The development of colorectal cancer (CRC) is strongly associated with the imbalance of various intracellular signal transduction cascades, including protein kinase B (AKT), mitogen-activated protein kinase 1 (MAPK), signal transducer and activator of transcription 3 (STAT3), as well as crosstalk between these signaling networks. At present, anti-tumor agents are often single-targeted and therefore are not always therapeutically effective. Moreover, long-term use of these anti-tumor agents often generates drug resistance and potential side effects. These problems highlight the urgent need for the development of novel and more effective anti-cancer drugs.
Colorectal cancer (CRC) is the third most common type of cancer affecting the gastrointestinal tract worldwide (
The development of colorectal cancer is a complex and multi-stage process, characterized by various interactions between environmental carcinogens, genetic modifications and the host immune system (
RPMI-1640, Dulbecco's modified Eagle's medium (DMEM) with high glucose, Fetal bovine serum (FBS), penicillin-streptomycin (catalog no. SV30010), 0.25% trypsin-EDTA, Pierce RIPA buffer, Pierce BCA Protein Assay kit (catalog no. 23227), and SuperSignal™ West Pico Chemiluminescent substrate (catalog no. 34080) were all purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit (MTS assay; catalog no. G5430) was provided by Promega Corporation (Madison, WI, USA). Matrigel was obtained from BD Biosciences (Franklin Lakes, NJ, USA). TumorTACS
Authentic plant material was purchased from Guo Yi Tang Chinese herbal medicine store (Fujian, China). Identification of the Hedyotis diffusa Willd (HDW) herb was confirmed by Dr Wei Xu (Department of Pharmacology, Fujian University of Traditional Chinese Medicine, Fujian, China). Ethanol extract of HDW (EEHDW) was prepared as previously described (
For
Human CRC cell lines, HCT-8, HT-29, HCT-116 and SW620, were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HCT-8 and HCT-116 cells were cultured in RPMI-1640. HT-29 and SW620 cells were cultured in DMEM. All cell media were supplemented with 10% (v/v) FBS, 100 U/ml penicillin and 100 µg/ml streptomycin and cultured at 37°C, with 5% CO2 in a humidified incubator.
Cell viability was assessed using MTS assay. The cells (10,000/well) were incubated in 96-well plates with culture medium at 37°C for 12 h and then treated with various concentrations of EEHDW (0, 0.5, 1 and 2 mg/ml) for 24 and 48 h; the control cells were treated with DMSO without EEHDW at 37°C for 24 and 48 h. Next, 10 µl MTS was added to each well, and the samples were incubated for 1 h at 37°C. The resulting absorbance was measured at 490 nm using an ELISA reader (BioTek Instruments, Inc., Winooski, VT, USA).
A total of 20-week-old athymic BALB/c nu/nu male mice (initial body weight, 20±2 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and housed under pathogen-free conditions (22°C; 12-h light/dark cycle). Food and water were given
HT-29 cells (5×106) mixed with Matrigel (1:1) were subcutaneously injected in the right flank area of athymic nude mice to initiate tumor growth. After 5 days of xenograft implantation, the mice were randomly divided into two groups (n=10) and given daily intra-gastric administration with 1 g/kg EEHDW or saline for 6 days per week for a total of 16 days. The diameters of the tumors were measured every second day with digital calipers and the tumor volume (V) was calculated using the formula: V=(width)2 × length × π/6. At the end of experiment, the mice were sacrificed using 100 mg/kg pelltobarbitalum natricum (Sigma-Aldrich; Merck KGaA), and the tumor tissues were removed and fixed in 4% paraformaldehyde (China National Medicines Corporation Ltd., Beijing, China) or stored at −80°C. Blood was collected aseptically from the orbital sinus. Blood collection tubes were allowed to stand at room temperature for 5 h prior to serum collection by centrifugation at 2,000 × g for 20 min at room temperature, and then stored at −80°C.
Tumor tissues were analyzed by IHC as previously described (
Apoptosis in tumor tissues were analyzed by TUNEL staining using TumorTACS
A total of three tumors were randomly selected from the control and the EEHDW group, washed with PBS 3 times and homogenized in Pierce RIPA buffer containing protease inhibitor and phosphatase inhibitor cocktails. The samples were then centrifuged at 17,000 × g for 20 min at 4°C, and the resulting protein concentrations were determined using BCA Protein Assay reagent kit. A total of 50 µg protein for each sample was loaded onto 10% SDS-PAGE and resolved at 20 V for 10 min, at 80 V for 30 min and at 120 V for 1 h. The proteins were then transferred onto nitrocellulose membranes. Following blocking with 5% non-fat milk powder at room temperature for 2 h with the 5% non-fat milk powder dissolved using TBS with Tween-20 (TBST, pH8.0) containing 0.1% Tween, the membranes were incubated with cytochrome C, caspase-3, caspase-9, PARP and β-actin (all 1:1,000) primary antibodies overnight at 4°C, and then incubated with the aforementioned HRP-conjugated anti-rabbit secondary antibodies to bind the antibodies of cytochrome C, caspase-3, PARP and β-actin, or anti-mouse secondary antibody to bind the caspase-9 antibody (all 1:5,000) for 1 h at room temperature. The membranes were then subjected to enhanced chemiluminescence (ECL) detection using SuperSignal™ West Pico Chemiluminescent substrate. Image Lab™ software (version 3.0; Bio-Rad Laboratories, Inc.) was used for densitometric analysis.
Blood were collected and stored at −80°C until further analysis. The level of IL-1β, IL-6, IL-4, IL-10 and TNF-α in the serum was measured using IL-1β (cat. no. F10770), IL-6 (cat. no. F10830), IL-4 (cat. no. F10810), IL-10 (cat. no. F10870) and TNF-α (cat. no. F11630) ELISA kits (Xitang Biological Technology Co., Ltd., Shanghai, China), respectively, according to the manufacturer's protocol. Briefly, 100 µl diluted standard and test samples were added in each well. Plates were covered and incubated for 2 h at 37°C, then washed 5 times prior to incubation with 50 µl biotinylated antibody solution for 1 h at 37°C. The Plates were washed three times, and 100 µl streptavidin-HRP solution was distributed to all wells prior to incubation for 1 h at 37°C. After washing, the substrate was added and incubated for 15 min at room temperature in a darkened room. Finally, the reactions were stopped and the absorbance was measured at 450 nm. Wells with no biotinylated antibody solutions or streptavidin-HRP solution were used as the negative controls. The concentrations of the aforementioned cytokines were determined by comparing to serial dilutions of the purified standards.
A total of 8 tumors were randomly selected from the EEHDW and control groups, and homogenized. For analysis of phosphorylation of protein kinase B (AKT), mitogen-activated protein kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK), p38, p53 and p70S6K
All data were expressed as the mean ± standard deviation. Statistical analysis was performed using the SPSS software (version 17.0) for Windows (SPSS, Inc. Chicago, IL, USA) using one-way analysis of variance. Fisher's least significant difference (for equal variances) or Dunnett's correction (for unequal variances) were used in post-hoc tests. P<0.05 was considered to be statistically significant.
The effect of EEHDW on the viability of various CRC cell lines was determined using MTS assay. EEHDW treatment at 0.5, 1 and 2 mg/ml for 24 or 48 h was able to significantly reduce the viability of HT-29, SW620, HCT-8 and HCT-116 cells) in a dose- and time-dependent manner compared with the control (
The
To further investigate how EEHDW is able to inhibit tumor growth, IHC staining was performed to detect the expression of Pim-1, Bcl-2 and Bax in mice. The percentage of Pim-1, Bcl-2 or Bax-positive cells in the control group was, 37.71±7.31, 27.33±3.67 and 18.50±3.83% respectively, whereas in EEHDW-treated mice the percentage was 21.75±4.57, 16.00±4.31 and 35.00±4.32%, respectively (P<0.05;
Dense infiltration of cytokine-producing immune cells is frequently observed in cancer tissues: Each immune cell subset and cytokine involved in the activation of intracellular pathways sustains the growth of cancer cells (
To investigate the underlying mechanisms of EEHDW in inhibiting tumor growth, the effect of EEHDW on the activation of various CRC-associated signal transduction cascades was determined. The effect of EEHDW treatment on the activation and phosphorylation of AKT, Erk1/2, JNK, p38, p70S6K p53 and STAT3 in CRC xenograft tumor tissues and HT-29 cells was determined using Bio-Plex Phosphoprotein assay.
Treatment with EEHDW was able to significantly decrease the levels of phosphorylated AKT, Erk1/2, JNK, p38, p70S6K and STAT3 in tumor tissues (P<0.05;
CRC is a serious global health problem with high morbidity and mortality rates. To date, chemotherapy has been the main therapeutic treatment for the majority of patients with CRC (
Cancer development is tightly regulated by multiple intracellular signaling pathways, including AKT, mitogen-activated protein kinase (MAPK) and STAT3 pathways, which are usually functionally redundant (
The MAPK signaling pathway is one of the main pathways involved in cell survival and proliferation. In mammals, there are three major subfamilies of MAPK, including ERK, JNK and p38. Activation of MAPK signaling is regulated by a central three-tiered kinase core, which consists of MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K) and MAPK (
The development of CRC is often characterized by complex interactions within the host immune system, ultimately resulting in the uncontrolled growth of transformed cells. For instance, chronic inflammation is an independent risk factor for the development of CRC, as well as other malignancies, including gastric cancer, hepatocellular and prostate carcinoma (
Natural products, including those used in TCM, have long been used to treat multiple diseases, including cancer (
In the present study, MTS was used to demonstrate that EEHDW reduced cell viability in different CRC cell lines in a dose- and time-dependent manner. It was also observed that EEHDW was able to significantly reduce tumor weight and volume
In conclusion, EEHDW exerts its anti-cancer activities via altering multiple targets. Therefore, EEHDW may be developed as a promising multi-potent anti-cancer agent for the clinical treatment of CRC.
The present study was sponsored by the Research Fund for the Doctoral Program of Higher Education of China (grant no. 20133519110003) and the Developmental Fund of Chen Keji Integrative Medicine (grant nos. CKJ2014013 and CKJ2015007).
colorectal cancer
ethanol extract of
traditional Chinese medicine
EEHDW inhibits growth of colorectal cancer cells and xenograft tumors in mice. (A) The cells were treated with various concentrations of EEHDW (0, 0.5, 1 and 2 mg/ml) for 24 and 48 h. Cell viability was assessed using MTS assay. Data are shown as the mean ± standard deviation from three independent experiments. *P<0.05 vs. untreated control cells. (B) The mice were administered intragastrically with 1 g/kg EEHDW or saline for 6 days per week for a total of 16 days. Tumor weight and volume were measured at the end of the experiment. Data are expressed as the mean ± standard deviation from 10 individual mice in each group. *P<0.01 vs. control mice. EEHDW, ethanol extract of
EEHDW inhibits cellular proliferation and induces apoptosis in xenograft tumors. (A) Staining for Ki-67 and TUNEL were performed to examine the
EEHDW regulates the expression of Pim-1, Bcl-2, Bax and angiogenic factors COX-2, iNOS, eNOS and HIF-1α. Tumor tissues were processed for immunohistochemical staining of Pim-1, Bcl-2, Bax, COX-2, iNOS, eNOS and HIF-1α. The photographs are representative images taken at a magnification of ×400. The percentage of positively stained cells was also quantified. Data are expressed as the mean ± standard deviation from 10 mice in each group. *P<0.05 vs. control mice. EEHDW, ethanol extract of
EEHDW regulates inflammatory cytokines in serum. The level of IL-1β, IL-6, IL-4, IL-10 and TNF-α in the serum was determined by ELISA assay. The concentrations of the cytokines were determined by comparing to serial dilutions of the purified standards. Data are expressed as the mean ± standard deviation from 10 individual mice in each group. *P<0.05 vs. control mice. EEHDW, ethanol extract of
EEHDW regulates multiple signaling pathways