each well of the experimental, and positive and negative control groups. Subsequent to a 4-h incubation at 37°C and subsequent centrifugation at 1,000 × g for 5 min at 37°C, 200 μl solution from every well was extracted, and 150 μl dimethylsulfoxide was added to the wells. After 15 min, the optical density (OD) at 490 nm was measured using an iMark microplate absorbance reader (Bio-Rad 550; Bio-Rad Laboratories, Inc., Hercules, CA, USA). The cell proliferation inhibition ratio was calculated using the following formula: Cell proliferation inhibition ratio = 1- (A490 of the experimental groups / A490 of the control groups) × 100. The A490 of the experimental groups = OD of the experimental groups - OD of the negative control groups. The A490 of the positive control groups = OD of the positive control groups - OD of the negative control groups.
The average half maximal inhibitory concentration of curcumin from six experiments was obtained by plotting the percentage of inhibition against the concentration of curcumin.
In total, 106/ml RPMI-8226 cells in the logarithmic phase were seeded into 25-cm2 culture bottles for the positive control and experimental groups, including the 10- and 15-μmol/l curcumin groups. The final volume of solution in each culture bottle was 5 ml. The positive control groups contained the cells and RPMI-1640 medium with 10% fetal bovine serum. The experimental groups contained the cells, curcumin and RPMI-1640 medium with 10% fetal bovine serum. The total RNA was isolated after 48 h from the cells in the culture bottle using TRIzol reagent (Invitrogen Life Technologies). Next, the RNA was reverse transcribed into cDNA and quantitative PCR (qPCR) was performed using the two-step method. Briefly, 25 μl reaction volume consisting of 12.5 μl of 2X PCR Buffer for KOD FX [ a PCR amplification enzyme (Qiagen, Venlo, Netherlands)], 5 μl 2 mM dNTPs, 2 μl of each primer, 0.1 μl KOD, 2.4 μl water and 1 μl DNA. The standard conditions for PCR were as follows: 95°C for 2 min, followed by 40 cycles at 95°C for 30 sec, 62°C for 1 min, and a final extension at 72°C for 5 min. All reactions were performed in a PerkinElmer 2400 thermocycler (Perkin Elmer Applied Biosystems, Foster City, CA, USA). The 2−ΔΔCt method was used to indicate the association between the expression of the target gene in the experimental group and the expression of the target gene in the positive control group. The sequences of the primers are shown in
For the western blot analysis, the cells were harvested and lysed, and the proteins were separated using a 12.5% SDS-PAGE gel. The proteins were then transferred to a Hybond-C membrane (Invitrogen Life Technologies). Next, the membrane was blocked with Blotto A (5% blocking grade dry milk in Tris-buffered saline and Tween 20; Invitrogen Life Technologies) and probed using a monoclonal mouse anti-human p53 primary antibody (dilution, 1:300; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) in Blotto A. The cells were incubated with the primary antibody at 4°C overnight, and then with monoclonal goat anti-mouse IgG-horseradish peroxidase-tagged secondary antibody (dilution, 1:1,000; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Detection of chemiluminescence was conducted using an enhanced chemiluminescence western detection reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and developed on a BioMax XAR film (Kodak, Rochester, NY, USA).
In total, 0.5×106/ml RPMI-8226 cells in the logarithmic phase were seeded into 6-well plates and cultured in four repeated wells. Control groups, and experimental groups, including 10 and 15 μmol/l curcumin groups, were used. The final volume of solution in each well was 1 ml. The control groups contained the cells and RPMI-1640 medium with 10% fetal bovine serum. The experimental groups contained the cells, curcumin and RPMI-1640 medium with 10% fetal bovine serum. The total protein was isolated after 48 h from the cells in each well of the plates using 100 μl lysis buffer with 1 mM EDTA, according to the manufacturer’s instructions. The ELISAs were conducted using the p53 kit (RAB0500, Sigma-Aldrich). The standard and sample groups were set, and the indicated reagents were added for the indicated time period according to the manufacturer’s instructions. The OD of each well was measured at 490 nm using a microplate reader. A standard curve was constructed according to the OD values of the standard groups and a formula was generated based upon this standard curve. The p53 protein concentration of each sample was calculated according to the formula: p53 concentration = 2318.3ODvalue − 241.19.
SPSS software version 17.0 (SPSS Inc., Chicago, IL, USA) was used to perform the statistical analysis. Data from the control and experiment groups were analyzed by an independent sample t-test. P<0.05 was considered to indicate a statistically significant difference.
The results revealed that the cells were in the logarithmic phase between 24 and 72 h. During this time period, the number of cells in the logarithmic phase decreased with increasing concentrations of curcumin. During the same period, the growth of cells treated with higher curcumin concentrations was slower (
In order to investigate the effect of curcumin on the proliferation inhibition of the MM RPMI 8226 cell line, the OD was measured at 490 nm following 24, 48 and 72 h of treatment with different concentrations of curcumin. The proliferation inhibition ratio following 24, 48 and 72 h of treatment with 10 μmol/l curcumin was 17.6, 29.2 and 33.8%, respectively. The difference in the OD value was statistically significant between the experimental and control groups (P<0.05). The proliferation inhibition ratio following 24, 48 and 72 h of treatment with 15 μmol/l curcumin was 25.8, 46.1 and 50.4%, respectively. The difference in the OD value was statistically significant between the experimental and control groups (P<0.05). The results revealed that a higher concentration of curcumin was more potent than a lower concentration of curcumin at the same time-point in the growth suppression of the RPMI 8226 cells, and that a longer duration of treatment was more potent than a shorter duration of treatment with the same concentration of curcumin in the growth suppression of the RPMI 8226 cells (
The qPCR results revealed that curcumin inhibited the growth of MM cells in a dose-dependent manner. Following a 48-h treatment with 10 and 15 μmol/l curcumin, the proliferation inhibition ratio was 29.2 and 46.1%, respectively. It has been reported that curcumin regulates the expression of the apoptosis-related proteins, Bax, Bcl-2 and p53, and that it regulates the apoptosis of tumor cells via the
The
As the expression of the
MM is a B-cell malignancy whereby plasma cells grow abnormally in the bone marrow and secret monoclonal immunoglobulin or an M protein fragment, which ultimately leads to relative organ or tissue injury. Although MM is sensitive to a variety of cytotoxic drugs in the initial and relapsed treatment periods, the relief is only temporary. Therefore, MM remains an incurable disease. A number of drugs, including bortezomib, a proteasome inhibitor, and thalidomide, an inhibitor of tumor necrosis factor production (
The results of the present study revealed that following treatment with curcumin, the growth of the MM RPMI 8226 cell line was inhibited in a concentration- and time-dependent manner, which was in agreement with the results of a study by Bharti
Curcumin also inhibits the proliferation, invasion, metastasis and angiogenesis of a number of cancers by interacting with a variety of cell signaling proteins (
Despite mutations in the
The present study demonstrated that with the intervention of curcumin, the growth of the MM RPMI 8226 cells was inhibited in a concentration- and time-dependent manner. Using qPCR to detect the mRNA expression of
The authors are grateful for the collaboration of the participating college and its staff. The authors would like to thank Dr Weiquan Lu from the Department of Cancer Prevention, Henan Cancer Hospital, Zhengzhou, China, for help with the statistical analysis.
Growth curve representing the growth of cells treated with different concentrations of curcumin. The initial number of cells in each well was the same (105/well). After 96 h, the number of cells in the wells treated with 0, 1, 2.5, 5, 7.5 and 10 μmol/l curcumin were >3×106, ~2.5×106, ~2×106, ~1.5×106, <1×106 and <5×105, respectively. The cells treated with 15, 20 and 40 μmol/l curcumin, however, barely grew.
Cells were treated with various concentrations of curcumin for 24, 48 and 72 h prior to the determination of cytotoxicity by an MTT cell proliferation assay. Each value is expressed as the mean ± standard deviation of six measurements.
p53 protein expression detected by western blotting following treatment with different concentrations of curcumin for 48 h.
Measured optical density (OD) values of the standard samples were constructed as horizontal ordinates, and the concentrations of each diluted standard sample were contructed as vertical coordinates. The standard curve was drawn according to the OD values of the standard groups and the formula was then generated based on the standard curve.
p53 protein concentration of each sample was calculated according to the formula: p53 concentration = 2318.3ODvalue − 241.19.
Sequence of primers.
Target gene | Sequence of primers |
---|---|
5′-CCACCATCCACTACAACTACAT-3′ | |
5′-TTTTGCTTCAGGGTTTCATC-3′ | |
5′-TACCTACTGATGGTGCTG-3′ | |
5′-GGATTTGGTCGTATTGGG-3′ |
p53 protein content of each sample (mean ± standard deviation; n=6).
Curcumin concentration, μmol/l | p53 protein, pg/ml |
---|---|
0 | 906.035±28.324 |
5 | 1226.024±36.536 |
10 | 1302.629±40.007 |
15 | 1481.220±45.510 |