Contributed equally
Anti-angiogenic therapy has been successfully applied to treat colorectal cancer (CRC). Ginsenoside Rg3, derived from the Chinese herb ginseng, has anti-vascularization effects and can inhibit tumor growth and metastasis, and can sensitize cancer cells to chemotherapy. Therefore, in the present study, we investigated whether Rg3 could be appropriate for CRC treatment. Growth of CRC cells was assessed by an MTT (methyl thiazolyl tetrazolium) assay
Colorectal cancer (CRC), one of the most commonly registered cancers worldwide, is associated with high mortality, especially for advanced and metastatic patients (
Aberrant angiogenesis is an essential step in the progression of CRC, which provides nutrients and oxygen for the survival, growth and metastasis of the tumor cells (
Ginsenoside, Rg3, one of the major active components of ginseng, displays anti-angiogenesis ability (
Therefore, in the present study, we attempted to verify whether Rg3 could be applied to the treatment of CRC in orthotopic xenograft models; the mechanisms involved were also investigated.
The human CRC cell lines, LoVo, SW620 and HCT116, were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS; Hyclone Laboratories, Inc., Logan, UT, USA), 100 U/m penicillin and 100 mg/ml streptomycin. The cultures were incubated at 37°C in a humidified atmosphere with 5% CO2. Cells were passaged every 2–3 days to obtain exponential growth.
Rg3 was purchased from Shanghai Jinsui Bio-Technology Co., Ltd., (Shanghai, China), fluorouracil (5-FU) was purchased from Shanghai Xudong Haipu Pharmaceutical Co., Ltd., (Shanghai, China) and oxaliplatin was purchased from Jiangsu Hengrui Medicine Co., Ltd. (Lianyungang, China).
Cellular growth was evaluated by an MTT (methyl thiazolyl-tetrazolium) assay (
Cells (1×104/well) were seeded in 96-well plates and grown to confluence. The monolayer culture was artificially scraped/wounded with a sterile micropipette tip to create a denuded zone of constant width. Each well was washed with phosphate-buffered saline (PBS) twice to remove the detached cells. Cell migration to the wounded region was observed using an XDS-1B inverted microscope (MIC Optical and Electrical Instrument, Chongqing, China) and photographed (×40 magnification). Images were captured at 0, 3, 6, 9 and 12 h to monitor the wound healing process. The wound areas were measured using ImageJ (NIH, Bethesda, MA, USA).
The protein levels of CD24, CD44 and EpCAM in colon cancer cells were measured by flow cytometry. Following treatment, the cells were harvested, fixed with 4% paraformaldehyde and were permeabilized using 0.1% Triton X-100. After washing with PBS three times, cells were incubated with anti-CD24 (FITC-conjugated; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-CD44 (PE-conjugated; Santa Cruz Biotechnology), and anti-EpCAM (PerCP-cy5.5-conjugated; Santa Cruz Biotechnology) antibodies, respectively, for 30 min at 4°C. Subsequently, the cells were analyzed using a Beckman Coulter FC500 flow cytometer (Beckman Coulter, Indianapolis, IN, USA).
Cells were harvested, washed twice with 2% fetal bovine serum (FBS)/PBS solution, and resuspended in 100
LoVo and HCT116 cells were used in this assay. LoVo cells were seeded at a density of 250 cells/well and HCT116 cells were seeded at a density of 500 cells/well in 24-well plates and treated with different concentrations of Rg3 3 days later. After treatment for 12 days, the cells were stained with 1% methylrosanilinium chloride and the numbers of visible colonies were counted. The relative clone formation ability was calculated as relative clone formation ability = (mean experimental clone number/mean control clone number) × 100%.
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's protocol. After spectrophotometric quantification, 1
Four-week-old female BALB/c athymic nude mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., (Shanghai, China) and received humane care according to the Soochow University Institutional Animal Care and Treatment Committee.
The orthotopic xenograft model was established as follows: Cells were injected into the left flanks of the mice in a total volume of 100
The mice were observed and weighed once per day during the study period. After 12 days, all mice were euthanized and the tumors were carefully resected and weighed. The tumor tissue was then formalin-fixed, paraffin-embedded, cut into 4-
The resection specimens were fixed in 10% buffered formalin and paraffin-embedded by routine processing. Sections were cut at a thickness of 4
Angiogenesis vascularity was defined as the number of vessels per field counted in the area of the highest vascular density, termed as microvessel density (MVD). Endothelial cells were marked with an anti-CD34 antibody. The CD34 antigen was localized in the cytoplasm and cellular membrane of vascular endothelial cells. Single endothelial cells, endothelial cell clusters, and microvessels in the tumors, clearly separated from adjacent microvessels, were counted. Peritumoral vascularity and vascularity in areas of necrosis were not scored. A vascular lumen was not a requirement for a structure to be counted as a microvessel. Branching structures were counted as one, unless there was a break in the continuity of the vessel, in which case it was counted as two distinct vessels. Areas with a higher density of CD34+ cells and cell clusters relative to adjacent areas were classified as 'hot spots'. The slides were initially screened at low power to identify the areas with the highest number of microvessels or vascularity hot spots. Microvessels were counted in ×400 magnification fields. MVD was defined as the number of manually counted vessel profiles per mm2, taken as the average from three hot-spot counts.
The study material was obtained from 129 patients with metastatic CRC whose tissue samples were available (mean age 62 years, range 26–82 years) and who were treated from January 2007 to July 2016 at the First Affiliated Hospital of Soochow University. Patient characteristics are detailed in
Each experiment was performed at least in triplicate. The results are expressed as the mean ± standard deviation. Kaplan-Meier curves were constructed, and the statistical analysis was carried out using the log-rank test. OS was defined as the time from the diagnosed date to the time of death from any cause. Statistical analysis was performed using an unpaired Student's t-test. P<0.05 was considered significant.
To investigate the effects of Rg3 on the biological behavior of CRC cells, MTT and wound healing assays were performed to evaluate cell growth and migration
It has been well accepted that there is a special subgroup of cancer cells in tumors, termed cancer stem cells (CSCs), which have been proven to preserve the abilities of extensive proliferation, self-renewal, multi-lineage differentiation, drug-resistance, high metastasis and high tumorigenic potential (
CD24, CD44 and EpCAM are the well accepted colorectal CSC markers (
In addition, we further confirmed the stemness of the cells using a plate clone formation assay. LoVo and HCT116 were treated with Rg3 at different low doses and clone formation ability was then evaluated by calculating the visible clones. As shown in
We used an established orthotopic xenograft model to evaluate the antitumor effect of Rg3
Rg3 is believed to inhibit angiogenesis in tumors; therefore, we investigated whether Rg3 could also affect vascularization of the CRC orthotopic xenografts. Endothelial cells in the tissue were positively stained using an anti-CD34 antibody (
To investigate the mechanisms involved in Rg3-repressed angiogenesis, we analyzed the expression of 41 angiogenesis-related genes (
Rg3 impaired the stemness of CRC cells and repressed angiogenesis; therefore, we next investigated whether Rg3 could improve the cytotoxicity of 5-Fu and oxaliplatin, two widely used first line pharmacotherapeutics in clinical treatments.
Separately, Rg3, 5-Fu and oxaliplatin could repress growth of the xenografts (
B7-H1 and B7-H3 belong to B7 family, and play important roles in tumor immune responses by integrating T cell receptor signaling to regulate T cell function. To confirm the relationship between outcomes of CRC patients and the levels of B7-H1 and B7-H3, we collected tissue samples from 129 patients with metastatic CRC, and assessed the levels of B7-H1 and B7-H3 using immunohistochemistry. The representative microscope images of immunohistochemical staining of B7-H1 and B7-H3 are shown in
The levels of B7-H1 and B7-H3 were then evaluated in Rg3-treated CRC orthotopic xenografts. As shown in
Ginsenoside Rg3 exhibits antitumor activity in various tumors (
CSCs, which have aberrant differentiation programs that generate progenitor cancer cells, play a crucial role in the formation of many solid tumors, including CRC. Deregulation of the pathways of self-renewal and differentiation in CSCs result in unlimited self-renewal and a subsequent excess of CSCs, which are the source of tumor formation (
A previous study proved that Rg3 plays a unique role in impairing angiogenesis in tumors by inhibiting the growth of vein endothelial cells (
Using real-time PCR, we further analyzed the expression of angiogenesis-related genes in Rg3-treated CRC cells, and found 22 pro-angiogenic genes
Much effort has been made towards discovering new anti-angiogenic agents. Bevacizumab is a partially humanized monoclonal antibody that binds to VEGF (
Rg3 is able to eliminate chemotherapy-resistant CSCs and preserve its anti-angiogenic ability; therefore, we speculated that Rg3 could be an effective supplement to chemotherapy regimens. 5-Fu is a pyrimidine class antagonist that interferes with the growth of cancer cells, and is currently a cornerstone in the therapeutic regimens of metastatic or advanced-stage CRC. Oxaliplatin, as a third-generation platinum drug, is commonly used in the adjuvant and palliative treatments of CRC. Therefore, we investigated whether Rg3 could be applied to treatments with 5-Fu and oxaliplatin. Using mouse orthotopic xenografts models, we proved that the combination of Rg3 and pharmacotherapies of 5-Fu or oxaliplatin presented stronger cytotoxicity against CRC than either chemotherapy alone. Therefore, Rg3 shows promise for future clinical applications.
Immune escape plays an important role in the development of tumors. B7-H1 is also known as programmed death ligand-1 (PDL-1) or CD274 and B7-H3 is also known as CD206. Both belong to the B7 family, and play important roles in tumor immune responses by integrating T cell receptor signaling to regulate T cell function. B7-H1 and B7-H3 are recognized as predictive and prognostic factors in various cancers. The interaction between B7-H1 and PD-1 inhibits the activation of tumor antigen-specific T cells, and induces immune tolerance of T cells to tumor cells, by which the tumor cells evade immune surveillance (
In the analysis of CRC tissue samples, we confirmed that high expression of B7-H1 and B7-H3 was significantly associated with worse outcomes of patients with CRC, which was consistent with previous studies. Moreover, by examining CRC orthotopic xenografts, we found that Rg3 could decrease the level of B7-H1 and B7-H3, suggesting that Rg3 might be able to promote antitumor immunity. However, considering that the nude mouse is a model of deficient T-cell function, we should be wary of making this conclusion based on the present data. Further investigations are required to confirm the anti-immune escape effect of Rg3.
Taken together, the results of this study showed that Rg3 not only inhibited the growth and migration of CRC, but also strengthened the cytotoxicity of 5-Fu and oxaliplatin
The present study was supported by the National Natural Science Foundation of China (grant nos. 81472296, 81602091, 81402176, 81402093, 81272542 and 81200369), the Six Major Talent Peak Project of Jiangsu Province (grant no. 2015-WSN-022), the Project of Invigorating Health Care through Science, Technology and Education, Jiangsu Provincial Medical Youth Talent (grant no. QNRC2016709), the Project of Jiangsu Provincial Commission of Health and Family Planning (grant no. H201518), the Science and Education for Health Foundation of Suzhou for Youth (grant no. kjxw2015003), and the Science and Technology Project Foundation of Suzhou (grant nos. SYS201464 and SYS201504).
Rg3 represses the growth and migration of colorectal cancer (CRC) cells. (A) Exposure to various concentrations of Rg3 resulted in a dose- and time-dependent growth inhibition on LoVo, SW620 and HCT116 CRC cells. (B) Treatment with 200
Rg3 represses the stemness of colorectal cancer (CRC) cells. (A) Rg3 repressed the expressions of CD24, CD44 and EpCAM at the mRNA level in LoVo cells. Cells were treated with 200
Rg3 represses growth and stemness of colorectal cancer (CRC) cells
Rg3 represses angiogenesis of colorectal cancer (CRC). (A) Immunohistochemical examination of LoVo and SW620 orthotopic xenografts using an anti-CD34 antibody. (B) Rg3 treatment decreased the microvessel density (MVD) levels in both LoVo and SW620 orthotopic xenografts. (C) Real-time PCR showing that Rg3 downregulated the expression of angiogenesis-related genes in LoVo cells. *P<0.05 and **P<0.01 indicate significant differences compared with the respective control groups.
Rg3 strengthens the cytotoxicity of fluorouracil (5-Fu) and oxaliplatin (OXA). (A) Iamges and weights of resected LoVo orthotopic xenografts treated by Rg3 and/or 5-Fu. (B) Photographs and weights of resected LoVo orthotopic xenograft s treated by Rg3 and/or OXA. **P<0.01 indicates significant differences compared with the respective control groups. ##P<0.01 indicates significant differences compared with the respective chemotherapy groups. @@P<0.01 indicates significant differences from the respective Rg3 groups.
Rg3 downregulates the levels of B7-H1 and B7-H3 in colorectal cancer (CRC). (A and B) Immunohistochemical examinations of B7H1 (A) and B7H3 (B) levels in human CRC tissue samples. (C and D) Kaplan-Meier curves for CRC overall survival according to tumor B7-H1 (C) and B7-H3 (D) expression status (low vs. high, P<0.01). (E and F) Immunohistochemical examination of B7H1 (E) and B7H3 (F) levels in LoVo orthotopic xenografts after Rg3 treatment.
Primers used in the present study.
Genes | Sense (5′–3′) | Antisense (5′–3′) | Product size (bp) |
---|---|---|---|
Cancer stem cell markers | |||
CD24 | CAGGGCAATGATGAATGAGAAT | CCTGGGCGACAAAGTGAGA | 233 |
CD44 | GTGATGGCACCCGCTATGTC | AACCTCCTGAAGTGCTGCTCC | 129 |
EpCAM | TAATCGTCAATGCCAGTGTACTTC | GCCATTCATTTCTGCCTTCAT | 100 |
Angiogenesis-related genes | |||
ANG | CAAGAATGGAAACCCTCACAGA | AAATGGAAGGCAAGGACAGC | 246 |
ANGPT1 | AGAGGTCAGAAGAAAGGAGCAAG | GTGAGTCAGAATGGCAGCGAG | 109 |
ANGPT2 | AGAGGAACAAAGGACCGTGAAAG | CTGTCAGATTGCAGTGGGAAG | 91 |
CCL1 | TGGATGGGTTCAGAGGCAC | GCAGGGCAGAAGGAATGGT | 147 |
CCL13 | AGGAGAAGTGGGTCCAGAATTAT | CTCAAATAAACTCCAAACCAGCAAC | 265 |
CCL5 | GAGAAGAAATGGGTTCGGGAGT | AGGACAAGAGCAAGCAGAAACAGGC | 109 |
CCL7 | GCTCAGCCAGTTGGGATTAAT | TCATGGCTTGTTTTCAGTTCAGTC | 164 |
COL18A | TCAGACCACGGCTCGATTTC | CTCAGCTCCCATTGCCTCA | 154 |
CSF3 | CCTTCGCCTCTGCTTTCCA | CGTTCTGCTCTTCCCTGTCTTT | 199 |
CXCL1 | CACCCCAAGAACATCCAAAGT | CCTTCAGGAACAGCCACCA | 210 |
CXCL2 | GCTTATTGGTGGCTGTTCCTG | ACACATTAGGCGCAATCCAG | 101 |
CXCL3 | GCCCAAACCGAAGTCATAGC | GAACCCTCGTAAGAAATAGTCAAAC | 271 |
CXCL5 | ACAGTGCCCTACGGTGGAAGT | CTCATCAAAGCAGGGAGTTCATA | 266 |
EGF | GGGTGACCGTTTGGGAGTT | ATCCACCACGTCGTCCATG | 335 |
PLG | GACTATCTGGTTTGTGGATGCGT | TTCTTCGTCCTCCTCACATTTT | 201 |
FGF-2 | CTGTCTGGTTTGCTGCTGTATCT | GGTTTCTGGGATTTGCTTTATTC | 95 |
FIGF | CATCCCATCGGTCCACTAGGT | CAGCCACCACATCGGAACA | 190 |
FLT4 | GCTGTGCCTGCGACTGTG | CGTGTCCTCGCTGTCCTTGT | 138 |
GM-CSF | ACACTGCTGCTGAGATGAATGA | AAAGGTGATAATCTGGGTTGCA | 218 |
IFNG | TCCAACGCAAAGCAATACATG | TTGCAGGCAGGACAACCAT | 137 |
IGF1 | GGTGGATGCTCTTCAGTTCGT | GCAATACATCTCCAGCCTCCTTAG | 182 |
IL10 | TGGTGAAGGAGGATCGCTAGA | CCTTGATGTCTGGGTCTTGGTT | 204 |
IL1A | TGACGACGCACTTGTAGCCAC | GCCAATGAAATGACTCCCTCT | 111 |
IL1B | ATTTGAGTCTGCCCAGTTCCC | AACCTTTCTGTTCCCTTTCTGC | 207 |
IL2 | CAGTAACCTCAACTCCTGCCAC | CTGGTGAGTTTGGGATTCTTGTA | 227 |
IL4 | CCCCTCTGTTCTTCCTGCTAG | TGTCCTTCTCATGGTGGCTGT | 181 |
IL8 | CTGGGTGCAGAGGGTTGTG | ACTGGCATCTTCACTGATTCTTG | 98 |
KDR | CCCAATAATCAGAGTGGCAGTG | CATAGACATAAATGACCGAGGCC | 163 |
MMP1 | GCTGAAAGTGACTGGGAAACC | TCTTGGCAAATCTGGCGTGT | 166 |
PDFGB | GCTGTTGAGGTGGCTGTAGATG | GTCGTGGCTGGGTTGGAAT | 281 |
PECAM1 | AGGTCAGCAGCATCGTGGT | GTGAAGTTGGCTGGAGGTG | 136 |
PGF | AAGGGAGCTGCTGTCTGCG | CTTGCGGAGTCAGGAGCCCGTAGGT | 192 |
PIGF | ACTGTGCCTTGCTTATGTTTGTT | CCAAGCCATGCTCCTACAAAG | 137 |
PLAUR | GCCGGGCTGTCACCTATT | CCACATCCAGGCACTGTTCTTC | 132 |
TEK | TAACTATGACTGTGGACAAGGGAG | GGCCGAGGTGAAGAGGTTT | 221 |
TGFB1 | CTGGCGATACCTCAGCAACC | CTAAGGCGAAAGCCCTCAAT | 126 |
THPO | TCTCAGACACTGCCGACATCA | GGGCTTTGGGTTTCAGGAGA | 112 |
TIMP1 | GGTTGTGGGACCTGTGGAAGTA | CCAAGATGTATAAAGGGTTCCAAG | 108 |
TIMP2 | CCCCTGTTCGCTTCCTGTATG | GCGTTCCACTCTGGGTCAAAT | 207 |
TPO | AAGCAAGCGCCTGGTGGA | CAGGAAGTTTGGAAAAAGACAGAAG | 156 |
VEGFA | CACCCACCCACATACATACATTT | CCTCCCAACTCAAGTCCACAG | 170 |
Internal control | |||
B2M | TCAAGAAGGTGGTGAAGCAG | AAGGTGGAGGAGTGGGTGTC | 112 |
Clinicopathological features of 129 patients with metastatic CRC.
Clinicopathological features | n | B7H3
|
B7H1
| ||||||
---|---|---|---|---|---|---|---|---|---|
Low (n) | High (n) | χ2 | P-value | Low (n) | High (n) | χ2 | P-value | ||
Sex | |||||||||
Male | 53 | 28 | 25 | 0.574 | 0.449 | 23 | 30 | 1.066 | 0.302 |
Female | 76 | 35 | 41 | 40 | 36 | ||||
Age (years) | |||||||||
>56 | 62 | 35 | 27 | 2.770 | 0.096 | 32 | 30 | 0.368 | 0.544 |
≤56 | 67 | 28 | 39 | 31 | 36 | ||||
BMI | |||||||||
>25 | 65 | 32 | 33 | 0.008 | 0.928 | 28 | 37 | 1.740 | 0.187 |
≤25 | 64 | 31 | 33 | 35 | 29 | ||||
Liver metastasis | |||||||||
No | 51 | 31 | 20 | 4.818 | 0.028 | 34 | 17 | 10.731 | 0.001 |
Yes | 78 | 32 | 46 | 29 | 49 |