Cancer is the second leading cause of death worldwide. Cancer continues to represent the largest cause of mortality in the world. China is confronted with an increasing incidence of cancer and cancer deaths annually. Mortality that results from the common forms of cancer will be unacceptably high in the 21th century. Despite many therapeutic advances in the understanding of the processes in carcinogenesis, overall mortality statistics are unlikely to change until there is a reorientation of the concepts for the use of natural products as new chemopreventive agents [
Natural products include thousands of compounds that exist in fruits, vegetables, plants, and herbs, and several clinical anticancer drugs have been derived from natural products. Thus, development of compounds with anticancer effects from natural products has currently become a very important topic. Natural compounds isolated from medicinal plants, as rich sources of novel anticancer drugs, have been of increasing interest since then [
Traditional Uighur Medicine, the main part of Traditional Chinese Medicine, has been used for pharmaceutical and dietary therapy for several millennia. Traditional Uighur medicine has its own traditional theory for prevention and treatment of cancer with prescriptions containing Abnormal Savda Munziq (ASMq) [
Dulbecco’s modified eagle medium (DMEM), RPMI1640, foetal calf serum (FCS), ethylenediamine tetraacetic acid (EDTA), ribonuclease A (RNase A), proteinase K, ethidium bromide, N-Lauroyl sarcosine, propidium iodide, trypsine-0.02% EDTA mixture were obtained from Sigma-Aldrich (Lyon, France). SV Total RNA Isolation System, Reverse Transcription System, dNTPs Mixture, Taq DNA polymerase, BenchTop 1kb DNA Ladder, DNA loading buffer were all from Promega, France. Oligonucleotides used in PCR were from Eurogentec S.A, Britsh. All other chemicals used were of analytical grade.
ASMq is composed of 10 kinds of herbs [
Caco-2 cells, a human colon cancer cell line, were obtained from the American Type Culture Collection (ATCC) and were grown in a high glucose concentration (4.5 g/L) DMEM medium supplemented with 10% FCS, 1% L-glutamine (200 mM), and 1% penicillin-streptomycin (100 IU–100
The viability of the cells was assessed by MTT (3,4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) assay, which is based on the reduction of MTT by the mitochondrial dehydrogenase of intact cells to a purple formazan product [
The cells were cultured (5 × 106 cells per mL) in 25 cm2 flasks, total volume 5 mL of medium per flask, in the presence of ASMq ethanol extract (0.5–7.5 mg/mL) for different time intervals (24 and 48 h) at 37°C. Controls were performed at the same time with DMEM. After incubation for 24 h, the cell layer was rinsed twice with 5 mL of PBS. To extract DNA, the cells were lysed by incubation for 5 min with 1 mL of lysis buffer (1% N-Lauroyl sarcosine, 20 mM Tris-HCl pH 8.0, 5 mM EDTA), and the cell lysates were collected and transferred into 15 mL corning tubes. Proteins were digested overnight by incubation with 100
For cell cycle analysis, 5 × 105 cells seeded in 3 mL total volume in 6-well multidishes were incubated as described above for 48 h. Flow cytometric analyses were conducted using a FACScan (Becton Dickinson, France). At the end of incubation, the cells were rinsed twice with PBS and trypsinized in trypsine-0.02% EDTA mixture. After centrifugation for 10 min at 600 ×g at 4°C, the supernatant was removed, the pellet resuspended in 300
Expression of apoptosis-related genes, bcl-2, bax, p21, and p53, was studied using reverse transcriptase-PCR (RT-PCR). 5 × 105 cells seeded in 3 mL total volume in 6-well multidishes were incubated with the presence of ASMq ethanol extract (0.5–7.5 mg/mL) for 48 h at 37°C. The housekeeping genes GAPDH were used as control. At the end of incubation, the cells were rinsed twice with PBS and trypsinized in trypsine-0.02% EDTA mixture. After centrifugation for 5 min at 500 ×g at 4°C, the supernatant was removed, and the pellet was used for RT-PCR studies. Total RNA was isolated using SV Total RNA Isolation System (Promega, France). cDNA was generated by Reverse Transcription System (Promega, France). 10
Oligonucleotides used in the gene expression studies.
GAPDH | sense: 5′-TTCATTGACCTCAACTACAT-3′ | 467 bp |
Bcl-2 | sense: 5′-TGCACCTGACGCCCTTCAC -3′ | 293 bp |
p53 | sense: 5′-AAACCTACCAGGGCAGCTA -3′ | 684 bp |
p21 | sense: 5′-CATGTCAGAACCGGCTGGGGATG-3′ | 355 bp |
Bax | sense: 5′-ACCAAGAAGCTGAGCGAGTGTC-3′ | 365 bp |
The data are expressed as mean ± standard deviation (SD) for at least three independent determinations in triplicate or quadruplicated for each experimental point. The statistical differences between treated groups and control groups were determined by Student’s
Caco-2 cells were used as a model system to examine the effect of ASMq ethanol extract on their growth. The growth inhibitory effect of ASMq ethanol extract was concentration and time dependent (Figure
Inhibitory effect of ASMq ethanol extract on Caco-2 cell growth. Cells were incubated with different concentrations of ASMq ethanol extract (0.5–7.5 mg/mL) at 37°C, 5% CO2 for 48 or 72 h. The cell growth was determined by the MTT assay. Results are given as mean ± SD from three independent experiments.
Results showed that Caco-2 cells treated with ASMq ethanol extract did not induce any DNA fragmentation at the concentration of 0.5–2.5 mg/mL at 48 h of incubation. In contrast, the apoptotic fragments were clearly detected when the cells were treated with a higher concentration of ASMq ethanol extract, 5.0 and 7.5 mg/mL, for 48 h (Figure
Induction of DNA fragmentation by ASMq ethanol extract in Caco-2 cells. Cells were incubated for 48 h at 37°C with different concentrations of ASMq ethanol extract (0.5–7.5 mg/mL). Lane 1–5: 0.5, 1.0, 2.5, 5.0, and 7.5 mg/mL of ASMq ethanol extract, respectively. Lane 6: cells with no ASMq ethanol extract treatment. Lane M: 250 bp DNA marker.
Flow cytometry analysis performed on Caco-2 cells after 48 h of incubation with ASMq ethanol extract (0.5–7.5 mg/mL) indicated an alteration in the percentage of cells in each stage of the cell cycle: G0/G1, S and G2/M, as compared to the control (Table
Effect of ASMq ethanol extracts on cell cycle distribution in Caco-2 cells (mean ± SD).
Groups | Percentage of cells in each stage (%) | |||
sub-G1 (M1) | G0/G1 (M2) | S (M3) | G2/M (M4) | |
Control | 0.53 ± 0.13 | 69.79 ± 0.51 | 14.60 ± 1.50 | 15.29 ± 1.05 |
0.5 mg/mL | 0.36 ± 0.13 | 65.35 ± 2.23 | 18.38 ± 0.71 | 16.21 ± 1.65 |
1.0 mg/mL | 0.47 ± 0.18 | 65.70 ± 1.77 | 16.62 ± 0.97 | 17.51 ± 2.59 |
2.5 mg/mL | 0.55 ± 0.12 | 67.12 ± 0.83 | 16.09 ± 0.42 | 16.59 ± 1.46 |
5.0 mg/mL | 5.74 ± 1.51* | 61.73 ± 0.80* | 16.23 ± 0.19 | 16.09 ± 0.45 |
7.5 mg/mL | 13.56 ± 0.65* | 54.69 ± 3.24* | 15.81 ± 0.13 | 16.18 ± 0.53 |
Cell cycle analysis on Caco-2 cells after treatment with ASMq ethanol extract for 48 h. (a) Control without ASMq ethanol extract treatment; (b) Cells treated with 0.5 mg/mL of ASMq ethanol extract; (c) Cells treated with 1.0 mg/mL of ASMq ethanol extract; (d) Cells treated with 2.5 mg/mL of ASMq ethanol extract; (e) Cells treated with 5.0 mg/mL of ASMq ethanol extract; (f) Cells treated with 7.5 mg/mL of ASMq ethanol extract.
As the results shown in Figure
Expression of apoptotic genes in Caco-2 cells treated with ASMq ethanol extracts for 48 h. Column 1, control cells with no treatment; Column 2, cells treated with 1.0 mg/mL of ASMq ethanol extract; Column 3, cells treated with 2.5 mg/mL of ASMq ethanol extract; Column 4, cells treated with 5.0 mg/mL of ASMq ethanol extract; Column 5, cells treated with 7.5 mg/mL of ASMq ethanol extract; Representative data from three individual analyses.
Cancer causes significant morbidity and mortality and is a major public health problem worldwide. An effective cancer prevention program, diet, herb and exercise may decrease the incidence of cancer. Herbs, herbal formulations, and herb-derived compounds are known to have curative potential [
Apoptosis, also called the programmed cell death characterized by several morphological and biochemical events, is now known as an important type of cell death in response to cytotoxic treatment. It is a general physiological process to remove unwanted cells without damaging the neighboring cells and inducing inflammatory responses. In recent years, many studies have demonstrated that the dysregulation of apoptosis process is involved in the development of neoplastic transformation and tumor growth. The induction of apoptosis in tumor cells has been shown to be the most common anticancer mechanism conjoint by many cancer therapies. Thus, to find the potential therapeutic antitumor drugs with potent and selective apoptotic effect would be valuable. The administration of many natural compounds with anticancer effect has been shown to be capable of inducing the apoptotic death of cancer cells [
In the present study, we found that ASMq ethanol extract displayed a significant inhibitory effect on the proliferation of Caco-2 cell lines in a dose and time dependently manner. Furthermore, a high-concentration of ASMq ethanol extract (more than 5.0 mg/mL) resulted in significant induction of apoptosis in Caco-2 cells, as evidenced by DNA fragmentation and sub-G1 peak of apoptotic markers detection. More interestingly, the Caco-2 cells treated with ASMq ethanol extract in the concentration of 7.5 mg/mL for 48 h exhibited a dramatic accumulation of cells in sub-G1 phase (13.56%).
Many genes such as p53, p21, and genes in Bcl-2 family have been demonstrated to play important roles in deciding the initiation and execution of apoptosis in tumor cells exposed to radiation or anticancer drugs. It has been demonstrated that Bcl-2 family members, such as Bcl-2 itself and Bax, are mediators of apoptosis [
The present study suggested that the anticancer effect of ASMq ethanol extract was mediated through multiple pathways. ASMq ethanol extract inhibits cell growth and induces DNA fragmentation and apoptosis in a concentration-dependent manner. Induction of apoptosis is possibly related with Bcl-2 and Bax dependent pathway, but independent of p53 and p21 gene expression. In addition, as a herbal medicine, ASMq ethanol extract has its unique properties of low cost, easy oral consumption, and a long history of use by the Uighur population, all of which are indicative of its potential application as an anticancer agent.
This research was supported by the Program for New Century Excellent Talents in University (NCET), Ministry of Education of the People’s Republic of China and by a research grant from Science and Technology Department of Xinjiang Uighur Autonomous Region, China (no. 200733146-4).