Colorectal cancer (CRC) is the third most frequent type of cancer in males and the second in females, and the fourth most common cause of oncological mortality worldwide (
Traditional Chinese medicine (TCM) has been used to treat cancer for thousands of years in China. TCM combined with modern treatments may improve symptoms, enhance quality of life, prevent recurrence and metastasis, and prolong patient survival. Additionally, TCM has potential advantages in patients who are not suitable candidates for radiotherapy and chemotherapy (
Hedyotis diffusa Willd (HDW) is a well-known herbal medicine, which exhibits a variety of bioactivities, including anti-inflammatory, antioxidative, immune-modulating and anticancer properties (
Dulbecco's modified Eagle's medium (DMEM), RPMI-1640 medium, fetal bovine serum (FBS), penicillin-streptomycin, 0.25% trypsin-EDTA, DreamTaq Green PCR Master Mix and Pierce Bicinchoninic Acid (BCA) Protein Assay kit were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). MTT was purchased from Beijing Solarbio Science and Technology Co., Ltd. (Beijing, China). Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) Cell Proliferation and Tracking kit and Annexin V-Fluorescein Isothiocyante (FITC) Apoptosis Detection kit were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). RNAiso Plus and PrimeScript RT Reagent kit with gDNA Eraser (Perfect Real Time) was purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Radio immunoprecipitation assay (RIPA) lysis buffer and blocking buffer were purchased from Beyotime Institute of Biotechnology (Haimen, China). Rabbit polyclonal antibodies against β-actin (20536–1-AP), Survivin (0508-1-AP), protein kinase B (AKT; 10176-2-AP) and extracellular-signal-regulated kinase (ERK; 16443-1-AP) were purchased from Proteintech Wuhan Sanying Biotechnology (Wuhan, China). Rabbit polyclonal antibodies against Bcl-2-associated X-protein (Bax; D220073), B-cell lymphoma 2 (Bcl-2; D160117), cyclin-dependent kinase 4 (CDK4; D220396) and proliferating cell nuclear antigen (PCNA; D120014) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Rabbit polyclonal antibodies against Cyclin D1 (sc-753), phospho-AKT (p-AKT; sc-135650) and phospho-ERK (p-ERK; sc-16982-R) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (E030120-01) was purchased from Earthox LLC (Millbrae, CA, USA). Culture flasks and plates were purchased from NEST Biotechnology Co., Ltd. (Wuxi, Jiangsu, China). All other chemicals used, unless otherwise stated, were purchased from Sigma Aldrich; Merck KGaA (Darmstadt, Germany).
HDW was purchased from the Guo Yi Tang Chinese Herbal Medicine Store (Fujian, China). Using a procedure described previously (
The human colorectal cancer cell lines SW620, HT-29, HCT116 and HCT-8 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). SW620 and HT-29 cells were cultured in DMEM supplemented with 10% (v/v) FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. HCT116 and HCT-8 cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) FBS, 100 U/ml penicillin and 100 µg/ml streptomycin. All of the cell lines were cultured at 37°C in a humidified incubator containing 5% CO2.
An MTT assay was used to assess the cell viability. SW620, HT-29, HCT116 and HCT-8 cells were incubated in 96-well plates at a density of 1×105 cells/ml in 100 µl culture medium for 12 h and treated with various concentrations (0, 150, 300 and 500 µg/ml) of HDW extract for 24 h at 37°C. In addition, SW620 cells were treated with various concentrations (0, 12.5, 25, 50, 75 and 100 µg/ml) of CEHDW for 24 h at 37°C in an additional MTT assay. Subsequently, the medium was replaced with 100 µl MTT (0.5 mg/ml in PBS) and cells were incubated at 37°C. After 4 h, the MTT solution was removed and 100 µl DMSO was added to solubilize the purple-blue formazan precipitate. The resulting absorbance was measured at 570 nm using an ELISA reader (model ELX800; BioTek Instruments, Inc., Winooski, VT, USA).
SW620 cells were seeded into 6-well plates at a density of 3×105 cells/ml in 2 ml culture medium. Following treatment with various concentrations (0, 50, 75 and 100 µg/ml) of CEHDW for 24 h at 37°C, cells were harvested and diluted with fresh medium without CEHDW, and subsequently reseeded in 6-well plates at a density of 1,000 cells/well in 2 ml. The medium was replaced with fresh medium every 4 days. After 10 days, colonies were fixed with 10% formaldehyde for 15 min at room temperature, stained with 0.01% crystal violet for 10 min at room temperature and photographed with digital camera.
The CFDA-SE probe was used to determine the cell proliferation (
SW620 cells were treated with various concentrations (0, 50, 75 and 100 µg/ml) of CEHDW for 24 h at 37°C. Cell apoptosis was determined using flow cytometry. Annexin V/PI staining was performed prior to analysis using a FACSCaliber instrument, according to the manufacturer's protocol. In this assay, the annexin V/PI double-negative population indicates viable cells, and the annexin V-positive/PI-negative or annexin V/PI double-positive population represents cells undergoing early or late apoptosis, respectively.
SW620 cells were treated with various concentrations (0, 50, 75 and 100 µg/ml) of CEHDW for 24 h at 37°C. RNA from cell samples was isolated with RNAiso Plus. Oligo-dT-primed RNA (1 µg) was reverse-transcribed using PrimeScript RT Reagent kit with gDNA Eraser (Perfect Real Time), according to the manufacturer's protocol. The resultant cDNA was used to determine the amount of Survivin, PCNA, Cyclin D1, CDK4, Bcl-2 and Bax mRNA using PCR with DreamTaq Green PCR Master Mix. PCR was performed using the 3-step method, with a denaturation stage at 95°C for 30 sec, an annealing stage at an appropriate temperature (55°C for Survivin, CDK4, Bcl-2 and Bax, and 58°C for PCNA, Cyclin D1 and GAPDH) for 30 sec and an extension stage at 72°C for 30 sec for 30 cycles. GAPDH was used as an internal control. The primers were synthesized by Invitrogen; Thermo Fisher Scientific, Inc., and the sequences are listed in
SW620 cells were treated with various concentrations (0, 50, 75 and 100 µg/ml) of CEHDW for 24 h at 37°C. Cells were washed with PBS three times and lysed with RIPA lysis buffer containing EASYpack protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and PhosSTOP (Roche Diagnostics). The concentrations of resultant protein were quantified using the BCA Protein Assay kit. Proteins (50 µg) were separated by SDS-PAGE and transferred onto nitrocellulose membranes (EMD Millipore Corporation, Darmstadt, Germany). The membranes were blocked with blocking buffer for 2 h at room temperature and incubated with antibodies against Survivin, PCNA, Cyclin D1, CDK4, Bcl-2, Bax, AKT, ERK, p-AKT, p-ERK or β-actin (all 1:1,000 dilution) for 16 h at 4°C, and then washed three times with Tris-buffered saline with Tween-20 (TBST), respectively. Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibodies for 1 h at room temperature. Cells were washed again in TBST and membranes were visualized using a BeyoECL Plus instrument. Image Lab™ software (version 3.0; Beyotime Institute of Biotechnology) was used for densitometric analysis and quantification of western blots.
Data were analyzed using the SPSS package for Windows (version 17.0; SPSS Inc., Chicago, IL, USA) using one-way analysis of variance. Fisher's least significant difference and Dunnett's test were used as post-hoc tests. P<0.05 was considered to indicate a statistically significant difference.
The inhibitory effects of CEHDW, PEEHDW, NBEHDW and EAEHDW on the viability of the CRC cell lines were determined using an MTT assay. As presented in
Following CEHDW treatment, SW620 demonstrated the most drug sensitivity as presented in
In order to investigate the underlying molecular mechanism of the proliferation suppressing activity of CEHDW, its effect on apoptosis in SW620 cells was assessed using annexin V/PI staining followed by FACS analysis. As presented in
To further explore the underlying molecular mechanism of the proliferation inhibition effect of CEHDW, RT-PCR and western blot analysis were performed to determine the expression of Survivin, PCNA, Cyclin D1, CDK4, Bcl-2 and Bax at the mRNA and protein levels. RT-PCR results revealed that CEHDW treatment decreased mRNA expression of the pro-proliferative PCNA, Cyclin D1 and CDK4 and anti-apoptotic Bcl-2 and Survivin, while also increasing expression of the pro-apoptotic Bax (
To gain further insight into the association between CEHDW and the proliferation and apoptosis of CRC cells, AKT and ERK signaling molecules were investigated. As presented in
As a multi-component herb, the active ingredients of HDW are distinct between various extracts due to different polarity. Ursolic acid (UA) and oleanolic acid (OA) are hypothesized to be the major active ingredients of HDW and have been demonstrated to possess anticancer activity (
The unlimited proliferation and apoptosis resistance of cancer cells facilitates the continuous growth and progression of tumors (
Numerous disordered genes and aberrant activation of signaling pathways regulate the growth of cancer. For instance, Survivin is a protein that is able to block apoptosis to prevent cell death and prolong cell survival (
The results of the present study demonstrate that CEHDW exhibits a potent inhibitory effect on CRC cell growth, which is mediated by its pro-apoptotic and anti-proliferative activity. Furthermore, the effect of CEHDW is mediated through the AKT and ERK signaling pathways (
The present study was supported by the Research Fund for the Doctoral Program of Higher Education of China (grant no. 20133519110003), Project Funding for the Training of Young and Middle-aged Backbone Personnel of Fujian Provincial Health and Family Planning Commission (grant no. 2016-ZQN-67) and the Developmental Fund of Chen Keji Integrative Medicine (grant nos. CKJ2014013 and CKJ2015007).
colorectal cancer
chloroform extract of Hedyotis diffusa Willd
traditional Chinese medicine
protein kinase B
extracellular-signal-regulated kinase
Effect of polar fractions of HDW on viability of human colorectal cancer cells. (A) SW620, (B) HT-29, (C) HCT116 and (D) HCT-8 cells were treated with CEHDW, PEEHDW, NBEHDW or EAEHDW for 24 or 48 h. Cell viability was determined using an MTT assay. The data were normalized to the viability of the untreated control cells (100%). Data are expressed as the mean ± standard deviation from at least three independent experiments. *P<0.05 vs. untreated control cells. HDW,
CEHDW treatment suppresses viability of SW620 cells. SW620 cells were treated with the indicated concentrations of CEHDW for 24 h. (A) Cell viability was determined using an MTT assay. The data were normalized to the viability of the untreated control cells (100%). Data are expressed as the mean ± standard deviation from at least three independent experiments. *P<0.05 vs. untreated control cells. (B) Morphological changes were observed using phase-contrast microscopy. Images were captured at a magnification of ×200 and are representative of three independent experiments. (C) Cell survival was determined using a colony formation assay. Images are representative of three independent experiments. (D) The proliferation of SW620 cells was determined using a CFDA-SE assay, and the proliferation was calculated by determining the alterations in fluorescence following CEHDW treatments for 24 h. (E) The alterations in fluorescence intensity are represented relative to that of the control. Data are expressed as the mean ± standard deviation from at least three independent experiments. *P<0.05 vs. untreated control cells. CEHDW, chloroform extract of
CEHDW treatment induces apoptosis of SW620 cells. (A) SW620 cells were treated with various concentrations of CEHDW for 24 h, and cells were collected and stained with annexin V/PI followed by FACS analysis. (B) Quantification of FACS analysis. Data are expressed as the mean ± standard deviation from at least three independent experiments. *P<0.05 vs. untreated control cells. CEHDW, chloroform extract of
CEHDW treatment regulates expression of Survivin, PCNA, cyclin D1, CDK4, Bcl-2 and Bax in SW620 cells. Cells were treated with various concentrations of CEHDW for 24 h. (A) mRNA expression and (B) protein expression levels of Survivin, PCNA, Cyclin D1, CDK4, Bcl-2 and Bax were evaluated by RT-PCR and Western blot analysis, respectively. GAPDH and β-actin were used as the internal controls for the RT-PCR and western blotting, respectively. Densitometric analysis. The data were normalized to the mean (C) mRNA or (D) protein expression of untreated control (100%). *P<0.05 vs. internal controls. CEHDW, chloroform extract of
CEHDW treatment inhibits phosphorylation of AKT and ERK in SW620 cells. (A) The levels of AKT and ERK phosphorylation in SW620 cells were determined by western blotting. Images are representative of three independent experiments. (B) Densitometric analysis. The data were normalized to the mean protein expression of untreated control (100%). *P<0.05 vs. untreated control cells. CEHDW, chloroform extract of
Schematic diagram of the potential underlying molecular mechanisms of CEHDW inhibiting proliferation of SW620 cells. HDW inhibits AKT and ERK phosphorylation, which subsequently upregulates Survivin, PCNA, Cyclin D1, CDK4, Bcl-2 and downregulates Bax, promoting survival and proliferation and decreasing apoptosis of SW620 cells. CEHDW, chloroform extract of
Primer sequences for reverse transcription-polymerase chain reaction.
Gene | Primer sequence (5′-3′) | Product size, bp |
---|---|---|
Survivin | Forward, 5′-CTGGGCTATGGGTGAGGTTC-3′ | 686 |
Reverse, 5′-CCCTAGAATCAGACAGCCGAC-3′ | ||
PCNA | Forward, 5′-GCTGACATGGGACACTTA-3′ | 165 |
Reverse, 5′-CTCAGGTACAAACTTGGTG-3′ | ||
Cyclin D1 | Forward, 5′-TGGATGCTGGAGGTCTGCGAGGAA-3′ | 573 |
Reverse, 5′-GGCTTCGATCTGCTCCTGGCAGGC-3′ | ||
CDK4 | Forward, 5′-GGTCAAAGATTTTGCCCAAC-3′ | 138 |
Reverse, 5′-CCGAAGTTCTTCTGCAGTCC-3′ | ||
Bcl-2 | Forward, 5′-CAGCTGCACCTGACGCCCTT-3′ | 231 |
Reverse, 5′-GCCTCCGTTATCCTGGATCC-3′ | ||
Bax | Forward, 5′-TGCTTCAGGGTTTCATCCAGG-3′ | 276 |
Reverse, 5′-TGGCAAAGTAGAAAAGGGCGA-3′ | ||
GAPDH | Forward, 5′-CGACCACTTTGTCAAGCTCA-3′ | 228 |
Reverse, 5′-AGGGGTCTACATGGCAACTG-3′ |
PCNA, proliferating cell nuclear antigen; CDK4, cyclin dependent kinase 4; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein.